Table of contents
Inventories, ecology and ethology (oral communications)
Phlebotomine sand fly fauna in the Federal District of Brazil
Aline Machado Rapello, Andrey José de Andrade, Douglas de Almeida Rocha, Jônatas C.B.
Ferreira, Renata VelôsoTimbó, Marcos Takashi Obara, Rodrigo Gurgel Gonçalves
Identification of host preferences from sand flies captured in endemic leishmaniasis
regions of Ecuador
Estefanía Palacios, Estefanía Espinosa, Gabriela Vela, Denis Augot, Jérôme Depaquit,
Sonia Zapata
Diversity of phlebotomine sand fly assemblage in a tropical forest of southern Mexico
Jorge J. Rodríguez-Rojas, Eduardo A. Rebollar-Téllez
Phlebotomine sand flies (Diptera: Psychodidae) of Texas, United States
Stavana E. Strutz, Cory L. Schlesener, Ryan Baggerly, Jérôme Depaquit, Camille Parmesan
EU-ECDC/EFSA VectorNet Project: Distribution of sand fly species (Diptera: Psychodidae),
community analysis and pathogen detection in Balkans
Vit Dvorak, Ozge Erisoz Kasap, Gizem Oguz, Nazli Ayhan, S. Vaselek, J. Omeragic, I.
Pajovic, F. Martinkovic, O. Mikov, J. Stefanovska, D. Petric, D. Baymak, Yusuf Ozbel,
Jérôme Depaquit, Vladimir Ivovic, Petr Volf, Bulent Alten
First data on sand fly presence in Slovenia
Alexandra Popovič, Eva Praprotnik, Vladimir Ivović
Distribution and travel distance of Phlebotomus papatasi (Diptera: Psychodidae) in
a zoonotic cutaneous leishmaniasis focus, the Northern Negev, Israel
Laor Orshan, Shirly Elbaz, Yossi Ben-Ari
Phlebotomine sand flies (Diptera: Psychodidae) in Morocco: Results of entomological
surveys in five regions of Morocco, Marrakech, Al Haouz-Immintanoute, Tlat Hanchan,
and Had Dra
Mohamed Echchakery, Fouad Ouanaimi, Samia Boussaa, Ali Boumezzough
First Phlebotominae in Botswana
Andreas Krüger
Uncharted phlebotomine sand flies in Gabon
Nil Rahola, Judicael Obame, Boris Makanga, Diego Ayala, Jérôme Depaquit, Christophe
Paupy
Inventories, ecology and ethology (posters)
Ecological niche modelling of phlebotominae sand flies and the relation to the distribution
of cutaneous leishmaniasis in an endemic region of South-Eastern Brazil
Blima Fux, Viviane Coutinho Meneguzzi, Claudiney Biral dos Santos, Carina Margonari
Gustavo Rocha Leite, Aloísio Falqueto
First recording of Sciopemyia vattierae (Diptera: Psychodidae: Phlebotominae) in the
State of Acre, Southeast of the Brazilian Amazon
Márcia Moreira de Ávila, Andreia Fernandes Brilhante, Eunice Aparecida Bianchi Galati,
Reginaldo Peçanha Brazil
Sand fly fauna (Diptera: Psychodidae) in an endemic area of American cutaneous leishmaniasis
of Brazilian Amazon
Márcia Moreira de Ávila, Andreia Fernandes Brilhante, Eunice Aparecida Bianchi Galati,
Reginaldo Peçanha Brazil
Environmental factors and the occurrence of Nyssomyia whitmani and Lutzomyia cruzi
in the Brazilian Central West
Wagner de Souza Fernandes, Anny Keli Aparecida Alves Cândido, Leandro Machado Borges,
Aline Etelvina Casaril, Jucelei de Oliveira Moura Infran, Elisa Teruya Oshiro, Suellem
Petilim Gomes, Antônio Conceição Paranhos Filho, Eliane de Mattos Piranda, Alessandra
Gutierrez de Oliveira
Normalized Difference Vegetation Index (NDVI) in the characterization of sand flies
environments
Helen Rezende de Figueiredo, Anny Keli Aparecida Alves Cândido, César Claudio Cáceres
Encina, Everton Falcão de Oliveira, Jaíza Santos Motta, Jucelei de Oliveira Infran,
Aline Etelvina Casaril, Elisa Teruya Oshiro, Antonio Conceição Paranhos Filho, Alessandra
Gutierrez de Oliveira
Preliminary information on insular sand flies, in Paraná River, Brazil
Barbara Andreo dos Santos, Kárin Rosi Reinhold-Castro, Herintha Coeto Neitzke-Abreu,
Edilson Colhera Cristóvão, Thaís Gomes Versignassi Silveira, Ueslei Teodoro
Entomological surveillance in Leishmania transmission areas on the border between
Brazil and Bolivia
Elizabeth F. Rangel, Adriana Zwetsch, Antônio Luís F. Santana, Rodrigo E. Godoy, Júlia
S. Silva, José O. Silva, Paulo S. Almeida, Zaida E. Yadon
Analysis of feeding preference of sand flies captured in peridomestic habitats in
Panama
Ana Rosa Caballero, Anayansi Valderrama
Why sand fly samplings of a single night are insufficient? An analysis in an urban
area of northeast Argentina based on light traps
Maria Soledad Santini, Maria Soledad Fernández, Maria Gabriela Quintana, Regino Cavia,
Oscar Daniel Salomón
Identification of natural breeding sites of sand flies in an endemic zone of cutaneous
leishmaniasis in Argentina
Mariana Manteca Acosta, Maria Soledad Santini, Maria Eugenia Utgés, Oscar Daniel Salomón
Update on Phlebotominae fauna from the Chaco region, Argentina
Enrique Alejandro Szelag, Jose Dilermando Andrade Filho, Juan Ramón Rosa, María Gabriela
Quintana, Oscar Daniel Salomón
Phlebotominae: spatial-temporal distribution in Corrientes city, Argentina
Pablo E. Berrozpe, Maria Soledad Santini, A.V. Araujo, D. Lamattina, Oscar Daniel
Salomon
Distribution, abundance, and genetic variability of Lutzomyia longipalpis (Diptera:
Psychodidae) in Tartagal city, Salta, Argentina
María Gabriela Quintana, María Soledad Santini, Andrea Gómez Bravo, Ana Denise Fuenzalida,
Mariana Manteca Acosta, Angélica Pech-May, Oscar Daniel Salomón
Updated distribution records of phlebotomine sand flies (Diptera: Psychodidae) of
Spain
Javier Lucientes, Rosa Estrada, Vladimir Oropeza-Velasquez, Sarah Delacour-Estrella,
Pedro María Alarcón-Elbal, José Ignacio Ruiz-Arrondo, Ricardo Molina
Presence of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, in northern Spain:
first record for the Cantabrian Region and second for the Iberian Peninsula
Javier Lucientes, Rosa Estrada, Vladimir Oropeza-Velasquez, Sarah Delacour-Estrella,
Pedro María Alarcón-Elbal, José Ignacio Ruiz-Arrondo, Ricardo Molina
Blood feeding behavior of Phlebotomus perniciosus collected in the human leishmaniasis
focus of southwest Madrid, Spain, during the period 2012–2015
Estela González, Ricardo Molina, Ana Tello, Andrés Iriso, Ángeles Vázquez, Maribel
Jiménez
Ecological aspects of phlebotomine sand flies in Gran Canaria (Canary Islands, Spain)
and risk of Leishmania transmission
Francisco Morillas Márquez, Montserrat Gállego Culleré, M.J. Morillas Mancilla, V.
Diaz Saez, G. Merino Espinosa, Bernard Pesson, C. Muñoz Batet, V. Corpas López, Joaquina
Martín Sánchez
First study of phlebotomine sand flies (Diptera, Psychodidae), vectors of Leishmania
sp., in Castelo Branco District, Central East region, Portugal
M.L. Vilela, Daniela De Pita-Pereira, Thais de Araujo-Pereira, J.M. Cristovão, Carla
Maia, Leana Campino, M. Magalhães, Elisabeth F. Rangel, Maria Odete Afonso
Phlebotomine sand fly species distribution in Croatia and implications in Leishmania
transmission
Sanja Bosnić, Gioia Bongiorno, Cristina Khoury, Trentina Di Muccio, Luigi Gradoni,
Marina Gramiccia, Michele Maroli
Sand flies (Diptera: Psychodidae) of Mediterranean Africa: Inventory and distribution
Adel Rhim, Youmna M’Ghirbi, Jacques Brunhes, Ali Bouattour
An entomological survey for sand flies in two counties of Taiwan
Chizu Sanjoba, Yusuf Ozbel, Jiamei Sun, Mehmet Karakus, Kwang-Poo Chang, Chi-Wei Tsai,
Tai-Chuan Wang, Yoshitsugu Matsumoto
An inventory of phlebotomine sand flies from Cambodia
Thibault Vallecillo, Eva Krupa, Julian Gratiaux, Idiyana Rahima Abdou el Aziz, Kimsour
Kang, Kalian Ouk, Mathieu Loyer, Frédérick Gay, Arezki Izri, Jérôme Depaquit
Laboratory studies (oral communications)
Keynote – The unparalleled efficiency of Leishmania transmission by sand fly bites
Ranadhir Dey, Vanessa Atayde, Amritanshu Joshi, Hamide Aslan, Lais da Silva, Shannon
Townsend, Claudio Meneses, Hira Nakhasi, Martin Olivier, Jesus Valenzuela, Shaden
Kamhawi
A comparison of vector competence in different sand fly species to transmit Leishmania
donovani
Jovana Sadlova, Jitka Myskova, Katerina Pruzinova, K. Homola, M. Yeo, Petr Volf
Leishmania donovani in Phlebotomus argentipes: comparison of development and transmission
of amastigote- and promastigote-initiated infections
Tereza Lestinova, Jovana Sadlova, Jitka Myskova, Jan Votypka, V. Yeo, Petr Volf
Establishing, expanding and certifying a closed working colony of Phlebotomus argentipes
(Diptera: Psychodidae) for xenodiagnostic studies at the kala azar medical research
center, Muzaffarpur, Bihar, India
Puja Tiwary, Shakti Kumar Singh, O.P. Singh, David Sacks, Shyam Sundar, Edgar Rowton,
Phillip Lawyer
Leishmania tropica development in Phlebotomus sergenti: The effect of temperature,
gregarines and geographic origin of sand flies
Jana Hlavacova, Magdalena Jancarova, Jan Votypka, Petr Volf
Lutzomyia umbratilis population captured in the south of the Negro River is refractory
to interaction with Leishmania guyanensis
R.P. Soares, P.M. Nogueira, N.F.C. Secundino, E.F. Santos, C.M. Ríos-Velásquez, F.A.
Pessoa
Lutzomyia migonei is a permissive vector competent for Leishmania infantum
Katerina Pruzinova, Vanessa Cristina Fitipaldi Veloso Guimarães, Jovana Sadlova, Vera
Volfova, Sinval Pinto Brandão Filho, Petr Volf
Leishmania proteophosphoglycans regurgitated from infected sand flies accelerates
dermal wound repair and exacerbates leishmaniasis via insulin-like growth factor 1-dependent
signalling
Emilie Giraud, Tamsyn Derrick, Oihane Martin, Rod J. Dillon, Tereza Leštinová, Petr
Volf, Ingrid Műller, Paul A. Bates, Matthew E. Rogers
Attraction of Lutzomyia sp. (Diptera: Psychodidae: phlebotomine) to volatile organic
compounds from the skin odour of individuals residing in an endemic area for tegumentary
leishmaniasis
D.S. Tavares, P.R.R. Mesquita, V.R. Salgado, F.M. Rodrigues, J.C. Miranda, A. Barral
Blood derived haem as a potential elicitor of anti-leishmanial activity in the gut
of the female sand fly Lutzomyia longipalpis
José R. Silva, Emma Shawcross, Rod J. Dillon
Bacterial communities associated with the digestive tract of wild populations of Lutzomyia
evansi: a vector of Leishmania in Colombia
Rafael José Vivero, Gloria Ester Cadavid-Restrepo, Sandra I. Uribe Soto, Claudia Ximena
Moreno Herrera, Ivan D. Velez
Systematics and phylogeny (posters)
What we know of the classification, evolution, and dispersion of Leishmania parasites
and sand flies?
Mohammad Akhoundi, Katrin Kuhls, Arnaud Cannet, Jan Votýpka, Pierre Marty, Pascal
Delaunay, Denis Sereno
Illustrated identification key to females of Phlebotominae recorded in the Central-West
Region of Brazil using only head and spermathecae
Douglas de Almeida Rocha, Eunice Aparecida Bianchi Galati, Andrey José de Andrade
First record of Psychodopygus francoisleponti Zapata, Depaquit & Léon 2012 (Diptera:
Psychodidae) in Acre State, Brazil
Andreia Fernandes Brilhante, Márcia Moreira de Ávila, Rodrigo Espíndola Godoy, Jailson
Ferreira de Souza, Cristiane de Oliveira Cardoso, Eunice Aparecida Bianchi Galati
Morphological and morphometric characters to distinguish females of three sympatric
species of the genus Trichophoromyia (Diptera: Psychodidae: Phlebotominae) in a Brazilian
Amazonian area
Andreia Fernandes Brilhante, Priscila Bassan Sábio, Eunice Aparecida Bianchi Galati
Metaphase karyotyping organization of Lutzomyia cruzi – preliminary result
Mirella Ferreira da Cunha Santos, Natália Camargo Braga, Douglas Araújo, Lucas Osti
de Freitas, Wagner Fernandes, Elisa Teruya Oshiro, Alessandra Gutierrez de Oliveira
Phylogeography and genetic variability of populations of Lutzomyia longipalpis (Diptera:
Psychodidae) inferred from ND4 gene
Angélica Pech-May, Janine Ramsey, Domingo Liotta, Magali Giuliani, Pablo Berrozpe,
María Gabriela Quintana, Oscar Daniel Salomón
It is time to use a non destructive method for DNA extraction from phlebotomine sand
flies
Julian Gratiaux, Eva Krupa, Thibault Valecillo, Denis Augot, Véronique Lehrter, Jean-Charles
Gantier, Jean-Yves Rasplus, Jérôme Depaquit
Systematics and phylogeny (oral communications)
Keynote – Fossil contribution in the classification of Psychodidae
Dany Azar
Geometric and linear morphometry as a tool for discriminating cryptic female specimens
of Psychodopygus genus Chagasi series
Rodrigo Espíndola Godoy, Elizabeth Ferreira Rangel, Eunice Aparecida Bianchi Galati
LutzodexTM – a digital key for sand flies (Diptera: Phlebotominae) using Android App
Douglas de Almeida Rocha, Maxwell Ramos de Almeida, Andrey José de Andrade
Phlebotomus (Paraphlebotomus) chabaudi Croset, Abonnenc & Rioux, 1970 and Phlebotomus
riouxi Depaquit, Killick-Kendrick & Léger, 1998: synonyms or closely related species?
Véronique Lehrter, Jérôme Depaquit
Beware of Sergentomyia from Southeastern Asia due to untimely synonymies and a need
to describe new species
Jérôme Depaquit
Epidemiology, laboratory studies & modern tools (posters)
The aminosugar galactosamine reduces the trypsinolytic activity of Lutzomyia longipalpis
(Diptera: Psychodidae) and promotes Leishmania mexicana and Leishmania infantum development
within the sand fly gut
T. Lima-Silva, L.K. Castro, A. Bortolini, Marcos H. Pereira, R.N. Araújo, N.F. Gontijo,
Mauricio R.V. Sant’ Anna
Evaluation of different diets for feeding larvae of Nyssomyia neivai (Diptera: Psychodidae:
Phlebotominae)
Antonio Carlos Ferrari Júnior, Kleiton Maciel dos Santos, Magda Freitas Fernandes,
Wedson Desidério Fernandes, Herintha Coeto Neitzke-Abreu, Maria Elizabeth Moraes Cavalheiros
Dorval, Alessandra Gutierrez de Oliveira, Eunice Aparecida Bianchi Galati
Is there oviposition pheromone in Nyssomyia neivai (Diptera: Psychodidae)?
Thais Marchi Goulart, Camila Feitosa de Castro, Wanderson Henrique Cruz Oliveira,
Flávia Benini da Rocha Silva, Vicente Estevam Machado, Dennys Ghenry Samillan Ortiz,
Christiann Davis Tosta, Mara Cristina Pinto
Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum
using a murine model and artificial feeder
Arnaud Cannet, Mohammad Akhoundi, Michel Gregory, Pierre Marty, Pascal Delaunay
Exploring the migration of kinetoplastid parasites in sand flies; why are hypopylarian
parasites backward in coming forward?
Raquel J. Vionette-Amaral, C.T. Nogueira, M. Ginger, Rod J. Dillon
Molecular and serological methods for evaluating blood meal sources in phlebotomines
sand flies (Diptera: Psychodidae)
Mauricio Baum, Edilene Alcântara de Castro, Elias Seixas Lorosa, Mara Cristina Pinto,
Thais Marchi Goulart, Walter Baura Magda Clara Vieira da Costa-Ribeiro
Host feeding preference and molecular screening of Leishmania infection in wild-caught
sand flies in an endemic focus Aydın, Turkey
Mehmet Karakuş, Metin Pekağırbaş, Samiye Demir, Hasan Eren, Seray Töz, Yusuf Özbel
Anthropophilic behaviour and detection of Leishmania spp. in Sergentomyia minuta collected
in the human leishmaniasis focus of Madrid, Spain
Estela González, Ana Tello, Ricardo Molina, Andrés Iriso, Ángeles Vázquez, Maribel
Jiménez
Molecular detection of Leishmania tropica parasites kDNA from naturally infected sand
flies in a new foothill endemic area, southeast Iran
M.D. Moemenbellah-Fard, K. Azizi, M.R. Fakoorziba, T. Dabaghmanesh, M. Ahmadyousefi-Sarhadi
Epidemiology of cutaneous leishmaniasis in the municipality of Brasiléia, Acre State:
Study on the sandy fly fauna
Thais De Araujo-Pereira, Daniela De Pita-Pereira, Mariana Boité, Daniella Alves Martins,
Taina A.N. Da Costa-Rego, Israel De Souza Pinto, Regina Barbosa Moreira, Andressa
A. Fuzari, José Dilermano Andrade-Filho, Marcia Oliveira, Reginaldo Brazil, Constança
Britto
Seasonal dynamics, evolution of Leishmania infantum infection rates, and host-feeding
preferences of Phlebotomus perniciosus in the focus of human leishmaniasis in the
Madrid region, Spain (2012–2014)
Ricardo Molina, Estela González, Sonia Hernández, Inés Martín-Martín, Maribel Jiménez
Molecular tools for the identification of phlebotomine sand flies and detection of
Leishmania spp. parasites in Misiones province, Argentina
Sofía L. Moya, Magalí G. Giuliani, Mariana Manteca Acosta, Oscar D. Salomón, Domingo
J. Liotta
Epidemiology and control (oral communications)
Keynote – Can Sergentomyia spp. play a role in the transmission of human and animal
leishmaniases?
Carla Maia
Molecular analysis of parasite, vector and blood meal DNA from field-caught sand flies
in a Moroccan focus of cutaneous leishmaniasis: Genetically heterogenous Leishmania
tropica in Phlebotomus sergenti as a mono-specific and multi-host feeding vector
Malika Ajaoud, Nargys Es-Sette, Rémi N. Charrel, Abderahmane Laamrani-Idrissi, Myriam
Riyad, Meryem Lemrani
Sand flies abundance, ecology and oviposition preferences in Bihar, India
Rajesh B. Garlapati, Shanta Mukherjee, Rahul Chaubey, Tahfizur Rahaman, Piyoosh Babele,
Akanksha Chowdhury, Suman Prakash, Vinod Kumar, Mukesh Kumar, Gregory Franckowiak,
Dan Somers, Lindsay Briley, Katelyn Wagner, Jenna Hulke, McCall Calvert, Larisa Polyakova,
David Poche, Richard Poche
Keynote – Phlebotomine flies vectors of arbovirus: review and recent data
Rémi N. Charrel
Sand fly fever in Iran: from the past up to the isolation of Dashli virus (a new Sicilian
like virus)
Vahideh Moin-Vaziri, Cigdem Alkan, M. Badakhshan, N. Rahbarian, Xavier de Lamballerie,
Rémi N. Charrel
Sand fly fauna of Palmas, state of Tocantins, Brazil: occurrence in different environments
and natural infection by trypanosomatids
Tâmara Dias Oliveira Machado, Tauana de Sousa Ferreira, Alcinei de Souza Santos Junior,
Nathyla Morgana Cunha Sales, Renata Velôzo Timbó, Tamires Emanuele Vital, Thaís Tâmara
Castro Minuzzi-Sousa, Andrey José de Andrade, Marcos Takashi Obara, Rodrigo Gurgel-Gonçalves
First detection of an unknown Trypanosoma DNA in a phlebotomine sand fly collected
from southern Thailand
Atchara Phumee, Apiwat Tawatsin, Usavadee Thavara, Theerakamol Pengsakul, Suwich Thammapalo,
Jérôme Depaquit, Frédérick Gay, Padet Siriyasatien
Overview and an update of the current knowledge and perspectives on sand fly research
in Mexico
Eduardo A. Rebollar-Téllez, Sergio I. Ibáñez-Bernal, Jorge J. Rodríguez-Rojas, David
A. Moo-Llanes, Angélica Pech-May, Ana C. Montes de Oca-Aguilar, Oscar Mikeri-Pacheco,
Miriam Berzunza-Cruz, Ingeborg Becker-Fauser, Janine Ramsey, Carlos Ibarra-Cerdeña,
Ángel Rodríguez-Moreno, Christopher Stephens, Victor Sánchez-Cordero, Alfredo Castillo-Vera,
Camila González, Wilfredo Arque-Chunga, Javier Escobedo-Ortegón, Silvia Pasos-Pinto,
Laura Sánchez-García
Abundance of Lutzomyia longipalpis (Diptera, Psychodidae) in a kennel and its surroundings
on a highly endemic visceral leishmaniosis area in São Paulo State, Brazil
Andre A. Cutolo, K.B.S. Briguente, G. Motoie, C.E.J. Pigozzi, B.L. Neves, I. Menz,
V.L. Pereira-Chioccola
Canine visceral leishmaniasis in the São Paulo metropotian area dissociated of Lutzomyia
longipalpis: Pintomyia fischeri as potential vector of Leishmania infantum chagasi
Fredy Galvis Ovallos, Eunice A.B. Galati
The emergence and spread of leishmaniases in the borders of Argentina, Brazil, Paraguay
and Uruguay
Oscar Daniel Salomón, María Gabriela Quintana, María Soledad Santini, Nilsa González-Britez,
Nidia Martínez, Antonieta Rojas de Arias, Vanete Thomaz-Soccol, André Luiz Gonçalves,
Alceu Bisetto Júnior, Gabriela Willat, Luis Calegari, Yester Basmadjian, Zaida E.
Yadon, and the IDRC Project #107577 team
Evaluation of the synthetic sex pheromone, (S)-9-methylgermacrene-B, for recruitment
and monitoring of Lutzomyia longipalpis (Diptera: Psychodidae) in an environmental
reserve in Rio de Janeiro, Brazil
Vanessa De Araujo Barbosa, Andressa Alencastre Fuzari Rodrigues, James Gordon Campbell
Hamilton, Reginaldo Peçanha Brazil
Synthetic pheromone and long lasting insecticidal nets (LLINs) as a new control strategy
for Lutzomyia longipalpis (Diptera: Psychodidae), the vector of Leishmania (Leishmania)
infantum
Vanessa De Araujo Barbosa, Cristian Ferreira De Souza, James Gordon Campbell Hamilton,
Reginaldo Peçanha Brazil
Identifying the Yeast community in the sand fly Phlebotomus perniciosus: towards a
strategy for yeast-mediated biological control of vector-borne diseases
Elena Martin, Ilaria Varotto Boccazzi, Gioia Bongiorno, Leone De Marco, Luigi Gradoni,
Nicoletta Basilico, Stefano Comazzi, Irene Ricci, Sara Epis
Targeting sand fly control by the use of systemic insecticides presented to mammalian
reservoir hosts of ZCL and VL: A review of recent studies
Richard M. Poché, Daniel Hartman, Larisa Polyakova, Rajesh Babu Garlapati, David Poché
Systemic insecticides used in dogs: potential candidates for sand fly control?
Sonia Ares Gomez, Albert Picado
Repellent efficacy of a new combination of fipronil and permethrin against the main
vector of canine visceral leishmaniasis in the Americas (Lutzomyia longipalpis)
Andre A. Cutolo, Fredy Galvis Ovallos, E.S. Neves, S. Sossai, M.M.F. Vieira, F.O.
Silva, S.T. Chester, B. Fankhauser, M.D. Soll
Molecular and biochemical characterization of insecticide resistance in Phlebotomus
and Lutzomyia sand flies
Scott A. Bernhardt, David S. Denlinger, Zachariah Gompert, Joseph S. Creswell
Evaluation of the spatial relationship between area of insecticide treatment and location
of Leishmaniasis cases using geographical information systems in Adana, Turkey
Hakan Kavur, Ozan Artun, Kenan Koca
Manipulation of sand fly distributions within the peridomestic environment, and implications
for the control of vector borne disease
Erin Dilger, Graziella Borges-Alves, Vicky Carter, M.G. Herededia, C.M. Nunes, L.M.
Garcez, Reginaldo Peçanha Brazil, James Gordon C. Hamilton, Orin Courtenay
KalaCORE research on the efficacy of control measures against Phlebotomus orientalis,
the principal vector of Visceral Leishmaniasis in East Africa
Dia-Eldin Elnaiem, Omran F. Osman, Wossenseged Lemma, Hanan A.A. Elhadi, Bakri Y.M.
Nour, Noteila M. Khalid, Mulat Yimer, Jorgi Alvar, Orin Courtenay
Visceral Leishmaniasis on the Indian Subcontinent: modelling the dynamic relationship
between vector control schemes and vector life cycles
David M. Poché, William E. Grant, Hsiao-Hsuan Wang
Epidemiology and control (posters)
Dynamics of Laroussius populations and Leishmania infection rate of female sand flies
in an endemic visceral leishmaniasis region, Tunisia, North Africa
Meriem Benabid, Adel Rhim, Rania Ben Romdhane, Manel Zerzri, Aïda Bouratbine
Epidemiologic survey of phlebotomine vectors in a canine leishmaniasis endemic area
in Spain
Rita Velez, C. Ballart, E. Domenech, J. Cairó, Montserrat Portús, Montserrat Gállego
Evidence for stable endemic sand fly populations in the light of migration streams
into Austria
Adelheid G. Obwaller, Mehmet Karakus, Wolfgang Poeppl, Seray Toz, Yusuf Ozbel, Horst
Aspöck, Julia Walochnik
Absence of Leishmania-infected phlebotomines in gallery forests of the Federal District
of Brazil
Aline Machado Rapello, Thaís Tâmara Castro Minuzzi-Sousa, Tamires Emanuele Vital,
Tauana Ferreira, Renata Velôzo Timbó, Andrey José de Andrade, Rodrigo Gurgel Gonçalves
Vectors of the subgenus Leishmania (Viannia) in the Tapajós national forest reserve
located in the lower Amazon Region of Brazil
Adelson Alcimar de Souza, Thiago Vasconcelos dos Santos, Yara Lins Jennings, Edna
Aoba Ishikawa, Iorlando Barata, Maria das Graças Silva, José Aprígio Lima, Jeffrey
Shaw, Ralph Lainson, Fernando Silveira
Natural transovarial and transstadial transmission of Leishmania infantum in Rhipicephalus
sanguineus (Acari: Ixodidae)
Kourosh Azizi, Qasem Asgari, Mohammad Djaefar Moemenbellah-Fard, Aboozar Soltani,
Tahereh Dabaghmanesh
Molecular epidemiology of phlebovirus in four provinces in Morocco
Nargys Es-Sette, Malika. Ajaoud, Rémi N. Charrel, Meryem Lemrani
Phleboviruses circulating in sand flies in Emilia-Romagna region (Northern Italy)
in 2013–2015
Mattia Calzolari, Romeo Bellini, Paolo Bonilauri, Marco Pinna, Francesco Defilippo,
Michele Dottori, Paola Angelini
Isolation of Piura virus, an insect-specific negevirus, from Lutzomyia evansi in Colombia
María Angélica Contreras-Gutiérrez, Hilda Guzman, Marcio R.T. Nunes, Sandra Uribe,
Rafael Vivero, Iván Darío Vélez, Nikos Vasilaskis, Robert B. Tesh
Characterization of susceptibility of Phlebotominae (Diptera: Psychodidae) to the
insecticide, alpha-cypermethrin
Douglas de Almeida Rocha, Andrey José de Andrade, Luciana Moura Reinaldo, Marcos Takashi
Obara
Evaluation of the level of knowledge of public health professionals regarding the
vector of visceral leishmaniasis and its control measures
Anna Ariel Polegato Martins, Mariana Fuga, Alessandra Gutierrez de Oliveira, Mirella
Ferreira da Cunha Santos
Comparison of various recombinant salivary proteins as epidemiological markers for
dog exposure to Phlebotomus perniciosus in different localities in Italy, Portugal
and Spain
Laura Willen, Tatiana Kostalova, Nikola Polanska, Tereza Lestinova, Carla Maia, Petra
Sumova, Michaela Vlkova, Eleonora Fiorentino, Aldo Scalone, Gaetano Oliva, Fabrizia
Veronesi, José Manuel Cristóvão, Orin Courtenay, Lenea Campino, Luigi Gradoni, Marina
Gramiccia, Cristina Ballart, Montserrat Gállego, Petr Volf
Can we identify Leishmania super-spreaders to reduce transmission to sand fly vectors?
Aurore Lison, Steve Reed, Orin Courtenay
Vector control using long-lasting insecticidal nets against kala-azar in Bangladesh
Chizu Sanjoba, Yusuf Ozbel, Bunpei Tojo, Eisei Noiri, Yoshitsugu Matsumoto
Analysis of gene expression in a Lutzomyia longipalpis-derived cell line
Luzia M.C. Cortes, Barbara C.A. Melo, Franklin Souza-Silva, Bernardo A.S. Pereira,
Felio J. Bello, Otacilio C. Moreira, Daniela de Pita-Pereira, Constança Britto, Carlos
R. Alves
Modern tools for sand flies studies (oral communications)
Leishmania HASP and SHERP genes are required for in vivo differentiation, parasite
transmission and host virulence attenuation
Johannes S.P. Doehl, Jovana Sádlová, Hamide Aslan, Sonia Metangmo, Jan Votýpka, Shaden
Kamhawi, Petr Volf, Deborah F. Smith
A glance at what Leishmania infantum chagasi expresses inside Lutzomyia longipalpis
Erich Loza Telleria, Thais Lemos da Silva, João Ramalho Ortigão Farias, Yara Maria
Traub-Csekö
Lutzomyia longipalpis TGF-β has a role in Leishmania infantum chagasi survival in
the vector
Tatiana Di-Blasi, E. Loza-Telleria, C. Marques, R. Macedo-Couto, M. Neves, A.J. Tempone,
M. Ramalho-Ortigão, Yara Maria Traub-Csekö
Novel method to quantify Leishmania metacyclic promastigotes delivered by individual
sand fly bite reveals the efficiency of parasite transmission
Émilie Giraud, Oihane Martin, Matthew Rogers
Blood feeding effect on Phlebotomus papatasi SP15 and SP44 salivary transcripts
Nasibeh Hosseini-Vasoukolaei, Amir Ahmad Akhavan, Mahmood Jeddi-Tehrani, Farah Idali,
Ali Khamesipour, Mohammad Reza Yaghoobi-Ershadi, Shaden Kamhawi, Jesus G. Valenzuela
Phlebotomus orientalis salivary proteins and antigens
Iva Rohousova, Alon Warburg, Petr Volf
Parity/nulliparity and sand fly salivary gland-gene expression
Nasibeh Hosseini-Vasoukolaei, Amir Ahmad Akhavan, Mahmood Jeddi-Tehrani, Farah Idali,
Ali Khamesipour, Mohammad Reza Yaghoobi-Ershadi, Shaden Kamhawi, Jesus G. Valenzuela
Different approaches for further application of ALDI-TOF mass spectrometry for species
identification of phlebotomine sand flies
Kristýna Hlavackova, Vit Dvorak, Petr Halada, Petr Volf
MALDI-TOF protein profiling as a method of choice for high-throughput species identification
of sand flies – an example from the Balkan
Vit Dvorak, Kristýna Hlavackova, Petr Halada, Bulent Alten, Vladimir Ivovic, J. Omeragic,
I. Pajovic, F. Martinkovic, O. Mikov, J. Stefanovska, Petr Volf
New generation sequencing (NGS) as a tool for identification of pooled sand flies
Nazli Ayhan, Vit Dvorak, Cigdem Alkan, Petr Volf, Rémi N. Charrel
Inventories, ecology and ethology (oral communications)
Phlebotomine sand fly fauna in the Federal District of Brazil
Aline Machado Rapello1, Andrey José de Andrade1,2, Douglas de Almeida Rocha1, Jônatas
C.B. Ferreira1, Renata VelôsoTimbó1, Marcos Takashi Obara1, Rodrigo Gurgel Gonçalves1
1Laboratório de Parasitologia Médica e Biologia de Vetores, Área de Patologia, Faculdade
de Medicina, Universidade de Brasília, Brasil
2Laboratório de Parasitologia Molecular, Departamento de Patologia Básica, Universidade
Federal do Paraná, Brasil
aline_rapello@hotmail.com
The Federal District (FD) is located in the Midwest Region (MR) of Brazil, which includes
the States of Goiás, Mato Grosso and Mato Grosso do Sul. The FD has the lowest phlebotomine
species richness of the MR (n = 29), which corresponds to 11% of the 273 registered
species in Brazil. Some of these species such as Lutzomyia longipalpis, Nyssomyia
whitmani and Bichromomyia flaviscutellata have been incriminated as important vectors
of Leishmania species. This study updated the list of phlebotomine species in the
FD by sampling sand flies in gallery forests in the area. Sand flies were captured
in four areas, Água Limpa Farm (FAL), Biological Reserve of Contagem (REBIO), Brasilia’s
National Park (PNB) and Botanic Garden of Brasília (JBB), in May and September, 2014.
The entire capture effort entailed 1,280 HP-light trap nights and 16 Shannon trap
sessions. A total of 1,209 sand flies were captured and 18 species were identified.
The overall capture success was 18%, being higher in FAL (27%) and in September (20%).
Most sand flies were captured in REBIO (n = 664) and in FAL (n = 472). In PNB, 64
sand flies were captured and nine were captured in JBB. The most captured species
was Bi. flaviscutellata (n = 668), followed by Psathyromyia pradobarrientosi (n = 285).
Nyssomyia whitmani (Leishmania brasiliensis vector) and Bi. flaviscutellata (L. amazonensis
vector) were found in three gallery forests sampled. Even with the great capture effort,
Lu. longipalpis was not captured, indicating that this species might be restricted
to domiciliary areas in FD. Brumptomyia guimaraesi, Br. brumpti, Micropygomyia ferreirana,
Pa. pradobarrientosi, Pa. campograndensis and Evandromyia bourrouli were reported
for the first time in FD, expanding the known geographical distributions of these
sand flies in Brazil. Pa. pradobarrientosi is reported for the first time in Brazil.
Now, 35 species are registered in FD. phlebotomine species richness in FD is relevant
when it is compared to Goiás (47 species), a state 59 times bigger than FD (area ~
5.780 Km2) or even when compared to France, where there are six species registered
and whose territory is about 111 times larger than FD.
Identification of host preferences from sand flies captured in endemic leishmaniasis
regions of Ecuador
Estefanía Palacios1, Estefanía Espinosa1, Gabriela Vela1, Denis Augot2, Jérôme Depaquit2,
Sonia Zapata1
1Instituto de Microbiología, Universidad San Francisco de Quito, Ecuador
2Université de Reims Champagne-Ardenne, ANSES, EA4688 – USC « Transmission Vectorielle
et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
szapata@usfq.edu.ec
Leishmaniasis is endemic in 22 of 24 Ecuadorian provinces. The disease is only recorded
under its tegumentary form (cutaneous and mucocutaneous). Some wild animals play a
crucial role in parasite transmission. However, a few reservoirs only have been identified
in the past. Entomological collections were performed between 2012 and 2015 in five
provinces of the northern part of the country where leishmaniasis is endemic (Bolívar,
Esmeraldas, Orellana, Manabí and Pichincha). A total of 3,103 specimens were collected,
of which 7.7% were engorged females. Ten species were collected in total including
known Leishmania vectors. Amplification of the PNOC nuclear gene was performed to
identify the source of blood meals from 50 specimens. The host preferences of sandflies
are: Choloepus hoffmanii, Choloepus hoffmani, Potos flavus, Bos taurus, Pecari tajacu,
Capra hircus, Equus caballus, Cebus capucinus, Tapirus terrestris and Homo sapiens.
Of these, the first two had already been reported as reservoirs, while Cebus capucinus
is suspected to be a reservoir of Leishmania. Moreover, we found anthropophylic behavior
of two sand flies species in the Amazonia basin which are not related with Leishmania
transmission. This study contributes to the understanding of the transmission cycle
of Leishmania and identifying potential new sites of transmission due to the presence
of vectors, reservoirs and humans.
Diversity of phlebotomine sand fly assemblage in a tropical forest of southern Mexico
Jorge J. Rodríguez-Rojas1, Eduardo A. Rebollar-Téllez1,2
1Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento
de Zoología de Invertebrados, Laboratorio de Entomología Médica, Avenida Universidad
S/N, Ciudad Universitaria, 66451 San Nicolás de los Garza, Nuevo León, México
2Universidad Autónoma de Nuevo León, Centro de Investigación en Ciencias de la Salud.
Avenida Carlos Canseco S/N, Colonia Mitras Centro, 64460 Monterrey, Nuevo León, México
jorge.rodriguezrj@uanl.edu.mx; eduardo.rebollart@uanl.edu.mx
Knowledge of biotic components of a particular place must be understood in its true
dimension, so the report of the species present in a given community is of fundamental
value in terms of biodiversity. The main aim of this study was to estimate the diversity
of phlebotomine sand flies in an endemic focus of cutaneous leishmaniasis in southern
Mexico. Field work was carried out in a tropical forest (18°59′46″ N, 088°09′27″ W;
19 m above sea level) from August 2013 to July 2014. Sampling was conducted during
three consecutive nights per month. In each trapping night, 48 traps were operated
from 1800 to 2400 h. The traps were CDC light traps (incandescent and LEDs colors
white, blue, red and green), as well as Disney traps, Shannon traps, Sticky traps
and Delta traps. Collection of sand flies were carried out in four transects, using
each transect as a randomized block design. Specimens were prepared for permanent
slide mounting using Euparal® and subsequent identification was accomplished using
different morphological structures described in taxonomic papers. Measures of alpha
community diversity were based on the quantification of the number of species (species
richness) and the community structure as well as the dominance and evenness. To evaluate
the number of species present in the area, we used the estimators Chao 2, Jacknife
2 and the equation of Clench. Heterogeneity was calculated with Shannon’s entropy
index and true diversity. Dominance was evaluated by Simpson and Berger-Parker, and
also evenness index and Margalef index were evaluated as well. With a total capture
effort of 1,728 night-traps, 16,101 phlebotomine sand flies were collected, representing
two genera and 13 species. The most abundant species were Lu. cruciata (Coquillett)
(42.33%), Lu. shannoni (Dyar) (32.68%), Brumptomyia mesai (Sherlock) (9.75%) and Lu.
ovallesi (Ortíz) (9.03%). Less abundant species were Lu. carpenteri (Fairchild and
Hertig), Lu. cayennensis maciasi (Fairchild and Hertig), Lu. cratifer (Fairchild and
Hertig), Lu. deleoni (Fairchild and Hertig), Lu. olmeca olmeca (Vargas and Díaz-Nájera),
Lu. permira (Fairchild and Hertig), Lu. steatopyga (Fairchild and Hertig), Lu. trinidadensis
(Newstead) and Lu. sp., which altogether accounted for only 5.66% of the total. Two
species were observed (Lu. permira and Lu. sp) as “doubletons”. According to diversity
estimates, 100% (Chao 2) and 85% (Jacknife2) of potential species in the study area
were calculated. Species accumulation curves using Clench’s equation, presented a
good fit to the predictive model (a = 4.47, b = 0.34, r
2 = 0.966, slope = 0.001) with 13 species, representing 100% of the species observed.
Alpha diversity shows that Shannon entropy and true diversity were: H′ = 1.42 and
1D = 4.14 respectively, whereas the Simpson dominance index was λ = 0.31 and Berger-Parker
d = 0.42, while equitativity index was J = 0.56 and Margalef index was D
Mg = 1.24. Four species of medical importance, namely Lu. cruciata, Lu. shannoni,
Lu. ovallesi and Lu. olmeca olmeca were collected and represented 85.81% of the total.
This inventory of phlebotomine sand flies is an important activity to enhance our
knowledge of sand fly assemblages and guilds. The understanding of the population
dynamics of sand flies could be an important factor for the implementation of strategies
for epidemic control of these insect-borne diseases.
Phlebotomine sand flies (Diptera: Psychodidae) of Texas, United States
Stavana E. Strutz1, Cory L. Schlesener1, Ryan Baggerly1, Jérôme Depaquit2, Camille
Parmesan3
1University of Texas at Austin, USA
2University of Reims Champagne-Ardenne, Reims, France
3Plymouth University, UK
stavana@utexas.edu
The phlebotomine sand fly fauna of Texas is poorly documented, which is somewhat surprising
given that Texas has at least eight of the thirteen species known to occur within
the United States and has active cutaneous leishmaniasis cases. The diversity of sand
flies and the presence of leishmaniasis suggest that Texas is an especially important
region to survey. Four of the species found in the United States may be potential
vectors of leishmaniasis and three of these species have been documented within Texas.
The recent spread of cutaneous leishmaniasis into northern portions of Texas and Oklahoma
has increased the importance of documenting sand fly distributions. While it is important
to document species’ ranges for biodiversity purposes, it is even more pressing now
that human health risks have increased. Most surveys have been conducted in southern
and central portions of Texas near historic cutaneous leishmaniasis foci. We surveyed
86 sites across the state and found at least four different genera (Dampfomyia, Lutzomyia,
Micropygomyia, and Psathyromyia). Of these genera, seven species were tentatively
identified with some species identification remaining ambiguous.
EU-ECDC/EFSA VectorNet Project: Distribution of sand fly species (Diptera: Psychodidae),
community analysis and pathogen detection in Balkans
Vit Dvorak1, Ozge Erisoz Kasap2, Gizem Oguz2, Nazli Ayhan3, S. Vaselek4, J. Omeragic5,
I. Pajovic6, F. Martinkovic7, O. Mikov8, J. Stefanovska9, D. Petric4, D. Baymak10,
Yusuf Ozbel11, Jérôme Depaquit12, Vladimir Ivovic13, Petr Volf4, Bulent Alten2,14
1Charles University, Parasitology Department, Prague, Czech Republic
2Hacettepe University, Department of Biology, Ecology Division, Beytepe-Ankara, Turkey
3Aix Marseille University, Medical Faculty, Virology Laboratories, Marseille, France
4Novi Sad University, Faculty of Agriculture, Novi Sad, Serbia
5University of Sarajevo, Veterinary Faculty, Sarajevo, Bosnia & Herzegovina
6Podgorica University, Veterinary Faculty, Podgorica, Montenegro
7University of Zagrep, Veterinary Faculty, Zagrep, Croatia
8National Center for Infectious and Parasitic Diseases, Sofia, Bulgaria
9Ss. Cyril and Methodius University, Faculty of Veterinary Medicine, Skopje, Macedonia
(FYROM)
10National Institute of Public Health, Pristina, Kosovo
11Ege University, Parasitology Department, Izmir, Turkey
12Université de Reims Champagne-Ardenne, Faculty of Pharmacy, ANSES, Reims, France
13Primorska University, Veterinary Faculty, Koper, Slovenia
14Hacettepe University, Institute of Science and Engineering, Beytepe, Ankara, Turkey
kaynas@hacettepe.edu.tr
VectorNet “A European network for sharing data on the geographic distribution of arthropod
vectors, transmitting human and animal disease agents” project is supported by the
EU-ECDC/EFSA consortium and coordinated by Avia-GIS, Belgium. This study shows some
of the results and achievements of the sand fly-team efforts in eight Balkan countries
in 2015 in the framework of the VectorNet Project. Eight countries (Bosnia & Herzegovina,
Montenegro, Croatia, Bulgaria, Macedonia, Serbia, Kosovo, Slovenia), including 267
locations and 36 cities were studied by the sand fly team with the aim of determining
the altitudinal and trans-sectional distribution of species, identifying species and
detecting possible pathogens in the Balkans. Sand flies were collected with light
traps for a total of 951 trap nights during the field missions. From this study, 12
species were identified and a total of 9,096 specimens collected from fieldwork. The
results show that Phlebotomus neglectus (74%) is the dominant species in Balkan countries
and this species was collected from all eight countries together with Ph. tobbi (10%).
These two species comprise 84% of total sand fly abundance. Other species include:
Ph. perfiliewi s.l (6.13%), Sergentomyia minuta (3.56%), Ph. perniciosus (1.57%),
Ph. papatasi (1.35%), Ph. simici (0.9%), Ph. mascitti (0.45%), Ph. sergenti (0.1%),
Ph. alexandri (0.07%), and Ph. balcanicus (0.03%) (found only in Montenegro). We also
calculated some of the important community parameters such as similarity, richness,
species diversity, species evenness and dominance for each country. The most similar
countries in terms of species composition are B&H-Kosovo (0.77), Bulgaria-Kosovo (0.85),
Serbia-Kosovo (0.86), Croatia-Macedonia (0.80) and Bulgaria-Macedonia (0.80). From
these results, it appears that Macedonia and Serbia are ecotones (transition areas)
in the Balkans. The highest species diversities were observed in Macedonia (1.366),
Bulgaria (1.247) and Serbia (1.169), respectively. The lowest was B&H with 0.386 values.
In contrast, B&H, like other less diverse countries, has the highest dominance value
(81.4%). In pathogen detection studies, two novel viruses in B&H and Macedonia and
Leishmania infantum parasites in B&H, Macedonia and Kosovo were detected. To the best
of our knowledge, most of the information derived from this study was new to the Balkan
countries.
First data on sand fly presence in Slovenia
Alexandra Popovič, Eva Praprotnik, Vladimir Ivović
Faculty of Mathematics, Natural Sciences and Information Technologies, University
of Primorska, Slovenia
vladimir.ivovic@famnit.upr.si
Distribution of phlebotomine sand flies around Mediterranean basin is mostly well
investigated and documented. Nevertheless, there are some regions where fauna of these,
medically very important insects was studied many years ago or never. Slovenia is
one of the smallest member countries of EU sited on the south of Austria and on the
east of Italy but despite its small size it has very heterogeneous relief. The northern
part of the country is composed of alpine and the southern part of Mediterranean and
Karst landscape. Being the bridge between eastern and western part of the Northern
Mediterranean, this region hosts unknown sand fly species and these are the first
faunistic data. In order to evaluate Slovenia as a potential leishmaniasis endemic
region we investigated presence of the disease vectors, particularly in the coastal
part of the country. During the high season 2015, 565 specimens were collected and
five species identified. Ph. neglectus and Ph. perniciosus, well known and proven
vectors of L. infantum in the Mediterranean, were the most abundant (76% and 16% respectively).
Ph. papatasi and Ph. mascitti, known and potential vectors of several phleboviruses
and Leishmania parasites, were also present but in smaller numbers (3% and 4.8%).
Medically not important species Sergentomyia minuta was also recorded (0.2%). During
the study period the peak of sand fly abundance was in the beginning of July, gradually
decreasing towards the end of August. It was expected to find all of recorded sand
fly species and the biggest surprise was relatively high abundance of Ph. mascitti
evenly present in all collection sites and always close to animal shelters.
Distribution and travel distance of Phlebotomus papatasi (Diptera: Psychodidae) in
a zoonotic cutaneous leishmaniasis focus, the Northern Negev, Israel
Laor Orshan1, Shirly Elbaz1, Yossi Ben-Ari2
1Laboratory of Entomology, Ministry of Health, Jerusalem, Israel
2Israel Nature and Parks Authority, Jerusalem, Israel
Laor.Orshan@MOH.health.gov.iI
In recent years, endemic transmission of zoonotic cutaneous leishmaniasis (ZCL) has
spread to new regions in Israel. In the southern part of the country, the new foci
of ZCL caused by Leishmania major are located in the cultivated plains of the northwestern
Negev. The agricultural pest Meriones tristrami, the main reservoir animal in this
area, is very common as well as Phlebotomus papatasi, the only known vector species
of L. major in Israel. A sand fly study was conducted in the summer of 2013 in and
around a small cooperative community. The aim was to understand from where and how
far away sand flies reach the residential area. Sand flies were collected from 55
sites in four categories of land use using CO2-baited modified CDC light traps. To
study the flight distances sand flies were marked in the field by spraying the vegetation
in five sites with sugar solutions containing different food dyes. The catch was counted,
identified, Leishmania DNA was detected in pooled female samples and the presence
of marked specimens was noted. Sand flies were abundant throughout the long summer,
showing one seasonal peak in the warmest months August and September. L. major DNA
was detected throughout the season except in June in 30/55 sites sampled. Infection
rates increased towards the end of the season and the estimated risk of exposure was
highest in September. Sand fly densities were low in the residential area and very
high in the surrounding agricultural fields. The maximum dispersal distances were
1.91 km for females and 1.53 km for males. The maximum range recorded for females
was limited by the distances between the marking sites and the most distant trap.
The calculated mean distance traveled indicating the typical dispersal distances of
the population was 0.75 km. The overall results indicated the existence of dense and
mobile sand fly populations. In the agricultural fields there seemed to be numerous
development sources and suitable resting sites for sand flies scattered over large
areas. Sand flies apparently moved in all directions. Typically, Leishmania-infected
Ph. papatasi females probably could reach the residential area from distances greater
than 0.75 km.
Phlebotomine sand flies (Diptera: Psychodidae) in Morocco: Results of entomological
surveys in five regions of Morocco, Marrakech, Al Haouz-Immintanoute, Tlat Hanchan,
and Had Dra
Mohamed Echchakery1, Fouad Ouanaimi1, Samia Boussaa1,2, Ali Boumezzough1
1Équipe Écologie Animale et Environnement-Lab L2E (URAC 32), Université Cadi Ayyad,
Faculté des Sciences Semlalia, Marrakech, Morocco
2I SPITS-Institut Supérieur des Professions Infirmières et des Techniques de Santé,
Marrakech, Morocco
mohamedechchakery@gmail.com
In Morocco, 23 species have been described, which belong into two genera (14 of the
genus Phlebotomus and nine of genus Sergentomyia). Only five species are involved
in the transmission of leishmaniasis in Morocco: Phlebotomus papatasi vector of Leishmania
major, Ph. sergenti vector of L. tropica; Ph. ariasi vector of L. infantum and Ph.
longicuspis, Ph. perniciosus, potential vectors of L. infantum. Cases of cutaneous
leishmaniasis (CL) due to L. tropica have gradually increased in the towns of Al Haouz,
Chichaoua (Imintanoute), Essaouira (Tlat Hanchan-Had Dra) tropica. The objectives
of this research were to inventory the phlebotomine sand flies species, and to identify
the environmental factors that influence the abundance of species in different locations.
Sand flies were captured with CDC light traps and sticky traps (castor-oil paper traps)
placed at intradomicilary and peridomicilary sites for one night (18:00 h until 06:00 h)
every 15 days, from May 2014 to June 2015. A total of 1,678 individuals were captured:
260 in Marrakech, 320 in Al Haouz, 380 in Imintanoute, 435 Tlat Hanchan and 283 in
Had Dra. Relative abundance of species was as follows: In Marrakech, Ph. papatasi
(40.3%), Ph. sergenti (17.7%), Se. fallax (12.9%), Se. minuta (22.6%) and Ph. longicuspis
(6.5%). In Al Haouz, Ph. papatasi (26.90%), Ph. sergenti (46.36%), Ph. perniciosus
(2.43%), Ph. longicuspis (4.3%), Ph. alexandri (1.3%), Se. fallax (9.3%), Se. minuta
(6.2%), Sergentomyia dreyfussi (3.12%). In Imintanoute, Ph. sergenti (56.40%), Ph.
longicuspis (9.71%), Ph. ariasi (6.48%), Ph. papatasi (15.42%), Ph. perniciosus (0.85%),
Ph. alexandri (2.36%), Se. fallax (2.12%), Se. minuta (6.30%), Se. dreyfussi (0.36%).
In Tlat Hanchan, Ph. sergenti (62.40%), Ph. longicuspis (12.60%), Ph. alexandri (4.54%),
Ph. perniciosus (6.72%), Ph. kazeruni (0.26%), Ph. langeroni (0.36%), Ph. bergeroti
(0.16%), Se. minuta (6.6%), Se. antennata (6.36%). In Had Dra, Ph. sergenti (70.6%),
Ph. longicuspis (11.6%), Ph. alexandri (5.6%), Ph. perniciosus (6.16%), Ph. langeroni
(0.12%), Ph. bergeroti (0.13%), Se. minuta (7.6%), Se. antennata (5.22%). The abundance
of species varied significantly. Sand fly population densities were highest in summer
followed by fall, spring and winter. The eco-epidemiological scenarios in our endemic
foci are associated with the presence of domestic mammals and poultry, sources of
blood for sand flies which sustains the vector population and the risk of infection
by Leishmania, The proximity of livestock manure, and cattle and sheep sheds to houses
and Climatic conditions including rainfall, light, temperature, relative humidity,
and air movement, are very important factors influencing the abundance of sand flies
in endemic foci.
First Phlebotominae in Botswana
Andreas Krüger
Military Hospital Hamburg, Dept. Tropical Medicine, Hamburg, Germany, and Okavango
Research Institute, Maun, Botswana
krueger@bnitm.de
Regarding the distribution of phlebotomine sandflies in the Afrotropical region Botswana
(southern Africa) appeared as a blank spot on the map, although there are several
reports from the surrounding countries, namely South Africa, Namibia, Zambia and Zimbabwe.
The lack of data was probably due to the non-endemicity of leishmaniasis in this country.
In a pilot vector survey with a CDC light trap, carried out during the wet season
2014–15 in Maun, northern Botswana, 41 sand fly specimens, belonging to four species,
were detected: Sergentomyia (Grassomyia) inermis and Se. (Sergentomyia) “bedfordi
group”. The latter comprised of specimens of Se. (Ser.) congolensis, Se. (Ser.) caliginosa,
and Se. (Ser.) salisburiensis. None of these species are known vectors of human leishmaniasis
parasites. Regarding the habitat, trap catches were all done beside a termite hill
at a fan height of 50 cm, about 10–200 m off Thamalakane river. Sergentomyia inermis
is a new record for the entire southern African fauna, and it remains to be confirmed
whether older record of the closely related Se. squamipleuris from South Africa are
correct. Molecular taxonomic analyses are underway to further characterize the taxa.
Uncharted phlebotomine sand flies in Gabon
Nil Rahola1,2, Judicael Obame2, Boris Makanga2, Diego Ayala1,2, Jérôme Depaquit3,
Christophe Paupy1
1Unité MIVEGEC, UMR 224-5290 IRD-CNRS-UM, Centre IRD de Montpellier, BP 64501, 34394
Montpellier, France
2Centre International de Recherches Médicales de Franceville (CIRMF), BP 769, Franceville,
Gabon
3Université de Reims Champagne Ardenne, ANSES, SFR Cap santé, EA 4688-USC « Transmission
Vectorielle et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
nil.rahola@ird.fr
Despite the former mention of an autochthonous case of visceral leishmaniasis, Gabon
does not currently constitute an endemic country for this disease. As a result, the
sand fly fauna in this country remains poorly documented and prospected. An exhaustive
review of literature reports only four species in Gabon. Since 2012 we have managed
to collect more than 10,000 sand flies through a large mosquito survey that consisted
in 1850 CDC-miniature light traps installed in two different forest sites and 700
CDC-miniature light traps installed in two different caves. Over 2,000 specimens were
collected in forest sites and 8,000 in caves. In addition, some collections were performed
using other CDC-miniature light traps in anthropic environments such as small savannahs
villages, forest villages and towns. These collections allowed us to expand the current
checklist of phlebotomine sand flies of Gabon from four to 25 species, with already
at least one species new to science (and two others being described). The forest had
the highest species diversity and allowed us to discover and describe a new species
of Phlebotomus with a quite singular morphology (Phlebotomus (Legeromyia) multihamatus)
and also the unknown male of Spelaeomyia moucheti. This discovery led us to perform
a molecular analysis of the whole genus Spelaeomyia. On the other hand, in caves,
sand flies were very abundant but of low diversity. With these new collections we
will be able to bring new morphological data, make some re-descriptions of some specimens
such as the female of Sa. moucheti or Sergentomyia lumsdeni, but also to consider
the possibility of the creation of a new subgenus of the genus Sergentomyia. This
update of the phlebotomine sand flies of Gabon encourages further surveillance in
this country. Even if only one case of leishmaniasis has been reported in Gabon, the
role of sand flies in the transmission of such parasites or other infectious agents,
as well as their trophic preferences, should be evaluated.
Inventories, ecology and ethology (posters)
Ecological niche modelling of Phlebotominae sand flies and the relation to the distribution
of cutaneous leishmaniasis in an endemic region of South-Eastern Brazil
Blima Fux1, Viviane Coutinho Meneguzzi1, Claudiney Biral dos Santos2, Carina Margonari3,
Gustavo Rocha Leite1, Aloísio Falqueto1
1Unidade de Medicina Tropical da Universidade Federal do Espírito Santo. Av. Mal.
Campos 1468, 29043-900, Vitória, ES, Brasil
2Núcleo de Entomologia da Secretaria da Saúde do Estado do Espírito Santo. Av. Mal.
Campos 1468, 29043-900, Vitória, ES, Brasil
3Centro de Pesquisa Rene Rachou. Av. Augusto de Lima 1715, 30190-002, Belo Horizonte,
MG, Brazil
blimafux@yahoo.com.br
Cutaneous leishmaniasis (CL) is caused by a protozoan of the genus Leishmania, which
is transmitted by bites of phlebotomine sand flies. The State of Espírito Santo (ES),
an endemic area in the Southeast of Brazil, has shown considerably high sand fly prevalence
in recent decades, allowing the spread of the disease to unaffected areas. Computer
tools, such as ecologic niche modelling (ENM), are useful for predicting potential
disease risk. In this study, ENM was applied to species of sand flies and CL cases
in ES to identify the principal vector and risk areas of the disease, aiming to understand
the early origin and spread of this disease. Sand flies were collected in 466 rural
localities between 1997 and 2013 during the three hours after evening twilight using
a combination of active and passive capture. Insects were identified to the species
level, and the localities were georeferenced. All autochthonous cases of CL treated
at the University Hospital Cassiano Antonio Moraes (HUCAM) between 1978 and 2013 were
evaluated. Twenty-one climate databases were selected from WorldClim. Maxent was used
to construct potential distribution models for Lu. intermedia, Lu. whitmani, Lu. migonei,
Lu. lenti, Lu. choti and CL cases. ENMTools was used to overlap the species and the
CL case models. The Kruskal-Wallis and qui-quadrado tests were performed, adopting
a 5% significance level. The 249,783 specimens captured represented 43 species. Of
the 1,423 autochthonous cases recorded, 10.8% presented mucosal lesions. The area
under the curve (AUC) was considered acceptable for Lu. intermedia, Lu. whitmani,
Lu. migonei, Lu. lenti, Lu. choti and the CL cases. Topography was considered relevant
to the construction of the models for all the species identified. Lutzomyia intermedia
and Lu. migonei showed relevance with some variables, such as topography, BIO13, BIO12,
and altitude. In order, variables such as topography, BIO13, and BIO15 were important
to L. lenti and L. whitmani. For L. choti, topography, BIO4, BIO18, BIO17, and altitude
presented as relevant variables. The overlay test identified Lu. intermedia as the
main vector of CL in the study area. There is evidence of the existence of a primitive
wild cycle of LTA in Atlantic forest areas in southeastern Brazil. It is possible
that L. braziliensis has been transferred from the Amazon region to the Atlantic forest
areas, for thousands of years through forest corridors linking the two biomes. There
were differences between the contagion regions of patients, indicating that there
was CL expansion to the east of ES, possibly caused by the intensification of migration
to the urban center. Spatial modelling tools enable an analysis of the association
among environmental variables, vector distributions, and CL cases in ES. Further,
they allow better understanding of the factors related to the CL geographical spread
in colonized areas of the Southeast of Brazil.
First recording of Sciopemyia vattierae (Diptera: Psychodidae: Phlebotominae) in the
State of Acre, Southeast of the Brazilian Amazon
Márcia Moreira de Ávila1, Andreia Fernandes Brilhante2, Eunice Aparecida Bianchi Galati2,
Reginaldo Peçanha Brazil3
1Instituto Federal do Acre (IFAC), Brasil
2Faculdade de Saúde Pública da Universidade de São Paulo, Brasil
3Fundação Oswaldo Cruz, Brasil
marcia.avila@ifac.edu.br
Sand flies (Diptera, Psychodidae, Phlebotominae) of various species are implicated
in the transmission of Leishmania protozoans to humans and other vertebrates. Currently,
85 sand fly species representing several genera are known to occur in the Brazilian
state of Acre. In this study, the occurrence of Sciopemyia vattierae (Le Pont & Desjeux,
1992), heretofore restricted to Peru (PE), Colombia and Bolivia, is reported in Brazil.
During collections undertaken with CDC-light traps from December 2014 to January 2016
in forest and peridomilary areas of a rural settlement and in forested areas of an
urban park located in the city of Rio Branco, Acre, three males and five females of
Sc. vattierae were collected. The presence of papillae on flagellomere III distinguishes
Sc. vattierae and Sciopemyia sordellii from the other species of the genus, in which
the papillae are absent. The distinction of males of these two species was based on
morphometric characters (lengths of FI and aedeagal ducts) and morphological characteristics
were used to distinguish the females (In Sc. vattierae, the terminal knob clearly
separated from the spermatheca and whereas it is sessil in Sc. sordellii). Thus, with
this finding the geographical distribution of Sc. vattiearae in South America and
the number of species of sand flies in Acre state are enlarged.
Sand fly fauna (Diptera: Psychodidae) in an endemic area of American cutaneous leishmaniasis
of Brazilian Amazon
Márcia Moreira de Ávila1, Andreia Fernandes Brilhante2, Eunice Aparecida Bianchi Galati2,
Reginaldo Peçanha Brazil3
1Instituto Federal do Acre (IFAC), Brasil
2Faculdade de Saúde Pública da Universidade de São Paulo, Brasil
3Fundação Oswaldo Cruz, Brasil
marcia.avila@ifac.edu.br
Cutaneous leishmaniasis is a zoonosis with wide geographic distribution and with different
Leishmania species as etiological agents. As in most states of the Brazilian Amazon
regions, the emergence of the disease in Acre seems to be related to anthropic actions
and human occupation. This study aimed to identify the sand fly fauna in Rio Branco,
capital of the state of Acre, Brazil. The sand flies were captured in a rural area
and in an urban park of the municipality using light traps (CDC-type) once a month,
from December 2014 to January 2016. We collected a total 2,210 sand flies belonging
to 13 genera and 37 species. The most frequent was Trichophoromyia auraensis (43.2%)
followed in descending or by Trichophoromyia sp. (27.4%), Pressatia calcarata (7.3%),
Pressatia sp. (4.1%), Evandromyia saulensis (6.1%), Ev. walkeri (3.9%), Psychodopygus
carrerai carrerai (3.6%), Bichromomyia flaviscutellata (2.7%), Nyssomyia whitmani
(1.6%) and Migonemyia migonei (0.1%). Of the species found in Rio Branco, three are
known vectors of Leishmania in the Amazon region: Ny. whitmani, Bi. flaviscutellata
and Mg. migonei. The results show that the sand fly fauna is diverse and includes
incriminated and proven vectors of Leishmania. This information will be of use to
the epidemiological surveillance team of the Rio Branco municipality in establishing
control actions in the urban and rural areas where there are reported leishmaniasis
cases.
Environmental factors and the occurrence of Nyssomyia whitmani and Lutzomyia cruzi
in the Brazilian Central West
Wagner de Souza Fernandes1, Anny Keli Aparecida Alves Cândido2, Leandro Machado Borges1,
Aline Etelvina Casaril1,3, Jucelei de Oliveira Moura Infran3, Elisa Teruya Oshiro3,
Suellem Petilim Gomes1, Antônio Conceição Paranhos Filho2, Eliane de Mattos Piranda3,
Alessandra Gutierrez de Oliveira1,3
1Federal University of Mato Grosso do Sul, Postgraduate Program in Infectious and
Parasitic Diseases, Campo Grande, MS, Brazil
2Federal University of Mato Grosso do Sul, Geoprocessing Laboratory for Environmental
Applications, Campo Grande, MS, Brazil
3Federal University of Mato Grosso do Sul, Laboratory of Parasitology/CCBS, MS, Brazil
alessandra.oliveira@ufms.br
Biological and ecological relationships between vectors and their pathogens are important
for understanding the epidemiology of vector transmission disease. Some sand flies
species are vectors of Leishmania, among of them, Lutzomyia cruzi and Nyssomyia whitmani
incriminated as vectors of Leishmania (Leishmania) infantum and Leishmania (Viannia)
braziliensis, respectively. The municipality of Camapuã (Mato Grosso do Sul state,
Brazil) is considered an endemic area for visceral and tegumentary leishmaniasis.
We related, descriptively, the phytophysiognomy of ecotopes and the presence of Lu.
cruzi and Ny. whitmani in Camapuã. A total of 24 captures were carried out, bimonthly,
using Falcão automatic light traps intra and peridomicile, from May 2014 to April
2015. We calculated the Normalized Difference Vegetation Index (NDVI) from images
obtained by LANDSAT 8. These images were classified according to the NDVI as follows,
water (−1.00/+0.00), exposed soil (+0.00/+0.30), pothole vegetation (+0.30/+0.50),
savannah (+0.50/+0.60) and forest (+0.60/+1.00). In total, 2,005 sand flies from nine
species were collected. Nyssomyia whitmani (55.3%) and Lu. cruzi (41.3%) were the
most representative among all collected species. Nyssomyia whitmani predominated in
periurban neighborhoods. In these areas, the NDVI average values were higher than
0.50, demonstrating the presence of dense vegetation surrounding dwellings, consequently,
the capture sites were shaded and there was considerable amount of organic matter
in the soil. Lutzomyia cruzi was more frequent in urban neighborhoods, with NDVI average
values below 0.50. We observed the preference of Ny. whitmani for preserved areas
while Lu. cruzi was more commonly found in urban areas.
Financial support: FUNDECT and CAPES.
Normalized Difference Vegetation Index (NDVI) in the characterization of sand flies
environments
Helen Rezende de Figueiredo1, Anny Keli Aparecida Alves Cândido2, César Claudio Cáceres
Encina2, Everton Falcão de Oliveira2, Jaíza Santos Motta2, Jucelei de Oliveira Infran3,
Aline Etelvina Casaril1,3, Elisa Teruya Oshiro3, Antonio Conceição Paranhos Filho2,
Alessandra Gutierrez de Oliveira1,3
1Federal University of Mato Grosso do Sul, Postgraduate Program in Infectious and
Parasitic Diseases, Campo Grande, MS, Brazil
2Federal University of Mato Grosso do Sul, Geoprocessing Laboratory for Environmental
Applications, Campo Grande, MS, Brazil
3Federal University of Mato Grosso do Sul, Laboratory of Parasitology/CCBS, MS, Brazil
alessandra.oliveira@ufms.br
The biology and ecology of sand flies can suffer environmental influences as well
as its interaction with vertebrate hosts in a particular breeding site. In Brazil,
the State of Mato Grosso do Sul has lost native vegetation due to intense urbanization
and this change has modified the habitat of sand flies and consequently the epidemiology
of leishmaniasis. We characterized the environments of the urbanized area of Aquidauana
(MS) associating land cover changes to the frequency of sand flies. Every fifteen
days, 48 captures were carried out in intra and peridomicile areas with automatic
light traps, from April 2012 to March 2014. We used the Normalized Difference Vegetation
Index (NDVI) obtained from Landsat 5 and 8 to define types of vegetation in the collection
sites and their surroundings. It was collected a total of 3,179 specimens in 10 species.
Lutzomyia longipalpis presented 93.02% of the total, followed by Evandromyia walkeri
(4.25%) and other species 2.74%. Lutzomyia longipalpis was observed in all months
and collection sites including areas with higher degrees of anthropization. The NDVI
was efficient to characterize the environments. It was possible to observe the relationship
between the amount of phytomass by NDVI and frequency, abundance, distribution and
diversity of sand flies.
Financial support: FUNDECT and CAPES.
Preliminary information on insular sand flies, in Paraná River, Brazil
Barbara Andreo dos Santos1, Kárin Rosi Reinhold-Castro1, Herintha Coeto Neitzke-Abreu2,
Edilson Colhera Cristóvão3, Thaís Gomes Versignassi Silveira1, Ueslei Teodoro1
1Programa de Pós-Graduação em Ciências da Saúde, Universidade Estadual de Maringá,
Brasil
2Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Brazil
3Secretaria Municipal de Saúde de Porto Rico, Núcleo de Entomologia, Brasil
karindecastro@yahoo.com.br
Leishmaniasis is a neglected disease with worldwide distribution, caused by the protozoan
parasite Leishmania; which is transmitted to humans by the bites of infected female
phlebotomine sand flies. Nowadays, 988 species of sand flies have been described.
American species comprise 529 of these (512 contemporary and 17 fossils). In Brazil,
19 species are suspected or implicated in the transmission of Leishmania to humans.
Among these, the most common are Nyssomyia neivai (Pinto), Migonemyia migonei (França),
Ny. whitmani (Antunes & Coutinho), Pintomyia pessoai (Coutinho & Barreto) and Pi.
fischeri (Pinto). The aim of this study was to gain preliminary knowledge of these
insects on some islands in the Paraná River that belong to municipalities of Porto
Rico, Querência do Norte, and São Pedro do Paraná, Paraná State, Brazil. Sand fly
collections were made with Falcão light traps in the edges of and within forests,
domestic animal shelters, and domiciles. The collections on Bandeira, Carioca and
Fina Islands were conducted in June, August and October 2012; on Mutum Island, from
October 2013 to October 2014; on Japonesa and São José Catarino Islands, from November
2013 and January, June, and September 2014; on Floresta Island, in December 2013,
January, June, and September 2014; on Chapéu Velho Island, in December 2013, January,
April, and June 2014. In Cruzeiro Island, in January, June, September, and October
2014; on Santa Rosa Island, in December 2013, January, April, and August 2014. On
Mutum Island, which has a large number of permanent residents, the collections were
conducted during one night each month, from 18:00 to 6:00 h, during 13 months. On
the other islands, with smaller numbers of residents, one night’s collection during
each season (fall, winter, spring, and summer), from 19:00 to 7:00 h, during one year.
The sand flies collected were sacrificed with chloroform and placed in tubes containing
80% alcohol for their preservation and subsequent identification. A total of 55,761
sand flies were collected. In 2012, collections included 19,818 specimens of Ny. neivai
were collected, (16,746 females and 3,072 males), and two females of Ny. whitmani
and a female Psathyromyia shannoni (Dyar). In 2013 and 2014, 35,943 specimens of Ny.
neivai were collected (31,414 females and 4,529 males) and one Brumptomyia brumpti
(Larrousse). Ny. neivai was the predominant species, accounting for 99.9% of the collected
specimens. A previous study also reported the capture of Br. cunhai (Mangabeira).
In the inhabited islands, a large number of insects were collected in domestic animal
shelters, which are close to domiciles. The presence of these animals insures blood
sources, which sustain sand fly growth and density in these ecotypes, increasing the
risk for locals and tourists who frequent the islands. The small number of species
collected in relation to the 49 described in the Paraná state may be due to the isolation
of the islands and the inability of other species to adapt to the insular environment.
Ny. neivai is a proven vector of cutaneous leishmaniasis (CL) and also the dominant
species in the studied islands and the most common in all ecotypes, especially in
domestic animal shelters and human domiciles. The capacity of a vector to transmit
pathogens is related to its density. Thus, our results indicate the necessity of strategies
to reduce human-vector contact.
Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Entomological surveillance in Leishmania transmission areas on the border between
Brazil and Bolivia
Elizabeth F. Rangel1, Adriana Zwetsch1, Antônio Luís F. Santana1, Rodrigo E. Godoy1,2,
Júlia S. Silva1, José O. Silva3, Paulo S. Almeida3, Zaida E. Yadon4
1Fundação Oswaldo Cruz (FIOCRUZ), Laboratório Interdisciplinar de Vigilância Entomológica
em Diptera e Hemiptera/Lab. de Referência Nacional em Vigilância Entomológica, Taxonomia
e Ecologia de Vetores de Leishmanioses, Brasil
2Departamento de Epidemiologia, Faculdade de Saúde Pública, Univer. de São Paulo,
SP, Brasil
3Secretaria de Estado de Saúde de Mato Grosso do Sul, Laboratório Regional de Saúde
de Dourados, Brasil
4Communicable Diseases and Health Analysis Department, Pan American Health Organization,
Washington, DC, USA
adrianaz@ioc.fiocruz.br
The leishmaniases are emerging and re-emerging diseases in American countries. In
Brazil, American Cutaneous Leishmaniasis (ACL) is registered in all states and American
Visceral Leishmaniasis (AVL) in most of them, both with significant impact on human
health. Bolivia records leishmaniasis in six of its nine departments. In the valley
of “Yungas”, cutaneous and mucocutaneous leishmaniasis are public health issues, with
20% prevalence. The AVL, rare in the Andean countries, has been recorded in Bolivia
with ten sporadic cases to date. Few studies have been conducted in relation to the
Leishmania vectors in Bolivia, including border areas with Brazil. The study aimed
to identify the species of sand fly vectors on the Brazil-Bolivia border; to know
the spatial distribution associated with the environment and risk factors for transmission.
Monitoring stations were established (MS) on both sides. In Brazil, in Corumbá (AVL
– intense transmission area), for each MS, traps were set in peridomicilary sites
(animal shelters) and in sites next to the forest. Captures (October and November/2015)
were done with CDC light traps installed in areas surrounding houses and close to
forest, and operated for 12 h from the start of the evening, for four consecutive
nights. Six MS were set in a military field, in the main access to Bolivia (City of
Arroyo Concepción), next to the main bridge that has a large flow of people, animals
and vehicles. Four MS were located on small farms, one just a kilometer from the border.
In Bolivia, in the city Arroyo Concepción, Department of Santa Cruz, the only MS studied
was the one close to MS6 (in Brazil), using the same methodology, with two nights
of captures. At this site, CDC light traps were installed in October close to a small
river and in a henhouse; and in November only near the river. The methods for taxonomic
identification follow the proposal by Galati (2003). Preliminary results from 120 h
of captures in Brazil and 36 h in Bolivia include twelve identified sand fly species:
Brumptomyia brumpti, Evandromyia aldafalcaoae, Ev. corumbaensis, Ev. sallesi, Lutzomyia
cruzi, Micropygomyia peresi, Psathyromyia aragaoi, Pa. bigeniculata, Pa. dendrophyla,
Pa. punctigeniculata and Sciopemyia sordellii, totaling 1,394 specimens from Brazil;
and Br. brumpti, Ev. corumbaensis, Lu. cruzi, Mi. peresi and Mi. quinquefer, totaling
22 specimens from Bolivia. Noteworthy is the predominance of Lu. cruzi in all MS,
including the home environment. Lutzomyia cruzi is the vector of AVL in endemic areas
in the Central Region of Brazil. Possibly there is no geographical barrier to sand
fly dispersion across the border between the two countries, may explain the record
of Lu. cruzi on the Bolivian side. It is of particular interest that to date, the
Lu. longipalpis appears to be absent at the monitoring stations in both Brazil and
Bolivia, suggesting that Lu. cruzi is participating in the AVL transmission in Bolivia,
as it is in Brazil.
Financial support: Small Grant Programme Tropical Diseases Research/Pan American Health
Organization 2015. No: IOC-013-PPE-15; Instituto Oswaldo Cruz/ FIOCRUZ, RJ, Brazil;
FAPESP, SP, Brazil.
Analysis of feeding preference of sand flies captured in peridomestic habitats in
Panama
Ana Rosa Caballero1, Anayansi Valderrama2
1Panama Latin University, Panama
2Institute Conmemorative Gorgas, Panama
avalderrama@gorgas.gob.pa
Leishmaniasis comprises a group of parasitic diseases caused by about 13 species and
subspecies of protozoans of the genus Leishmania. Parasites of this genus are transmitted
to humans by the bites of phlebotomine sand flies. In Panama, vector species that
exhibit the highest degree of anthropophily and relevance in the transmission of Leishmania
in Panama include Lutzomyia panamensis, Lu. trapidoi and Lu. ylephiletor. Knowledge
of preferences in their hematophagy can provide indirect information on potential
reservoir hosts and facilitate better understanding of leishmaniasis ecology under
natural conditions. For this, molecular techniques involving the amplification of
a region of cytochrome b to were use to identify mammalian and avian sources of blood
meals in midguts of relevant Lutzomyia species to determine blood-feeding preferences.
An analysis was applied by simple correspondence resulting in a χ2: 57.24, df: 18,
p < 0.05. It was observed that Lu. trapidoi and Lu. panamensis prefer to feed on humans
and swine, while Lu. ylephiletor shows a preference for dogs or other mammals in general.
This preliminary information helps clarify ecological aspects Leishmania transmission
in perdomicilary hábitats, providing valuable knowledge for prevention of leishmaniasis.
Why sand fly samplings of a single night are insufficient? An analysis in an urban
area of northeast Argentina based on light traps
Maria Soledad Santini1, Maria Soledad Fernández2, Maria Gabriela Quintana3,4, Regino
Cavia2, Oscar Daniel Salomón4
1Centro Nacional de Diagnóstico e Investigación en Endemo-epidemias (CeNDIE), ANLIS,
Ministerio de Salud de la Nación, Buenos Aires, and Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Argentina
2Dto. de Ecología, Genética y Evolución, FCEN, UBA and Instituto de Ecología, Genética
y Evolución de Buenos Aires (UBA-CONICET), Buenos Aires, Argentina
3Instituto Superior de Entomología (INSUE-UNT), Tucumán, and CONICET, Argentina
4Instituto Nacional de Medicina Tropical (INMeT), Ministerio de Salud de la Nación,
Puerto Iguazú, Misiones, and CONICET, Argentina
mariasoledadsantini@gmail.com; msantini@anlis.gov.ar
In recent years, in Argentina, entomological surveys to study the occurrence and abundance
of sand flies of epidemiological interest have become more frequent. Indicators of
infestation levels, as the proportion of sampled sites where a particular sand fly
species occurs, or its abundance, were usually estimated, as well as other community
characteristics, such as the number of species present (richness). Some of these surveys
cover large areas, so the number of nights that lights traps remains active, as well
as the total number of light traps, has a cost in relation to human and material resources.
The aim of this work was to study how the estimation of the proportion of sampled
sites where a particular species occurred, the species abundances – trap success –
and the richness changed when the light traps remained active for more than one night.
We analyzed data from four entomological surveys conducted in the city of Puerto Iguazú,
northeastern Argentina (summer 2011, fall and winter 2012 and spring 2014) at 49–55
sampling sites selected with worst-scenario criteria. In each site, a light trap for
capturing Phlebotominae was active during two consecutive nights in summer 2011, fall
and winter 2012 and three consecutive nights in spring 2014. Lutzomyia longipalpis
and Nyssomyia whitmani (Leishmania infantum and L. braziliensis main vectors in the
study area, respectively), were the species more frequently captured. For these vectors,
the estimation in the percentage of sites with presence increased between 23% and
75% (depending on the species and the season) with two nights compared to the estimation
of a single night. The pattern for the average number of captures showed that the
estimation did not change substantially but the coefficients of variation slightly
decreased. The richness increased with the number of trap nights in all samples. Our
results show the importance of avoiding single-night sampling due to the increase
in the estimated percentage of sites with occurrence of vectors with a two-night protocol
and in the richness also with a three night protocol. As more nights are sampled,
the probability of detection of sand flies of different species increases (even medically
important species), and the variability between nights is compensated. However, due
to cost restrictions, complementary studies are in progress to determine the optimal
minimum number of nights to estimate these and other variables in different environments
and seasons. Also, other characteristics of the survey, such as the type of trap (or
traps) used and the main objective of the survey should be considered. Currently in
Argentina, where operationally possible, the general recommendation is a three-night
capture protocol.
Identification of natural breeding sites of sand flies in an endemic zone of cutaneous
leishmaniasis in Argentina
Mariana Manteca Acosta1, Maria Soledad Santini2,3, Maria Eugenia Utgés2, Oscar Daniel
Salomón1,3
1Instituto Nacional de Medicina Tropical (INMeT), Ministerio de Salud de la Nación,
Puerto Iguazú, Misiones, Argentina
2Centro Nacional de Diagnóstico e Investigación en Endemo-epidemias (CeNDIE), ANLIS,
Ministerio de Salud de la Nación, Buenos Aires, Argentina
3Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
mariasoledadsantini@gmail.com
The location of the microhabitats where immature stages of phlebotomine sand flies
develop is one of the least-known aspects of sand fly biology because these stages
are difficult to detect in their terrestrial breeding sites. This project was developed
in a rural area (Misiones, northeastern Argentina) where epidemic outbreaks of cutaneous
leishmaniasis due to Leishmania braziliensis took place during 2004–2005, with Nyssomyia
whitmani as the main vector. The houses in this zone are built typically on stilts,
with floors elevated above the ground, providing a microhabitat with suitable and
stable temperatures, high humidity and a resting place for domestic and even synanthropic
animals. The objective of this work was to detect the presence of natural sand fly
breeding sites both in chicken sheds and under the stilt houses, presumably the more
suitable places for phlebotomine larvae. From October 2015 to April 2016, a total
of 439 emergence traps were set to sample these two microhabitats (designated as “chicken
shed” and “below house”) at each of eight farms. The area of each microhabitat was
divided into squares of 40 × 40 cm, and an emergence trap was set inside each square.
The traps were checked fortnightly and relocated every 40 days within the same square,
considering the average lab-based time spent for development of the immature stages.
Emergent adults were collected in 40 (9%) of the total traps placed, 21 from “chicken
shed” sites and 19 from “below house” sites. We collected 79 sand flies: 77 Ny. whitmani,
one Pyntomyia monticola and one Expapillata firmantoi. The proportion of Ny. whitmani
positive emergence traps in chicken sheds was not significantly higher than the proportion
from “below house” sites (χ2 = 0.82, df = 1, p > 0.05). For the chicken shed microhabitat,
the total density was 2.7 individuals per square, and for the below house microhabitat
only 1.1 individuals per square. However, we did not find significant differences
between the average number of individuals per square in “chicken shed” sites (0.03,
DS 0.04) and “below house” sites (0.02, DS 0.02) (F = 1.26, df = 1, p = 0.28). Nyssomyia
whitmani was collected during each month of the study, with a peak in November–December
2015, consistent with previous adults captures. Along the northeastern border of Argentina,
the chicken shed- and below house-microhabitats are suspected as potential natural
breeding sites for phlebotomine sand flies, mainly Ny. whitmani, vector of Leishmania
braziliensis, the causative agent of cutaneous leishmaniasis in this area.
Update on Phlebotominae fauna from the Chaco region, Argentina
Enrique Alejandro Szelag1,3,4, Jose Dilermando Andrade Filho, Juan Ramón Rosa3, María
Gabriela Quintana1,4,5, Oscar Daniel Salomón1,5
1Instituto Nacional de Medicina Tropical, Misiones, Argentina
2Centro de Referência Nacional e Internacional para Flebotomíneos, Grupo de Estudos
em Leishmaniose, Centro de Pesquisas René Rachou, Belo Horizonte, Brazil
3Instituto de Medina Regional, Chaco, Argentina
4Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET). C.A.B.A,
Argentina
5Instituto Superior de Entomología “Dr. Abraham Willink”, San Miguel de Tucumán. Argentina
odanielsalomon@gmail.com
Previous studies showed that the Phlebotominae fauna in Argentina consists of 31 species
distributed in 14 of the 24 provinces. This work aims to provide an update on the
Phlebotoinae fauna and species distribution in the Chacoan region. Biogeographically,
the Chaco region is divided by its annual rainfall gradient into two clearly differentiated
subregions: the Eastern region (Wet Chaco) and the Western region (Dry Chaco). The
first presents a damp Atlantic rainfall pattern where biomes form parks and savanna
with abundant rainfall in summer and fall. On the other hand, the Western region has
a dry continental and semi-arid rainfall pattern. Captures were conducted monthly
in the two biogeographic regions using CDC miniature light placed 1.5 m above the
ground, and operating for 12 h (from 7 pm to 7 am). In Western Chaco region, captures
were made during the period 2006–2008 for two consecutive nights each month in Nueva
Población (S24°58′18″–W61°21′25″). In the Eastern Chaco region, captures were made
monthly for overnight from 2009 to 2013 in the towns of Margarita Belen (S27°26′34″–W58°54′13″),
Colonia Benitez (S27°19′16″–W58°59′53″), Resistencia (S27°16′48″–W59°1′11″), Tres
Isletas (S26°20′24″–W60°25′52″). In the Transitional Area (Wet Chaco/Dry Chaco) of
Pampa del Indio (S25°52′29.9″; W59°49′25.5″), monthly collections for two consecutive
nights were made from 2013 to 2015. At each collection site, three light traps were
set, one intradomicilary, one extradomicilary and one in a nearby peridomestic habitat.
Species previously recorded for Argentina: Brumptomyia avellari, Br. brumpti, Br.
guimaraesi, Br. pintoi, Evandromyia evandroi, Ev. cortelezzii, Ev. sallesi, Expapillata
firmatoi, Micropygomyia oswaldoi, Mi. peresi, Mi. quinquefer, Migonemyia migonei,
Martinsmyia alphabetica, Nyssomyia neivai, Ny. whitmani, Lutzomyia longipalpis, Oligodontomyia
spp., Pintomyia bianchigalatiae, Pi. fischeri, Pi. pessoai, Pi. misionensis, Pi. monticola,
Pi. torresi, Pi. damascenoi, Psathyromyia pascalei, Pa. lanei, Pa. punctigeniculata,
Pa. baratai, Pa. bigeniculata, Sciopemyia sordellii, Trichophoromyia auraensis. New
records for Argentina: – Ev. aldafalcaoae Margarita Belén: 1 male 06-II-2012; Resistencia:
1 male 04-XII-2013. Pampa del Indio: 1 male, 2 females 10-I-2013; 1 male, 1 female
11-III-2014; 4 males, 3 females 09-IV-2015. Caught in peridomestic site (associated
with kennels and pigsty), intradomestic site (veranda) and edge of forest. – Ev. corumbaensis:
Colonia Benítez: 1 male 19-III-2012; Resistencia: 2 males 09-IV-12 and 17-I-2013.
Western biogeographic region – Nueva Población: 7 males 21-XI-2006, 09-I-2007, 21-III-2007,
01-X-2007 and 13-XII-2007; 8 females 25-IV-2007, 01-X-2007, 13-XII-2007 and 15-XII-2008,
captured in peridomestic and extradomestic sites. – Ev. termitophila: Nueva Población:
1 male 10-X-2008; Tres Isletas: 1 female 04-IV-2011, captured in intradomestic site
and peridomestic sites associated with a pigsty and a henhouse. New record for Chaco:
– Pa. bigeniculata: Margarita Belen, 6 males 15-XII-2011, 2 males 27-III-2013 captured
in peridomestic (pigsty and henhouse) and extradomestic sites. These new records extend
the southern limit of known distribution for these species, including the southernmost
record on the continent, and also extend the total number of known species in the
country to 34.
Financial support: Alberto J. Roemmers Argentina Foundation and Bunge & Born Foundation.
Phlebotominae: spatial-temporal distribution in Corrientes city, Argentina
Pablo E. Berrozpe1,2,5, Maria Soledad Santini2,3,5, A.V. Araujo4,5, D. Lamattina1,
Oscar Daniel Salomon1,2,5
1Instituto Nacional de Medicina Tropical (INMeT), Ministerio de Salud de la Nación,
Puerto Iguazú, Misiones, Argentina
2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
3Centro Nacional de Diagnóstico e Investigación en Endemo-epidemias (CeNDIE), ANLIS,
Ministerio de Salud de la Nación, Buenos Aires, Argentina
4Universidad Nacional del Nordeste, Corrientes
5Red de Investigación de las Leishmaniasis en Argentina
mariasoledadsantini@gmail.com
Reports of cases of visceral leishmaniasis (VL) in canine reservoirs in Corrientes
city prompted a search for sand fly vectors in 2008. The presence of three species
was confirmed: Lutzomyia longipalpis, vector of the causative agent of VL, Nyssomia
neivai and the species complex Evandromyia cortelezzi-sallesi, vectors of the causative
agents of Tegumentary Leishmaniasis (TL). Based on this information, this study was
designed to study the spatio-temporal distribution of the Phlebotominae of Corrientes,
in order to establish seasonal (temporal) and environmental (spatial) profiles sand
fly relative abundance as indicators of risk of exposure to sand fly bites. Relying
on this information, it is hoped that distribution models can be developed and validated
in other endemic areas. Sociodemographic and environmental data (taken from satellite
images) were used to define three strata: rural immersed in a peri-urban matrix, peri-urban
and urban. The sampling period was from September 2014 to August 2015, two samplings
per season, and consisted of placing REDILA-BL minilight traps in six peridomiciliary
sites of each identified stratum for three consecutive, rainless nights. The sites
were selected under “worst case scenario” criteria. Captured sand fly specimens were
kept dry until diaphanized with lactophenol and identified using an optical microscope,
following Galatti (2005). Spring: 662 Phlebotominae, 66% from rural stratum, 28% from
peri-urban and 6% from urban stratum. Species: Rural stratum – Lu. longipalpis (91%),
Nyssomyia. neivai (7.5%), Migonemyia migonei (1.4%) and a single female Ev. cortelezzi-sallesi.
Peri-urban stratum – Lu. longipalpis (83.7%), Ny. neivai (15.7%) and a single female
Mg. migonei. Urban stratum – Lu. longipalpis (100%). Summer: 2208 Phlebotominae, 77%
from rural, 21% from peri-urban and 2% from urban stratum. Species: Rural stratum
– Lu. longipalpis (30%), Ny. neivai (57%), Mg. migonei (10.5%) and Ev. cortelezzi-sallesi
(2%) and 3 female Micropigomyia quinquefer. Peri-urban stratum – Lu. longipalpis (50%),
Ny. neivai (40%), Mg. migonei (9%) and a single female Ev. cortelezzi-sallessi. Urban
stratum – Lu. longipalpis (100%). Autumn: 723 Phlebotominae, 91% from rural stratum,
6% from peri-urban and 3% from urban stratum. Species: Rural stratum – Lu. longipalpis
(11%), Ny. neivai (80%), Mg. migonei (8.5%) and three female Ev. cortelezzi-sallesi.
Peri-urban stratum – Lu. longipalpis (68%), Ny. neivai (25%), Mg. migonei (7%). Urban
stratum – Lu. longipalpis (100%). Winter: 80 Phlebotominae, 56% from rural stratum,
15% from peirurban and 19% from urban stratum. Species: Rural stratum – Lu. longipalpis
(27%) and Ny. neivai (73%). Peri-urban stratum – Lu. longipalpis (80%), Ny. neivai
(20%). Urban stratum – Lu. longipalpis (100%). The spatial distribution reveals that
Lu. longipalpis is the only species present in all three environments. The remaining
species were restricted to peri-urban and mostly to the rural stratum. The temporal
distribution shows a bimodal abundance curve with a peak in late spring and the other
in late summer. This study identifies risk strata and moments of higher probability
of contact between humans and vectors for the study area. Differences in abundances
between environmental strata could indicate that populations of phlebotomine species
behave as metapopulations with recolonizations, in times of greater abundance, from
the rural stratum to the other strata, and that Lu. longipalpis is the species with
best colonization success.
Distribution, abundance, and genetic variability of Lutzomyia longipalpis (Diptera:
Psychodidae) in Tartagal city, Salta, Argentina
María Gabriela Quintana1,2,3,4, María Soledad Santini2,4,5, Andrea Gómez Bravo2,6,
Ana Denise Fuenzalida1,2,3, Mariana Manteca Acosta1,2, Angélica Pech-May1,2,4,7, Oscar
Daniel Salomón1,2,4
1Instituto Nacional de Medicina Tropical-MSN, Argentina
2REDILA, Argentina
3Instituto Superior de Entomología-UNT, Argentina
4CONICET, Argentina
5Centro Nacional de Diagnóstico e Investigaciones en Endemoepidemias-ANLIS-MSN, Argentina
6Fundación Mundo Sano, Argentina
7Instituto Nacional de Salud Pública/Centro Regional de Investigación en Salud Pública,
Tapachula, Chiapas, México
gabrieladealquintana@gmail.com
Lutzomyia longipalpis was first reported in northeastern Argentina in 2004, and from
there began its spread southwards. Three human cases of visceral leishmaniasis (VL)
were reported without the confirmed presence of Lu. longipalpis between 2008 and 2011
in the province of Salta, located in the northwest of the country. In the northeast,
the first human cases were reported in 2006 and to date there have been 153 cases
reported. In 2013, phlebotomine sand fly collections were made in the city of Tartagal,
Salta, when the presence of Lutzomia longipalpis was first reported in northwestern
Argentina. Subsequently, intensive sampling was done in this city in order to study
the distribution pattern and stratify the potential transmission risk of Lesihmania
infantum. The city was divided into 400 × 400-meter quadrants and 66 sites/households
were selected according to “worst case scenario” criteria. Collections were made for
three consecutive nights with CDC-type miniature light traps. In order to correlate
and characterize those sites with high sand fly abundance, the following variables
were explored: micro variables/macro-habitats taken in situ (10 m2), and environmental
variables taken from high-resolution image. At the same time, samples of Lu. longipalpis
males from two different sites approximately 1500 m apart were analyzed to study the
genetic variability of the populations (ND4 gene). A total of 709 sand flies were
collected comprising the following species: Evandromyia cortelezzii-sallesi complex
(486), Migonemyia migonei (139), Lutzomyia longipalpis (82) and Nyssomyia neivai (2).
Of the sampled sites, 54 yielded sand flies, and 16 sites were positive for Lu. longipalpis
(24%). Of the Lu. longipalpis, the ratio of males to females was 1:1; 25% of the females
were gravid, and 27% with blood in abdomen. Regarding the variables explored in relation
to the abundance of Lu. longipalpis, correlation was found with the following: soil
coverage and the number of dogs (micro-habitats); square meters of waterlogged areas
(macro-habitats); and the environmental variables were: normalized difference vegetation
index (NDVI) average of a 50 m2 buffer around the trap, and the normalized difference
water index (NDWI) within 5 m2. Genetic variability between sand fly populations at
the two analyzed sites was high, with 14 different haplotypes, with only one shared
by both populations. These preliminary results reveal an incipient colonization and
interesting population variability that demand intensified, especially in the border
area with Bolivia to and elucidate the route of entry and/or dispersion of the main
vector of Lesihmania infantum in northwestern Argentina.
Financial support: Bunge & Born Foundation, Ministry of Health of Argentina and Mundo
Sano Foundation.
Updated distribution records of phlebotomine sand flies (Diptera: Psychodidae) of
Spain
Javier Lucientes1, Rosa Estrada1, Vladimir Oropeza-Velasquez1, Sarah Delacour-Estrella1,
Pedro María Alarcón-Elbal1,2, José Ignacio Ruiz-Arrondo1, Ricardo Molina3
1Departamento der Patología Animal, Facultad de Veterinaria, Instituto Agroalimentario
de Aragón (IA2) (Universidad de Zaragoza-CITA), Spain
2Universidad Agroforestal Fernando Arturo de Meriño, Jarabacoa, Dominican Republic
3Laboratorio de Entomología Médica, Servicio de Parasitología, Centro Nacional de
Microbiología, Instituto de Salud Carlos III. Madrid, Spain
jlucien@unizar.es
The Spanish Bluetongue Entomological Surveillance Programme was started in 2004 to
monitor bluetongue vector populations at weekly intervals in the country. This viral
disease is mainly transmitted among vertebrate hosts (principally domestic and wild
ruminants) by several species of biting midges of the genus Culicoides (Diptera: Ceratopogonidae).
The traps used were CDC miniature blacklight traps (Model 1212; John W. Hock Company,
Gainesville, FL) were placed outside selected sheep or cattle farms and was run for
one night each week from dusk until dawn. All collected arthropods were transported
to the laboratory and preserved in 70% ethanol. Upon examination, species of medical
and veterinary interest were separated from other insects. The captured phlebotomine
sand flies were stored into separate tubes with 70% ethanol and taxonomic identification
was made according to Lewis (1982) and Gállego et al (1992). Seven species were identified:
Sergentomyia minuta, Phlebotomus papatasi, Ph. perniciosus, Ph. ariasi, Ph. langeroni,
Ph. sergenti and Ph. mascitti. Of these, Ph. perniciosus is the most widespread vector
of Leishmania infantum in the country. In the present work we present the updated
distribution maps of sand flies species for the period 2004–2015 in peninsular Spain
and Balearic Islands. No catches have been recorded in Canary Islands.
Financial support: Ministry of Agriculture, Food and Environment of Spain.
Presence of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908, in northern Spain:
first record for the Cantabrian Region and second for the Iberian Peninsula
Javier Lucientes1, Rosa Estrada1, Vladimir Oropeza-Velasquez1, Sarah Delacour-Estrella1,
Pedro María Alarcón-Elbal1,2, José Ignacio Ruiz-Arrondo1, Ricardo Molina3
1Departamento de Patología Animal, Facultad de Veterinaria, Instituto Agroalimentario
de Aragón (IA2) (Universidad de Zaragoza-CITA), Spain
2Universidad Agroforestal Fernando Arturo de Meriño, Jarabacoa, Dominican Republic
3Laboratorio de Entomología Médica, Servicio de Parasitología, Centro Nacional de
Microbiología, Instituto de Salud Carlos III, Madrid, Spain
jlucien@unizar.es
Framed within the Spanish Bluetongue Entomological Surveillance Programme, CDC miniature
black-light (UV) traps (Model 1212) were used in selected sheep and cattle farms nationwide.
All collected arthropods were transported to the laboratory and preserved in 70% ethanol.
Upon taxonomic identification, species of medical and veterinary interest were separated
from other insects. The captured phlebotomine sand flies were identified according
to Lewis (1982) and Gállego et al. (1992). During an entomological survey carried
out in 2009, one male and five females of Phlebotomus (Transphlebotomus) mascittii
were caught in the province of Cantabria, north-central Atlantic coast of Spain, specifically
in the municipalities of Coo, Cobreces and San Juan de Soba. These are the first documented
records of the occurrence of this species in the Cantabrian Region. Phebotomus mascittii
was originally described from Italy and subsequently found in several Mediterranean
regions from Spain in the west to Turkey in the east, and also in central European
countries such as Switzerland, France, Belgium, Germany, and Austria. In the case
of Spain, seven specimens of this species were collected in 1983 in the provinces
of Barcelona and Gerona in the Autonomous Community of Catalonia, northeastern part
of the Iberian Peninsula. Consequently, the presence of this phlebotomine sand fly
is confirmed in Spain after been found for the second time in the Iberian Peninsula.
Although the species has never been proven to be a vector of Leishmania, the scarcity
of catches in the country makes this a relevant finding.
Financial support: Ministry of Agriculture, Food and Environment of Spain.
Blood feeding behavior of Phlebotomus perniciosus collected in the human leishmaniasis
focus of southwest Madrid, Spain, during the period 2012–2015
Estela González1, Ricardo Molina1, Ana Tello2, Andrés Iriso3, Ángeles Vázquez2, Maribel
Jiménez1
1Medical Entomology Unit, Parasitology Service, National Centre of Microbiology, Institute
of Health Carlos III, Majadahonda, Madrid, Spain
2Zoology and Physical Anthropology Department, Faculty of Biological Science, Complutense
University of Madrid, Madrid, Spain.
3Zoonosis and Biological Risk Section, General Directorate of Public Health, Madrid
Regional Health Authority, Community of Madrid, Madrid, Spain
mjimenez@isciii.es
Seasonal entomological surveys performed monthly for three consecutive years from
May to October at four stations neighboring an urban focus of human leishmaniasis
in Madrid (Spain) have shown that Phlebotomus perniciosus, collected with both sticky
and CDC light traps, is the only vector of Leishmania infantum in the area, with mean
densities reaching 193.60 specimens/m2, as determined by sticky trap collections.
In the same study the analysis of blood preferences of Ph. perniciosus revealed that
they feed mainly on rabbits (Oryctolagus cuniculus) followed by hares (Lepus granatensis).
Direct xenodiagnoses of leishmaniasis carried out on hares and wild rabbits from the
focus proved that these lagomorphs are infective to colonized Ph. perniciosus, although
in different proportions. The host preferences of sand flies collected in an entomological
survey performed during the years 2012 and 2015 along the area of the aforementioned
focus were studied. Phlebotomus perniciosus females collected by both sticky and CDC
light traps in 29 sampling stations located in five municipalities (Fuenlabrada, Leganés,
Getafe, Parla, and Humanes de Madrid) were analyzed. Blood meal identification was
carried out by amplification of a fragment of 359 bp of vertebrate cytochrome b gene,
sequencing and comparison with sequences deposited in the GenBank®. Moreover, RFLP-PCR
of cyt b gene was applied in order to further discriminate between species and, especially,
between mixed blood meals. A total of 535 blood-engorged Ph. perniciosus collected
in Leganés (n = 401), Fuenlabrada (n = 126), Parla (n = 4), Getafe (n = 3), and Humanes
de Madrid (n = 1) were studied. In 456 of them (85.24%) the blood meal was successfully
identified. Blood meal identification shows that female Ph. perniciosus feed predominantly
on hares (n = 341, 63.74%), most of them collected in Leganés. Rabbit blood meals
were also found but in a lower proportion (n = 63, 11.77%). Such results could be
related to the availability and abundance of these lagomorphs within the green area
located close to this town. Also, dog (n = 12, 2.24%), human (n = 11, 2.05%), horse
(n = 8, 1.5%), cat (n = 7, 1.31%), wild boar (n = 4, 0.74%), sheep (n = 3, 0.56%),
rhea (n = 2, 0.38%), and partridge bird (n = 1, 0.19%) blood meals were identified
among all the stations. Mixed blood meals were also detected: hare/rabbit (n = 2,
0.37%), rabbit/turkey (n = 1, 0.19%) and wild boar/chicken (n = 1, 0.19%). These behavioral
observations show evidence of the opportunistic feeding behavior of Ph. perniciosus
female sand flies. The diversity of host-feeding choices observed in this study could
be explained by the availability of the different vertebrates. On one hand, high populations
of hares and rabbits are described in the studied area. On the other hand, some colonies
of feral cats are present in the zone, and other vertebrates such as horse, wild boar,
rhea and chicken, blood of which was detected in sand flies collected in a farm school
located in the urban area of Fuenlabrada town. These results can be explained as a
consequence of different variables: the preference of sand flies for a specific host,
the availability and abundance of the vertebrates, and the accessibility of sand flies
to the different hosts. Feeding on various hosts species indicates an opportunistic
behavior of Ph. perniciosus and can influence Leishmania transmission in the area.
Such information is essential for planning and developing effective strategies to
control vector-borne diseases with the features of leishmaniasis.
Ecological aspects of phlebotomine sand flies in Gran Canaria (Canary Islands, Spain)
and risk of Leishmania transmission
Francisco Morillas Márquez1, Montserrat Gállego Culleré2,3, M.J. Morillas Mancilla1,
V. Diaz Saez1, G. Merino Espinosa1, Bernard Pesson4, C. Muñoz Batet5,6, V. Corpas
López1, Joaquina Martín Sánchez1
1Dpt. Parasitología, Fac. Farmacia, Univ. Granada, Spain
2Lab. Parasitologia, Fac. Farmacia, Univ. Barcelona, Barcelona, Spain
3ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic – Univ. Barcelona,
Barcelona, Spain
4Strasbourg, France
5Servei Microbiologia, Hosp. Santa Creu i San Pau, Barcelona, Spain
6Institut d’Investigació Biomèdica Sant Pau, Barcelona, Spain
fmorilla@ugr.es
There is a rising interest in phlebotomine sand flies due to their role as vectors
of Leishmania and several phleboviruses, the emergence of said infectious agents and
the spread of these Diptera to regions of Europe where their presence had not been
reported before. The presence of phlebotomine sand flies was not reported on the Canary
archipelago until 1982, when a new sand fly species was found on the island of Gran
Canaria (Phlebotomus fortunatarum). Subsequent studies confirmed this endemism and
reported the presence of other species in low numbers. On the other hand, canine leishmaniasis
cases have been described on the Canary Islands for several decades, although always
in dogs that had visited the Iberian Peninsula or other endemic regions, and a human
Toscana virus infection has been reported as well. Moreover, in 2005 a visceral leishmaniasis
case was diagnosed in a patient who had not left Gran Canaria for at least three years.
This background strongly suggests the necessity of a survey of the phlebotomine sand
flies of the Canary Islands, therefore the present study was conducted on the island
of Gran Canaria. Sand fly captures were conducted in September 2013 using sticky-paper
and CDC-light traps. Eight sampling stations with one or two light traps (10 traps/night),
and 575 sticky traps (71.2 m2) were placed over three days in 30 sampling stations
distributed over the main bioclimatic levels of the island. Using the sticky traps,
738 sand flies were captured and identified as (sorted by decreasing abundance): Sergentomyia
fallax (50.5%), Se. minuta (30.4%), Ph. fortunatarum (11.4%), and Ph. sergenti (1.6%).
Forty-four specimens belonging to genus Sergentomyia and one belonging to Phlebotomus
could not be identified to species due to damage. Ph. sergenti is reported for the
first time. Taking into account the bioclimatic levels, most captures were carried
out in the Thermo-Canary (73.0%), followed by the Meso-Canary (25.2%). No sand flies
were captured in CDC-light traps. In conclusions: 1. The only proven vector of L.
tropica on the island is Ph. sergenti, which was captured in low densities (0.17 specimens/m2).
2. Captures were not achieved using CDC-light traps. 3. Other sand fly species with
proven vectorial roles in the Mediterranean or Morocco, such as Ph. perniciosus, Ph.
ariasi, or Ph. papatasi, were not found in this study. 4. As a consequence, risk for
the transmission of Leishmania on the island is very low.
Financial support: Ministerio de Economía y competitividad (Madrid, Spain), project
GL2010-22368-C02-02.
First study of phlebotomine sand flies (Diptera, Psychodidae), vectors of Leishmania
sp., in Castelo Branco District, Central East region, Portugal
M.L. Vilela1, Daniela De Pita-Pereira2, Thais de Araujo-Pereira2, J.M. Cristovão3,
Carla Maia3, Leana Campino3, M. Magalhães4, Elisabeth F. Rangel1, Maria Odete Afonso3
1Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Instituto
Oswaldo Cruz, FIOCRUZ, Brasil
2Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ,
Brasil
3Unidade de Ensino e Investigação em Parasitologia Médica (UEI PM), Global Health
and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical, Universidade
de Nova Lisboa (IHMT, UNL), Portugal
4Laboratório de Informação em Saúde, Instituto de Comunicação e Informação Científica
e Tecnológica em Saúde, LIS/ICICT/FIOCRUZ, Brasil
danypyta@gmail.com
Portugal is an endemic region of human and canine leishmaniasis due to Leishmania
infantum. Dogs are the main reservoir, although other animals have been found infected
with this parasite. Phlebotomus perniciosus and Ph. ariasi are the proven vectors
in well studied leishmaniasis foci in Portugal. However, several regions of the country
have never been studied with regard to phlebotomine sand fly species, as is the case
with Castelo Branco District (CBD) (39°49′19″ N, 7°29′27″ W; 409 m – altitude), in
the east central region, bordering Spain, where there is 12.5% prevalence of canine
leishmaniasis. This study is the first phlebotomine survey in CBD, the main objectives
of which were to characterize the phlebotomine sand fly fauna, including bioecological
and vector aspects. Sand fly captures were performed monthly from 25 May to 1 November,
2015, with CDC light-traps, in domestic and peridomestic biotopes for three-four consecutive
nights in seven of the 11 municipalities of the CBD, including 11 parishes and 24
localities. Of the 24 localities, 13 produced sand flies. The present and visible
animals within 20 m of the traps, in descending order of quantity, were chickens,
ducks, geese, dogs, cats, cattle and goats, rabbits, horses, pigs, pigeons, doves,
swallows, turkeys and one pheasant. The total number of captured sand flies of both
sexes was 151. The relative abundance of each species found was: Ph. perniciosus –
52.99% (80/151), Ph. ariasi – 25.16% (38/151), Ph. sergenti – 21.19% (32/151) and
Sergentomyia minuta – 0.66% (1/151). For females, the numbers gravid and engorged
were recorded as well as monthly densities, and for males, the degree of rotation
of the external genitalia of males was noted. The bodies of females morphologically
identified and preserved, minus genitalia, will be screened for L. infantum infection
by kDNA PCR. Blood meal identification will be accomplished by cytochrome b PCR and
sequencing. To characterize areas of greater risk due to the presence of Leishmania
vectors, demographic, climatic and phlebotomine databases will be integrated in Geographic
Information Systems (GIS) to produce risk maps. Preliminary results confirm that four
of the five sand fly species known to occur in Portugal are present in CBD. Of these,
Ph. sergenti appears to be spreading northward, and Ph. perniciosus and Ph. ariasi
are the most abundant.
Financial support: Programa Ciências sem Fronteiras, CNPq, Brasil.
Acknowledgements: To IOC, FIOCRUZ, Brasil and UEI PM, IHMT, GHTM/FCT, UNL, Portugal,
to the Presidents of the Parishes and the Staff (Sertã, Vila de Rei, and Fundada)
and to the Population of the different Localities, without whom it would not have
been possible to perform the phlebotomine captures.
Phlebotomine sand fly species distribution in Croatia and implications in Leishmania
transmission
Sanja Bosnić1, Gioia Bongiorno2, Cristina Khoury2, Trentina Di Muccio2, Luigi Gradoni2,
Marina Gramiccia2, Michele Maroli2
1Croatian Veterinary Institute, Laboratory of Parasitology, Zagreb, Croatia
2Unit of Vector-borne Diseases & International Health, Istituto Superiore di Sanità,
Rome, Italy
gioia.bongiorno@iss.it
Leishmaniasis was reported as endemic in Croatia as early as 1930, but it is only
since the early 2000s that human (both visceral and cutaneous) and canine leishmaniasis
(CanL) foci have been well documented from coastal and insular territories of central
and southern Dalmatia. Leishmania isolates from infected dogs were identified as belonging
to the Leishmania infantum zymodeme (Z) MON-1, the widespread agent of Mediterranean
zoonotic visceral leishmaniasis. We report on a 2005–2011 phlebotomine survey conducted
to confirm species composition and seasonality in three central-southern counties
of Dalmatia previously investigated (Šibenik-Knin, Split-Dalmatia and Dubrovnik–Neretva);
to assess the current species distribution in the westernmost Istria county, including
the major part of the Istrian peninsula, for which available information dates back
60 years; and to search for natural Leishmania infections. Further, we performed Leishmania
sp. identification from CanL cases originating from the same counties and diagnosed
during 2000–2005. Sand fly collections, carried out in the frame of bluetongue disease
surveillance, used black-light suction traps employed for Culicoides monitoring. Fifteen
localities in four Croatian counties were investigated. Specimens were preserved in
ethanol pending morphological identification and DNA extraction. Canine Leishmania
strains were identified by Multi Locus Enzyme Electrophoresis. Sand flies were trapped
from late May through early December. One thousand specimens were collected and seven
species identified. Among Phlebotomus sand flies, Phlebotomus perfiliewi was the most
abundant species (54.6%), followed by Ph. neglectus (28.2%), Ph. tobbi (8.9%), Ph.
perniciosus (5.4%), Ph. papatasi (0.6%) and Ph. mascittii (0.1%). Sergentomyia minuta
accounted for 2.2%. A difference in prevalence distribution was detected, with Ph.
perniciosus most prevalent in Istrian peninsula (49/56, 87.5%), Ph. perfiliewi in
central counties (542/830, 65.3%) and Ph. neglectus in the southernmost county (79/114,
69.3%). A subset of 369 Larroussius females (76.2% Ph. perfiliewi) organized in pools
(1–27 specimens/pool) according to species, site and date of collection, was analysed
for Leishmania DNA presence. All pools were found negative. A total of 18 canine Leishmania
strains was isolated and identified as belonging to three zymodemes of L. infantum:
15 strains belonged to ZMON-1, the commonest L. infantum zymodeme, two to ZMON-27
and one to ZMON-34, the latter two being uncommon viscerotropic and dermotropic zymodemes
found in other Mediterranean countries. The long-lasting sand fly season (May-December)
and the high Ph. perniciosus prevalence, the main L. infantum vector, in the westernmost
Istriacounty may represent a warning signal of re-emerging leishmaniasis. Leishmania
infantum was confirmed as the CanL agent in Croatia.
Financial support: Partially by the FP7-UE EDENext collaborative project, Contract
Number: 261504.
Sand flies (Diptera: Psychodidae) of Mediterranean Africa: inventory and distribution
Adel Rhim1, Youmna M’Ghirbi1, Jacques Brunhes2, Ali Bouattour1
1Université Tunis El Manar, Institut Pasteur de Tunis, Service d’entomologie médicale,
Tunisie
2IRD, Montpellier, France
adel.rhaim@pasteur.rns.tn
Sand flies are very small bloodsucking Diptera belonging to the suborder Nematocera
and family Psychodidae. They are distributed worldwide but scarce in North America
and Australia and absent in the Nordic countries. They are most common in tropical
and subtropical areas of Africa, South America, Europe and Asia. Phlebotomus and Sergentomyia
species are widely distributed in Mediterranean African countries where they may be
involved in the transmission agents of several human and animal diseases. However,
environmental changes may influence the composition of the sand fly fauna.in a particular
area. For this reason it seemed useful to update the list of sand flies and their
distribution in North African countries (Tunisia, Morocco, Algeria, Libya and Egypt).
To develop software for the identification of sand flies of the Mediterranean Africa
region, it was necessary to update the list of these insects and their distributions.
We based our efforts on bibliographic data available in the international literature,
with a focus on information pertaining to the presence sand fly species in the studied
region, taking into account all recent publications including results of recent investigations
made by our laboratory (Institute Pasteur of Tunis). The result is a list of 36 species
of sand flies belonging to two genera: Phlebotomus (18 species comprising the five
subgenra: Paraphlebotomus, Adlerius, Larroussius, Phlebotomus, and Transphlebotomus)
and Sergentomyia (18 species comprising four subgenera: Sintonius, Parrotomyia, Sergentomyia,
Grassomyia). Furthermore, the most recent work has reported for the first time the
presence of a female of Phlebotomus (Larroussius) chadlii Rioux, Juminer and Gibily
1966 in Tunisia and Algeria, as well as a new record of Phlebotomus (Paraphlebotomus)
riouxi Dépaquit, Léger, Killick-Kendrick, 1988 in Tunisia. The latter species is morphologically
very close to Ph. (Par.) chabaudi Croset, Abonnenc, Rioux 1970 and, in fact, does
not show phylogenetic or biological characteristics that would distinguish it from
Ph. chabaudi, and therefore is considered synonymous. Finally, a confirmation of the
presence in Tunisia of Se. (Sin.) clydei Sinton, 1928, was reported after being absent
for several years. A female of Ph. (Lar.) langeroni Nitzeluscu, 1930 has been reported
in Tunisia; which also occurs in Egypt. Similarly, after a recent inventory of sand
flies in Libya, Se. fallax and Se. antennata were also reported.
An entomological survey for sand flies in two counties of Taiwan
Chizu Sanjoba1, Yusuf Ozbel2, Jiamei Sun1, Mehmet Karakus2, Kwang-Poo Chang3, Chi-Wei
Tsai4, Tai-Chuan Wang4, Yoshitsugu Matsumoto1
1Department of Molecular Immunology School of Agricultural and Life Sciences, University
of Tokyo, Tokyo, Japan
2Department of Parasitology Ege University of Faculty of Medicine, Bornova, Izmir,
Turkey
3Department of Microbiology/Immunology, Chicago Medical School/Rosalind Franklin University
of Medicine and Science, North Chicago, IL 60064, USA
4Department of Entomology, National Taiwan University Taipei, Taiwan
yusuf.ozbel@ege.edu.tr
The first occurrence of phlebotomine sand flies in Taiwan was noted in Tatung Township
of I-lan County in 1940. In 1966, sand fly collections were carried out in 19 townships
in six Counties of Taiwan. Among 1,558 specimens collected, eight species were detected
but only four of them could identified as follows: Phlebotomus kiangsuensis, Sergentomyia
iyengari taiwanensis, Se. barraudi, Se. squamipleuris. A morphological identification
key for Taiwanese sand flies was also prepared and published in 1970. In another study
completed in 1996, 979 sand fly specimens were collected from 16 townships of nine
counties and the same Sergentomyia species were found while no Phlebotomus was caught.
In the latest study carried out in Fushin Township of Taoyuan County, 102 specimens
were collected and only Se. iyengari taiwanensis was found. Researchers also checked
the females for the presence of Leishmania parasites but none of them were positive.
Besides sand fly studies, three indigenous cutaneous leishmaniasis cases were reported
from different counties in 2008 and 2009. In the present study, we aimed to carry
out an entomological survey for morphological and molecular identification of sand
fly species in the areas where leishmaniasis cases were reported previously. The sand
fly collection was done in the Liquid area of Kaohsiung and Fushin county of Taoyuang
between 11 and 16 September 2014 using CDC Light traps, mouth aspirators and sticky
papers coated with castor oil. These areas were chosen because of their favorable
topology and climate as habitats of sand flies and because of the report of patients
at the site of collection in Taoyuang. The identification was done based on morphological
characters, as well as molecular techniques using the cytochrome b gene region. The
female specimens were first kept in Marc André solution overnight for clarification
and after separating the head from the body, slides were prepared for morphological
identification. Male specimens were directly mounted after separating the head from
the body. The DNA isolation was done using three legs of the specimens. A total of
66 sand fly specimens (36 male & 30 female) was collected in five nights of trapping.
The preliminary results of morphological and molecular identification showed that
one Phlebotomus species, Ph. kiangsuensis, and three Sergentomyia species are present
in the study areas. The identification studies are still ongoing. In conclusion, because
Taiwan has a tropical climate and many places are suitable for breeding of sand flies,
the studies related to sand fly fauna and natural Leishmania infection need to be
continued in “endemic” areas of Taiwan
An inventory of phlebotomine sand flies from Cambodia
Thibault Vallecillo1, Eva Krupa1, Julian Gratiaux1, Idiyana Rahima Abdou el Aziz1,2,
Kimsour Kang2, Kalian Ouk3, Mathieu Loyer1, Frédérick Gay4, Arezki Izri2, Jérôme Depaquit1
1Université de Reims Champagne Ardenne, ANSES, SFR Cap santé, EA 4688 – USC « Transmission
Vectorielle et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
2Parasitologie-Mycologie, CHU Avicenne, Université Paris 13, Bobigny, UMR 190, Unité
des Virus Émergent, Marseille, France
3Université Chenla, Phnom Penh, Cambodia
4Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, AP-HP, Groupe Hospitalier
Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France
thibault.vallecillo@gmail.com
The sand fly fauna of Cambodia remains poorly documented. Because of the lack of local
Leishmania transmission, very few studies related to Cambodian phlebotomine sand flies
are available in the literature. Only six species of sand flies have previously been
recorded: Sergentomyia barraudi, Se. pertubans, Se. bailyi, Se. sylvatica, Se. khawi
and Grassomyia indica. None belong to the genus Phlebotomus. However, in connection
with the emergence of the first autochthonous cases in Thailand and Vietnam, we carried
out an inventory of the sand flies from the country. Sand flies were sampled using
CDC miniature light traps in different places in the country including two main biotopes:
villages with animals and caves. Specimens were identified according to their original
descriptions. In addition, DNA barcoding based on cytb sequences was performed. The
identification of males of the genus Sergentomyia has not been done yet due to difficulty
in identifying them. We identified 10 species belonging to four genera: Idiophlebotomus
nicolegerae, Phlebotomus stantoni, Ph. kiangsuensis, Se. barraudi s.l., Se. bailyi,
Se. sylvatica, Se. khawi, Se. anodontis, Se. hibernus and Gr. indica. The molecular
data are in agreement with the morphological identifications. The status of Sergentomyia
barraudi s.l. and Se. khawi must be carefully examined taking into account both morphology
and the division of each species in two molecular populations. Finally, we recorded
five species new to the fauna of Cambodia.
Laboratory studies (oral communications)
Keynote – The unparalleled efficiency of Leishmania transmission by sand fly bites
Ranadhir Dey1, Vanessa Atayde2, Amritanshu Joshi1, Hamide Aslan3,4, Lais da Silva3,
Shannon Townsend3, Claudio Meneses3, Hira Nakhasi1, Martin Olivier2, Jesus Valenzuela3,
Shaden Kamhawi3
1Laboratory of Emerging Pathogens, Center for Biologics Evaluation and Research, Food
and Drug Administration, Silver Spring, MD 20993, USA
2Infectious Diseases and Immunity in Global Heath Program, Research Institute of the
McGill University Health Centre, 1001 Boulevard Decarie, Montreal, QC H4A 3J1, Canada
3Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,
Maryland 20852, USA
4Faculty of Health Science, Selahaddin Eyyubi University, Diyarbakir, Turkey
skamhawi@niaid.nih.gov
Transmission of Leishmania parasites by vector sand fly bites continues to fascinate
us with its complexity. To date, various researchers have demonstrated that sand flies
do not simply inject Leishmania parasites into the skin. Instead, the infectious inoculum
includes various vector-derived factors that are equally vital for early parasite
survival and establishment, enhancing virulence of leishmaniasis. This heightened
virulence has been attributed to the immunomodulatory properties of vector-derived
factors, the most renowned being saliva and the promastigote secretory gel (PSG).
Here, I report on two newly identified vector-derived factors, Leishmania exosomes
and sand fly gut microbiota, that contribute to the unique inflammatory environment
favoring parasite survival at the bite site. Our studies show that exosomes are secreted
in abundance by parasites developing in the midgut. These exosomes accumulate within
the gut lumen and are egested into the mammalian host where they enhance the virulence
of Leishmania parasites. Gut microbiota are also egested into the mammalian host triggering
a distinctive inflammatory response dominated by activation of the inflammasome and
IL1β production. The high level of IL1β at the bite site drives persistent recruitment
of neutrophils and is essential for successful visceralization of L. donovani parasites.
It is clear that the collective specific and shared effects of vector-derived factors
continue to define the outcome of Leishmania transmission by vector bite rendering
it unparalleled in efficiency and impossible to reproduce in its entirety.
A comparison of vector competence in different sand fly species to transmit Leishmania
donovani
Jovana Sadlova1, Jitka Myskova1, Katerina Pruzinova1, K. Homola1, M. Yeo2, Petr Volf1
1Department of Parasitology, Faculty of Science, Charles University, Prague, Czech
Republic
2Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases,
London School of Hygiene and Tropical Medicine, London, UK
sadlovaj@natur.cuni.cz
Development of Leishmania infections in sand fly vectors is a complex, often species-specific
process. Factors controlling vector competence during the early phase of infection
are closely associated with bloodmeal digestion where Leishmania are exposed to midgut
digestive enzymes and persistence of an acellular chitin-containing envelope (the
peritrophic matrix, PM). After escape from the PM parasites attach to the midgut epithelium
enabling them to avoid expulsion from the midgut with remnants of the digested bloodmeal
during defecation. The aim of our study was to assess the influence of the PM and
bloodmeal digestion on the course of L. donovani infections in both refractory and
susceptible sand fly’s by measuring key developmental parameters of infection. We
compared development of L. donovani in two susceptible sand fly species (Phlebotomus
orientalis, Ph. argentipes) and two refractory ones (Ph. papatasi, Sergentomyia schwetzi).
Amastigote-initiated infections were performed using a membrane feeding method; parasite
numbers, their physical location in situ, presence and proportion of morphological
forms were all examined by fluorescence microscopy at 2, 3, 6 and 10 days post bloodmeal
ingestion. In addition, we studied various parameters of bloodmeal digestion. These
included volume of ingested bloodmeal, time to defecation of bloodmeal remnants, time
of formation and degradation of the peritrophic matrix (PM) and its morphology, and
finally trypsin and chymotrypsin enzyme activity in the midgut. Parasites were also
exposed to midgut lysates in-vitro to further investigate their effects on Leishmania
differentiation from amastigote to promastigote morphological forms. Parasites produced
massive infections with colonization of the stomodeal valve and presence of infective
metacyclic stages in susceptible Ph. orientalis and Ph. argentipes vector species.
These two natural vectors showed significantly lower trypsin activity and relatively
slower formation of the PM than refractory species. In contrast, L. donovani infections
were not maintained and did not progress past defecation of blood remnants in either
refractory vector species (Se. schwetzi and Ph. papatasi). With regard to Se. schwetzi,
parasites remained enclosed within the PM, whereas in Ph. papatasi parasites reached
the abdominal midgut. The loss of infections in both refractory sand fly species is,
therefore, caused by different mechanisms. It was apparent for Se. schwetzi, that
the time period between degradation of the PM and defecation of bloodmeal remnants
was extremely short, promastigotes were unable to escape from the PM before defecation.
In contrast, in Ph. papatasi, infections survived high activities of midgut proteases
and escaped from the PM, but failed to attach to the midgut epithelium and so were
ejected during defecation. Procyclic promastigotes remained the prevailing morphological
form in both refractory species. The findings provide insight into factors affecting
Leishmania development and help predict vector competency of different sand fly species.
Leishmania donovani in Phlebotomus argentipes: comparison of development and transmission
of amastigote- and promastigote-initiated infections
Tereza Lestinova1, Jovana Sadlova1, Jitka Myskova1, Jan Votypka1, V. Yeo2, Petr Volf1
1Department of Parasitology, Faculty of Science, Charles University, Prague, Czech
Republic
2Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases,
London School of Hygiene and Tropical Medicine, London, UK
terka.kratochvilova@seznam.cz
All Leishmania species share a digenetic life cycle characterized by motile promastigote
stages that develop in the gut of phlebotomine sand flies, and by non-motile amastigotes
residing inside mononuclear phagocytic cells of vertebrate hosts. Under laboratory
conditions, both aforementioned Leishmania stages can initiate experimental infections
of sand flies. While promastigotes can be simply cultivated in vitro, ingestion of
promastigote-initiated forms by sand fly females is unnatural. In contrast, amastigote-initiated
infections are natural, but associated with ethical concerns and some other disadvantages,
which make their usage unfavorable. Assessing amastigote dose by direct feeding on
infected hosts is difficult and those derived from organs of infected animals requires
frequent animal sacrifice and are unavoidably contaminated with host material. Alternatively,
cultivation of axenic amastigote-like forms is relatively easy, however, a large number
of studies infer considerable differences when comparing axenic amastigotes with “real”
macrophage- or lesion-derived amastigotes. For these reasons, the cultivation of amastigotes
via macrophages, or macrophage-like cell lines remains the gold standard although
it is relatively time-consuming and laborious. In our study, we address an important
omission in the current knowledge of experimental Leishmania infections in sand flies,
namely the extent to which promastigote-initiated experimental infections differ from
those initiated with amastigotes. We compared the development of promastigote- against
amastigote-initiated L. donovani infections in the natural vector Phlebotomus argentipes
and, for the first time, the subsequent effect on transmission to BALB/c mice. Promastigotes
from log-phase cultures and amastigotes grown ex vivo in macrophages, derived from
bone marrow of mice, were mixed with heat-inactivated rabbit blood (106 parasites
per 1 mL of blood) and Ph. argentipes infected by feeding through chick-skin membranes.
Infections initiated with promastigotes had a markedly quicker onset in Ph. argentipes.
Ingested amastigotes underwent a substantial initial reduction in numbers before they
start to multiply. As expected, in the early phase of infection (day 1-2 post bloodmeal)
experimental groups differed in representation of morphological forms; procyclic promastigotes
prevailed in amastigote-initiated infection while short promastigotes were dominant
in promastigote-initiated infection. More importantly, in mature infections (day 8-9
post bloodmeal), no significant differences were observed either in intensity of infection,
in situ distribution of parasites, or in numbers of metacyclic forms. The efficiency
of Leishmania transmission, the most important marker of successful parasite development
in the vector, was also comparable between both experimental groups. We conclude that
use of promastigote stages for sand fly infections does not alter the final outcome
of L. donovani development in Ph. argentipes. Nevertheless, for studies specific to
early phase of Leishmania development in sand flies, i.e. before defecation of blood
remnants, amastigotes grown in macrophages should be used.
Establishing, expanding and certifying a closed working colony of Phlebotomus argentipes
(Diptera: Psychodidae) for xenodiagnostic studies at the kala azar medical research
center, Muzaffarpur, Bihar, India
Puja Tiwary1, Shakti Kumar Singh1, O.P. Singh1, David Sacks2, Shyam Sundar1, Edgar
Rowton3, Phillip Lawyer2
1Department of Medicine, Institute of Medical Sciences, Banaras Hindu University,
Varanasi, Uttar Pradesh, India
2Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland, USA
3Headquarters, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
plawyer349@verizon.net
The Kala Azar Medical Research Center (KAMRC), Muzaffarpur, Bihar, India is a field
station of the Institute of Medical Sciences, Banaras Hindu University. This pilot
project is preliminary and essential to a larger follow-on effort aimed at defining
the ability of specific human-subject groups across the infection spectrum to serve
as reservoirs of Leishmania donovani infection to sand flies in areas of anthroponotic
transmission such as Bihar state. This is possible only via xenodiagnosis of well-defined
subject groups using live vector sand flies. The objective was to establish, at the
KAMRC, a robust, self-sustaining, working colony of Phlebotomus argentipes Annandale
and Brunetti, closed to infusion with wild-caught material and certified safe for
human xenodiagnostic use. Prior to this endeavor, no laboratory colony of this vector
existed in India that met the stringent biosafety requirements of this human-use study.
Requisite for initiating and establishing a permanent sand fly colony were: construction
of a proper insectary facility, procurement of equipment and supplies, as well as
training of personnel to perform the complex, labor-intensive procedures required
to support the colony. All this was accomplished in seven months, March to September,
2014, under a pilot grant from the Bill and Melinda Gates Foundation. From September
through mid-December, 2014, sand flies were collected from VL-endemic regions of Muzaffarpur
district, Bihar (26.07uN 85.45uE) in rural villages selected previously based on reported
VL cases. As a result of this first effort, a small colony was initiated and maintained
for three generations but it did not achieve the critical mass necessary to be self-sustaining
before the end of the collecting season. Then in March, 2015, a village was identified
in which residual spraying had not been done recently and where sand fly density was
consistently high enough to enable trapping large numbers of sand flies to build the
colony. From March through mid-December, 2015, sand flies were collected in human
dwellings and cattle sheds using 30 light traps over 254 nights (7,620 trap nights).
A total of 68,601 flies were collected (37,397 males; 31,204 females). From 13,348
females set up in isoline vials for oviposition, 2,598 clutches averaging 28 eggs
each were harvested, approximately 90% of which hatched). Progeny were reared according
to standard methods, providing a continuous critical mass of F1 males and females
to stimulate optimal social feeding behavior. With the construction of a large feeding
cage and use of a unique, custom-made rabbit restrainer, the desired level of blood-feeding
on un-anesthetized rabbits was achieved for the colony to be self-sustaining and expanded
to working level. Presently in its 10th generation, the colony yields 1,500–2,500
blood-fed females per week for egg production. Because the colonized sand flies will
be used for xenodiagnosis on humans, the colony was closed to further infusion with
wild-caught material in December, 2015, and steps were taken via PCR to insure the
purity of the colony as Ph. argentipes and to certify it free of pathogens of potential
or actual concern to humans.
Leishmania tropica development in Phlebotomus sergenti: the effect of temperature,
gregarines and geographic origin of sand flies
Jana Hlavacova, Magdalena Jancarova, Jan Votypka, Petr Volf
Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna
7, 128 43 Prague 2, Czech Republic
janehlavac@seznam.cz
Phlebotomus sergenti Parrot, 1917 is a widely distributed Old World species recognized
as the main vector of Leishmania tropica. It also serves as a host of gregarine Psychodiella
sergenti, insect parasite specific for this sand fly species. In mosquitoes, it was
shown that gregarines can affect development of other parasites, like microsporidia
or arboviruses. Therefore, we studied the effect of co-infection of gregarine Psychodiella
sergenti on L. tropica in Ph. sergenti. We found that Leishmania developed similarly
well in both sand fly groups, causing the same rate of heavy late stage infections
in gregarine-infected and non-infected sand flies. Next, we studied the impact of
larval rearing temperature (27 and 32 °C) on Ph. sergenti susceptibility to L. tropica
and gregarine Psychodiella sergenti. Conditions experienced during larval development
are known to affect fitness of adults in many insect species. It was previously shown
that rearing temperature of larvae can affect susceptibility of mosquitoes to arboviruses.
However, no analogous studies have ever been performed on sand flies. Here, we demonstrated
that temperature significantly affects development of the gregarine Ps. sergenti in
Ph. sergenti. Fourth instar larvae maintained at 32 °C were significantly less infected
by gregarines than the control group maintained at 27 °C. In adults, the difference
between two groups tested was even more pronounced: All 117 adults emerged from larvae
kept at 27 °C gregarine positive, but only three of 120 adults from the group kept
at 32 °C were gregarine positive. In contrast, L. tropica thrived comparably well
in females obtained from both temperatures tested; no differences were observed either
in rates of infected sand flies or in intensity of Leishmania infection. Additionally,
we observed that larvae kept at higher temperatures develop faster and produce smaller
adults. We hypothesize that higher temperature tested may modulate immune response
which, together with faster development of sand flies, could intensify loss of gregarines.
Finally, as Ph. sergenti has a broad geographic distribution and molecular heterogeneity,
which were previously suggested to affect vector competence to L. tropica, we infected
laboratory-reared Ph. sergenti originating from Turkey and Israel to compare their
susceptibility. In both tested groups, L. tropica developed equally well, causing
heavy late-stage infections with the colonization of the stomodeal valve. This indicates
that the different geographic origins of Ph. sergenti are not reflected by a different
vector competence to L. tropica.
Lutzomyia umbratilis population captured in the south of the Negro River is refractory
to interaction with Leishmania guyanensis
R.P. Soares1, P.M. Nogueira1, N.F.C. Secundino1, E.F. Santos2, C.M. Ríos-Velásquez2,
F.A. Pessoa2
1Centro De Pesquisas René Rachou/Fundação Oswaldo Cruz, Belo Horizonte – MG, Brasil
2Centro de Pesquisas Leônidas e Maria Deane/Fiocruz, Manaus – AM, Brazil
rsoares@cpqrr.fiocruz.br
Lutzomyia umbratilis is the vector of Leishmania guyanensis in Northern South America.
This vector has been found naturally infected with this parasite only east of the
Negro River and north of the Amazonas River. However, populations of Lu. umbratilis
are present also in areas south of these rivers, which are natural geographic barriers.
An interesting aspect is that this sand fly species has never been found infected
or to be transmitting L. guyanensis in these areas. Genetic differences among Lu.
umbratilis populations suggest that they may be in the process of speciation. However,
no studies of the parasite-host interactions are available. Here, the interaction
of L. guyanensis (MHOM/BR/75/M4147) with Lu. umbratilis captured at two sites, Rio
Preto da Eva and Manacapuru (north and south of the Negro River, respectively), was
evaluated. Procyclic and metacyclic attachment was quantitated in vitro after interaction
with the midguts of field-collected Lu. umbratilis. Midguts (11 per group) were incubated
for 20 min with procyclic and metacyclic promastigotes (2 × 107 cells/mL) and the
number of attached parasites counted. No attachment of parasites was observed in the
midguts of any of the insects from Manacapuru. On the other hand, a high binding of
PNA+ parasites was observed in the midguts of flies from Rio Preto da Eva and this
attachment was more pronounced than that observed for PNA- (P < 0.001). These data
suggest that the population of Lu. umbratilis south of the Negro River is refractory
to interaction with L. guyanensis, corroborating previous epidemiological information
on L. guyanensis transmission in different regions of the Amazon.
Financial support: CNPq and FAPEMIG.
Lutzomyia migonei is a permissive vector competent for Leishmania infantum
Katerina Pruzinova1, Vanessa Cristina Fitipaldi Veloso Guimarães2, Jovana Sadlova1,
Vera Volfova1, Sinval Pinto Brandão Filho2, Petr Volf1
1Department of Parasitology, Faculty of Science, Charles University, Prague, Czech
Republic
2Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz
(Fiocruz), Recife, Pernambuco, Brazil
katerina.pruzinova@gmail.com
In Latin America, Leishmania infantum is primarily transmitted by Lutzomyia longipalpis,
but the role of Lu. migonei as a potential vector has been repeatedly discussed. Lu.
migonei is well known for its opportunistic feeding habits and anthropophilic behaviour
and repeatedly has been found to be naturally infected by L. infantum. The vectorial
role of this sand fly species has been suggested especially in areas with a record
of human and canine cases of visceral leishmaniasis but where the proven vector is
absent. However, the vector competence of L. migonei has not yet been proven experimentally.
In our study, we evaluated for the first time the susceptibility of Lu. migonei to
L. infantum and used Lu. longipalpis as a positive control. Females of laboratory-reared
Lu. migonei and Lu. longipalpis were fed through a chick-skin membrane on heat-inactivated
rabbit blood containing 106 promastigotes/mL. A viscerotropic L. infantum strain (MHOM/BR/76/M4192)
and dermotropic L. infantum strain (ITOB/TR/2005/CUK3) were used in the experiments.
Blood-fed females were dissected at one, five and eight days post-infection (PI) and
checked microscopically for the presence, intensity and localization of Leishmania
infections. In addition, morphological forms of promastigotes were analysed. Both
L. infantum strains developed well in Lu. migonei with high infection rates and intensities
of infections. Leishmania colonized the cardia region and stomodeal valve in most
Lu. migonei females from day 5 PI and no significant differences were found in comparison
with the development in Lu. longipalpis. Metacyclic forms were observed in both sand
fly species. Our study clearly demonstrated that L. infantum develops late-stage infections
in Lu. migonei, fully comparable to those found in Lu. longipalpis. This, together
with its known anthropophily, abundance in VL foci and natural infection by L. infantum,
constitute important evidence that Lu. migonei is a vector of this parasite in Latin
America. Since it has been reported that Lu. migonei supports the development of other
Leishmania species, namely L. braziliensis and L. amazonensis, we propose that this
sand fly species is a permissive vector susceptible to various Leishmania and Viannia
species. Our results contribute to a better understanding of the epidemiology of VL
caused by L. infantum in South America.
Leishmania proteophosphoglycans regurgitated from infected sand flies accelerates
dermal wound repair and exacerbates leishmaniasis via insulin-like growth factor 1-dependent
signalling
Emilie Giraud1, Tamsyn Derrick1, Oihane Martin1, Rod J. Dillon2, Tereza Leštinová3,
Petr Volf3, Ingrid Műller4, Paul A. Bates2, Matthew E. Rogers1
1Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical
Medicine, WC1E 7HT, UK
2Division of Biomedical and Life Sciences, Lancaster University, LA1 4YB, UK
3Department of Parasitology, Charles University, Vinicna 7, Prague 2, 128 44 Czech
Republic
4Department of Medicine, Section of Immunology, Imperial College London, W2 1PG, UK
emilie.Giraud@lshtm.ac.uk
The promastigote secretory gel (PSG) is a matrix of filamentous proteophosphoglycan
secreted by Leishmania promastigotes inside the sand fly gut, which facilitates the
transmission and infection of the mammalian host. The early host response to PSG has
not been characterised. One thousand Leishmania mexicana metacyclic promastigotes
were inoculated into BALB/c mouse ears, with or without PSG. The Affymetrix Mouse
GeneChip revealed differential expression of 7,927 transcripts (FC > 1.5, 5% FDR)
to PSG, i.e. 27% of the mouse genome. We found that PSG was associated with an early
up-regulation of transcripts involved in inflammation, inflammatory cell recruitment,
epithelial cell proliferation and fibrosis. In vitro- and in vivo-experiments revealed
that PSG significantly accelerated wound healing. Insulin-like growth factor 1 (IGF1)
is linked to macrophage alternative activation and wound repair. Dermal expression
of IGF1 was enhanced following an infected sand fly bite and was acutely responsive
to the PSG but not to parasites or sand fly saliva. Antibody blockade of IGF1 ablated
the gel’s ability to promote wound closure in mice and significantly reduced the virulence
of L. mexicana infection delivered by sand fly bite. These results show that PSG strongly
influences multiple stages of the wound healing process in skin following Leishmania
transmission; resulting in accelerated healing and, via IGF1-signalling, provides
an environment that promotes parasite survival and growth.
Attraction of Lutzomyia sp. (Diptera: Psychodidae: phlebotomine) to volatile organic
compounds from the skin odour of individuals residing in an endemic area for tegumentary
leishmaniasis
D.S. Tavares1, P.R.R. Mesquita3, V.R. Salgado2, F.M. Rodrigues3, J.C. Miranda2, A.
Barral2
1Post-graduation Program of Pathology, Faculty of Medicine, Federal University of
Bahia, Oswaldo Cruz Foundation, Fiocruz, Salvador, Bahia, Brazil
2Laboratory of Immunoparasitology, Center of Researches Gonaçalo Moniz, Oswaldo Cruz
Foundation – Fiocruz, Rua Waldemar Falcão, 121 – Candeal, 40296-710 Salvador, Bahia
3Chemistry Institute, Department of General and Inorganic Chemistry, Federal University
of Bahia, Brazil
divast.bio@gmail.com
The olfactory sense of insects is the most important tool for their orientation. Hence,
blood-feeding insects find their hosts by following olfactory cues. Female phlebotomine
sand flies feed on vertebrate hosts to obtain nutrients from blood required to mature
their eggs. Some species are competent vectors of Leishmania and infect humans via
their bites. However, little is known about what attracts these insects to humans.
Some workers have investigated this question for mosquitoes of medical importance,
like Anopheles sp. and Aedes sp., but very few studies have been developed with phlebotomine
sand flies. Volatile organic compounds (VOCs) present in human odours have been shown
to represent important cues for insects, when seeking their sources for blood meal.
Octenol, for instance, is an alcohol present in human sweat and it has been shown
to enhance attraction of Lutzomyia neivai, L. intermedia and Lu. longipalpis under
both field and laboratory conditions. Heptanol, octanol and nonanol are also present
in human sweat and have been shown to attract Lu. longipalpis and Lu. neivai in wind
tunnel assays. The main objective of this study was to evaluate phlebotomine sand
flies’ attraction to VOCs from human skin odour of male individuals residing in an
area endemic for tegumentary leishmaniasis. To accomplish this, 33 individuals, from
Corte de Pedra, Southeast of Bahia, Brazil, between 18 and 60 years of age, were invited
to participate by allowing collection of hair from their legs, after it had retained
odours from the skin. Headspace Solid Phase Microextraction (HS-SPME) was applied
associated with Gas Chromatography and Mass Spectrometry (GC-MS) for VOCs extraction
and detection, respectively. Females of Lutzomyia sp. were captured in the field with
automatic light traps, and subjected to attraction behaviour bioassays in a transparent
acrylic wind tunnel. For each test, three female Lutzomyia sp. were placed in a releasing
chamber for a 20 min-acclimatation, in the dark and with no food sources. The chamber
was then placed 110 cm downwind from the odour source. Each tested VOC was delivered
(200 μL) on a filter paper (4 × 4 cm). The trials were 2 min-long and activation and
attraction behavior were recorded. At least 10 replicates were conducted for each
VOC tested. Forty-two VOCs were identified and until now six of them (phenylacethaldehyde,
tetradecane, hexadecane, eicosane, pentadecane, 6-methyl-5-hepten-2-one), plus hexane
and octenol (negative and positive control, respectively) were tested through wind
tunnel assays for sand fly attraction. As expected, octenol (positive control) significantly
induced activation and attraction of female Lutzomyia sp. (P = 0.0237). Significant
activation was also observed for phenylacethaldehyde (P = 0.0044), eicosane (P = 0.0257)
and 6-methyl-5-hepten-2-one (P = 0.0352). Significant attraction was observed for
phenylacethaldehyde (P = 0.0328) and eicosane (P = 0.0257). It has been reported that
skin human odours are a product of resident bacterial metabolism. Except for eicosane,
the tested VOCs are reported as products of the metabolism of bacteria from human
skin. Bacillus subtilis is known to produce phenylacethaldehyde and hexadecane and
Streptomyces sp. is produces phenylacethaldehyde and 6-methyl-5-hepten-2-one. Phenylacethaldehyde
is also produced by Staphylococcus sp. The results so far are promising as a means
of enhancing phlebotomine sand fly captures in the field, which may be very helpful
for monitoring sand fly activity. In addition, the enhanced capture of these insects
can also provide material for laboratory research on prevention and control of leishmaniasis.
Blood derived haem as a potential elicitor of anti-leishmanial activity in the gut
of the female sand fly Lutzomyia longipalpis
José R. Silva1, Emma Shawcross2, Rod J. Dillon2
1Lab Integrado de Bioquímica Hatisaburo Masuda, Núcleo de Pesquisas em Ecologia e
Desenvolvimento Sócio-Ambiental, Universidade Federal do Rio de Janeiro, Campus Macaé,
CEP: 27971-550 Macaé, Brazil
2Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster
University, LA1 4YQ Lancaster, UK
r.dillon@lancaster.ac.uk
Establishment and development of a transmissible Leishmania infection in the gut of
the female sand fly requires that the parasite either is resistant to the anti-parasitic
immune molecules present or that the molecules are not expressed at sufficient levels
in the gut to cause suppression of the Leishmania. Our previous studies showed that
reactive oxygen species (ROS) are a component of the innate immune response in the
gut of phlebotomine sand flies. We suggested that sand flies tolerate the presence
of Leishmania by differential response of the ROS system. The aim of the present study
was to examine the role of blood-derived haem and the influence of the peritrophic
matrix on ROS production in the gut. Feeding chitinase-supplemented blood to the sand
flies resulted in increased hydrogen peroxide concentration compared to blood-fed
controls. Chitinase destroys peritrophic matrix (PM) integrity and this result suggests
that the PM limits ROS production by the blood during blood digestion. Initiating
a L. mexicana infection with chitinase supplemented blood resulted in a significant
decline of parasite population in the gut. In order to check the role of ROS during
this infection, a group of sand flies was fed ad libitum following emergence on uric
acid-supplemented sucrose solution prior to infection. The supplement “rescued” (restored)
the Leishmania infection to populations similar to controls. We will describe further
experiments regarding the role of haem during Leishmania growth within the digesting
bloodmeal. Results will be discussed in the light of previous studies showing that
the presence of the PM also protects Leishmania from proteolysis due to digestive
enzymes.
Financial support: CNPq Brazil and BBSRC UK.
Bacterial communities associated with the digestive tract of wild populations of Lutzomyia
evansi: a vector of Leishmania in Colombia
Rafael José Vivero1,+
,2,3,4, Gloria Ester Cadavid-Restrepo4, Sandra I. Uribe Soto2,3, Claudia Ximena Moreno
Herrera4, Ivan D. Velez3,+
1PhD Student in Biotechnology, Universidad Nacional de Colombia, Sede Medellín, Colombia
2Grupo de Investigación en Sistematica Molecular, Universidad Nacional de Colombia,
Sede Medellín, Colombia
3PECET (Programa de Estudio y Control de Enfermedades Tropicales), Universidad de
Antioquia, Medellín, Colombia
4Grupo de Microbiodiversidad y Bioprospección, Laboratorio de Biología Celular y Molecular,
Universidad Nacional de Colombia, Sede Medellín, Colombia, Colombia
idvelez@pecet-colombia.org
Lutzomyia (Lutzomyia) evansi is a phlebotomine sand fly endemic to the Caribbean coast
of Colombia with epidemiological significance as the main Leishmania vector in that
region. Although the intestinal microbiota of these insects may include pathogenic
and non-pathogenic microorganisms, there is little knowledge of the bacterial diversity
present in the digestive tract of wild populations. In this study, conventional microbiological
methods and molecular tools, were used to assess the composition of bacterial communities
within the digestive tracts of immature and adult stages of wild Lu. evansi from the
department of Sucre (Caribbean coast of Colombia). Different molecular techniques
for the identification of bacteria were used, such as, ribosomal intergenic spacer
analysis (RISA) and analysis of the 16S rRNA gene. We also detected the presence of
Wolbachia endosymbiont bacteria and Leishmania parasites by PCR and DNA sequence analysis
in Lu. evansi and other species. The culture-dependent technique showed that the intestinal
bacteria belonging to Acinetobacter, Enterobacter, Pseudomonas, Ochrobactrum, Shinella
and Paenibacillus were the dominant bacteria isolated in larvae; Lysobacter, Microbacterium,
Streptomyces, Bacillus and Rummeliibacillus in pupae; and Staphylococcus, Streptomyces,
Brevibacterium, Acinetobacter, Enterobacter and Pantoea in adult stages. Statistical
analysis reveals that the fingerprint pattern of the PCR – TGGE bands varies significant
between bacterial communities of immature and adult Lu. evansi. Results show 20% infection
by Wolbachia in samples of Lutzomyia tested. Endosymbiotic Wolbachia were found in
three species: Lu. cayennensis and Lu. dubitans, with positive pools = 3; 8.5% for
both species, and Lutzomyia evansi, with one positive pool = 1; 2.8%. Two distinct
Wolbachia genotypes (strains) were found, wLev in Lu. dubitans, Lu. cayennensis, and
Lu. evansi; while wLcay was found only in Lu. cayennensis. The analyses by microbiological
and molecular approaches revealed significant variation in the bacterial communities
associated with the digestive tract of Lu. evansi, depending on the developmental
stage and the blood intake of females. Evidence of Wolbachia infections in natural
Lutzomyia populations warrant further investigation on the possible effects of this
bacteria in Leishmania transmission.
Systematics and phylogeny (posters)
What we know of the classification, evolution, and dispersion of Leishmania parasites
and sand flies?
Mohammad Akhoundi1, Katrin Kuhls2, Arnaud Cannet3, Jan Votýpka4,5, Pierre Marty1,3,
Pascal Delaunay1,3, Denis Sereno6,7
1Service de Parasitologie-Mycologie, Hôpital de l’Archet, Centre Hospitalier Universitaire
de Nice, Nice, France
2Division of Molecular Biotechnology and Functional Genetics, Technical University
of Applied Sciences Wildau, Wildau, Germany
3Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Université de Nice-Sophia
Antipolis, Nice, France
4Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Prague, Czech
Republic
5Department of Parasitology, Faculty of Science, Charles University in Prague, Prague,
Czech Republic
6MIVEGEC, UMR CNRS-IRD-Université de Montpellier Centre IRD, Montpellier, France
7UMR177, Centre IRD de Montpellier, Montpellier, France
m.akhoundi@yahoo.com
Leishmaniases are vector-borne diseases caused by obligate protozoan parasites of
the genus Leishmania (Trypanosomatida: Trypanosomatidae). Leishmaniasis is transmitted
by bites of infected female sand flies, whose hosts are animals such as canids, rodents,
marsupials, hyraxes, or humans. The aim of our study is to describe the major evolutionary
historical events among Leishmania, sand flies, and the associated animal reservoirs
in detail, in accordance with the geographical evolution of the Earth, which has not
been previously discussed on a large scale. Classification of Leishmania and sand
flies has always been a controversial matter, and the increasing number of species
currently described further complicates this issue. Despite several hypotheses on
the origin, evolution, and distribution of Leishmania and sand flies in the Old and
New Worlds, no consistent agreement exists regarding dissemination of the actors that
play roles in leishmaniasis. For this purpose, we summarize here three centuries of
research on sand fly and Leishmania descriptions, as well as a complete description
of Leishmania and sand fly fossils and the emergence date of each Leishmania and sand
fly group during different geographical periods, from 550 million years ago until
now. We discuss critically the different approaches that were used for Leishmana and
sand fly classification and their synonymies, proposing an updated classification
for each species of Leishmania and sand fly. We update information on the current
distribution and dispersion of different species of Leishmania (53), sand flies (more
than 800 at genus or subgenus level), and animal reservoirs in each of the following
geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy
and Australian. We propose an updated list of the potential and proven sand fly vectors
for each Leishmania species in the Old and New World. Finally, we address a classical
question about digenetic Leishmania evolution: which was the first host, a vertebrate
or an invertebrate?
Illustrated identification key to females of Phlebotominae recorded in the Central-West
Region of Brazil using only head and spermathecae
Douglas de Almeida Rocha1, Eunice Aparecida Bianchi Galati2, Andrey José de Andrade3,4
1Núcleo de Medicina Tropical, Universidade de Brasília, Brasil
2Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo,
Brasil
3Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina,
Universidade de Brasília, Brasil
4Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal
do Paraná, Brasil
dougalmeidarocha@gmail.com
Phlebotomine sand flies (Diptera: Phlebotominae) present wide distribution and may
be found in distinct ecosystems. Worldwide there are 966 species, 273 of which are
recorded from Brazil and 125 of those are from the Central-West Region (CWR). However,
only 20 species have been incriminated or are suspected in Leishmania transmission
in the country; seven of which occur in the CWR. The proposed taxonomic classification
of American sand flies based on phylogenetic analysis separates the tribe Phlebotomini
into four subtribes: Brumptomyiina, Sergentomyiina, Lutzomyiina, and Psychodopygina,
elevating subgenera to genera. With the use of molecular methods, mainly in Leishmania
detection, thoracic structures and a major portion of the abdomen are removed for
DNA extraction, thus important morphological characters used in species identification
are lost. To compensate for this, we present here an identification key to females
of sand flies using only characteres of the head and spermathecae, using Galati’s
proposed taxonomic classification. A total of 125 species included in 16 genera recorded
in the CWR were grouped by subtribe and later separated by species, without the relationship
between them. Based on the subtribes, all morphological characters were included in
a data base for each species. Twenty photos of diagnostic structures of some species
were taken and included. The sand fly fauna of the CWR was chosen because it had been
reviewed and used recently, first, in four dichotomies to distinguish between subtribes
of Phlebotomini resulting in four entrances to Brumptomyiina, 10 for Sergentomyiina,
37 for Psychodopygina, and 40 for Lutzomyiina, total in 94 dichotomies. Thoracic characters
such as coloration, setal distribution and number, legs and wing venation were excluded,
in consequence of which some species of the genera Evandromyia (2 spp.), Psathyromyia
(4 spp.), Pintomyia (3 spp.) Psychodopygus (3 spp.) and Lutzomyia (5 spp.) may be
indistinguishable. Thus giving other characters specific to genera, like spermatheca
form and sclerotization, posterior spurs of the ascoids, antennal papilla higher priority
in separating and disingquishing genera (e.g. Psychodopygus, Psathyromyia and Lutzomyia)
and including the majority of species to minimize entrances in the key. This proposal
may be used by only researchers and secretary health of the CWR of Brazil. Sixteen
genera were tested and the key was validated.
First record of Psychodopygus francoisleponti Zapata, Depaquit & Léon 2012 (Diptera:
Psychodidae) in Acre State, Brazil
Andreia Fernandes Brilhante1, Márcia Moreira de Ávila2, Rodrigo Espíndola Godoy1,
Jailson Ferreira de Souza3, Cristiane de Oliveira Cardoso4, Eunice Aparecida Bianchi
Galati1
1Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, Brazil
2Instituto Federal do Acre, Rio Branco, Brazil
3Gerência de Endemias, Prefeitura Municipal de Xapuri, Xapuri, Acre, Brazil
4Centro de Ciências da Saúde e do Desporto, Universidade Federal do Acre, Rio Branco,
Brazil
brilhanteaf@usp.br
The Guyanensis series Barretto, 1962 of the Psychodopygus genus (Psychodidae, Phlebotominae)
comprises seven species, Psychodopygus guyanensis (Floch and Abonnenc, 1941), Ps.
geniculatus (Mangabeira, 1941), Ps. lainsoni (Fraiha and Ward, 1974), Ps. corossoniensis
(Le Pont and Pajot, 1978), Ps. dorlinsis (Le Pont and Desjeux, 1982), Ps. luisleoni
(León et al., 2009) and Ps. francoisleponti Zapata, Depaquit and Léon 2012. Both sexes
of this group are characterized by the absence of anepimeral setae; in the males the
gonostyle has one large terminal spine and three atrophied subapical spines and in
the females the individual spermathecal ducts are longer than the spermathecae. The
males may be distinguished by morphological differences in their parameres and the
females are morphologically very similar, except Ps. lainsoni, which has a pale mesonotum.
Ps. francoisleponti, described from Ecuador, is reported here for the first time in
Acre State, Brazil. In entomological surveys carried out in Amazonian forested areas
of Xapuri municipality, Acre state, Brazil, using CDC light traps, from August 2013
to July 2015, two male specimens of Ps. francoisleponti were collected in February
2014. The specimens of Acre present morphometry of FI, Labrum-epipharynx, gonocoxite,
gonostyle, paramere, parameral sheath and epandrial lobe similar to that of the type-specimens,
however, their aedeagal ducts (635 μm; 639 μm) are shorter than those of the type-specimens
(715–815 μm). Previously, Ps. francoisleponti was registered only in Ecuador. This
new record for the state of Acre, significantly extends the geographical distribution
of this species.
Morphological and morphometric characters to distinguish females of three sympatric
species of the genus Trichophoromyia (Diptera: Psychodidae: Phlebotominae) in a Brazilian
Amazonian area
Andreia Fernandes Brilhante, Priscila Bassan Sábio, Eunice Aparecida Bianchi Galati
Faculdade de Saúde Pública da Universidade de São Paulo, São Paulo, Brazil
brilhanteaf@usp.br
Members of the genus Trichophoromyia Barretto, 1962, comprised of 40 species, are
abundant through Amazonian region. Both sexes of this genus are characterized by the
absence of ventro-cervical sensillae, and presence of Newstead’s sensilla in palp
II; the males present large terminalia, equal to or greater than the length of the
thorax, the gonostyle with four strong, short spines, with the apical commonly shorter
than the length of the gonostyle; the females present maxillary laciniae with two
rows of external teeth and spermathecae with more than 20 rings, with the terminal
knob being sessile or detached from the spermatheca. This latter characteristic is
present in most species of the genus and the females are indistinguishable. In a study
undertaken to investigate the sand fly fauna in several sites in an endemic focus
of cutaneous leishmaniasis in a Brazilian Amazonian area (Xapuri municipality, Acre
state), three species Trichophoromyia octavioi (Vargas, 1949), Th. auraensis (Mangabeira,
1942) and Th. ruifreitasi Oliveira et al., 2015 were collected, but their females
were not distinguishable based on information in the literature. At some sites, captured
males of Th. octavioi were predominantly collected together with a few specimens of
Th. ruifreitasi and it was noted the Th. octavioi labrum-epipharynx (LE) is longer
than that of Th. ruifreitasi. For females, this separation into two classes of LE
values was observed also. At several other sites where the three species were collected,
a gradient of LE values without a clear separation was observed. Subsequently, specimens
of Th. auraensis from other areas (deposited in the laboratory collection), where
the other two species are absent, were evaluated. With this strategy it was possible
to analyse the LE length as a diagnostic character besides others for the females.
Ten specimens of both sexes of each species were cleared and mounted between slide
and coverslip and measurements (in μm) of head, thorax and genitalia characters of
each were evaluated by analysis of variance. Multiple comparisons of the morphometric
characters were performed using the Gabriel test (F test, p < 0.05). The males can
be distinguished by the number of bristles in the clusters situated at the middle
region of the gonocoxite. Trichophorpmyia octavioi has ca. 14 long and thick bristles;
Th. auraensis has 26 long bristles, thinner than those of Th. octavioi. Trichophoromyia
ruifreitasi bears over 35 long bristles similar to those of Th. auraensi, and ventrally
to this group of bristles, another set of ca.12 spiniform setae. Among the morphometric
characters of the males, only the gonostyle length distinguished the three species:
Th. octavioi, Th. auraensis and Th. ruifreitasi measure 216 μm, 195 μm and 185 μm,
respectively (F = 46; p < 0.05). For females, only the length of the LE distinguish
the three species: Th. octavioi, Th. auraensis and Th ruifreitasi with values of 376 μm,
331 μm and 302 μm, respectively (F = 98; p < 0.05). Other male characters such as
ratio between the length of head/LE, LE/F1, head/F1; gonocoxite/gonostyle distinguish
only between two of the species. For females the length of head, clypeus and palpus
III also may be used to distinguish between two species. Although these results suggest
the possibility to distinguish the three species, an analysis with more robust samples
may reveal other diagnostic characters.
Metaphase karyotyping organization of Lutzomyia cruzi – preliminary result
Mirella Ferreira da Cunha Santos1, Natália Camargo Braga2, Douglas Araújo3, Lucas
Osti de Freitas2, Wagner Fernandes4, Elisa Teruya Oshiro4, Alessandra Gutierrez de
Oliveira4
1State University of Mato Grosso do Sul (UEMS), School of Medicine, Campo Grande,
Brazil
2Federal University of Mato Grosso do Sul (UFMS), Center of Biological and Health
Sciences, Campo Grande, Brazil
3Federal University of Mato Grosso do Sul (UFMS), Biology laboratory, Campo Grande,
Brazil
4Federal University of Mato Grosso do Sul (UFMS), Parasitology laboratory, Campo Grande,
Brazil
mirella.santos@uems.br
Visceral Leishmaniasis (VL) is a disease caused by a flagellate protozoan, Leishmania
infantum (family Trypanosomatidae). In the Americas, the main vector of this disease
is the female sand fly of the species Lutzomyia longipalpis. A closely related species,
Lutzomyia cruzi is implicated as the vector of L. infantum in some regions of Brazil,
including the state of Mato Grosso do Sul. Because the females of these two species
are morphologically indistinguishable, the aim of this study was to describe the organization
of the metaphase karyotyping of Lu. cruzi. The cerebral ganglia of 44 fourth-instar
larvae were dissected, fixed, mounted on slides, stained by conventional Giemsa and
photographed for karyotype and chromosome analysis. In the slide images it was possible
to count the chromosomes of Lu. cruzi, n = 10, whereas Lu. longipalpis is n = 8. The
establishment of proper cytologic preparations to confirm this preliminary finding
is needed, since this taxonomic tool could help entomologists differentiate and identify
females of both species. Moreover, due to frequent VL epidemics in the state of Mato
Grosso do Sul, the study of these vectors may help public authorities promote more
effective preventive measures.
Phylogeography and genetic variability of populations of Lutzomyia longipalpis (Diptera:
Psychodidae) inferred from ND4 gene
Angélica Pech-May1,2, Janine Ramsey2, Domingo Liotta3, Magali Giuliani1, Pablo Berrozpe1,
María Gabriela Quintana1,4, Oscar Daniel Salomón1
1Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación, Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET), Puerto Iguazú, Misiones,
Argentina
2Instituto Nacional de Salud Pública / Centro Regional de Investigación en Salud Pública,
Tapachula, Chiapas, México
3Universidad Nacional de Misiones, Posadas, Argentina
4Universidad Nacional de Tucumán-CONICET, Instituto Superior de Entomología, San Miguel
de Tucumán, Argentina
apechmay@gmail.com
Lutzomyia longipalpis is the most important vector in the transmission of Leishmania
infantum in the Americas; it is widely distributed from southern Mexico to northern
Argentina. This species is actually a complex of at least four sister species. In
Argentina, it is distributed in the provinces of Misiones, Formosa, Corrientes, Entre
Ríos and Salta. We report here the preliminary analysis of phylogeography and genetic
variability of Argentinian populations of Lutzomyia longipalpis inferred from the
ND4 gene. For the phylogeographic analysis, we used Genbank sequences of the mitochondrial
ND4 gene from Brazil, Colombia, Venezuela, Honduras, Costa Rica, and Guatemala, in
addition to our 73 sequences from Argentina: Clorinda (Clo), Corrientes (Corr), Puerto
Iguazú (Ig), San Ignacio (SI), Santo Tome (ST) and Tartagal (Tar). Phylogenetic analysis
indicates that Lu. longipalpis populations from Argentina divide into two clades,
(1) Corr, ST, Clo, Ig, Tar and SI; (2) Tar, ST and SI. These two clades were separated
from Colombian, Venezuelan, Honduran, Costa Rican, and Guatemalan populations. The
second Argentinian clade was grouped with populations from Jacobina and Lapinha in
Brazil. In terms of Inter-population genetic diversity, 68 polymorphic sites were
determined and 35 haplotypes were identified, with a range of 4–14 haplotypes per
population. Haplotype diversity was high Hd = 0.858 ± 0.039, while nucleotide diversity
and nucleotide polymorphism were low (π = 0.014 ± 0.001 and θ = 0.022 ± 0.002, respectively).
Neutrality tests were not significant for population expansion. ST and Tar had the
highest genetic diversity among the six populations, while Ig and Corr had the lowest.
Population expansion was found in Corr and Ig, while ST and Tar had population subdivisions.
The population structure of Lu. longipalpis with the ND4 fragment showed high genetic
differentiation among Argentina populations (Fst = 0.452, p < 0.0001). High genetic
differentiation was observed between SI and Corr populations (Fst = 0.86, p < 0.0001),
with a genetic distance of 0.017 ± 0.004. According to these preliminary results,
divergence patterns may be associated with climatic and physiographic discontinuities.
In order to understand better dispersal routes and clades involved in Argentinian
populations, samples from the neighboring countries Bolivia, Paraguay, Uruguay and
more samples of Brazil will be analyzed.
It is time to use a non destructive method for DNA extraction from phlebotomine sand
flies
Julian Gratiaux1, Eva Krupa1, Thibault Valecillo1, Denis Augot1, Véronique Lehrter1,
Jean-Charles Gantier2, Jean-Yves Rasplus3, Jérôme Depaquit1
1Université de Reims Champagne Ardenne, ANSES, SFR Cap santé, EA 4688 – USC « Transmission
Vectorielle et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
2Laboratoire des Identifications Fongiques et Entomologiques (LIFE), Mennecy, France
3INRA, UMR 1062 CBGP Montferrier-sur-Lez, France
julian_51@hotmail.fr
Before the first application of molecular biology to phlebotomine sand fly systematics
in the late 80’s, all the specimens were mounted in toto. Depending of the goals of
the studies (epidemiology, systematics, etc.), the mounting media differed. In epidemiological
studies carried out in the field just to identify the specimens and determine if their
guts contain Leishmania, a very quick mounting in Marc-André medium was sufficient,
thus precliding any possible storage of the specimen without remounting. For systematics
applications, the first step every time is lysis of the soft tissues in KOH in order
to observe internal characters such as spermathecae, cibarium or pharynx. Then, the
specimens are washed in an acid solution like Marc-André in order to clear the tissues
without destroying of the characters used for the identification. Then, it is possible
to mount the specimen in an aqueous medium like chloral gum or CMCP-9 low viscosity
mountant, correctly exhibiting spermathecae. However, these are non-permanent media
due not enable long-term storage. For long-term storage of mounted specimens, the
classical protocol is to dehydrate the specimens through an ethanol series of increasing
concentrations, then beech creosote, and finally mount them in Canada balsam. The
use of molecular techniques for systematics requires DNA extraction from a single
sand fly. On the one hand, it is necessary to obtain enough DNA to carry out the molecular
studies. On the other hand, it is necessary to preserve enough parts of each specimen
to identify it. The process of isolating organs (head, genitalia, wings) is difficult,
time-consuming and there is always a possibility of damaging or losing some of them.
We propose a non-destructive method of DNA extraction coupled with easy processing
and mounting of the whole specimen. Briefly, we use Qiagen reagents. The sand fly
is placed in ATL buffer and proteinase K at 56 °C overnight. Then the buffer AL and
ethanol are added after a step at 70 °C. Then the whole sand fly is removed and the
DNA extraction is done on a column according to a home-made protocol. The sand fly
is then cleared and dehydrated in a 1.5 mL vial in successive reagents as follows:
10% KOH, distilled water, Marc-André solution including acid fuchsin, distilled water,
70% ethanol, then 95% ethanol. Once cleared and dehydrated, the specimen is mounted
in Euparal for long-term storage.
Systematics and phylogeny (oral communications)
Keynote – Fossil contribution in the classification of Psychodidae
Dany Azar
Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing
210008, People’s Republic of China; Lebanese University, Faculty of Science II, Fanar,
Natural Sciences Department, Fanar – Matn, PO Box 26110217, Lebanon
azar@mnhn.fr
The family Psychodidae comprises nematoceran flies of more than 2,700 described species.
At present it is largely accepted that this family comprises six recent subfamilies,
namely Horaiellinae, Bruchomyiinae, Phlebotominae, Psychodinae, Sycoracinae and Trichomyiinae.
Phlebotomine sand flies (Phlebotominae) are a group of Diptera with blood-sucking
adaptations. They are either considered as belonging to the family Psychodidae, or
to separately distinct family Phlebotomidae forming with the Psychodidae the superfamily
of Psychodoidea. Nevertheless the latter opinion is not supported by any phylogeny.
The fossil record of Psychodidae is relatively rich (around 150 described species),
and representatives of this group were found in Cretaceous Lebanese, Spanish, Taimyr,
New Jersey, French Charente Maritime and Burmese amber, and in Cenozoic Baltic, Saxonian,
Ukrainian, Oise, Mexican, Saint Domingo and Indian ambers. The earliest definite phlebotomine
record is from the Lower Cretaceous Lebanese amber, but this group could be older
as Phlebotominae are well diversified in this material. Moreover several fossil psychodids
that could be assigned to Psychodinae (especially the Cretaceous ones) have developed
mouthparts and phlebotomine-type genitalia to the point that their exact subfamilial
assignment is rather difficult and can lead to confusion. Recently several fossil
flies that could be assigned to Tanyderidae if only the wings are taken into account,
could be attributed to psychodids as well when considering the remaining parts of
the body especially head, mouthparts and genitalia structures. One can observe all
states of gradual change of the characters from Tanyderidae to Psychodidae when taking
into consideration the fossils. It is noteworthy that Hennig (1973) placed Tanyderids
and Psychodidae as a sister groups within the Psychodomorpha, while Wood & Borkent
(1989) and Oosterbroek & Courtney (1995) placed Tanyderidae within the Ptychopteromorpha.
Support for a relationship between the Tanyderidae and Psychodidae is not completely
unexpected – wing venation characters, including five radial veins reaching the wing
margin, even have been used to unite these taxa in the past considered that fossil
Tanyderidae and Psychodidae are very similar, so much so that confusion of fossil
taxa has occurred. Recent molecular analyses support a tight relationship between
these two families. Based on the fossil record and molecular data, it appears likely
that sooner or later the Tanyderidae and Psychodidae will be united in a single family.
Geometric and linear morphometry as a tool for discriminating cryptic female specimens
of Psychodopygus genus Chagasi series
Rodrigo Espíndola Godoy1,2, Elizabeth Ferreira Rangel2, Eunice Aparecida Bianchi Galati1
1Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo,
Av. Doutor Arnaldo, 715, Pinheiros, São Paulo, SP – 01255-00, Brasil
2Laboratório Interdisciplinar em Vigilância Entomológica em Diptera e Hemíptera, Instituto
Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro
21040-360, Brazil
rodrigoeg@usp.br
Psychodopygus is a genus of great medical-veterinary importance as some species have
been incriminated as vectors of American cutaneous leishmaniasis agents. Among these
species, Psychodopygus complexus and Ps. wellcomei of the Chagasi series are vectors
of Leishmania (Viannia) braziliensis in Northern Brazil. However, the great morphological
similarity between Chagasi series females has been a problem for their species identification.
Thus our study aimed to use geometric and linear morphometric analyses to distinguish
the species of the Chagasi series based on both sexes. Five species from different
Brazilian states: Psychodopygus chagasi (Amazonas and Rondônia), Ps. complexus (Tocantins
and Pará), Ps. squamiventris maripaensis (Amapá), Ps. squamiventris squamiventris
(Roraima) and Ps. wellcomei (Pará) were investigated. For the geometric morphometry,
thirteen landmarks on the wings of 239 females and 108 males were evaluated through
the principal components, canonical variables and centroid size analyses. The linear
morphometry was evaluated by 58 characters of 419 specimens (155 males and 264 females).
The differences in the characters’ means were assessed using ANOVA with Tukey’s post-test
(parametric data) and Kruskal-Wallis with Dunn’s test (non-parametric). We also performed
canonical variables and discriminant function analyses to cluster the female specimens
through linear morphometry data using 16 characters of 182 specimens. The results
of geometric morphometry analyses showed: the centroid size was significantly different
(p < 0.05) when comparing all species except between Ps. complexus and Ps. maripaensis
(both males and females). The discriminant analysis showed correct classification
for all pairs of species except in female Ps. chagasi and Ps. wellcomei (89.6%). In
males, 100% correct classification was obtained between Ps. mariapensis and Ps. complexus,
Ps. wellcomei and Ps. chagasi, and also between Ps. complexus and Ps. wellcomei. For
linear morphometry the characters that differ significantly between the five species
were: in males, labrum length, gonocoxite width, aedeagal duct length, and in females,
head and clypeus length. The common spermathecal duct and sternite 9 width also differentiated
all the species (Ps. wellcomei was not evaluated for these characters). Discriminant
function of linear data showed that all species had fully correct classification,
except for Ps. maripaensis that had 95.5%. As for the cross-validation test, only
Ps. complexus (98.8%) and Ps. maripaensis (90.9%) did not show completely correct
classification. In conclusion, the discriminant analysis using both geometric and
linear morphometric data can be used as a tool to help in the correct species identification
of the Chagasi series, including their females.
Financial support: Grant 2015/02282-9, São Paulo Research Foundation (FAPESP); Instituto
Oswaldo Cruz/FIOCRUZ.
Lutzodex
TM
– a digital key for sand flies (Diptera: Phlebotominae) using Android App
Douglas de Almeida Rocha1, Maxwell Ramos de Almeida2, Andrey José de Andrade3,4
1Núcleo de Medicina Tropical, Universidade de Brasília, Brasil
2Real Comércio e Indústria de Alumínio Ltda, Brasil
3Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina,
Universidade de Brasília, Brasil
4Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal
do Paraná, Brasil
dougalmeidarocha@gmail.com
Phlebotomine sand flies are small insects included in the subfamily Phlebotominae
in which nowadays there are about 966 species described in worldwide. Of these, 515
have been recorded in the Neotropical region, of which 273 (53%) occur in Brazil.
The Central-West region (CWR) of Brazil comprises the states of Mato Grosso, Mato
Grosso do Sul, Goiás, and the Federal District, and the combined sand fly fauna of
the region includes 125 species. In the Americas, sand fly taxonomy is based on the
classifications and keys proposed by Young & Duncan (1994) and Galati (2003). Recently,
the Brazilian Leishmaniasis Control Program of the Ministry of Health adopted the
latter work, which is reviewed annually, in the routine of secretaries of health.
Some identification keys, using what is called cybertaxonomy, have been proposed due
to advantages they afford, especially when working with large taxa such as sand flies,
thus reducing the laborious identification procedures. The data’s choice is easy and
the identification may be directed to a specific location, and the generated keys
may be reviewed if errors are found; new taxa and/or characters may also be added.
The present study applied the development of an Android App to further phlebotomine
sand fly identification. Then, an identification key for male and female sand flies
recorded in the CWR was made using the above-mentioned classifications and assaying
slides recorded in the region. Based on the key, a database was prepared in tabular
format using the Excel program. The App, named LutzodexTM, received information included
in the database with morphological characters. This tool does not use conventional
dichotomies, since up to seven options of response to a question may be found. In
practically all the responses, the images of structures can be observed for easy identification
and at any time during the process one can check the possible species, ie, species
that have the same synapomorphies. Also, it is possible to verify the geographic distribution
in Brazil and if the species is incriminated in Leishmania transmission based on the
literature. The question answered can be analysed at any time. The answers may be
deleted and the correct identification follows based on the last question answered.
Every answer of the remaining number of species is presented. Digital keys decrease
the number of questions, facilitating the identification of species. LutzodexTM is
being registered by the National Industrial Property Institute and will be available
to researchers, students and institutions, free of charge, and will be available in
the Google Play Store.
Phlebotomus (Paraphlebotomus) chabaudi Croset, Abonnenc & Rioux, 1970 and Phlebotomus
riouxi Depaquit, Killick-Kendrick & Léger, 1998: synonyms or closely related species?
Véronique Lehrter, Jérôme Depaquit
EA 4688 – USC Anses « VECPAR », SFR Cap Santé, UFR de Pharmacie, Université de Reims
Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims, France
veronique.lehrter@univ-reims.fr
Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger, 1998, was described as a species
closely related to Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970. They differ
mainly by the size and number of setae on the basal lobe of the coxite. Moreover,
molecular studies carried out on several populations in Algeria and Tunisia based
on mitochondrial genes cytochrome b (cytb) and cytochrome oxidase I (COI) supported
the typological validity of these two species. However, specimens recently studied
by Tabbabi et al. (Med. Vet. Entomol. 2014; 28 Suppl. 1:51–59), were morphologically
identified as Ph. riouxi, Ph. chabaudi, and intermediate specimens. Their cytb and
nuclear gene elongation factor-1alpha (EF-1α) sequences showed that they belong to
the same clade and the authors suggested that Ph. riouxi could be synonymized with
Ph. chabaudi. In order to know if Ph. riouxi really is a junior synonym of Ph. chabaudi,
we carried out a molecular study using the same molecular markers as those used by
Tabbabi et al. and a part of 28S rDNA more (D1 and D2 domains). Surprisingly, we highlighted
many new haplotypes very different from those obtained by Tabbabi et al. We didn’t
have access to “intermediate” specimens so are not yet able to make a definite conclusion.
However, there is no relation between morphotypes and haplotypes. The only observable
link was between the haplotypes and the geographical origin of the populations (northern
or southern) without highlighting any introgression. Consequently, as we await further
studies, it seems better to consider Ph. riouxi and Ph. chabaudi as valid typological
species.
Beware of Sergentomyia from Southeastern Asia due to untimely synonymies and a need
to describe new species
Jérôme Depaquit
Université de Reims Champagne Ardenne, ANSES, SFR Cap santé, EA 4688 – USC « Transmission
Vectorielle et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
jerome.depaquit@univ-reims.fr
Southeast Asia has long been considered free of autochthonous transmission of Leishmania.
Consequently, there are few data related to phlebotomine sand flies from this part
of the World. However, the first autochthonous South-East Asian cases of leishmaniasis
recorded in Vietnam, then in Thailand in 1996, due to Leishmania “siamensis” and,
surprisingly, to L. martiniquensis explain a growing interest in the sand fly fauna
of Southeast Asia, which remains poorly documented. Only a few studies were carried
in the 1960s to the 1980s and more recently in Thailand. Except for the species described
from the Southeast Asia by Raynal, all other species recorded in this area were described
from India by Sinton. The paleobiogeography of India differs from that of SE Asia.
Moreover, some surprising synonymizations were proposed by both Lewis and Quate. Sergentomyia
hivernus is a valid species very curiously synonymized with Se. iyengari. The latter
species has never been found in South-Eastern Asia and surprisingly, Se. gemmea, proposed
as a possible vector of leishmaniasis in Thailand, is a very uncommon species, possibly
confused with those previously mentioned.
Epidemiology, laboratory studies & modern tools (posters)
The aminosugar galactosamine reduces the trypsinolytic activity of Lutzomyia longipalpis
(Diptera: Psychodidae) and promotes Leishmania mexicana and Leishmania infantum development
within the sand fly gut
T. Lima-Silva, L.K. Castro, A. Bortolini, Marcos H. Pereira, R.N. Araújo, N.F. Gontijo,
Mauricio R.V. Sant’ Anna
Laboratório de Fisiologia de Insetos Hematófagos, Departamento de Parasitologia, Instituto
de Ciências Biológicas, UFMG, Belo Horizonte, MG, Brasil
mellima9@yahoo.es
Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to
the genus Leishmania and these haemoflagellates are transmitted to their vertebrate
hosts by minute insects called sand flies. The etiological agent of lethal visceral
leishmaniasis in Brazil (Leishmania infantum) is transmitted to the vertebrate hosts
by the bites of Lutzomyia longipalpis (Diptera, Psychodidae). Sand fly males and females
are plant feeders but females must obtain a blood meal from vertebrate hosts in order
to produce eggs. Leishmania develop within the sand fly gut and during blood digestion,
ingested parasites must survive the onslaught of sand fly digestive enzymes. Trypsin
is the key enzyme involved in blood digestion in arthropod vectors. During their life
cycle inside their sand fly vector, Leishmania are most susceptible to trypsin attack
during their transition from the amastigote form present in newly digested macrophages
to procyclic promastigotes, which are responsible for commencing parasite replication
inside the insect vector. This study aimed to manipulate the digestive physiology
of Lu. longipalpis to increase their susceptibility and favour the development of
L. mexicana and L. infantum inside the sand fly gut. Galactosamine (an amino sugar)
was capable of reducing Lu. longipalpis tripsinolytic activity in a dose-dependent
manner. This activity was specific to galactosamine as other similar molecules such
as galactose, glicosamine and N-acetylgalactosamine were not able to reduce the sand
fly tripsinolytic activity in the same way observed for galactosamine. An excess of
amino acids supplemented with the bloodmeal and 15 mM galactosamine was able to partially
abrogate the reduction of the tripsinolytic activity caused by galactosamine, suggesting
that this phenomenon might be related to an impairment of amino acid detection by
sand fly enterocytes when galactosamine is mixed with blood during an artificial bloodmeal.
In mosquitoes, TOR signaling is the upstream activator of early trypsin synthesis
in the midgut and amino acids function as stimulus for TOR signaling (see: TOR signaling
is required for amino acid stimulation of early trypsin protein synthesis in the midgut
of Aedes aegypti mosquitoes by Brandon et al., for details). Galactosamine reduces
the trypsinolytic activity in the gut of Aedes aegypti and other nematoceran flies.
In an attempt to increase Lu. longipalpis midgut infection by two Leishmania species
(L. mexicana strain M379 and L. infantum strain BH401), we infected 7-day-old sand
flies through chick skin membranes on a Hemotek artificial feeding device. Two groups
of Lu. longipalpis ingested different amounts of galactosamine (15 and 30 mM final
concentration) added to heparinized human blood and the guts of infected sand flies
were dissected five and eight days after the infection. Parasite count was carried
out using a haemocytometer and the parasite load from insects infected with Leishmania
seeded in human blood supplemented with galactosamine was compared with control insects
infected with Leishmania without galactosamine. The results showed that administration
of 15 and 30 mM galactosamine was sufficient to increase the number of promastigote
forms of L. mexicana and L. infantum in galactosamine-treated insects in comparison
to their controls. The reduction of trypsin production within Lu. longipalpis gut
and the delay in blood digestion generated by galactosamine ingestion benefited Leishmania
growth within the sand fly gut. The inhibition of the trypsinolytic activity is part
of a protocol that has been developed in our lab to generate heavily infected sand
flies to be used as an ideal challenge in vaccine trials against L. infantum.
Evaluation of different diets for feeding larvae of Nyssomyia neivai (Diptera: Psychodidae:
Phlebotominae)
Antonio Carlos Ferrari Júnior1, Kleiton Maciel dos Santos1, Magda Freitas Fernandes1,
Wedson Desidério Fernandes1, Herintha Coeto Neitzke-Abreu2, Maria Elizabeth Moraes
Cavalheiros Dorval3, Alessandra Gutierrez de Oliveira3, Eunice Aparecida Bianchi Galati4
1Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourados
(UFGD), Brasil
2Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados (UFGD), Brasil
3Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do
Sul (UFMS), Brasil
4Faculdade de Saúde Pública, Universidade de São Paulo (USP), Brasil
magdamattosfer@gmail.com
Studies of vector competence of sand fly species for Leishmania spp. require sufficient
numbers of adult live females for experimental infection. In this study we aimed to
establish a laboratory colony of Nyssomyia neivai using different larval diets to
assess which diet produces the most synchronous emergence of adults in the greatest
numbers for vector competence studies. Collections of wild sand flies were conducted
with white Shannon traps in a forest fragment in Rio Brilhante municipality, Mato
Grosso do Sul, Brazil, in September 2014. To obtain F1 adults, wild-caught males and
females were kept together in breeding cages (metal frame and voile fabric), packed
in polystyrene boxes coated internally with stone plaster cream and supplied with
chilled towels for humidity. Apple slices were offered as a source of carbohydrate
to enhance survival of the flies. For blood feeding, anesthetized hamsters (Mesocricetus
auratus), were exposed for two hours inside the mating cage. The engorged females
were placed individually in oviposition vials until they laid eggs. Then the eggs
were transferred to culture plates for rearing the immature forms. Cohorts of larvae
were fed on five different diets as follows: Diet 1 = vegetable soil + quail faeces;
Diet 2 = yeast − Saccharomyces cerevisiae + B vitamins; Diet 3 = flake food for ornamental
fish; Diet 4 = combination of diets 1 and 3; Diet 5 = combination of diet 1 and 2.
The diets were prepared in equal parts, triturated and sieved. Each diet was tested
in 10 replicates, making a total of 50 tests. The mean number of eggs per replicate
was 70.3 for Diet 1; 70.5 for Diet 2; 70.6 for Diet 3; 70.2 for Diet 4 and 70.1 eggs
for D 5. The gonotrophic cycle Ny. neivai was four days. A total of 173 adults emerged
with an overall average generation time of 51 days. Time to adult emergence on Diet
1 was 25 days with 64 adults emerging, (35 males and 29 females); Diet 2 produced
only one male; Diet 3 produced 52 adults (18 males, 25 females and nine not identified
as to sex); Diet 4 produced 46 specimens (14 males and 32 females); and Diet 5 produced
10 adults (one male and nine females). Diet 4, although in 3rd place in the total
of adults that emerged, produced the largest number of F1 females compared to other
diets. There was a significant difference between Diet 1 and Diet 3, 4 and 5 (p < 0.05
Tukey Test). Larvae fed on Diet 2 developed to 4th instars faster than those fed on
the other diets, however, all but one larva died before pupation and only one adult
emerged. Thus, we concluded that Diet 1 (vegetable soil + quail faeces) was the best
for the adult emergence of Ny. neivai, producing sufficient F1 females in 25 days
for use in Leishmania transmission experiments.
Financial support: This work was supported by Fundect, and CAPES, the Brazilian Government
for the qualification of human resources.
Is there oviposition pheromone in Nyssomyia neivai (Diptera: Psychodidae)?
Thais Marchi Goulart1, Camila Feitosa de Castro2, Wanderson Henrique Cruz Oliveira2,
Flávia Benini da Rocha Silva2, Vicente Estevam Machado2, Dennys Ghenry Samillan Ortiz1,
Christiann Davis Tosta3, Mara Cristina Pinto2
1Departamento de Biologia Animal, Instituto de Biologia, UNICAMP/Brasil
2Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Câmpus
Araraquara, UNESP/Brasil
3Instituto Federal de São Paulo – IFSP, campus Matão/Brasil
marap@fcfar.unesp.br
Oviposition pheromone has already been described in Lutzomyia longipalpis. However,
except for a biological evidence in Lu. renei there are no study related to this issue
to other Latin America sand fly species. Oviposition pheromone increases the number
of eggs laid by females. This fact is important to improve sand fly colonies of sand
flies at laboratory conditions and also for possible baits to collect gravid females
in field. The aim of this study was to search for biological evidences of an ovipositon
pheromone in Nyssomyia neivai. Oviposition chambers used in the tests were made of
250 mL plastic pots (9 cm diameter and 5 cm height) with a thin layer of plaster of
Paris on the bottom. In the first trial the area of the plaster of Paris was divided
into six equal parts. A Falcon tube of 50 mL was cut off in the middle and fixed vertically
over one part of the plaster of Paris. Three engorged females and three males were
placed into the tube. After oviposition another new five engorged females and five
males were placed into the oviposition chamber and five days later the number of eggs
was recorded in each part of the pot. Experiment was replicated five times and the
number of the initial eggs for each pot was respectively: 245, 246, 144, 155 and 114.
In all five pots there was clearly a highest number of eggs in the place of the previous
oviposition (p < 0.01). After this positive biological clue we decided to compare
the attraction of gravid females of Ny. neivai to conspecific eggs and Aedes aegypti
eggs. Almost the same described methodology was used, but at this time the plaster
of Paris was divided into four quadrants. A total of 20 A. aegypti eggs were placed
in an opposite quadrant to 50 Ny. neivai eggs. Three sand fly couples were introduced
and the eggs were recorded after five days. This experiment was replicated five times.
There was no statistical difference among the number of laid eggs into the quadrants
(p > 0.05). Other experiments will be performed to investigate if volatiles of A.
aegypti could have disrupted the attraction of a possible oviposition pheromone of
Ny. neivai or if a visual and/or tactile response of females could be the real stimulus.
Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum
using a murine model and artificial feeder
Arnaud Cannet1, Mohammad Akhoundi2,*, Michel Gregory1,*, Pierre Marty1,2, Pascal Delaunay1,2
1Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Université de Nice-Sophia
Antipolis, France
2Service de Parasitologie-Mycologie, Hôpital de l’Archet, Centre Hospitalier Universitaire
de Nice, France
*Equal contribution in the present study
delaunay.p@chu-nice.fr
Leishmaniasis is protozoan disease caused by digenetic Leishmania parasites that circulate
between vector sand flies harboring the extracellular-stage promastigotes and the
mammalian hosts harboring the intracellular amastigotes. The goal of the present study
was to screen experimental infections of Phlebotomus perniciosus with bioluminescent
Leishmania infantum using a murine model and artificial feeder. We developed a real-time
PCR-based method to determine the number of Leishmania promastigotes ingested by individual
infected flies. Of 1,840 newly emerged female sand flies, 428 fed on mice infected
with bioluminescent L. infantum. After death, each fly was individually analyzed by
RT-PCR. Only a single female was Leishmania positive at six days post bloodmeal. A
total of 1,070 female sand flies offered blood meals on an artificial feeder containing
the human blood infected with Leishmania parasites as follows: A blood meal containing
1.107/mL LUC-promastigotes was offered to 270 females, 75 (28%) of which engorged
engorged. Of the engorged flies, 44 (59%) were positive by RT-PCR analysis, with a
relative average of 50,551 Leishmania parasites. A second group of 800 female sand
flies was offered a blood meal infected with 2.106/mL promastigotes, of which 57 (7%)
female flies successfully fed. Of the blood-fed flies in the second group, 22 (39%)
were positive with a relative average of 6,487 parasites.
Exploring the migration of kinetoplastid parasites in sand flies; why are hypopylarian
parasites backward in coming forward?
Raquel J. Vionette-Amaral, C.T. Nogueira, M. Ginger, Rod J. Dillon
Faculty of Health and Medicine, Division of Biomedical and Life Science, Lancaster
University, UK
r.vionettedoamaral@lancaster.ac.uk
The development of human pathogenic Leishmania in the gut of sand flies is split between
suprapylarian and peripylarian states. Suprapylarians such as L. infantum and L. mexicana
of subgenus Leishmania usually confine their developmental cycle to the midgut and
foregut. Leishmania braziliensis of subgenus Viannia are peripylarian, developing
first in the hindgut and migrating forward to the midgut and then foregut. In contrast,
members of the subgenus Sauroleishmania are thought to be hypopylarian. Hence they
are mainly confined to the hindgut and transmission is either by the faecal route
or ingestion of the infected sand fly by the vertebrate. We are exploring the hypopylarian
state using L. tarentolae (subgenus Sauroleishmania) and Crithidia fasciculata with
the permissive sand fly Lutzomyia longipalpis. L. tarentolae is an intracellular parasite
of reptiles that uses phlebotomine sand fly vectors. It is not clear whether infection
in lizards occurs through cutaneous transmission, via the bite or faeces of the vector,
or through direct ingestion of the sand fly. Once ingested by the vector, the parasites
multiply as promastigotes in the gut. It is currently unknown what developmental forms
of L. tarentolae exist in the vector. C. fasciculata are extracellular monogenic flagellates
that naturally infect members of the Culicidae. Female Lu. longipalpis (Jacobina strain)
were infected with the two species and observed during a period of 10 days post infection.
We analysed the percentage of infection, mortality rate, morphology, and localization
of the parasites in the gut. We also investigated the effect of sand fly feeding mode
on C. fasciculata infection. For infection with C. fasciculata, the flies were fed
with culture medium or with sucrose solution 10% v/v 0.5% bromophenol blue containing
the parasites either through a chicken-skin membrane or cotton wool. The infection
with L. tarentolae was done by feeding sand flies on sheep blood containing the parasites.
Gut infections for both kinetoplastids were observed including parasite adhesion to
the hindgut cuticle. The results of these infection studies will be used to explore
hyopylarian vs. peripylrian and suprapylarian development in this permissive sand
fly and to understand why these parasites may be backward in coming forward.
Molecular and serological methods for evaluating blood meal sources in phlebotomines
sand flies (Diptera: Psychodidae)
Mauricio Baum1, Edilene Alcântara de Castro1, Elias Seixas Lorosa2, Mara Cristina
Pinto3, Thais Marchi Goulart3, Walter Baura1, Magda Clara Vieira da Costa-Ribeiro1
1Federal University of Paraná, Biological Sciences Sector, Polytechnic Center, Curitiba,
Paraná, Brazil
2Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
3State University of Campinas, Institute of Biology, Department of Animal Biology,
Campinas, São Paulo, Brazil
magdacostaribeiro@gmail.com
American cutaneous leishmaniasis (ACL) is a noncontagious infectious disease caused
by different species of protozoa of the genus Leishmania, which infect skin and mucosa.
In Brazil, ACL is widely distributed in all regions. It is very difficult to determine
the reservoirs of this parasite in several areas in which it occurs. The blood meal
source of sand flies provides valuable information about vector/host interactions
and allows better understanding of Leishmania transmission mechanisms. The precipitin
test based on Ag-Ab reactions is used largely for blood meal source identification
and allows the identification of a wide variety of hosts as long as the specimens
are tested with several antisera. This is an easily performed, inexpensive method
with relatively good sensitivity and easily interpreted results. The identification
of multiple blood meal sources through precipitin tests permits verification of whether
or not a species exhibits opportunistic feeding behavior, but does not allow reservoir
identification at the species level. Other limitations include the requirement to
produce specific antibodies to a wide range of potential hosts, and the inability
to discover unpredicted reservoirs. Recently, molecular approaches have been developed
in order to identify vector blood meal sources, such as those based on the amplification
and sequencing of the preponociceptin (PNOC) gene. Successful blood meal identification
via molecular means depends on the amount of blood ingested and length of time it
has been in the in the insect midgut expose to digestive enzymes. Our data show that
it is possible to identify the blood meal source using the PNOC gene up to 24 h after
a blood meal. Finally, the current method based on PNOC gene amplification revealed
a promising tool for identify in the blood-meal sources of female sand flies in an
endemic area of American cutaneous leishmaniasis. Investigations of the blood-meal
source of sand flies have great ecological and epidemiological significance because
they enable correct identification of the mammalian reservoirs and vector feeding
preferences.
Host feeding preference and molecular screening of Leishmania infection in wild-caught
sand flies in an endemic focus Aydın, Turkey
Mehmet Karakuş1, Metin Pekağırbaş2, Samiye Demir3, Hasan Eren2, Seray Töz1, Yusuf
Özbel1
1Ege University, Faculty of Medicine, Department of Parasitology, Izmir, Turkey
2Adnan Menderes University, Faculty of Veterinary, Department of Parasitology, Aydın,
Turkey
3Ege University, Faculty of Science, Department of Biology, Izmir, Turkey
metinpekagirbas@gmail.com
Leishmaniasis is an arthropod-borne disease, which affects more than two million people
worldwide annually. There are three main clinical forms of leishmaniasis (mucocutaneous,
cutaneous and visceral) and both cutaneous and visceral forms exist endemically in
Turkey. The aims of the study were to detect natural Leishmania infection and feeding
preferences of probable vector species by DNA-based techniques for a better understanding
of the epidemiology of the disease in this area. Entomological sampling was done between
28–31 August and 01–03 October 2015 in five villages of Aydın province, where leishmaniasis
cases, both visceral and cutaneous, were previously reported. Besides species level
identification of the sand fly specimens, molecular screening using kinetoplast DNA
and the ITS1 region of nuclear DNA was carried out using pooled groups of sand flies
to determine natural Leishmania infection and individual blood meal analysis was conducted
in order to determine probable host species. A total of 1,059 sand fly specimens belonging
to 2 genera (Phlebotomus and Sergentomyia) and 5 subgenera (Phlebotomus, Paraphlebotomus,
Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Ph. neglectus
(39%) was the most abundant species in the study area followed by Ph. tobbi (18%)
among all Phlebotomus specimens. Leishmania infection was detected in Ph. neglectus
(3 pools), Ph. tobbi (3 pools) and Se. dentata (1 pool). The Leishmania species found
in Phlebotomus specimens was identified as L. infantum by species-specific real time
ITS1 PCR. A total of 33 specimens were used for blood meal analysis and mostly human
(8/33) and dog (10/33) DNA was found in Ph. neglectus and Ph. tobbi. The detection
of natural Leishmania infection in wild caught Ph. neglectus and a high percentage
(24.2%) of that species with of human DNA in engorged specimens suggest that Ph. neglectus
is probably an important vector in Aydın. The data obtained by this study could help
public health authorities take necessary and appropriate precautions.
Financial support: Partially by The Scientific and Technological Research Council
of Turkey (TÜBİTAK) Project No: 114S999.
Anthropophilic behaviour and detection of Leishmania spp. in Sergentomyia minuta collected
in the human leishmaniasis focus of Madrid, Spain
Estela González1, Ana Tello2, Ricardo Molina1, Andrés Iriso3, Ángeles Vázquez2, Maribel
Jiménez1
1Medical Entomology Unit, Parasitology Service, National Centre of Microbiology, Institute
of Health Carlos III, Majadahonda, Madrid, Spain
2Zoology and Physical Anthropology Department, Faculty of Biological Science, Complutense
University of Madrid, Madrid, Spain
3Zoonosis and Biological Risk Section, General Directorate of Public Health, Madrid
Regional Health Authority, Community of Madrid, Madrid, Spain
mjimenez@isciii.es
Anthropophilic behaviour as well as detection of DNA from Leishmania major and L.
tarentolae-like has recently been reported in Sergentomyia minuta from Mediterranean
countries. As a result of such findings questions on the role of the Sergentomyia
genus in Leishmania spp. transmission needs to be clarified. Since 2010 an unusual
increase of human leishmaniasis cases due to L. infantum has taken place in urban
areas of the southwest Madrid region, Spain, mainly in the town of Fuenlabrada. Phlebotomus
perniciosus is the only proven vector in this focus however Se. minuta is the second
most abundant species in the area with a mean average density of 106.81 specimens/m2.
With the aim of providing more information about the potential role of Se. minuta
in the transmission of Leishmania spp. in the focus, we carried out an analysis of
blood-meal preferences and the detection of Leishmania spp. in this sand fly species.
A total of 107 Se. minuta females (n = 84 blood-fed and n = 23 unfed) collected throughout
the transmission seasons of 2012–2015 with both sticky (n = 81) and CDC light traps
(n = 26) were studied. Traps were placed in different stations located in four municipalities
of the southwestern Madrid region. Blood-meal identification was conducted in 84 blood-fed
Se. minuta females collected with both sticky (n = 81) and light (n = 3) traps. Amplification
of a fragment of 359 bp of vertebrate cytochrome-b gene was carried out. Afterwards,
the samples were sequenced and comparison with sequences deposited in GenBank® was
performed by BLAST. In 16 (19%) out of 84 specimens amplification was successful and
the analysis revealed that Se. minuta collected with sticky traps had fed mainly on
gecko (Tarentola mauritanica, n = 8) and lizard (Podarcis hispanica, n = 2), followed
by rabbit (Oryctolagus cuniculus, n = 1). Surprisingly, in the remaining n = 5 (31.3%)
specimens from sticky (n = 3) and light traps (n = 2), human (Homo sapiens) blood
was found. Detection of Leishmania spp. was carried out by amplification of a 120
bp fragment of the conserved region of kDNA. Samples were further analysed following
a specific L. infantum PCR based on the amplification of a fragment of 702 bp of cpb
gene. Leishmania spp. was detected in 10 out of 84 blood-fed Se. minuta using kDNA-PCR:
8 were from the group of 68 Se. minuta with an unidentified blood meal source and
the other 2 were from the group of 16 specimens with an identified blood meal (one
had fed on gecko and one on human). In addition, 1 out of 23 unfed Se. minuta successfully
studied was positive for Leishmania spp. by kDNA-PCR. Further analysis by PCR of the
cpb gene excluded L. infantum infection in all the samples. Moreover, amplification
of the internal transcribed spacer regions 1 (ITS1) and ITS1-RFLP was performed in
kDNA-PCR positive samples (n = 11). Amplification of ITS1 region and further sequencing
was successfully achieved in four DNA samples named CM145, CM146, CM148, and CDC290.
Sequences obtained were analyzed and homologies with the available sequences data
in GenBank® were carried out by BLAST. The four sequences presented between 93 and
99% identity with sequences of Leishmania spp. (LC028233.1 and LC028235.1) from Portugal,
Leishmania spp. (LC031456.1) from Spain and L. tarentolae-like (LC086296.1). With
the present work we describe for the first time the anthropophilic behavior of a L.
tarentolae-like positive Se. minuta collected in the focus of human leishmaniasis
in Madrid and conclude that there is no evidence that Se. minuta is the vector of
L. infantum in the Madrid focus.
Molecular detection of Leishmania tropica parasites kDNA from naturally infected sand
flies in a new foothill endemic area, southeast Iran
M.D. Moemenbellah-Fard1, K. Azizi1, M.R. Fakoorziba1, T. Dabaghmanesh2, M. Ahmadyousefi-Sarhadi2
1Research Centre for Health Sciences, Department of Medical Entomology and Vector
Control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran
2Department of Medical Entomology, School of Health, Shiraz University of Medical
Sciences, Shiraz, Iran
dabaghmanesh@gmail.com
Human dermal leishmaniasis caused by Leishmania tropica is a serious public health
concern in Iran and the adjacent countries of the Middle East and Central Asia. It
is usually known as the urban oriental sore. The sand fly Phlebotomus sergenti, which
transmits the protozoan parasites to man, is the principal vector. Little is known
about the sand fly fauna or their distribution in southeast Iran and no research has
been conducted on natural infection of sand flies in Jiroft, Kerman province, Iran.
The main aim of this study was to determine the sand fly faunal composition, its’
frequency distribution and the occurrence of natural infection with Leishmania parasites
to incriminate the likely vectors of dermal leishmaniasis in Jiroft. Sand flies were
caught with sticky traps during a six month period in 2013. They were identified to
species level using taxonomic keys. They were then subjected to nested polymerase
chain reaction method and the results were analyzed to confirm Leishmania infection.
A total of 3,751 sand flies, belonging to 21 species in two genera (8 spp. in the
Phlebotomus genus, and 13 spp. in the Sergentomyia genus) were identified, of which
males were 65.5% and 63.8% were exophilic. The two species most frequently captured
were Ph. papatasi (39.4%) and Ph. sergenti (17.1%). The latter was confirmed as being
naturally infected with L. tropica (3.33%). It was thus concluded that Ph. sergenti
which was found by PCR to be infected with L. tropica is the principal vector of human
dermal leishmaniasis in this endemic focus.
Epidemiology of cutaneous leishmaniasis in the municipality of Brasiléia, Acre State:
Study on the sandy fly fauna
Thais De Araujo-Pereira1, Daniela De Pita-Pereira1, Mariana Boité2, Daniella Alves
Martins1, Taina A.N. Da Costa-Rego1, Israel De Souza Pinto3, Regina Barbosa Moreira4,
Andressa A. Fuzari5, José Dilermano Andrade-Filho3, Marcia Oliveira4, Reginaldo Brazil5,
Constança Britto1
1Laboratório de Biologia Molecular e Doenças Endêmicas/IOC, Brasil
2Laboratório de Pesquisa em Leishmanioses/IOC, Brasil
3Centro de Referência Nacional e Internacional para Flebotomíneos/Centro de Pesquisa
René Rachou/Fiocruz, Brasil
4Laboratório Interdisciplinar de Doenças Medicas/IOC, Brasil
5Laboratório de Doenças Parasitárias/IOC, Brasil
pereirathata@gmail.com
Despite an increase in cases in recent years and 1,009 notifications of cutaneous
leishmaniasis in 2013, there are few studies concerning the situation with cutaneous
leishmaniasis (CL) in Acre State, Brazil. In this study we investigated the heterogeneity
of sand flies captured in the municipality of Brasiléia to bring insights into the
epidemiology of CL in the Acre State. Following the species identification we evaluated
Leishmania infection rates, the blood meal sources and correlated the parasite species
found in the sand flies with the parasite species detected in the lesions of patients
from the same region. Sand fly collections were made from September 2013, using light
traps and manual capture in Shannon trap and taxonomically identified. In parallel,
biopsy imprints were obtained from patients with CL lesions living in Brasiléia. Parasite
detection in insects was performed individually, in non-blood-fed females, using a
multiplex PCR with primers for Leishmania genus (kDNA) and sand fly gene (cacophony).
For the accurate identification of Leishmania species, PCR targeting the parasite
hsp70 gene followed by sequencing was done. A total of 5,969 sand flies (2,170 males;
3,705 non-blood-fed females (889 used for molecular diagnosis); 94 blood-fed females
(for the blood meal research) were collected at seven trapping sites on four separate
occasions. So far, we have identified 14 sand fly genera and 63 species by morphological
identification. To determine natural infection rates, 489 females were processed individually
and Leishmania DNA was identified in 22 specimens (4.5%). Clinical samples were obtained
from 23 CL patients. From these, 8 (34.8%) were infected by L. (V.) braziliensis,
one (4.3%) corresponded to L. (V.) guyanensis. Infections in 10 patients (43.5%) were
not identified to species level, and 4 (17.4%) were found not infected. With the conclusion
of this study, we expected to provide new elements for better understanding the CL
transmission cycle in the study area.
Financial support: CNPq, FAPERJ, CAPES, Fiocruz.
Seasonal dynamics, evolution of Leishmania infantum infection rates, and host-feeding
preferences of Phlebotomus perniciosus in the focus of human leishmaniasis in the
Madrid region, Spain (2012–2014)
Ricardo Molina, Estela González, Sonia Hernández, Inés Martín-Martín, Maribel Jiménez
Medical Entomology Unit, Parasitology Service, National Centre of Microbiology, Institute
of Health Carlos III, Majadahonda, Madrid, Spain
rmolina@isciii.es
Since 2010 the number of cases of human leishmaniasis in south western Madrid, Spain
has increased while the prevalence of canine leishmaniasis remains unchanged or lower
compared nearby areas. Phlebotomus perniciosus is the only vector identified in the
area in high numbers. The objective of this work was to carry out a detailed and intensive
entomological survey to obtain information on Ph. perniciosus seasonal trends; sand
fly densities, monthly Leishmania infantum infection rates and blood-meal preferences
in this exceptional human focus of leishmaniasis that is affecting several urban areas
and most particularly, Fuenlabrada town. The studies were carried out during the transmission
seasons of 2012, 2013 and 2014. The entomological surveys were performed monthly from
May to October at 4 sites located beside the urban area of the focus. Sticky traps
(20 × 20 cm) (n = 20) and CDC light traps (n = 2) were placed at each of the 4 sites
station each month during two consecutive nights (CDC traps were replaced every day).
Temperature and relative humidity (RH) data were registered every 10 min using data
loggers hanging on each light trap. For phenology studies data recorded from dusk
to dawn were used. In the seasonal study 45,127 Ph. perniciosus (75.34%), the predominant
sand fly species in the area, were collected. Ph. perniciosus average density obtained
with sticky traps in the three surveys was 193.6 specimens/m2. The density showed
two annual peaks one in June and one in August 2012, while in 2013 a small peak in
July and a bigger one in September were observed. Only one annual peak was observed
in September 2014. Average relative abundance of Ph. perniciosus obtained from light
trap collections was 94.24%. Statistical analysis showed that there was a negative
correlation between numbers of sand flies captured (by either light or sticky traps)
and RH (mean, maximum and minimum). Sand fly captures were positively correlated with
temperature (mean, maximum and minimum) in both light and sticky traps. Ph. perniciosus
females (n = 3,203) were dissected and 117 (3.7%) of them were found infected with
L. infantum during the three years of the study. In 2012 we dissected 735 females
and 19 (2.6%) of them were found infected. In 2013 we dissected 864 females and 57
(6.6%) of them were infected. In 2014 we dissected 1,604 females and 41 (2.6%) of
them were infected. All isolates were characterized by PCR of ITS regions as L. infantum.
Furthermore, 13.31% and 7.78% of blood-fed and unfed females respectively were found
to be infected with L. infantum by PCR examination. Blood-meal identifications were
conducted with females collected with both sticky and CDC traps. Amplification of
a fragment of 359 bp of vertebrate cytochrome-b gene, sequencing and comparison with
sequences deposited in GenBank was performed. The analysis of blood preferences revealed
that sand flies feed mainly on rabbits (Oryctolagus cuniculus) follow by hares (Lepus
granatensis). Characterization of promastigotes isolated from sand flies fed on both
lagomorphs proved that were infected by L. infantum. These data are in concordance
with results obtained from xenodiagnostic studies in hares and wild rabbits from the
focus. In conclusion, the present entomological study highlights the exceptional nature
of the Leishmania outbreak occurring in southwest Madrid region. It is confirmed that
Ph. perniciosus is the only vector in the area and that it is present in high densities
with high rates of infection. Rabbits and hares were the main blood sources for this
sand fly species. These results reinforce the need for an extensive and permanent
surveillance in this region, and others of similar characteristics, in order to control
vectors and wild reservoirs.
Financial support: In part by FP7-UE EDENext, grant 261504.
Molecular tools for the identification of phlebotomine sand flies and detection of
Leishmania spp. parasites in Misiones province, Argentina
Sofía L. Moya1,2, Magalí G. Giuliani1, Mariana Manteca Acosta1, Oscar D. Salomón1,2,
Domingo J. Liotta1,3
1Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación (INMeT-MSAL),
Puerto Iguazú, Misiones, Argentina
2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
3Laboratorio de Biología Molecular Aplicada (LaBiMAp-FCEQyN-UNaM), Posadas-Misiones-Argentina
sofialorian@gmail.com; odanielsalomon@gmail.com
In Argentina, Tegumentary Leishmaniasis (TL) is endemic in nine provinces, while the
number of Visceral Leishmaniasis (VL) cases have increased sharply in the last years
and expanded to new areas, Misiones province having the highest incidence. Because
the disease spread depends largely on the geographical distribution of the vectors,
simultaneous characterization of vectors and parasites using molecular tools will
contribute to the eco-epidemiological knowledge of this disease through a simplified
experimental approach. Procedures for sand fly identification based on morphology
often alter the nature of the sample and inhibit subsequent analysis by molecular
methods, such as those required for detection and identification of parasites. In
this context, the aim of this work was evaluate two protocols: PCR-Sequencing of cacophony-IVS6,
as a complementary method in the taxonomic identification of sand flies, and PCR-RFLP
of ITS-1 for detection and identification of Leishmania spp. Female sand flies were
captured with light traps and identified to species level by dissection and examination
of the morphology of the spermathecae according to available taxonomic keys. Sand
fly identification also was carried out through DNA extraction of the remaining sand
fly body, followed by amplification and sequencing of cac-IVS6. After the evaluation
and alignment of sequences, datasets were analyzed by Neighbor-Joining (NJ) using
the Kimura 2 parameters model. Detection and identification of Leishmania parasites
was done according to the ITS-1 PCR-RFLP protocol in 39 of the blood-fed captured
females. Amplification and sequencing of cac-IVS6 was achieved in 107 female sand
flies. The multiple alignment revealed a conserved exon and a polymorphic intragenic
region. NJ consensus tree showed that the intron polymorphism is sufficient for differentiation
of sequences belonging to sand flies of different genera (Lutzomyia, Nyssomyia, Migonemyia
and Evandromyia). Sequences belonging to the same genus (Ny. whitmani and Ny. neivai,
both proven to be vectors of L. braziliensis) clustered together making differentiation
between species impossible. Further, the protocol of cac-IVS6 allowed the identification
of four sand flies whose identities could not be achieved by morphological means due
to the condition of the specimens. Detection of Leishmania parasites was possible
in 15% of the samples (6/39), but the results of the RFLP assay for species identification
was different than expected. Because of this, ITS-1 products were sequenced and analyzed
for identity with BLASTn, resulting in six sequences of L. infantum (detected in Ny.
whitmani and Mg. migonei sand flies). In conclusion, even though a sequencing protocol
was required for Leishmania species identification, both protocols were applied in
the same biological sample with positive results, allowing simultaneous identification
of sand fly and the parasite present. It is important to note that the detection of
Leishmania DNA itself is not enough to declare a sand fly species a vector of Leishmania,
although these findings may serve as a first step to define the direction of future
eco-epidemiological studies in Misiones province.
Epidemiology and control (oral communications)
Keynote – Can Sergentomyia spp. play a role in the transmission of human and animal
leishmaniases?
Carla Maia
Global Health and Tropical Medicine, Medical Parasitology Unit, Instituto de Higiene
e Medicina Tropical, Universidade de Nova Lisboa, Portugal
carlamaia@ihmt.unl.pt
Leishmaniases are parasitic diseases caused by protozoa of the genus Leishmania. The
parasites, which infect various wild and domestic mammals, are usually transmitted
by the bite of phlebotomine sand flies belonging to the genus Phlebotomus (in the
Old World) or Lutzomyia (in the New World) genera. On the other hand, Sergentomyia
sand flies, which are widely distributed throughout the Old World, have been proven
as vectors of reptile Leishmania. Since it is generally accepted that most species
of Sergentomyia are not anthropophilic, they are not usually regarded as vectors of
infectious agents (including Leishmania) to humans. However, based upon literature
reviews, a consideration of the role of Sergentomyia in the circulation of mammalian
Leishmania becomes apparent as Leishmania DNA has been identified in several species.
These include the molecular detection of L. major in Se. sintoni in Iran, Se. garnhami
in Kenya, Se. darlingi in Mali, and Se. minuta in Portugal. Furthermore L. donovani
has been detected in Se. babu in India, L. infantum in Se. dubia, Se. magna and Se.
schewtzi in Senegal, and L. siamensis in Se. gemmea in Thailand. Finally, more recently,
L. tropica has been found in Se. ingrami and Se. hamoni in Ghana and Leishmania sp.
related to Chinese Leishmania sp. previously isolated from human and canine leishmaniases
have been detected in Se. minuta in Portugal. Nevertheless, PCR positivity alone should
not be used for incrimination of Sergentomyia sand flies as Leishmania vectors since
the detection of DNA does not give any information about the parasites’ viability
or the presence as virulent metacyclic promastigotes in these invertebrate vectors.
In fact, and although L. infantum DNA was detected in Se. schwetzi from Senegal, the
refractoriness of this African species to some species of Leishmania that infect humans
(including L. donovani, L. infantum and L. major) has also been recently demonstrated.
In any case, the refractoriness of this particular Sergentomyia species does not necessarily
extend to the whole of the genus. In this line of reasoning, the competence and permissiveness
of the different species of Phlebotomus to different Old World Leishmania has also
been observed. Future work must be done to unravel whether any Sergentomyia spp. fulfills
all the criteria that will undoubtedly incriminate it as a vector for Old World Leishmania
with medical and veterinarian importance. These include, (i) the isolation and/or
genetic typing of parasites from several unambiguously identified wild female flies
not containing blood meals; (ii) the detection of infective forms of Leishmania on
naturally infected females and/or on colonized sand flies infected experimentally;
(iii) demonstration of any biting activity on human reservoirs and (iv) experimental
demonstration of vectorial competence for transmission of parasites as a result of
blood-feeding on a mammal.
Molecular analysis of parasite, vector and blood meal DNA from field-caught sand flies
in a Moroccan focus of cutaneous leishmaniasis: Genetically heterogenous Leishmania
tropica in Phlebotomus sergenti as a mono-specific and multi-host feeding vector
Malika Ajaoud1,2, Nargys Es-Sette1, Rémi N. Charrel3, Abderahmane Laamrani-Idrissi4,
Myriam Riyad2,5, Meryem Lemrani1
1Laboratoire de Parasitologie et Maladies Vectorielles, Institut Pasteur du Maroc,
Casablanca, Morocco
2Centre d’Études Doctorales des Sciences de la Santé, Faculté de Médecine et Pharmacie,
Casablanca Morocco
3Aix Marseille University, IRD French Institute of Research for Development, EHESP
French School of Public Health, EPV UMR_D 190 “Émergence des Pathologies Virales”,
& IHU Méditerranée Infection, APHM Public Hospitals of Marseille, Marseille, France
4Service de Parasitologie, Direction d’Épidémiologie et de Lutte contre les Maladies,
Ministère de la Santé, Rabat, Morocco
5Équipe de Recherche sur les Leishmanioses Cutanées, Faculté de Médecine et Pharmacie,
Casablanca, Morocco
meryem.lemrani@pasteur.ma
Phlebotomus (Paraphlebotomus) sergenti is at least one of the confirmed vectors for
the transmission of cutaneous leishmaniasis caused by Leishmania tropica and is distributed
widely in Morocco. This form of leishmaniasis is considered largely to be anthroponotic,
although dogs found infected with L. tropica, suggest that it is zoonotic in some
rural areas. This survey aimed at (i) studying the presence of Leishmania in field
caught Ph. sergenti, (ii) investigating genetic diversity within L. tropica and (iii)
identifying the host-blood feeding preferences of Ph. sergenti. A total of 4,407 sand
flies were collected in three rural areas of Azilal province, using CDC miniature
light traps. Samples collected were found to consist of 13 species: Phlebotomus spp.
and 3 Sergentomyia spp. The most abundant species was Ph. sergenti, accounting for
45.75% of the total. 965 female Ph. sergenti were screened for the presence of Leishmania
sp. by ITS1-PCR-RFLP, giving a positive rate of 5.7% (55/965), all being identified
as L. tropica. Nucleotide heterogeneity of PCR-amplified ITS1-5.8S rRNA gene-ITS2
was noted. Analyses of 31 sequences obtained segregated them into 16 haplotypes, of
which 7 contained superimposed peaks at certain nucleotide positions, suggestive of
heterozygosity. Ph. sergenti was found to have fed on a large variety of vertebrate
hosts, as determined by cytochrome-B sequencing of the DNA from the blood meals of
64 engorged females. Our findings supported the notion that Ph. sergenti is the primary
vector of L. tropica in this focus, and that the latter is genetically very heterogeneous.
Furthermore, our results might be suggestive of a certain level of heterozygosity
in the L. tropica population. This finding, as well as the feeding of the vectors
on different animals are of interest for further investigation.
Sand flies abundance, ecology and oviposition preferences in Bihar, India
Rajesh B. Garlapati1, Shanta Mukherjee1, Rahul Chaubey1, Tahfizur Rahaman1, Piyoosh
Babele1, Akanksha Chowdhury1, Suman Prakash1, Vinod Kumar1, Mukesh Kumar1, Gregory
Franckowiak2, Dan Somers2, Lindsay Briley2, Katelyn Wagner2, Jenna Hulke2, McCall
Calvert2, Larisa Polyakova2, David Poche2, Richard Poche2
1Genesis Laboratories India Private Limited, Patna, Bihar, India
2Genesis Laboratories Inc. Wellington, Colorado, USA
rajesh@genesislabs.com
Visceral leishmaniasis (VL) also known as Kala-azar in India, is transmitted to man
by Phlebotomus argentipes. VL is endemic in the Indian State of Bihar with an estimated
death rate of 20,000–40,000 per year. Little is known about sand fly behavior, ecology
and oviposition, thus a study into sand fly behaviour and to identify potential oviposition
sites was initiated in two villages in Saran district of Bihar state. CDC light traps
were installed to monitor sand flies in vegetation and in palm trees and emergence
traps were set over assumed oviposition sites. Another study was initiated in twenty
four villages, in two districts of Bihar, to study the abundance of sand flies. In
each village twelve traps were installed in three types of locations; houses, cattle
dwellings and vegetation. In each location type 4 traps were installed. Traps were
activated every two weeks. Trapped sand flies were transferred to the lab in Patna
and species identity recorded. Results from both studies indicated that Ph. argentipes
are prevalent in peridomestic habitats and are exophilic in behavior. These studies
also indicated a different dimension to sand fly dispersal in India and suggest that
new control measures should be explored in addition to current indoor residual spraying
to control VL cases in India.
Keynote – Phlebotomine flies vectors of arbovirus: review and recent data
Rémi N. Charrel
UMR “Émergence des Pathologies Virales” (EPV: Aix-Marseille Universite – IRD 190 –
Inserm 1207 – EHESP), Marseille, France & Institut hospitalo-universitaire Méditerranée
infection, APHM Public Hospitals of Marseille, Marseille, France
remi.charrel@univ-amu.fr
Seventy three years ago, the causative agents of sand fly fevers were documented by
Albert Sabin from blood samples collected in acutely ill US soldiers after landing
in Sicily, Italy, 1943. Naples and Sicilian viruses were causing the same disease
although belonging to distinct antigenic complexes. From WWII to 1970’s several viruses
were isolated from sand flies in the Old World. All these viruses were distributed
into 3 serocomplexes. During the last decade, new technologies as NGS has allowed
full genome sequencing for all sand fly-borne phleboviruses and genetic characterisation
has prevailed over serology; however, recent studies plead for resurrection of serology
as a pivotal tool for integrative and translational research activities. Multidisciplinary
research projects are the inescapable way to study viruses transmitted by sand flies.
Success rates of isolation of viral strains have placed field work as a priority when
investigating new territories far beyond the classic seroepidemiological approaches.
Recent results described by several teams will be presented and the points of interest
for entomologists will be presented and discussed. The last decade has provided exciting
results and innovative scientific approaches that demonstrate that collaborations
between entomologists, ecologists, veterinarians, infectious diseases specialists,
virologists, and parasitologists is the best manner to unravel the importance of microbes
transmitted by sand flies.
Sand fly fever in Iran: from the past up to the isolation of Dashli virus (a new Sicilian
like virus)
Vahideh Moin-Vaziri1, Cigdem Alkan2, M. Badakhshan3, N. Rahbarian1,4, Xavier de Lamballerie2,
Rémi N. Charrel2
1Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
2UMR “Émergence des Pathologies Virales” (EPV: Aix-Marseille University – IRD 190
– Inserm 1207 – EHESP) & Fondation IHU Méditerranée Infection, APHM Public Hospitals
of Marseille, Marseille, France
3Department of Medical Entomology and Vector Control, School of Public Health and
Institute of Public Health Research, Tehran University of Medical Sciences, Tehran,
Iran
4Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
vmvaziri@gmail.com; v.vaziri@sbmu.ac.ir
Phlebotominae sand flies are the vectors of phleboviruses such as Toscana, sand fly
fever Sicilian and sand fly fever Naples, which circulate at high rates in Mediterranean
countries and can cause human disease. Recent studies have shown a massive increase
of novel phleboviruses combined with an expanding geographic distribution of existing
phleboviruses in the Mediterranean area and in the Middle-East. In this context, there
is an urgent need to extend our knowledge of sand fly fever in Iran. Different sand
fly fever viruses such as; Sicilian virus, Karimabad, Salehabad, Tehran were reported
by pioneer researchers such as Professors Tesh, Javadian and Saidi since 1977. They
declared that Phlebotomus papatasi is the main vector in Iran. We conducted this study
to update the information that we have about circulation of viruses among sand flies
in Iran. Sand flies were collected using CDC light traps and by aspiration from different
cities and villages of 10 different provinces of Iran. Each specimen was identified
at species level by using reliable morphology keys. Sand flies were pooled based on
sex, trapping methods and species, then were sent to the French EPV laboratory in
Marseille for virus detection and isolation. A total of 4,770 sand flies (3,158 females
and 1,162 males) were collected during 2009–2011 and pooled in 315 groups. The most
prevalent species collected was Ph. papatasi (57.57%) followed by Sergentomyia spp.
(31.05%). Also collected were Ph. alexandri, Ph. tobbi, Ph. mongolensis, Ph. sergenti,
Ph caucasicus, Ph. major s.l., Ph. bergeroti and Ph. kandelakii. RT-PCR revealed that
a pool which consisted of 30 Sergentomyia spp., collected from Dashliboroun village,
Golestan province was positive with the primer pair N-Phlebo 1+/1−. Sequence results
showed proximity with sand fly fever Turkey virus (Accession number: GQ847513). Real-time
PCR also showed a positive result for another pool of 30 Ph. papatasi that were trapped
at the same location and on the same day. The novel isolate was named Dashli virus.
Further studies are needed to clarify the circulation of Dashli in nature and its
potential pathogenicity for human and animals.
Sand fly fauna of Palmas, state of Tocantins, Brazil: occurrence in different environments
and natural infection by trypanosomatids
Tâmara Dias Oliveira Machado1,3, Tauana de Sousa Ferreira1, Alcinei de Souza Santos
Junior3, Nathyla Morgana Cunha Sales3, Renata Velôzo Timbó1, Tamires Emanuele Vital2,
Thaís Tâmara Castro Minuzzi-Sousa1, Andrey José de Andrade1,5, Marcos Takashi Obara4,
Rodrigo Gurgel-Gonçalves1
1Laboratório de Parasitologia Médica e Biologia de Vetores, Área de Patologia, Faculdade
de Medicina, Universidade de Brasília, Brasil
2Laboratório Interdisciplinar de Biociências, Área de Patologia da Faculdade de Medicina,
Universidade de Brasília, Brasil
3Coordenação de Ciências Matemáticas e Naturais, Instituto Federal de Educação, Ciência
e Tecnologia do Tocantins, Palmas, Brasil
4Faculdade de Ceilândia da Universidade de Brasília, Ceilândia, Distrito Federal,
Brasil
5Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal
do Paraná, Brasil
machadoto@ifto.edu.br
The present study aimed to analyze the occurrence of sand flies in gallery forests
and housing units (HUs) in Palmas municipality, state of Tocantins, Brazil, and to
evaluate their natural infection by trypanosomatids. Four gallery forests and four
adjacent domiciliary areas (10 HUs in each) were sampled. In each area 20 HP light
traps were placed for three consecutive days. Captures with Shannon traps (3 h) were
also performed in forests. Captures were carried out in July (dry season) and November
(rainy season) of 2014. The total sampling effort was 960 HP-trap nights and eight
Shannon trap sessions. After identification, females captured in the dry season were
grouped for DNA extraction. Integrity of the samples was checked by a PCR designed
to amplify the cacophony gene IVS6 region in sand flies. Trypanosomatid detection
was performed by amplifying the SSU rDNA region. Positive samples were purified, sequenced
and compared with sequences deposited at GenBank. A total of 1,527 sand flies representing
30 species was captured: 949 (30 spp.) and 578 (22 spp.) specimens in July and November,
respectively. Nyssomyia whitmani was the most common species captured (n = 743), followed
by Evandromyia carmelinoi (n = 111), Bichromomyia flaviscutellata (n = 111), and Psathyromyia
hermanlenti (n = 102). In July a greater number of specimens was captured in gallery
forests (n = 762, 80%), mostly Ny. whitmani. In November, most specimens were found
in HUs (n = 551, 95%). Ny. whitmani was the most captured species in HUs (n = 336),
followed by Lutzomyia longipalpis (n = 84) and Ev. carmelinoi (n = 77). Lu. longipalpis
was found mainly in domiciliary areas (84/86) and Bi. flaviscutellata in gallery forests
(108/111). DNA was extracted from 569 females and grouped into 78 pools, ranging from
1 to 10 specimens. The nested SSU rDNA PCR identified five positive samples including
four Ny. whitmani and one Pintomyia christenseni, resulting in infection rates of
1.03% and 10%, respectively. Among the four sequences obtained from Ny. whitmani,
two forest specimens revealed 100% identity with Leishmania braziliensis, one HU specimen
revealed 100% identity with Trypanosoma sp. and the other forest specimen revealed
100% identity with Crithidia fasciculata sequence. This is the first record of C.
fasciculata in Ny. whitmani, showing its circulation in Tocantins, and L. braziliensis
was found in sand flies for the first time in Palmas. The sequence obtained from Pi.
christenseni from forest revealed 100% identity with L. braziliensis. In both periods
of the year, sand fly species were collected that have been incriminated as vectors
in gallery forests or in the domiciliary areas. However, the highest occurrence of
those species occurred during the dry season in gallery forests. Sand flies were more
frequent in houses in the rainy season. This was probably due to the moisture from
forests that can promote the retention of sand fly populations during the dry season
and provide the colonization of adjacent houses in the rainy season.
First detection of an unknown Trypanosoma DNA in a phlebotomine sand fly collected
from southern Thailand
Atchara Phumee1, Apiwat Tawatsin2, Usavadee Thavara2, Theerakamol Pengsakul3, Suwich
Thammapalo4, Jérôme Depaquit5, Frédérick Gay6, Padet Siriyasatien1,7
1Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok,
Thailand
2National Institute of Health, Department of Medical Sciences, Ministry of Public
Health, Nonthaburi, Thailand
3Faculty of Medical Technology, Prince of Songkla University, Songkhla, Thailand
4The Office of Disease Prevention and Control 12, Songkhla, Thailand
5Université de Reims Champagne Ardenne, ANSES, SFR Cap santé, EA 4688 – USC « Transmission
Vectorielle et Épidémiosurveillance de Maladies Parasitaires (VECPAR) », Reims, France
6Université Pierre et Marie Curie-Paris 6, CHU Pitié-Salpêtrière, AP-HP, Groupe Hospitalier
Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France
7Excellence Center for Emerging Infectious Diseases, King Chulalongkorn Memorial Hospital,
Thai Red Cross Society, Bangkok, Thailand
padet.s@chula.ac.th
Female sand flies are known to be the vectors of Leishmania parasites and viruses.
Several reports have also shown that these insects can also act as vectors for trypanosomes
of bats, lizards and snakes. We report here on the collection of sand fly specimens
from southern Thailand and our analysis to determine which species of sand fly were
present and whether or not trypanosomes were present. To demonstrate the presence
of the trypanosomes in the sand fly we used PCR sets annealed specifically to the
internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rDNA) gene
of trypanosomatids; in addition, the cytochrome b gene was used to identify the species
of sand fly. Among 45 samples of sand fly collected, 7 samples were shown to be Phlebotomus
stantoni, and one of these specimens also amplified positively in the PCR for Trypanosoma
sp. The study is the first detection of Trypanosoma sp. in a sand fly from Thailand.
Sequence of the amplified ITS1 and SSU rDNA gene indicated that this Trypanosome is
a suspected novel species. Further studies of this suspected new Trypanosoma species
including identification of the vertebrate host and the potential of this parasite
to be pathogenic are necessary.
Overview and an update of the current knowledge and perspectives on sand fly research
in Mexico
Eduardo A. Rebollar-Téllez1, Sergio I. Ibáñez-Bernal2, Jorge J. Rodríguez-Rojas1,
David A. Moo-Llanes3, Angélica Pech-May3, Ana C. Montes de Oca-Aguilar2, Oscar Mikeri-Pacheco9,
Miriam Berzunza-Cruz4, Ingeborg Becker-Fauser4, Janine Ramsey3, Carlos Ibarra-Cerdeña5,
Ángel Rodríguez-Moreno6, Christopher Stephens7,8, Victor Sánchez-Cordero6, Alfredo
Castillo-Vera9, Camila González10, Wilfredo Arque-Chunga1, Javier Escobedo-Ortegón11,
Silvia Pasos-Pinto4, Laura Sánchez-García4
1Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento
de Zoología de Invertebrados, Laboratorio de Entomología Médica, Avenida Universidad
S/N, Ciudad Universitaria, CP. 66451, San Nicolás de los Garza, Nuevo León, México
2Instituto de Ecología, A.C., Red Ambiente y Sustentabilidad, Laboratorio de Sistemática
y Ecología de Insectos con Interés Médico y Veterinario, Veracruz, México
3Centro Regional de Investigación en Salud Pública (CRISP), Instituto Nacional de
Salud Pública (INSP), Tapachula, Chiapas, México
4Unidad de Investigación en Medicina Experimental, Facultad de Medicina, Universidad
Nacional Autónoma de México (UNAM), Ciudad de México, México
5Centro de Investigación y Estudios Avanzados, Instituto Politécnico Nacional, Mérida,
Yucatán, México
6Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Ciudad de
México, México
7Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México (UNAM),
Ciudad de México, México
8Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México (UNAM),
Ciudad de México, México
9El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, México
10Universidad de los Andes (UNIANDES), Ciencias Biológicas, Colombia
11Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma
de Yucatán, Mérida, Yucatán, Mexico
eduardo.rebollartl@uanl.edu.mx
Cutaneous leishmaniasis is highly endemic in southern Mexico. The first clinical reports
were made by Danish physician Harald Seidelin in 1912 and other epidemiological work
by Beltran and Bustamante (1942) and a decade later, the outstanding work of Dr. Francisco
Biagi, identified the causative parasite as Leishmania mexicana (Biagi, 1953). During
field studies conducted in Quintana Roo, México, Biagi and his team, were able to
show that the sand fly Bichromomyia olmeca olmeca was the main vector. During the
following years; the knowledge of species distribution and taxonomy increased substantially.
To-date a total of 50 phlebotomine sand fly species have been recorded; which include
48 extant species and 2 extinct species. Of these 48 extant species, it is considered
that in southern Mexico only four sand fly species may play a significant role as
vectors of L. mexicana. Over the last years our research group has shown that geographical
distribution of leishmaniasis does not correlate with the distribution of the sand
fly Bi. olmeca olmeca. Furthermore, evidence gathered in the Yucatan Peninsula over
the last years, has shown that other sand fly species may also act as vectors. These
suspected vectors are Lutzomyia cruciata, Psathryromyia shannoni, and Psychodopygus
panamensis. Thus, based on infection and biting rates; it has been hypothesized that
transmission of L. mexicana in southern Mexico may be because of these four sand fly
species. Vector species in Central, Pacific coast and Northern Mexico, may vary as
well although these foci are less studied. In northern Mexico we have recorded a total
of 11 sand fly species, of which Lu. diabolica and Dampfomyia anthophora are the most
likely vectors. Even though natural infection of sand flies in northern Mexico has
not yet been demonstrated, the presence of L. mexicana in wild rodents has been detected.
Currently alpha diversity studies are underway with the aim of understanding sand
fly assemblages and during these studies it has been observed that taxonomical work
is still required to describe new species. Quantitative models of alpha and beta diversity
in particular sites, has shown that these methods are useful tools to describe richness,
abundances, heterogeneities, dominances and evenness. In addition; ecological niche
models (ENM) have also been employed to assess how sand fly species distribution may
shift under different scenarios of global warming. Overall; the main objective of
this work is to revise and present an updated status of sand fly vectors in México.
Abundance of Lutzomyia longipalpis (Diptera, Psychodidae) in a kennel and its surroundings
on a highly endemic visceral leishmaniosis area in São Paulo State, Brazil
Andre A. Cutolo1, K.B.S. Briguente1, G. Motoie1, C.E.J. Pigozzi2, B.L. Neves3, I.
Menz4, V.L. Pereira-Chioccola1
1Instituto Adolfo Lutz, Av. Dr Arnaldo, 351, 8° andar. São Paulo (SP), Brazil
2Faculdade de Ciências Agrárias de Andradina, Andradina (SP), Brazil
3Universidade do Oeste Paulista, Presidente Prudente (SP), Brazil
4Ingrid Menz Micro Empresa, Campinas (SP), Brazil
cutoloandre@yahoo.com
Lutzomyia longipalpis is the main vector of Leishmania infantum, the etiologic agent
of human and canine visceral leishmaniasis (cVL) in the Americas. From 1999 to 2013
there were 192 deaths among 2,204 human VL diagnosed cases, within 76 municipalities
while Lu. longipalpis was detected in 108 out of 645 cities of São Paulo state, in
southeastern Brazil. VL has been expanding its range and number of human and canine
cases has been increasing in several parts of South America. Dogs are the main reservoir
of the VL causing agent in the urban environment. In order to evaluate aspects of
the dynamics and behavior of Lu. longipalpis in a highly endemic VL area, sand flies
were captured and monitored from January 2012 to September 2013 at a kennel with approximately
100 dogs in the city of Dracena, SP. Automatic light traps were set up in at least
eight different points around the kennel area and its’ surroundings during 70 nights
on an average of 3.34 nights per month. Automatic aspirators were also used for sand
fly capture inside dog-houses from March to September 2013. During the evaluation
period a total of 4,439 sand flies were captured with Lu. longipalpis representing
4,425 (99.68%) of the captured specimens. Evandromyia cortellezzii and Ev. lenti were
also captured; 13 (0.29%) and 1 (0.02%) individuals respectively. Of the captured
sandflies, 2,714 (61.14%), originated from the automatic light traps with the following
distribution in decreasing order: 1,265 (46.51%) chicken coop and 968 (35.67%) from
the domicile porch of west neighbour farmstead, 133 (4.90%) from the kennel pen, 99
(3.65%) front orchard of neighbouring farmstead, 61 (2.25%) east neighbour backyard,
60 (2.21%) back of kennel main house, 55 (2.03%) front of kennel main house and others
73 (2.69%). Direct aspiration of kennel houses used as shelters by the dogs produced
1,725 (38.86%) of the total number of captured sand flies. Regarding seasonality,
the total number of sand flies captured monthly by automatic light traps respectively
from January to November 2012 were 302 (17.84%), 282 (16.66%), 160 (9.45%), 427 (25.22%),
102 (6.02%), 59 (3.08%), 42 (2.48%), 106 (6.26%), 70 (4.13%), 101 (5.97%) and 42 (2.48%),
and from January to September 2013 were 199 (19.49%), 271 (26.54%), 158 (15.48%),
162 (15.87%), 119 (11.66%), 12 (1.18%), 73 (7.15%), 26 (2.55%) and 1 (0.10%). The
higher abundance of sand flies seems to be related to the most hot and humid months
and also related to the sites with constant presence of hosts for blood meals. Lu.
longipalpis was present during all sampled months and trap sites, confirming its high
adaptability to the urban and human environment.
Canine visceral leishmaniasis in the São Paulo metropotian area dissociated of Lutzomyia
longipalpis: Pintomyia fischeri as potential vector of Leishmania infantum chagasi
Fredy Galvis Ovallos, Eunice A.B. Galati
Public Health Faculty, Sao Paulo University, Av Dr. Arnaldo 715, CEP 01246904, São
Paulo, SP, Brazil
galvisfregao@gmail.com
Zoonotic visceral leishmaniasis (ZVL) is an important public health problem in Brazil.
This disease is caused by infection with Leishmania infantum transmitted mainly by
the sand fly Lutzomyia longipalpis. After the first detection of autochthonous canine
visceral leishmaniasis (cVL) cases in São Paulo State in 1998, the enzootic has spread
in association with the geographical expansion of Lu. longipalpis. In Cotia and Embu
das Artes municipalities, in the São Paulo Metropolitan region (São Paulo State),
autochthonous canine cases have been identified since 2003 in the absence of Lu. longipalpis.
However, ecological studies in this area showed that Pintomyia fischeri is the predominant
species, representing about 90% of the specimens among 13 species captured. This result
has led to the suspicion that Pi. fischeri could be acting as vector of the VL agent.
Other evidence such as the attractiveness of dogs to this species and its blood feeding
habits support this hypothesis. In addition, specimens of Pi. fischeri captured in
another focus of cVL have been found to have L. infantum DNA, suggesting that they
are naturally infected with this parasite. In xenodiagnoses experiments Pi. fischeri
females were fed on dogs infected with L. infantum, their susceptibility to this parasite
and the development of potentially infective promastigotes forms was observed. However,
the vectorial competence of Pi. fischeri has not yet been demonstrated. These data
suggest a potential role for Pi. fischeri as a vector of L. infantum, however, further
studies demonstrating vectorial competence, blood feeding habits in a cVL focus and
survival in natural conditions are required.
The emergence and spread of leishmaniases in the borders of Argentina, Brazil, Paraguay
and Uruguay
Oscar Daniel Salomón1, María Gabriela Quintana1,2, María Soledad Santini3, Nilsa González-Britez4,
Nidia Martínez5, Antonieta Rojas de Arias6, Vanete Thomaz-Soccol7, André Luiz Gonçalves7,
Alceu Bisetto Júnior8, Gabriela Willat9, Luis Calegari10, Yester Basmadjian10, Zaida
E. Yadon11, and the IDRC Project #107577 team
Argentina, CONICET: 1Inst Nac de Med Tropical-MSN
2Inst Sup Entomología-UNT
3Centro Nac Diag e Inv Endemoepidemias-ANLIS-MSN, Paraguay
4Facultad de Cs de la Salud-Univ Nac del Este
5Ministerio de Salud Pública, SENEPA
6Centro Desarrollo Inv Científica, Brazil
7Dep Engen de Biopr e Biotecnologia, Univ Federal do Parana
8Secretaria de Saúde do Estado do Paraná, Uruguay
9Ministerio de Salud
10Facultad de Medicina, Univ de la República, USA
11Comm Dis and Health Anal Department, Pan American Health Organization
odanielsalomon@gmail.com; dsalomon@msal.gov.ar
Since 2014 in the border region of Argentina (Ar), Brazil (Br) and Paraguay (Py),
a multi-country project has been carried out to identify the main biological, environmental
and social drivers associated with the risk of transmission of leishmaniases, within
the theoretical frame of Eco-Health. Uruguay (Uy) joined the project since the emergence
of cVL in 2015. The entomological component of the project involves three-night sampling
with mini light traps in urban-periurban domestic units, rural-forest and rural-periurban
transects by; (a) transverse survey on grids of 400 × 400 m (censal or stratified
according to the size of the locality), (b) longitudinal, seasonal/monthly surveys
(10% of the original sites). More than 500 sites were sampled among the four countries.
The main results so far have shown: (a) the dominance of Lutzomyia longipalpis (VL
vector) in the urban stratum, both indoors and in peridomestic environments. It is
more frequent/abundant in the warmer seasons and places with a tendency to spread
to the rural non-sylvatic areas. The dominance of Nyssomyia whitmani (CL vector) in
the sylvatic-rural stratum, and in “green patches” within urban areas, persistent
in colder seasons and places. This spatial segregation was associated with variables
that could allow the modelling of the spatial distribution of vectors in time and
space at different scales. (b) The evidence-based delimitation of an area for environmental
interventions at macrohabitat scale of 400 × 400 m. (c) The genotipification of Leishmania
infantum and L. braziliensis as the main parasites circulating in the region, infecting
indoor and outdoor sand flies. In order to avoid averaging very diverse results the
actual figures of relative abundance will be shown in tables. However, related to
the current spread Lu. longipalpis we could distinguish different scenarios: (a) the
broad urban distribution of the vector, with cVL prevalence rates of 22–26%, and increasing
prevalence during the longitudinal survey: Foz do Iguaçu-Br and Puerto Iguazú-Ar.
(b) incipient colonization by Lu. longipalpis and 2–4% of cVL: Santa Terezinha de
Itaipu-Br and Puerto Libertad-Ar. (c) vectors and cVL restricted to small foci: Presidente
Franco-Py (rural-urban) and Ciudad del Este-Py (urban) with 2% cVL prevalence, and
three foci in Salto-Uy with cVL rates from 1 to 12% in the main focus. (d) Vectors
of CL (Ny. whitmani and a lower proportion Ny. neivai) without Lu. longipalpis, and
cVL only from imported cases in transects. (e) In Paysandú-Uy, no vectors were found,
so the southernmost border up to now of Lu. longipalpis-VL spread is Salto-Uy. The
analysis of the entomological component and the integration with the results from
other disciplinary components are currently in progress.
Financial support: IDRC-Canada Project #107577, Ministry of Health of Argentina, Brazil,
Paraguay and Uruguay; PAHO.
Evaluation of the synthetic sex pheromone, (S)-9-methylgermacrene-B, for recruitment
and monitoring of Lutzomyia longipalpis (Diptera: Psychodidae) in an environmental
reserve in Rio de Janeiro, Brazil
Vanessa De Araujo Barbosa1, Andressa Alencastre Fuzari Rodrigues1, James Gordon Campbell
Hamilton2, Reginaldo Peçanha Brazil1
1Laboratório Doenças Parasitárias – Instituto Oswaldo Cruz – Fiocruz, Av. Brasil,
4365, Pavilhão Arthur Neiva, 21040-900, Rio De Janeiro, RJ, Brasil
2Infectious Disease Transmission and Biology Group, Department of Biomedical and Life
Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, LA1 4YG,
UK
vanessabarbosa_bio@hotmail.com
The Serra da Tiririca State Park, located between the Municipalities of Niterói and
Marica in Rio de Janeiro State was created by a community movement to protect remnants
of Atlantic Forest that were threatened by large scale real estate speculation and
intense tourism in the region. Around the park, cases of cutaneous leishmaniasis occur
sporadically near residential areas and Lutzomyia longipalpis, the main vector of
Leishmania infantum, is found in the park. The vector is considered a species complex
because of the different sex pheromones that are produced by the members of the complex.
As pheromones are considered to be non-toxic substances, they can have an important
role in monitoring and vector control. Lu. longipalpis populations are frequently
sampled by light trap, Shannon trap and others, but collections may represent only
a small proportion of the actual population because of inefficiencies in the sampling
method. In forested areas, such as the Serra da Tiririca, the number of sand flies
captured with CDC light traps is very low, in 8 years of trapping using miniature
CDC light traps 8 male and 4 female Lu. longipalpis were captured suggesting that
in this park the numbers of Lu. longipalpis are low. The main species trapped were
Evandromyia tupynambai (451), Brumptomyia cunhai (172), Micropygomyia schreiberi (128)
and Br. nitzulescui (95). However sampling methodolgy could mask the real situation
with sand fly abundance and thus the transmission cycle in this region. Thus our objective
was to evaluate the synthetic pheromone (S)-9-methylgermacrene-B as a component of
a strategy for monitoring Lu. longipalpis in a wild environment, where their presence
is currently considered to be rare. In this study male and female Lu. longipalpis
were collected with miniature CDC type traps fitted with either lights and synthetic
pheromone (test) or light only (control). Test and control traps were placed 5 m apart.
GC/MS analysis confirmed that the population of Lu. longipalpis in the area was (S)-9-methylgermacrene-B.
In six trapping nights over 3 weeks, where the position of test and control traps
was alternated on each subsequent night, we observed a significant difference between
the number of Lu longipalpis captured in the test traps (synthetic pheromone present)
(mean = 127.2 ± 37.6) compared to those captured using only light attraction (mean = 17.7 ± 4.6),
(Paired T-test; P = 0.022). We concluded that synthetic sex pheromone (S)-9-methylgermacrene-B
is a valuable tool for monitoring Lu. longipalpis and possibly as a component of a
new control strategy for this species.
Synthetic pheromone and long lasting insecticidal nets (LLINs) as a new control strategy
for Lutzomyia longipalpis (Diptera: Psychodidae), the vector of Leishmania (Leishmania)
infantum
Vanessa De Araujo Barbosa1, Cristian Ferreira De Souza1, James Gordon Campbell Hamilton2,
Reginaldo Peçanha Brazil1
1Laboratório Doenças Parasitárias – Instituto Oswaldo Cruz – Fiocruz, Av. Brasil,
4365, Pavilhão Arthur Neiva, 21040-900, Rio De Janeiro, RJ, Brasil
2Infectious Disease Transmission and Biology Group, Department of Biomedical and Life
Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, LA1 4YG,
UK
vanessabarbosa_bio@hotmail.com
Lutzomyia longipalpis, the main vector of Leishmania infantum, is considered to be
a species complex and different members of the complex produce different pheromones.
Current strategies for controlling sand flies using residual spraying have been unable
to prevent the spread of the American visceral leishmaniasis (AVL) across Brazil.
Because of this, new cost-effective approaches are urgently needed to manage populations
of the sand fly vector. Long lasting insecticidal nets (LLINs) offer a new approach
to vector control because they could be an alternative to residual spraying at Lu.
longipalpis aggregation sites, such as chicken sheds. The synthetic sand fly pheromone,
(S)-9-methylgermacrene-B, can attract Lu. longipalpis in natural environments. Here,
we tested an “attract-and-kill” strategy for improving Lu. longipalpis control by
using the synthetic pheromone in conjunction with LLINs. This study was conducted
in Governador Valadares, Minas Gerais, an area considered endemic for AVL in Brazil.
Field experiments were performed with experimental chicken sheds baited with synthetic
sex pheromone to compare the use of LLIN with conventional insecticide spraying on
Lu. longipalpis mortality. Our results demonstrated that the addition of synthetic
pheromone resulted in greater numbers of male and female sand flies being caught at
experimental chicken sheds. This study showed that both treatments (residual insecticide
and impregnated netting) killed close to 100% of sand flies in 24 h following exposure.
After 2 months the lethal effect of both treatments was maintained at close to 100%.
After 4 months of exposure treatments, the lethal effect of netting diminished to
approximately 69% and residual insecticide to 89%. We concluded that insecticide impregnated
netting has potential as a tool for killing sand flies at aggregation sites, and synthetic
pheromone can increase their effectiveness attracting more sand flies to be killed
by the netting. More long-term field trials are needed to improve the effectiveness
of the netting and identify the feasibility of treating surfaces with impregnated
netting as part of visceral leishmaniasis control program.
Identifying the yeast community in the sand fly Phlebotomus perniciosus: towards a
strategy for yeast-mediated biological control of vector-borne diseases
Elena Martin1, Ilaria Varotto Boccazzi1, Gioia Bongiorno2, Leone De Marco3, Luigi
Gradoni2, Nicoletta Basilico4, Stefano Comazzi1, Irene Ricci3, Sara Epis1
1University of Milan – Department of Veterinary Medicine, Italy
2Istituto Superiore di Sanità – Unit of Vector-Borne Diseases and International Health,
Italy
3University of Camerino – School of Biosciences and Veterinary Medicine, Italy
4University of Milan – Department of Biomedical Sciences, Surgical and Dental, Italy
sara.epis@unimi.it
Vector-borne diseases (VBDs) are one of the greatest public health problems worldwide
and their control represents a key global public health challenge for the 21st century.
In particular leishmaniases are considered a worldwide re-emerging health problem
because they are spreading and this may be caused by climate and environmental changes.
In this context, the increased awareness of the environmental and the public health
problems caused by the excessive and uncontrolled use of insecticides to combat VBDs
is leading to the development of alternative control strategies. While bacteria associated
with arthropods are broadly studied and are the focus of several research programs
aimed at developing strategies to control VBDs, such as malaria, dengue, and trypanosomiasis,
arthropod-associated yeasts and their possible application in biocontrol have not
yet been intensely investigated. The objective of this work was to study the yeast
community associated with the sand fly Phlebotomus perniciosus, the main vector of
leishmaniasis in the western Mediterranean area, with the aim of investigating their
potential to interfere with Leishmania development in the insect. To reach this goal
we associated culture-based methodology with culture independent methods: we performed
yeast isolation and identification, 454 pyrosequencing, PCR screening and whole mount
FISH with specific probes to identify the yeast species. We isolated the yeast Wickerhamomyces
anomalus, a species known for its potential in biocontrol, from both male and female
Ph. perniciosus. This yeast was phylogenetically characterized and then tested against
toxin-susceptible yeast strains, demonstrating its’ killer phenotype. Finally, in
order to explore the possibility that this yeast could exert inhibitory/killing activity
against pathogens, we tested the in vitro activity of W. anomalus strains against
the pathogen Leishmania infantum. This study offers the basis for the development
of an environmentally-friendly and human health safe method for VBD control that could
be included in an integrated leishmaniasis control program.
Targeting sand fly control by the use of systemic insecticides presented to mammalian
reservoir hosts of ZCL and VL: A review of recent studies
Richard M. Poché1, Daniel Hartman1, Larisa Polyakova1, Rajesh Babu Garlapati2, David
Poché1
1Genesis Laboratories, Inc., Wellington, Colorado – 80549, USA
2Genesis Laboratories India Private Limited, Patna, India
richard@genesislabs.com
Zoonotic cutaneous leishmaniasis (zCL) and visceral leishmaniasis (VL) are diseases
transmitted by phlebotomine sand fly vectors throughout much of the world. Control
of the diseases is most practical by attempting to reduce the sand fly numbers using
available methods. Studies over the past 20 years have demonstrated good control of
sand flies by treating the reservoir hosts of zCL and VL with systemic insecticides.
Both larval and adult sand fly numbers may be greatly reduced through the use of such
products. Studies from India, Kazakhstan, Tunisia, and the USA conclude that the oral
treatment of animals with insecticides such as imidacloprid and fipronil can greatly
reduce these vector numbers. We present a review of data collected, along with recent
research, that show efficacy against the vector as high as 98% by treating animals
such as Norway rats, great gerbils, bandicoot rats, fat sand rats and cattle to control
sand fly numbers. Additional data have been compiled to show similar efficacy results
with other vectors, including mosquitoes and ticks.
Systemic insecticides used in dogs: potential candidates for sand fly control?
Sonia Ares Gomez, Albert Picado
Barcelona Institute for Global Health, Hospital Clinic – Universitat de Barcelona
Roselló 132, Barcelona, Spain
sonia.ares@isglobal.org
Phlebotomine sand flies are vectors of zoonotic visceral leishmaniasis (ZVL), a disease
in humans and domestic dogs caused by the protozoan, Leishmania infantum. Dogs, the
main reservoir, are required to maintain transmission. Interest in sand fly control
has been increasing in the past few decades because ZVL is adapting to new environments
and spreading to new geographical areas. New strategies to prevent human cases include
the protection of dogs with insecticides. Insecticide impregnated collars, for instance,
have shown to protect dogs from sand fly bites and also to reduce the risk of human
leishmaniasis. But the use of collars poses logistic challenges and may not be cost-effective
in ZVL endemic areas. Thus new sand fly control strategies should be evaluated. The
use of systemic insecticides in dogs has been postulated as an alternative or complement
to current vector control methods. The objective of the study is to find systemic
insecticides in dogs that could be used in sand fly control. We have reviewed the
systemic insecticides currently used in dogs and we have assessed their potential
use as sand fly control tools. The search was made in 4 steps. First, we listed the
systemic drugs used in dogs with endo- and ectoparasiticide activity from the following
sources: small animal formulary of the British small animal veterinary association
(BSAVA), Vademecum veterinario (Guíavet), and the web page parasitipidia.net. Second,
using the European Medicines Agency and U.S. Food and Drug Administration web pages
we looked for information such as product indications, clinical pharmacology, mechanism
of action, duration of action, and efficacy of the systemic drugs selected in the
first step. Third, using PubMed.gov we reviewed the research published about the active
ingredients and their potential use in sand fly control. Fourth, we ranked these systemic
drugs based on following criteria: (1) evidence about the product capability of killing
sand flies, (2) optimal plasma concentration levels in dogs to kill sand flies and
(3) at least one month of expected efficacy. A total of 13 commercial products and
10 systemic drugs were identified in the first three steps. Four of the commercial
products were a combination of two systemic drugs. After ranking the products only
five drugs (spinosad, fluralaner, afoxolaner, sarolaner, and moxidectin) were included
in the list of potential systemic insecticides in dogs to control sand flies. The
efficacy of those drugs as sand fly control measures should be evaluated in laboratory
and field studies. In order to have an impact on L. infantum transmission, thus on
the risk of ZVL in humans, these drugs should have a long-lasting efficacy against
sand flies and should be easy to administer (e.g. ideally per os). Mathematical modeling
can help estimate the efficacy of this potential new vector control strategy in ZVL
endemic areas.
Repellent efficacy of a new combination of fipronil and permethrin against the main
vector of canine visceral leishmaniasis in the Americas (Lutzomyia longipalpis)
Andre A. Cutolo1, Fredy Galvis Ovallos2, E.S. Neves3, S. Sossai3, M.M.F. Vieira3,
F.O. Silva1, S.T. Chester4, B. Fankhauser4, M.D. Soll4
1Merial Saúde Animal, Fazenda S. Francisco s/n°, Paulínia (SP) – 13140-970, Brazil
2Universidade de São Paulo, Faculdade de Saúde Pública, Av. Dr Arnaldo, 715, São Paulo
(SP) – 01246-904, Brazil
3Nowavet. R. Joaquim Lopes de Faria, 630, Viçosa (MG) – 36570-000, Brazil
4Merial Limited, 3239 Satellite Blvd. Duluth, GA – 30096, USA
cutoloandre@yahoo.com
A study was conducted to evaluate the efficacy of a new fipronil and permethrin combination
to repel Lutzomyia longipalpis sand flies when applied once topically on dogs. The
study was conducted using 16 beagle dogs and laboratory reared Lu. longipalpis sand
flies. Eight non-treated dogs were compared to eight dogs treated with a combination
containing 67.6 mg/mL fipronil + 504.8 mg/mL permethrin (Frontect®/Frontline Tri-Act®,
Merial). The treatments were applied topically once on Day 0. Each dog was exposed
to approximately 80 Lu. longipalpis female sand flies on Days 1, 14, 21 and 30. Percent
sand fly repellency on treated dogs was 95.7%, 94.3%, 81.7% and 72.2%, for Days 1,
14, 21 and 30, respectively. There was a significant difference (P ≤ 0.016) between
the treated and control groups on every assessment day. A single topical administration
of a new combination of fipronil and permethrin demonstrated a significant repellent
effect (i.e. >80%) for 21 days against Lu. longipalpis. The results suggest that treatment
with the fipronil-permethrin combination could be integrated into canine visceral
leishmaniasis prevention and control programs in endemic countries in the Americas.
Molecular and biochemical characterization of insecticide resistance in Phlebotomus
and Lutzomyia sand flies
Scott A. Bernhardt, David S. Denlinger, Zachariah Gompert, Joseph S. Creswell
Utah State University, Department of Biology, 5305 Old Main Hill, Logan, Utah 84341,
USA
scott.bernhardt@usu.edu
Identification of phenotypic traits associated with insecticide resistance in phlebotomine
sand flies is an essential component of vector control programs. Chemical insecticides
continue to be a critical tool of control programs in an effort to mitigate the spread
of leishmaniasis. Sand flies have been exposed for decades to a variety of insecticide
classes, which has increased evolutionary selective pressures of resistance. In many
insects, resistance arises either from target-site insensitivity and/or metabolic
detoxification. Target-site insensitivity involves single nucleotide polymorphisms
(SNPs) in the paralytic (para) and acetylcholinesterase-1 (ace-1) genes, which have
been implicated in conferring insensitivity to pyrethroids and organophosphates. Metabolic
resistance involves changes in the expression of carboxylesterases, cytochrome P450’s,
and glutathione S-transferases that are capable of binding, sequestering, metabolizing,
and conjugating insecticides. The primary objective of this research was to augment
the understanding of target-site insensitivity and metabolic detoxification at the
molecular level for pyrethroid and organophosphate resistance in Phlebotomus papatasi
and Lutzomyia longipalpis. We are in the process of characterizing para, ace-1, and
protein profiles in insecticide-susceptible and -resistant Ph. papatasi and Lu. longipalpis
laboratory colonies. To do so, we initially determined the diagnostic doses and times
for Ph. papatasi and Lu. longipalpis sand flies using the CDC bottle bioassay with
10 different insecticides covering four insecticide classes. Both species were determined
to be highly susceptible to DDT and carbamates, whereas with other insecticides, these
species varied substantially in their diagnostic dose and times. In addition, we have
exposed separate Ph. papatasi and Lu. longipalpis colonies to permethrin (pyrethroid)
and malathion (organophosphate) over multiple generations to induce resistance. At
a 60% malathion lethal concentration exposure, F7 generation Ph. papatasi are demonstrating
87.8% survival compared to a 58.7% survival in F5 generation Lu. longipalpis. At a
60% permethrin lethal concentration exposure, F16
Ph. papatasi demonstrate 79.6% survival compared to a 77.1% survival in F9 generation
Lu. longipalpis. Baseline molecular and biochemical characterization have also been
completed in the susceptible colonies. DNA sequence has been recovered surrounding
para 1,014th (kdr), 918th super-kdr region, ace-1 119th codons, and upstream (5′)
and downstream (3′) regions from these codons in the permethrin- and malathion-resistant-selected
Lu. longipalpis and Ph. papatasi colonies. Initial sequence data from the F3 generation
of the Lu. longipalpis permethrin-resistant-selected colony and from the F3 generation
of the Lu. longipalpis malathion-selected colony has revealed no SNPs in the para
and ace-1 genes. Initial sequence data from the F4 generation of the Ph. papatasi
permethrin-resistant-selected colony and from the F3 generation of the Ph. papatasi
malathion-resistant-selected colony has revealed no SNPs in the para and ace-1 genes.
We have begun to conduct genotyping-by-sequencing, utilizing an Illumina sequencing
platform, to identify genome-wide SNP markers associated with insecticide resistance
in Ph. papatasi and Lu. longipalpis. Our working hypothesis is that insecticide resistance
can potentially be more diverse than a single SNP or enzyme class and that resistance
may be polygenic with many genes contributing to the resistance phenotype. Results
from these analyses will provide significant insights into understanding the mechanisms
of insecticide resistance in field-collected sand flies where leishmaniasis is a serious
public health threat.
Evaluation of the spatial relationship between area of insecticide treatment and location
of Leishmaniasis cases using geographical information systems in Adana, Turkey
Hakan Kavur1, Ozan Artun1, Kenan Koca2
1Cukurova University, Karaisalı Vocational School, Adana, Turkey
2Dicle University, Faculty of Agriculture, Department of Soil and Plant Nutrition,
Diyarbakır, Turkey
hakankavur@yahoo.com
Insecticide spraying was conducted to control arthropod vector populations, especially
sand flies and mosquitoes, in 103 districts in downtown Adana, after people had reported
their mosquito and sand fly problems to the metropolitan municipality. People, from
103 locations telephoned the municipality 5,656 times in 2014. Leishmaniases are present
in Turkey in two clinical forms; visceral and cutaneous, and show a tendency for spreading
throughout the country. Numbers and distribution of cutaneous leishmaniasis cases
in Adana province were obtained from the Turkish Ministry of Health. The aim of the
present study was to compare the relationships between the locations of leishmaniasis
patients and insecticide spraying area by using the data in selected study sites in
Adana province. In this regard, city borders were drawn using ARCMAP10.0. Additionally
the map was separated into different layers for querying each province independently.
Altitude, leishmaniasis case numbers and insecticide application numbers of the selected
locations were entered into the database and relationships between them were evaluated.
Identification of the correlation levels of these data may provide useful information
to better guide control program interventions.
Manipulation of sand fly distributions within the peridomestic environment, and implications
for the control of vector borne disease
Erin Dilger1, Graziella Borges-Alves2, Vicky Carter2, M.G. Herededia2, C.M. Nunes3,
L.M. Garcez4, Reginaldo Peçanha Brazil5, James Gordon C. Hamilton6, Orin Courtenay1
1School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
2School of Life Sciences, Keele University, Staffordshire, ST5 5BG, UK
3UNESP, R. Clóvis Pestana, Dona Amelia, Araçatuba, São Paulo
4Instituto Evandro Chagas, Av. Almirante Barroso, Belem, Pará
5Fundaçao Oswaldo Cruz, Instituto Oswaldo Cruz, Lab Doenças Parasitarias, Rio de Janeiro,
Brazil
6Infectious Disease Transmission and Biology Group, Department of Biomedical and Life
Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, LA1 4YG,
UK
orin.courtenay@warwick.ac.uk
In Brazil, zoonotic visceral leishmaniasis (ZVL) is transmitted by the bite of Lutzomyia
longipalpis sandflies. Lu. longipalpis, are catholic in their biting habits and readily
take bloodmeals from a wide range of hosts on which they demonstrate complex mating
aggregation (lekking) dynamics. This results in non-linearities in the host-vector
interactions that influences ZVL transmission. A crucial component of vector control
is to reduce contact between vectors and infectious or susceptible hosts. Here, we
report on a series of investigations to better understand these dynamics and to identify
possible “push/pull” manipulation strategies towards reducing transmission. Preference
of sand flies for common peridomestic host types (dogs, humans and chickens) was investigated
in households under two separate scenarios (i) over a range of experimentally manipulated
maintenance host (chicken) densities, to examine zoopotentiation, and (ii) in households
with and without deltamethrin collars (Scalibor®) on dogs, to explore the effects
of individual based insecticidal interventions on sand fly abundance and displacement.
The results indicate significant shifts in sand fly host preference with changes in
the density of the maintenance host, whereby the number of flies caught in association
with dogs and people reduce as chicken numbers increase, and vice versa. This is likely
related to changes in host odour biomass in conjunction with pheromone mediated aggregation
behaviour. Similarly, deltamethrin dog collars also appear to confer a protective
effect to households beyond individual dog protection, as collar use is related to
a reduced absolute number of flies found in association with both dogs and inside
houses relative to control households. The implications of these findings in relation
to transmission are discussed in the wider context of host demography and vector control
options by illustrating potential benefits beyond just individual protection.
KalaCORE research on the efficacy of control measures against Phlebotomus orientalis,
the principal vector of Visceral Leishmaniasis in East Africa
Dia-Eldin Elnaiem1, Omran F. Osman2, Wossenseged Lemma3, Hanan A.A. Elhadi4, Bakri
Y.M. Nour5, Noteila M. Khalid6, Mulat Yimer7, Jorgi Alvar8, Orin Courtenay9
1Department of Natural Sciences, University of Maryland Eastern Shore, USA
2Department of Zoology, Faculty of Science University of Khartoum, Sudan
3College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia
4Department of Parasitology & Microbiology, Faculty of Medicine, Gedarif University,
Gedarif, Sudan
5Faculty of Medicine, Blue Nile Research and Training Institute, University of Gezira,
Sudan
6Department of Zoology, Khartoum College of Medical Science, PO Box 10995, Khartoum,
Sudan
7College of Medicine and Health Sciences, Bahir Dar University, Ethiopia
8DNDI, Geneva, Switzerland
9School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL,
UK
daelnaiem@umes.edu
Phlebotomus orientalis is the principal vector of visceral leishmaniasis (VL) throughout
East Africa. Due to poor knowledge of its behavior and bionomics and the limited attempts
to evaluate different control tools, little success has been achieved in controlling
this important vector of VL. KalaCORE is a DFID funded consortium initiative to support
endemic countries in achieving elimination (South Asia) or improving control of visceral
leishmaniasis (East Africa). The aims of KalaCORE entomological research in East Africa
is to evaluate cost-effective measures that can be used for control of Ph. orientalis
and suppression of transmission of VL in Sudan, the Republic of South Sudan and Ethiopia.
This includes a sound understanding of the vector bionomics, testing the efficacy
of old and new vector control tools against Ph. orientalis and evaluation of the acceptability
and compliance in use of different control measures under different socioeconomic
contexts. The project is conducted in two main study areas; one among resident population
in 6 VL endemic villages in Gedaref State, eastern Sudan and the other in agricultural
farms in NW Ethiopia where thousands of seasonal and migrant populations are believed
to contract the disease. In this presentation we will display the plan of the study
in each study area and discuss the research findings from the first year of the project.
Visceral Leishmaniasis on the Indian Subcontinent: modelling the dynamic relationship
between vector control schemes and vector life cycles
David M. Poché1, William E. Grant2, Hsiao-Hsuan Wang2
1Genesis Laboratories, Inc. Wellington, Colorado, USA
2Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station,
Texas, USA
davidp@genesislabs.com
Visceral leishmaniasis (VL) is a disease, caused by two known vector-borne parasite
species (Leishmania donovani and L. infantum) that are transmitted to man by phlebotomine
sand flies (Phlebotomus and Lutzomyia spp.). VL results in approximately 50,000 human
fatalities annually of which ≈67% occur on the Indian subcontinent. Indoor residual
spraying is the current method of sand fly control in India, but given that recent
research suggests Phlebotomus argentipes is more exophilic than once believed, alternative
means of vector control, such as the treatment of livestock with systemic insecticide-based
drugs, are being evaluated. We describe an individual-based, stochastic, life-stage-structured
model that represents a temperature-driven sand fly vector population within a village
in India and simulates the effects of vector control via fipronil-based drugs orally
administered to cattle, which targets both blood-feeding adults and larvae that feed
on host feces. Simulation results indicated that the efficacy of fipronil-based control
schemes in reducing sand fly abundance depended on the timing of drug applications
relative to seasonality of the sand fly life cycle. Taking into account cost-effectiveness
and logistical feasibility, two of the most efficacious treatment schemes reduced
population peaks occurring from April through August by ≈90% (applications 3 times
per year at 2-month intervals initiated in March) and >95% (applications 6 times per
year at 2-month intervals initiated in January) relative to no control. The cumulative
number of sand fly days occurring during the peak clinical VL period (April–August)
reduced by ≈83% and ≈97%, respectively, and during the summer months of peak human
exposure (June–August) by ≈85% and ≈97%, respectively. Future research involving an
extensive oviposition site survey and fipronil field trial would best validate the
simulation results. Although this model represents a sand fly population in India
exclusively, parameter values representing oviposition site and blood meal preference
and daily air and soil temperatures can be easily adjusted, suggesting that our model
could prove useful in a priori evaluation of the efficacy of fipronil-based drugs
in controlling leishmaniasis on the Indian subcontinent and beyond
Epidemiology and control (posters)
Dynamics of Laroussius populations and Leishmania infection rate of female sand flies
in an endemic visceral leishmaniasis region, Tunisia, North Africa
Meriem Benabid, Adel Rhim, Rania Ben Romdhane, Manel Zerzri, Aïda Bouratbine
Laboratoire de Parasitologie Médicale Biotechnologie et Biomolécules LR11IPT06, Institut
Pasteur de Tunis, Tunisia
meriem_benabid@yahoo.fr
In Tunisia, North Africa, visceral leishmaniasis (VL) remains primarily a pediatric
disease responsible for considerable infantile morbidity and mortality. It is endemic
in many foci and is mainly found in the northern and central areas of the country
where the climate is favorable for the development of sand flies species of the subgenus
Larroussius. Our interest was to study the dynamics of the Larroussius populations
and the infection rate of female sand flies in a VL “hot spot”, where there is a VL
incidence rate higher than the global average. An entomological survey was carried
out monthly for one year (from June 2010 to May 2011) in two VL foci located in north
eastern Tunisia: Hammed (35°59′51.13″ N/9°58′14.73″ E) and Khadhra (36°11′09.84″ N/10°02′58.39″ E)
both of which are reported as belonging to the Tunisian VL “hot spot” region. Sand
fly collections were done with light traps placed indoors and in animal sheds. Phlebotominae
male (M) and female (F) specimens were separated by microscopy. Male specimens were
identified at genus level according to morphological characters. Female specimens
caught during early September were conserved separately for molecular analysis. The
whole body of female sand flies was used for DNA extraction. DNA samples were analyzed
for Leishmania infection by kinetoplast DNA (kDNA) real-time PCR. Leishmania species
were identified by high-resolution melting (HRM) analysis of the 7SL RNA gene. Sand
fly species were characterized by cycle sequencing of mitochondrial DNA fragment (cytochrome
b). A total of 4,021 specimens were collected in the two sites (2,884 in Khadhra and
1,137 in Hammad). Sex-ratio of the combined sites was 1.2 (1,425 M/1,459 F in Khadhra
plus 775 M/362 F in Hammad). The proportion of male specimens belonging to the Larroussius
species group was higher in Khadhra than Hammad (59.1% versus 40.7%, P < 0.0001).
Phlebotomus perniciosus was the predominant species in the 2 sites (92% in Khadhra
and 85.9% in Hammad, P = ns). The number of male Larroussius specimens captured varied
according to period of capture and showed 2 peaks: a small one in July (Khadhra) or
in June (Hammad) and a more significant peak and spread over a longer period of time
during mid-August–October (Khadhra) and mid-July–November (Hammad). Sixty three female
specimens from the 2 sites were tested for Leishmania infection. Sixteen (25.4%) were
positive by kDNA qPCR. Primilinary results on 7 specimens identified Leishmania species
as L. infantum and sand fly species as Ph. perniciosus. This study showed an extended
transmission season and a high L. infantum infection rate in Ph. perniciosus females
in the hot spot VL region. These environmental characteristics may explain the high
endemicity of visceral leishmaniasis.
Epidemiologic survey of phlebotomine vectors in a canine leishmaniasis endemic area
in Spain
Rita Velez1,2, C. Ballart1,2, E. Domenech3, J. Cairó3, Montserrat Portús2, Montserrat
Gállego1,2
1ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic, Universitat
de Barcelona, Barcelona, Spain
2Laboratory of Parasitology, Faculty of Pharmacy, Universitat de Barcelona, Barcelona,
Spain
3Canis Veterinary Hospital, Girona, Spain
rita.velez@isglobal.org
Leishmaniasis caused by Leishmania infantum is an endemic vector-borne zoonosis in
the Mediterranean region. Canine leishmaniasis (CanL) has a major impact in domestic
dog populations, and it represents an important source of the parasite for human infection.
In this region, control of both human and canine leishmaniasis greatly depends on
an effort to reduce infection burdens in the animal host and phlebotomine vector surveillance.
Recently, a vaccine to prevent CanL has been released in Europe (France, Italy, Portugal
and Spain) and a project to evaluate its effectiveness in an endemic area of northern
Spain has being underway since mid-2015. Though it is known that Phlebotomus perniciosus
and Ph. ariasi are the vectors in L. infantum transmission in Spain, little is known
about their current distribution or the presence of other sand fly species in Northern
Spain. Therefore, a preliminary phlebotomine epidemiologic survey was conducted simultaneously
with a CanL seroprevalence study in order to characterize the area where the vaccine
field study would take place. A total of 20 dog kennels located in Girona province
(Catalonia, northern Spain) were sampled during September 2015. Dog densities in these
kennels ranged from 4 to 34 animals, with an average of 16 dogs per sampling site.
These were mainly located in rural areas at altitudes varying from 70 m to 400 m above
sea level. One to two CDC light traps were placed for one night in each sampling station,
from which 33 traps were recovered. All sand flies were preserved in 70% ethanol until
mounting in Hoyer’s medium for morphological identification under optical microscopy.
A total of 133 phlebotomine specimens were recovered, from which 60 were females (45%).
Sand flies were trapped in 13 sampling stations and the species identified in order
of decreasing abundance were Ph. perniciosus (53%), Ph. ariasi (32%) and Sergentomyia
minuta (15%). Ph. perniciosus was present in 50% of the sampling locations (frequency),
ranging from altitudes of 84 m to 343 m a.s.l. The frequency of Ph. ariasi was 30%
and it was found at altitude ranges of 139 m to 259 m a.s.l. Se. minuta was found
between 73 m and 285 m a.s.l. and presented a frequency of 45%. These preliminary
results allow an insight into the phlebotomine vectors present in an endemic CanL
region and support the selection of this location for carrying out a CanL vaccine
trial. A further characterization of these arthropod populations combined with results
from currently ongoing CanL seroprevalence studies will enable a better understanding
of this zoonosis and the identification of transmission risk factors in this geographic
area.
Financial support: European Union’s Horizon 2020 research and innovation programme
under the Marie Sklodowska-Curie grant agreement N° 642609.
Evidence for stable endemic sand fly populations in the light of migration streams
into Austria
Adelheid G. Obwaller1,4, Mehmet Karakus2, Wolfgang Poeppl3, Seray Toz3, Yusuf Ozbel3,
Horst Aspöck4, Julia Walochnik4
1Federal Ministry of Defence and Sports, Division of Science, Research and Development,
Vienna, Austria
2Ege University, Faculty of Medicine, Parasitology Department, Bornova, Izmir, Turkey
3Department of Dermatology, Medical University of Vienna, Austria
4Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology,
Infectiology and Immunology Medical University Vienna, Austria
julia.walochnik@meduniwien.ac.at
Sand flies (Diptera: Psychodidae: Phlebotominae) are the vectors of various pathogens
of medical-veterinary importance, including Leishmania spp., Bartonella spp. and Phleboviruses.
In Central Europe, leishmaniasis is a rare disease diagnosed almost exclusively in
travellers, soldiers or migrants coming from tropical or subtropical countries. Leishmaniases
are however a major public health problem in the Eastern Mediterranean region and
the Middle East, reinforced by war, which has resulted in a massive stream of refugees
into Europe. Sand fly trapping was performed at different capture sites in South-Eastern
Austria in July and August in the years 2012, 2013 and 2015. In several regions of
Austria sand fly populations were shown to be stable if not increasing. All individuals
found were identified as Phlebotomus (Transphlebotomus) mascittii Grassi 1908, of
which one unfed female individual was infected with L. infantum. A possible circulation
of Leishmania spp. might become an important issue in Central Europe.
Absence of Leishmania-infected phlebotomines in gallery forests of the Federal District
of Brazil
Aline Machado Rapello1, Thaís Tâmara Castro Minuzzi-Sousa1, Tamires Emanuele Vital2,
Tauana Ferreira1, Renata Velôzo Timbó1, Andrey José de Andrade1,3, Rodrigo Gurgel
Gonçalves1
1Laboratório de Parasitologia Médica e Biologia de Vetores, Área de Patologia, Faculdade
de Medicina, Universidade de Brasília, Brasil
2Laboratório Interdisciplinar de Biociências, Área de Patologia da Faculdade de Medicina,
Universidade de Brasília, Brasil
3Laboratório de Parasitologia Molecular, Departamento de Patologia Básica, Universidade
Federal do Paraná, Brasil
aline_rapello@hotmail.com
The aim of this study was to detect Leishmania-infected phlebotomines in gallery forests
of Brasília, Federal District of Brazil (FD), during the dry and the rainy seasons
of 2014. Sand flies were captured in four areas, Água Limpa Farm (FAL), Biological
Reserve of Contagem (REBIO), Brasilia’s National Park (PNB) and Botanic Garden of
Brasília (JBB), in May and September, 2014. The entire capture effort entailed 1,280
HP light traps and 16 Shannon traps. A total of 1,209 sand flies were captured and
dissected. Their heads and genitalia were cleared and mounted in Canada balsam for
species identification. Other female body parts (thorax, part of the abdomen, legs,
and wings) were placed in microtubes with PBS 1× (one specimen for each labeled tube)
and frozen. A variable number of female specimens (one to ten), belonging to the same
species and capture sites, were pooled for total DNA extraction using the Illustra
tissue and cells genomicPrep Mini Spin Kit. Integrity of the samples was checked by
a PCR designed to amplify the cacophony gene IVS6 region in sand flies. Trypanosomatid
detection was performed by amplifying the SSU rDNA and ITS-1 regions. Male phlebotomines
(614) from 14 species and females (594) from 13 species were identified. These included
Bichromomyia flaviscutellata and Nyssomyia whitmani, which are considered to be potential
vectors of Leishmania amazonensis and L. braziliensis, respectively. DNA was extracted
from 569 females grouped in 87 pools. Fragments that corresponded to the cacophony
gene were amplified in all of the samples, demonstrating the integrity of the extracted
DNA. All samples tested negative for Leishmania spp. Despite negative results, the
high population density of potential vector species in the studied areas, the presence
of Leishmania-infected small mammals in gallery forests of FD and the known adaptation
of Ny. whitmani in anthropic environments are risk factors for Leishmania transmission
in the area. Positive canine and human cases of leishmaniases in adjacent areas of
the studied gallery forests were registered, therefore negative results in the present
study do not implicate the absence of infected vectors in these gallery forests and
surroundings.
Vectors of the subgenus Leishmania (Viannia) in the Tapajós national forest reserve
located in the lower Amazon Region of Brazil
Adelson Alcimar de Souza1,†, Thiago Vasconcelos dos Santos1, Yara Lins Jennings1,
Edna Aoba Ishikawa2, Iorlando Barata1, Maria das Graças Silva1,†, José Aprígio Lima1,
Jeffrey Shaw3, Ralph Lainson1,†, Fernando Silveira1
1Parasitology Department, Evandro Chagas Institute, Belém, Pará State, Brazil
2Tropical Medicine Nucleus, Federal University of Pará, Belém, Pará State, Brazil
3Biomedical Sciences Institute, São Paulo University, São Paulo State, Brazil
†In memoriam
jayusp@hotmail.com
In the Brazilian Amazon region American cutaneous leishmaniasis (ACL) is caused by
at least seven Leishmania species Leishmania (Viannia) braziliensis, L. (V.) guyanensis,
L. (V.) lainsoni, L. (V.) shawi, L. (V.) naiffi, L. (V.) lindenbergi and L. (Leishmania)
amazonensis. The distribution of these species often overlaps within a habitat and
they are associated with complex transmission cycles that involve different vectors.
The national forest reserves, known locally as FLONAs (FLOresta NAcional) are preserved
by law. There are 65 registered FLONAs in Brazil and 13 are located in the Pará State
that includes the Tapajós FLONA. These reserves offer unprecedented opportunities
to study the natural transmission cycles of the different Leishmania species which
in this area of Brazil are dominated by L. (Viannia) species. In total 9,704 sand
flies (6,179 females/3,525 males) were captured over a 2 year period between May 2003
and October 2004 using CDC lights traps set at 1.5 m and 20 m (above ground level),
a Shannon trap and aspirations from tree bases. Female flies were dissected, identified
and cultures made of any flagellates found in their intestines. Isolates were identified
by MLEE and monoclonal antibodies. Species abundance was expressed for the four surveyed
ecotopes (ground, canopy, Shannon and tree base collections) with index of species
abundance (ISA) and standard index of species abundance (SISA). The top 10 SISA indices
were as follows: 1. Nysommyia umbratilis; 2. Ny. whitmani; 3. Trichophoromyia ubiquitalis;
4. Psychodopygus complexus/Ps.wellcomei; 5. Ps. davisi; 6. Ny. shawi; 7. Micropygomyia
rorataensis; 8. Vianamyia furcata; 9. Ny. anduzei; 10. Ps. paraensis. Of these 10
species, Viannia infections have previously been found in nine and in our study we
identified Viannia infections in species whose SISA ranking was 2 (Ny. whitmani),
6 (Ps. davisi), 11 (Lu. gomezi) and 13 (Ps. h. hirsutus). Natural flagellate infections
were found in 18 of 6,179 dissected females (infection rate: 0.29%) of the following
8 species: Ny. whitmani [SISA index 2] (6/486, 3 from CDC 1.5 m, 1 from CDC 20 m and
2 from Shannon), Mi. pilosa [SISA index 32] (4/19, 3 from CDC 1.5 m and 1 from CDC
20 m), Ps. davisi [SISA index 6] (2/388, from Shannon), Sc. sordellii [SISA index
29] (2/35, from CDC 1.5 m), Lu. gomezi [SISA index 11] (1/135, from Shannon), Ps.
h. hirsutus [SISA index 13] (1/100, from CDC 20 m), Evandromyia infraspinosa [SISA
index 32] (1/76, from CDC 1.5 m) and Ny. shawi [SISA index 6] (1/280, from CDC 1.5 m).
Of the 18 infections 8 were successfully cultured and 7 were Leishmana. Of the 7 infections
that were successfully identified L. (V.) s. shawi was found in Ny. whitmani (3) and
Lu. gomezi (1) and L. (V.) naiffi in Ps. davisi (2) and Ps. h. hirsutus (1). Our results
support the hypothesis that L. (Viannia) species may have more than one vector and
that Ps. davisi and Ps. h. hirsutus are important vectors of L. (V.) naiffi. The finding
of a L. (V.) s. shawi in a Lutzomyia, Lu. gomezi, raises the question of its potential
transmission by sand flies belonging to the other genera. The mere presence of 10
sand fly species previously linked to the transmission as well as others that have
been found infected elsewhere indicates how very complex cutaneous leishmaniasis transmission
is in the Tapajós FLONA. Based on our past experience, 6 infections in Mi. pilosa
and Ev. infraspinosa most probably belonged to a lizard or anuran trypanosome. SISA
values need to be interpreted with caution as to their importance as indicating vectors.
For instance the high SISA ranking of Mi. rorataensis was due to a large number of
males that were captured from the base of a tree trunk were it was the dominant species
and feeds preferentially on cold blooded vertebrates. With this in mind we consider
that SISA indices are important in evaluating vector potential and that our results
support this view.
Natural transovarial and transstadial transmission of Leishmania infantum in Rhipicephalus
sanguineus (Acari: Ixodidae)
Kourosh Azizi1, Qasem Asgari2, Mohammad Djaefar Moemenbellah-Fard1, Aboozar Soltani1,
Tahereh Dabaghmanesh3
1Research Centre for Health Sciences, Department of Medical Entomology and Vector
Control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran
2Basic Sciences in Infectious Diseases Research Center, Department of Parasitology
and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3Department of Medical Entomology and Vector Control, School of Health, Shiraz University
of Medical Sciences, Shiraz, Iran
dabaghmanesh@gmail.com
The visceral leishmaniasis parasite, Leishmania infantum, is naturally transmitted
through the bites of phlebotomine sand flies. Alternative routes of transmission have
been suggested. The main aim of this study was to verify the passage of L. infantum
kDNA in ticks, Rhipicephalus sanguineus, blood feeding on a parasitemic dog in Shiraz,
south of Iran. Overall, 180 Leishmania-free ticks were collected from the field and
then fed on lab rodents. They were then were divided into eight groups and allowed
to blood-feed on a dog, Canis familiaris, for fixed periods of time. These and all
third-generation stages of ticks were checked for L. infantum kDNA using conventional
PCR protocol at time intervals. The infection rate was significantly higher in female
than male ticks (P = 0.043). The rates were higher among nymphs (50%) than adult ticks
(41.7%). The kDNA of L. infantum was not detected in ticks 24 h post-feeding. It was,
however, positive among the second to fourth groups of nymphs (40, 50 and 55%) and
adult (40, 46.7 and 36.7%) ticks. Eggs and unfed larvae recovered from the third and
fourth adult groups (2w, 4w) were 100% PCR-positive. The data revealed the passage
of L. infantum kDNA in nymphs and adults of brown dog tick following fixed time intervals
post blood feeding on an infected dog. The natural transovarial and transstadial passage
of kDNA through ticks was shown.
Molecular epidemiology of phlebovirus in four provinces in Morocco
Nargys Es-Sette1, Malika Ajaoud1, Rémi N. Charrel2, Meryem Lemrani1
1Laboratoire de Parasitologie et de Maladies Vectorielles, Institut Pasteur du Maroc,
Casablanca, Morocco
2UMR EPV “Émergence des Pathologies Virales”, Aix Marseille Université, IRD U190,
INSERM U1207, IRBA, EFS, EHESP; Marseille, France & Fondation Méditerranée Infection,
APHM Public Hospitals of Marseille, Marseille, France
meryem.lemrani@pasteur.ma
Sand flies are vectors of protozoa, viruses, and bacteria. To investigate the transmission
of phleboviruses, a total of 8,753 sandflies were collected in four foci of leishmaniasis.
A total of 16 species were morphologically identified. Cell culture and Nested-PCR
screening for phleboviruses, using an assay targeting the polymerase gene, showed
positive results for 19 pools of sand flies belonging to different species that had
originated in four different foci, and were different from those commonly reported
in the literature. Sequencing of the corresponding products confirmed these results
and allowed identification of Toscana virus exclusively. Sequence analysis shows that
Moroccan Toscana virus belonging to genotype B and appear close to Toscana virus isolated
in France and Spain. This study reported the existence of the virus in the north,
center and the south of the country. The abundance and diversity of sand flies in
Morocco and Mediterranean climate, would support the continuous circulation of Toscana
virus in our country, posing a potential risk of emergence of this arbovirus.
Phleboviruses circulating in sand flies in Emilia-Romagna region (Northern Italy)
in 2013–2015
Mattia Calzolari1, Romeo Bellini2, Paolo Bonilauri1, Marco Pinna1, Francesco Defilippo1,
Michele Dottori1, Paola Angelini3
1Istituto Zooprofilattico Sperimentale Della Lombardia e dell’Emilia Romagna “B. Ubertini”
(IZSLER), Reggio Emilia, Italy
2Centro Agricoltura Ambiente “G. Nicoli”, Crevalcore, Italy
3Regione Emilia-Romagna, Bologna, Italy
mattia.calzolari@izsler.it
Phlebotomine sand flies are the biological vectors of a variety of viruses belonging
to the Phlebovirus genus. As several of these viruses, like Toscana virus, are important
agents of diseases in humans, defining the phleboviruses circulating in a particular
area is an important health issue. To monitor the presence of phleboviruses, a surveillance
system, based on sampling and testing of sand flies, was activated in the Emilia-Romagna
region (Northern Italy). The system detected the co-circulation of three different
phlebovirus: the well-known Toscana virus and other two previously unreported phleboviruses,
tentatively named Ponticelli virus and Sole virus. Sandflies were sampled in 151 geo-referenced
sites by suction traps, baited with carbon dioxide, and activated overnight. Sites
were placed mainly in the hilly areas of the Region, which are characterized by ecological
conditions particularly favorable to sand-flies. Between 2013 and 2015 a total of
90,506 sandflies were sampled. A subsample of 2,527 specimens were morphologically
identified: 2,441 (96.5%) were Phlebotomus perfiliewi and 86 (3.5%) were Ph. perniciosus.
This result is consistent with previous results obtained in Emilia-Romagna, which
show the overwhelming presence of the Ph. perfiliewi compared to Ph. perniciosus.
The largest number of sand flies (82,181) was collected in 2013. In this year we also
caught the largest number of specimens per trap per night, with more than 10,000 sand
flies in two different sites. A total of 87,492 sandflies, sorted in 321 pools, were
submitted to a Real Time PCR analysis that targeted the Toscana virus, and 35 of these
pools, from 17 sites, tested positive. Moreover 26,853 sandflies (in 110 pools) were
tested with a pan-phlebovirus PCR followed by the sequencing of produced amplicons,
giving 52 positive pools. The phylogenetic analysis made with the homologous sequences
of other phleboviruses available in Genbank, suggest the presence of two previously
unreported phleboviruses, highlighted by the presence of two well supported clades
in the resultant tree. One of these clades falls with the Salehabad serocomplex, and
the respective virus has been tentatively named Ponticelli virus, the other clade
falls in the Sand fly fever Naples serocomplex, and the respective virus has been
tentatively named Sole virus. Both viruses were detected in all the three years of
survey in different locations, Ponticelli virus in 11 sites and Sole virus in 10 sites.
Interestingly the sequence ascribed to Sole virus was also detected in sand flies
from the neighboring Lombardia Region. The isolation and detection of non-described
phlebovirus is consistent with the wide variety of new phleboviruses reported in last
ten years, especially in Mediterranean basin. Despite Toscana virus having been described
as the major cause of summer meningitis in Italy, France and Spain, this virus remains
a neglected pathogen. Moreover the discovering of new phleboviruses, reported in this
study, and in the Mediterranean basin, raises the issue of their infectious potential,
since several of these viruses are serologically detected in vertebrates and show
the ability to grow on Vero cells. The sympatric co-circulation of different phlebovirus
reported in this study, indicate a very dynamic and complex situation, which deserves
a more detailed investigation to characterize the circulation and the possible pathogenicity
to humans and animals of these uncharacterized viruses.
Isolation of Piura virus, an insect-specific negevirus, from Lutzomyia evansi in Colombia
María Angélica Contreras-Gutiérrez1,2, Hilda Guzman3, Marcio R.T. Nunes4, Sandra Uribe2,
Rafael Vivero1,2, Iván Darío Vélez1, Nikos Vasilaskis3, Robert B. Tesh3
1Programa de Estudio y Control de Enfermedades Tropicales – PECET – SIU-Sede de Investigación
Universitaria – Universidad de Antioquia, Medellín, Colombia
2Grupo de Investigación en Sistemática Molecular-GSM, Facultad de Ciencias, Universidad
Nacional de Colombia, sede Medellín, Medellín, Colombia
3Department of Pathology, University of Texas Medical Branch at Galveston, TX, US
4Center for Technological Innovation, Evandro Chagas Institute, Ministry of Health,
Ananindeua, Pará, Brazil
maria.contreras@pecet-colombia.org
Phlebotomine sand flies are known vectors of protozoa (Leishmania spp.), bacteria
(Bartonella bacilliformis), and a diverse group of viruses of public health and veterinary
importance, including members of the families Rhabdoviridae, Bunyaviridae, and Reoviridae.
However, the increasing interest in and discovery of, the diverse nature of the viral
microbiome in insects, indicates that phlebotomine sand flies are also naturally infected
with other types of insect-specific viruses. These viruses are widely distributed
in Diptera around the world. Here we report a new strain of Piura virus (CoR 10),
isolated in C6/36 mosquito cells from a pool of adult Lutzomyia evansi collected in
2013 from Sucre Department on the Caribbean coast of Colombia. Piura virus induced
a rapid cytopathic effect in a C6/36 cell culture. Genome sequencing of CoR 10 showed
a genomic RNA of 10500 Nt with poly-A tail positive strand, and polycistronic encoding
the viral mRNA in 3 ORFs. Nucleotide and amino acid sequence comparison with sequences
of other Negevirus available on Genbank showed that there was 99% identity between
CoR 10 and the prototype PIUV, meanwhile, a low nucleotide and amino acid identity
was obtained (60–70%) with other members of the Negevirus taxon. According to phylogenetic
analysis, CoR 10 is member of the genus Nelorpivirus, negevirus taxon, and is a new
strain of the recently described PIUV Clade, which includes isolates from Peru, Mexico,
and now Colombia. This is the first report of an insect-specific virus from Colombia,
and also, the first register of PIUV isolated from phlebotomine sand flies.
Characterization of susceptibility of Phlebotominae (Diptera: Psychodidae) to the
insecticide, alpha-cypermethrin
Douglas de Almeida Rocha1, Andrey José de Andrade2,4, Luciana Moura Reinaldo3, Marcos
Takashi Obara1
1Núcleo de Medicina Tropical, Universidade de Brasília, Brasil
2Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina,
Universidade de Brasília, Brasil
3Departamento de Estatística – Instituto de Ciências Exatas, Universidade de Brasília,
Brasil
4Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade Federal
do Paraná, Brasil
dougalmeidarocha@gmail.com
Leishmaniases are a group of infectious diseases primarily transmitted by infected
female phlebotomine sand flies. Chemical insecticides used against vector species
are one of the control measures for these diseases. In Brazil the pyrethroid alpha-cypermethrin
has been recognized by the Ministry of Health. Despite the continuous and intensive
control campaigns few studies have been carried out to detect changes in the susceptibility
of sand flies to insecticides. The objective of this study was characterize the susceptibility
profile of sand fly populations to the pyrethroid alpha-cypermethrin. Sand flies caught
in the field in six Brazilian municipalities and specimens from a laboratory colony
classified as the Susceptibility Reference Lineage (SRL) were evaluated using CDC
bottles with different concentrations of alpha-cypermethrin (3 μg/mL, 5 μg/mL, 7 μg/mL
and 9 μg/mL). An acetone control was used for comparison. Males and females from each
municipality were tested in the bottle bioassay (number of replicates = 3). A total
of 2,198 sand fly specimens were captured and Lutzomyia longipalpis was the species
most frequently found in all municipalities. It was observed that CDC bottles can
be used to evaluate the susceptibility of sand flies to insecticides and in the present
study we estimated that the discriminating dose to the SRL population was 2.38 μg/mL.
Kaplan-Meier survival curves showed that specimens of Montes Claros and Paracatu municipalities
were tolerant to alpha-cypermethrin, compared to specimens from Pirenópolis, Unaí,
Januária, and Belo Horizonte. The time taken to kill 50% of sand flies varied according
to the insecticide concentrations that they were exposed to: 40 min for 9 ug/mL, 50 min.
for 7 ug/mL, 60 min. for 5 ug/mL and 70 min. for 3 ug/mL. All sand fly populations,
including the specimens characterized as SRL, showed changes in susceptibility profiles,
indicating tolerance to alpha-cypermethrin.
Financial support: CAPES.
Evaluation of the level of knowledge of public health professionals regarding the
vector of visceral leishmaniasis and its control measures
Anna Ariel Polegato Martins1, Mariana Fuga1, Alessandra Gutierrez de Oliveira2, Mirella
Ferreira da Cunha Santos1
1State University of Mato Grosso do Sul (UEMS), School of Medicine, Campo Grande,
Mato Grosso do Sul, Brazil
2Federal University of Mato Grosso do Sul (UFMS), Parasitology laboratory, Campo Grande,
Mato Grosso do Sul, Brazil
mirella.santos@uems.br
The sand fly Lutzomyia longipalpis is the major vector of Leishmania (Leishmania)
infantum, the causative agent of visceral leishmaniasis in Central and South America.
A thorough understanding of the transmission mechanism of any infectious agent is
crucial to implementing an effective intervention strategy. An accurate identification
of sand fly species and of the epidemiological aspects concerning leishmania vectors
is therefore important, especially to public health professionals in endemic areas.
Based on theoretical public opinion research reference methodology, this study investigated
the level of knowledge of the vector of visceral leishmaniasis (VL) by social actors
directly involved in the prevention and control of the disease. A total of 67 public
health professionals, in 10 Basic Units of Family Health (UBSF) in Campo Grande, Mato
Grosso do Sul, Brazil, answered the questionnaire containing 11 questions regarding
sand flies, VL and control measures. Almost 97% of those who answered the questionnaire
considered VL relevant to public health, but only 80% declared knowing the main symptoms
of the disease. Some 88% referred to sand flies as responsible for the transmission
of VL, but were not capable of describing morphological features. 77% knew of some
measures of insect control and 67% had given some guidance on vector control to the
public. The directions that they reported that they had given to the population were
wrong in 55% of cases. These results demonstrate weakness between the theoretical
and practical understanding, since most professionals were not able to offer correct
instructions on leishmaniasis and its vectors, although they considered that they
had sufficient knowledge of the issue.
Comparison of various recombinant salivary proteins as epidemiological markers for
dog exposure to Phlebotomus perniciosus in different localities in Italy, Portugal
and Spain
Laura Willen1, Tatiana Kostalova1, Nikola Polanska1, Tereza Lestinova1, Carla Maia2,3,
Petra Sumova1, Michaela Vlkova1, Eleonora Fiorentino4, Aldo Scalone4, Gaetano Oliva5,
Fabrizia Veronesi6, José Manuel Cristóvão2, Orin Courtenay7, Lenea Campino2,8, Luigi
Gradoni4, Marina Gramiccia4, Cristina Ballart9,10, Montserrat Gállego9,10, Petr Volf1
1Dept. Parasitol., Charles University, Prague, Czech Republic
2Med. Parasitol. Unit, GHTM, IHMT, Universidade Nova de Lisboa, Portugal
3Fac. Med. Vet., Universidade Lusófona de Humanidades e Tecnologias, Lisboa, Portugal
4Istituto Superiore di Sanità, Rome, Italy
5Dept. Vet. Med. Animal Production, University Federico II, Naples, Italy
6Dept. Vet. Med., Universityof Perugia, Italy
7WIDER and School of Life Sci., University of Warwick, UK
8Dept. de Ciências Biomédicas e Medicina, Universidade do Algarve, Faro, Portugal
9Lab. Parasitol., Universitat de Barcelona, Spain
10ISGlobal, Hospital Clínic – UB, Barcelona, Spain
laura.willen@gmail.com
In Europe Phlebotomus perniciosus (Diptera: Psychodidae) is known as the main vector
of Leishmania infantum (Trypanosomatida: Trypanosomatidae), the parasite that causes
canine and human leishmaniasis. When hosts are bitten, the injected saliva of the
sand fly will elicit an antibody response in the host, which enables the measurement
of the frequency of sand fly – host contact. Since the use of whole salivary gland
homogenate (SGH) is a time-consuming and labour-intensive process, recombinant salivary
proteins and salivary peptides are being suggested as a valid replacement for SGH
in large-scale serological studies. Moreover, the use of peptides as epidemiological
markers in large-scale serology studies is beneficial because of the opportunity to
decrease the chance of cross-reactivity, and thus increasing the specificity of the
antibody response against antigens of Ph. perniciosus. However, it is essential to
determine if these antigens display the same sensitivity in the different localities
where Ph. perniciosus is present. In this study, 214 sera samples from naturally exposed
dogs from Campania (south Italy) and Umbria (central Italy), 341 sera samples from
Metropolitan Lisbon region (Portugal), and a total of 60 sera samples from Catalonia
and Balearic Islands (Spain) were tested with Ph. perniciosus 43 kDa yellow-related
recombinant protein (rSP03B), 42 kDa yellow-related recombinant protein (rSP03) and
SGH. Studies on salivary peptides that represent the most antigenic parts of the proteins
are in progress. In all the sampling areas, where Ph. perniciosus is the principal
vector of L. infantum, a strong correlation was observed between the antibody response
against SGH and rSP03B, suggesting that different populations of Ph. perniciosus share
similar antigenic properties of this salivary protein. Furthermore, no significant
differences were detected across regions, supporting the use of rSP03B as a universal
epidemiological marker throughout the geographical distribution of Ph. perniciosus.
In contrast, correlation between antibody response against SGH and rSP03 was only
found in sera from the two localities in Spain. This suggests that rSP03 is not suitable
as universal marker throughout the geographical distribution of Ph. perniciosus. In
order to confirm the presence of similar antigenic epitopes in the native yellow-related
protein and rSP03B, an inhibition blot was performed. This resulted in a clear inhibition
of the binding of IgG to the native protein after pre-incubating the sera with rSP03B.
The same procedure was performed with rSP03; in this case no inhibition of the binding
of IgG to the native protein was observed which ensures the specificity of the antibody
response against rSP03B. In conclusion, we propose rSP03B as a universal marker of
sand fly exposure throughout the geographical distribution of Ph. perniciosus.
Financial support: Partially by Charles University (GAUK – 1642314/2014), the EU grants
FP6-010284 EDEN and FP7-261504 EDENext, the Spanish projects AGL2004-06909-C02-01,
CGL2007-66943-C02-01/BOS and CG12010-22368-C02-01 and the European Union’s H2020 Programme
under the MSCA GA n° 642609.
Can we identify Leishmania super-spreaders to reduce transmission to sand fly vectors?
Aurore Lison1, Steve Reed2, Orin Courtenay1
1School of Life Sciences, University of Warwick, CV4 7AL, Coventry, UK
2Infectious Disease Research Institute, 1616 Eastlake Avenue East, Seattle, Washington,
USA
orin.Courtenay@warwick.ac.uk
Heterogeneities in the vector-host interactions leading to onward transmission will
determine the degree of aggregation in infection, and ultimately in the effort required
to reduce transmission. In longitudinal xenodiagnosis studies of natural infection
with Leishmania infantum, a large fraction of transmission events to the sand fly
vector Lutzomyia longipalpis is from a very small fraction of the infected canine
reservoir population. Most infected individuals are not significantly infectious,
and the available serological tests, such as those based on anti-Leishmania antigens
rK39 and rK28, are designed to detect current and historical exposure, thus may fail
to target the highly infectious population which is desirable to directly impact on
transmission. Targeting these “super-spreaders”, and the sand flies that they infect,
to manage visceral leishmaniasis incidence requires a different approach to current
suboptimal blanket control strategies, as these epidemiologically significant vector-host
interactions will otherwise require extremely high intervention coverage to successfully
include them. Increasing the specificity is also likely to reduce the likewise non-infectious
(and asymptomatic) infection dogs. The primary objective of this study was to test
existing and novel anti-Leishmania antigens and certain combinations in order to identify
and differentiate super-spreaders in the mixed reservoir population. The ELISA assays
were performed on archived sera collected from a naturally infected cohort population
of Brazilian dogs. Their transmission potential was measured by xenodiagnoses during
a two years longitudinal study. These dog sera samples are well characterized in terms
of infection, disease and infectiousness; and classified as infected, latent infected
and infectious, current and in their sampled lifetime. Here we present preliminary
results towards modeling the impact of such a new diagnostic test specific to reduce
transmission.
Vector control using long-lasting insecticidal nets against kala-azar in Bangladesh
Chizu Sanjoba1, Yusuf Ozbel2, Bunpei Tojo3, Eisei Noiri3, Yoshitsugu Matsumoto1
1Laboratory of Molecular Immunology, Graduate School of Agricultural and Life Sciences,
University of Tokyo, Tokyo, Japan
2Department of Parasitology, Ege University of Faculty of Medicine, Bornova, Izmir,
Turkey
3Division of Advanced Medical Science of Nephrology, the University of Tokyo Hospital,
Tokyo, Japan
asanjoba@mail.ecc.u-tokyo.ac.jp
Kala-azar is one of the major public health problems in Bangladesh, where the disease
has been endemic for many decades. Vector control takes on a role as an important
part in controlling the disease which is transmitted by phlebotomine sand flies, but
the most appropriate vector control measures is still a matter of debate. Here, the
efficacy of two long-lasting insecticidal nets (LLINs), Olyset® and Olyset® Plus against
field collected sand flies was evaluated in a kala-azar endemic area in Bangladesh.
Sand fly bioassays were conducted according to the WHO-approved cone test methodology
with modification. The major species of sand flies (91.28%) tested was Phlebotomus
argentipes. Sand flies were introduced into a plastic cone which had a piece of Olyset®
or Olyset® Plus netting attached over the wide-end for 3 min and then removed and
placed in a plastic cup and provided with sucrose solution soaked onto an absorbent
cotton pad. Mortality was recorded 24 h after the exposure. Approximately 20–25 sand
flies were used in each cone-test, which was repeated 4 times. The mortality of sand
flies recorded 24 h after the exposure was 100% in the Olyset® Plus group while the
mortality of sand flies in the Olyset® group was 83.63% (corrected mortality = (%
test mortality − % control mortality)/(100 − % control mortality) × 100). It is essential
to understand the knowledge, attitude and practice of people who live in an endemic
area towards Kala-azar in order to propose successful vector control strategies. Therefore,
a questionnaire-based survey was also carried out to understand whether or not vector
control using LLINs is a sustainable application. The questionnaire consisted of three
sections, socio-demographic characteristics, knowledge of kala-azar and history of
kala-azar and perceptions of kala-azar vector control. Based on the analysis of 1,393
households, the knowledge, attitude and practice of the people who live in the endemic
area about kala-azar was relatively low. Though utilization of LLINs is promising,
its ownership is noticeably low. Vector control using Olyset® Plus could be a potential
tool for reducing the morbidity rate of kala-azar in an endemic area in Bangladesh
but any control program would require community based education and acceptance.
Financial support: JST/JICA, Science and Technology Research Partnership for Sustainable
Development.
Analysis of gene expression in a Lutzomyia longipalpis-derived cell line
Luzia M.C. Cortes1, Barbara C.A. Melo1, Franklin Souza-Silva1, Bernardo A.S. Pereira1,2,
Felio J. Bello2, Otacilio C. Moreira1, Daniela de Pita-Pereira1, Constança Britto1,
Carlos R. Alves1
1Laboratório de Biologia Molecular e Doenças Endêmicas-IOC, Fiocruz, Av. Brasil, 4365
Manguinhos CEP 21045-900 – Rio de Janeiro, RJ – Brasil
2Universidad Antonio Nariño, Facultad de Medicina, Bogotá, Colombia
lmccortes@gmail.com
Parasites of the genus Leishmania are transmitted to mammals during blood feeding
by sand flies (Diptera: Psychodidae: Phlebotominae), which are widely distributed
in tropical and subtropical regions worldwide. Preventing the development of the parasites
in the gut of the vector sand fly is a feasible strategy to control the infection.
The success of this strategy will only be achieved with the full knowledge of the
interactive events that occur between the parasite and its vector, especially those
that take place in the sand fly gut. In vitro studies with insect cell lines can be
useful to shed light on some of these interactions, as they provide a more controlled
environment. In this context, it was previously demonstrated that a cell line obtained
from embryonic tissue of Lutzomyia longipalpis (Lulo cells) can be an appropriate
model for such studies. Therefore, we aimed to develop molecular and biochemical studies
of Lulo cells to further chatacterize this cell line’s features and, thus, its actual
application for studies with Leishmania spp. Recently, in a proteomic approach to
studying Lulo cells, we observed that promastigotes of Leishmania (Viannia) braziliensis
are able to bind to some proteins of the Lulo cells (data submitted for publication).
Based on this previous study, we analysed, by Real-Time PCR, the expression profile
of some Lulo cell genes, which we observed to be relevant for host-parasite interactions,
before and after incubation of Lulo cells with L. (V.) braziliensis during different
periods of time. The gene targets are based on the following Lu. longipalpis protein
sequences: enolase, zinc ion-binding protein, heat shock protein, oxidoreductase,
peroxiredoxin and putative Cu/Zn superoxide dismutase. As housekeeping reference genes,
we used the Lu. longipalpis GAPDH and RD 49 genes. The relative expression of the
target genes was assessed using the ΔΔCt methodology. Trials in different times of
interaction with the parasite Leishmania spp. will be conducted to simulate what happens
in the phlebotomine gut and to evaluate the possible role of these molecules of the
parasite during the process of access to vector. This study represents a step towards
the establishment of a new in vitro model of the interaction between Leishmania and
the Lulo cells in order to simulate the events that occur in the digestive tract of
infected insects.
Modern tools for sand flies studies (oral communications)
Leishmania HASP and SHERP genes are required for in vivo differentiation, parasite
transmission and host virulence attenuation
Johannes S.P. Doehl1, Jovana Sádlová2, Hamide Aslan3, Sonia Metangmo3, Jan Votýpka2,
Shaden Kamhawi3, Petr Volf2, Deborah F. Smith1
1Centre for Immunology and Infection, Department of Biology, University of York, York,
UK
2Department of Parasitology, Faculty of Science, Charles University, Prague, Czech
Republic
3Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,
Maryland, USA
johannes.doehl@gmail.com
Differentiation of extracellular Leishmania promastigotes within their sand fly vector,
termed metacyclogenesis, is considered to be essential for parasites to regain mammalian
host infectivity. Metacyclogenesis is accompanied by changes in the local parasite
environment, including secretion of complex glycoconjugates (the promastigote secretory
gel) and colonization and degradation of the sand fly stomodeal valve. Deletion of
the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known
to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite
mutants deficient in specific genes within the HASP/SHERP chromosomal region were
used to investigate their role in metacyclogenesis, parasite transmission and establishment
of infection. HASP/SHERP mutants stalled in metacyclogenesis in vivo, although still
capable of osmotaxis, failed to secrete promastigote secretory gel, correlating with
a lack of parasite accumulation in the thoracic midgut and failure to colonize the
stomodeal valve. These defects prevent parasite transmission to a new mammalian host.
Sand fly midgut homogenates modulate parasite behaviour in vitro, suggesting a role
for molecular interactions between parasite and vector in Leishmania development within
the sand fly. For the first time, stage-regulated expression of the small HASPA proteins
has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and
HASPA1 only in intracellular amastigotes of Leishmania. Replacement of HASPA2 into
the null locus background delays onset of pathology in BALB/c mice, despite its lack
of expression in amastigotes, a phenotype associated with significantly slower onset
and progression of pathology. This HASPA2-dependent effect is reversed by HASPA1 gene
addition, suggesting that the HASPAs may be involved in host immunomodulation.
A glance at what Leishmania infantum chagasi expresses inside Lutzomyia longipalpis
Erich Loza Telleria, Thais Lemos da Silva, João Ramalho Ortigão Farias, Yara Maria
Traub-Csekö
Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro – RJ, Brasil
ytraub@ioc.fiocruz.br
Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis the main
vector of visceral leishmaniasis. The parasite is acquired when a female sand fly
feeds on an infected host. The ingested parasites must resist blood digestion and
differentiate into promastigote forms to survive in the insect gut. In the early hours
of blood digestion, intense proteolytic activity caused by the insect digestive enzymes
occurs, as well as oxidative stress caused by the production of heme derived from
hemoglobin digestion. After blood digestion and degradation of the perithrophic matrix
the parasites migrate to the anterior part of the insect gut and differentiate into
metacyclic promastigotes. During the whole cycle inside the insect, the parasites
must survive the insect immune response and interaction with the gut microbiota. We
are investigating which L. i. chagasi molecules are expressed during the establishment
of infection inside the insect. Previous work has described Leishmania molecules that
interact with the insect, such as lipophosphoglycans and proteophosphoglycans, but
little is known regarding other genes expressed by Leishmania inside Lu. longipalpis.
We analyzed the transcriptomes of Lu. longipalpis infected with L. i. chagasi promastigotes
available in the Vector Base website. We identified Leishmania genes that are differentially
expressed at 6 h, 24 h and 144 h post infection. At 6 h post infection we found an
up-regulation of Leishmania amino acid permease and putative glycerol uptake protein
genes related to amino acid metabolism and gluconeogenesis. At 24 h post infection,
there was an increased expression of Leishmania HSP70, HSP60 and aldehyde dehydrogenase
genes involved in stress responses and lipid metabolism. At 144 h, there was a major
expression of Leishmania glucose transporter and amino acid transporter related to
carbohydrate and amino acid metabolism. In order to validate this information we performed
RTPCR using sand flies artificially infected with procyclic L. i. chagasi collected
at 6 h, 24 h, 72 h and 96 h post infection, or procyclic and metacyclic-like parasites
obtained from culture. The gene expression levels were calculated relative to a housekeeping
gene (actin). For initial gene expression assays, we selected genes that were previously
under investigation by our group. The expression level of FLAG/SMP1, which plays a
role in the attachment of Leishmania major to the gut of Phlebotomus papatasi, and
so far without a known function in Lu. longipalpis, was reduced at 24 h and 72 h,
which related to the course of insect digestion, before returning to control levels
at 96 h. In parasites from cultures, FLAG/SMP1 levels did not present significant
variation. The promastigote surface antigen GP63, a zinc dependent metalloprotease,
is notably increased in culture metacyclic-like parasites compared to procyclic parasites.
This increase was also observed in Leishmania-infected insects that increased GP63
expression above control levels at 96 h post infection. We are currently investigating
the expression of other genes that are differentially expressed by the parasite when
infecting Lu. longipalpis. In conclusion, Leishmania present a plethora of differentially
expressed genes that may favor the parasite survival in the constantly changing gut
micro-environment.
Financial support: Fiocruz/PROEP; Faperj/APQ1; CNPq- Ciência sem Fronteiras- BJT.
Lutzomyia longipalpis TGF-β has a role in Leishmania infantum chagasi survival in
the vector
Tatiana Di-Blasi, E. Loza-Telleria, C. Marques, R. Macedo-Couto, M. Neves, A.J. Tempone,
M. Ramalho-Ortigão, Yara Maria Traub-Csekö
Laboratório de Biologia Molecular de Parasitas e Vetores, IOC-FIOCRUZ, Rio de Janeiro,
Brazil
tati.di.blasi@gmail.com
Leishmaniases are caused by Leishmania protozoans and transmitted to the vertebrate
host mainly through bites of Lutzomyia (New World) or Phlebotomus (Old World) sand
fly species. Following blood digestion, the ingested parasites adhere to the sand
fly midgut epithelium, which is an important step to avoid parasite elimination together
with digested blood remnants. This intimate relationship with the parasite leads to
an immune response by the insect in the attempt to control infection. Previous studies
from our group have shown that Caspar silencing in Lutzomyia. longipalpis, decreases
the parasite load inside the vector, and that different bacterial challenges modulate
the expression of a defensin. Other groups also showed that Leishmania major infection
leads to an increase of defensin expression in Phlebotomus duboscqi and that high
levels of ROS in Lu. longipalpis decrease the number of L. mexicana promastigotes
96 h after infection. We found that a Lu. longipalpis TGF-β (Transforming Growth Factor
– β) was overexpressed 72 h post infection with L. infantum chagasi, when parasites
were starting to attach to the midgut epithelium. In an attempt to test the effect
of TGF-β depletion during the Leishmania infection, we fed Lu. longipalpis with L.
i. chagasi and anti-TGF-β polyclonal antibody. We observed that the antibody-fed group
had a significant increase in the number of parasites, in comparison to the control
group without antibody, at the end of digestion. These results indicate that the Leishmania
infection modulates TGF-β gene expression when the parasite contacts the insect digestive
tract, and that TGF-β blocking at the beginning of the infection increases the parasite
load. This indicates a possible role for TGF-β in controlling L. i. chagasi infection
inside the sand fly. Previous studies with the malaria vector Anopheles showed that
the modulation of a TGF-β expression could influence the production of NO and that
this modulation affects the Plasmodium infection cycle. We have successfully silenced
the TGF-β gene and are now investigating the effect of this silencing on the L. i.
chagasi infection, iNOS gene expression and NO production.
Novel method to quantify Leishmania metacyclic promastigotes delivered by individual
sand fly bite reveals the efficiency of parasite transmission
Émilie Giraud, Oihane Martin, Matthew Rogers
Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical
Medicine, WC1E 7HT, UK
matthew.rogers@lshtm.ac.uk
The composition of the infectious dose has never been directly determined from single
infective bites. We developed a real-time PCR-based method for determining the number
of Leishmania mexicana and L. infantum metacyclic promastigotes transmitted by individual
Lutzomyia longipalpis sands flies to living mice. Despite high variation in the numbers
of parasites delivered per bite, 76% of L. mexicana bites and 71% L. infantum bites
contained ≥75% metacyclics. Analysis of multiple bites from individual infected flies
revealed that approximately 60% could transmit ≥80% L. mexicana metacyclics for up
to 10 consecutive bites. Meta-analysis of the 10th bite in relation to the vector’s
midgut infection revealed that sand flies were more likely to repeatedly transmit
a high proportion of metacyclics if the midgut was significantly blocked with parasites
and a parasite-derived glycan-rich plug – the promastigote secretory gel. To test
the impact of the proportion of metacyclics for infection, we mimicked “high-quality”
(100% metacyclics) and “low-quality” (50% metacyclics), low dose L. mexicana transmissions
to BALB/c mice using needles. Doses enriched for metacyclic promastigotes were associated
with slower growing cutaneous lesions that harboured significantly more amastigotes
compared to low quality infections and demonstrated greater transmission potential
back to sand flies. This new method for interrogating sand fly infection and Leishmania
transmission highlights the efficiency of parasite transmission and reveals the composition
of the infectious dose as an important infection determinant.
Blood feeding effect on Phlebotomus papatasi SP15 and SP44 salivary transcripts
Nasibeh Hosseini-Vasoukolaei1,2, Amir Ahmad Akhavan1, Mahmood Jeddi-Tehrani3, Farah
Idali4, Ali Khamesipour5, Mohammad Reza Yaghoobi-Ershadi1, Shaden Kamhawi6, Jesus
G. Valenzuela6
1Department of Medical Entomology and Vector Control, School of Public Health, Tehran
University of Medical Sciences, Tehran, Iran
2Department of Medical Entomology and Vector Control, Health Sciences Research Center,
School of Public Health, Mazandaran University of Medical Sciences, Sari, Iran
3Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran,
Iran
4Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran,
Iran
5Center for Research and Training in Skin Diseases and Leprosy, Tehran University
of Medical Sciences, Tehran, Iran
6Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious
Diseases, National Institute of Health, Rockville, MD 20852, USA
nasibeh.hoseini@gmail.com
As the components of an insect’s meal can induce hormonal changes in blood sucking
insects and consequently gene expression, we verified whether or not blood feeding
can modulate expression of Phlebotomus papatasi salivary gland transcripts. Phlebotomines
were collected using aspirating tubes during 2012–2013 from Esfahan province, a hyperendemic
area of zoonotic cutaneous leishmaniasis in central Iran. Sand flies were identified
according to morphological characters using a valid systematic key. Females of Ph.
papatasi were categorized into four groups according to physiological stages: blood
fed, unfed, semi-gravid and gravid. In flies from each of these groups, the expression
of PpSP15 and PpSP44 salivary transcripts were assessed using a Real-Time PCR method.
Testing the effect of blood feeding on expression of PpSP15 in collected Ph. papatasi
showed 2.41 ± 0.09 fold change in unfed vs. 4.44 ± 0.21 in fed group (p < 0.05). Semi-gravid
flies expressed a significantly higher level of the PpSP15 transcript (2.74 ± 0.07)
than gravid flies (1.17 ± 0.02) (p < 0.05). The transcript level of PpSP44 showed
a 3.33 ± 0.25 fold increase in unfed vs. 4.58 ± 0.23 in the fed group (p < 0.05).
Semi-gravid flies expressed a significantly higher level of the PpSP44 transcript
(2.6 ± 0.05) than gravid flies (1.26 ± 0.02) (p < 0.05). The highest amount of PpSP15
and PpSP44 transcripts was observed in blood-fed sand flies and the lowest amount
in gravid flies. This difference was highly significant (p < 0.01) using the Kruskal-Wallis
statistical test. This induction of salivary transcripts following blood feeding of
sand flies suggests that an important role is played by salivation during feeding.
Higher gene expression in fed flies may be because of the subsequent need for regeneration
of salivary proteins after a meal. Lower mRNA expression in unfed flies may suggest
that sand fly has already deposited a sufficient amount of saliva that can be used
during the feeding process, down regulating salivary gene transcription. The result
of this study showed the correlation between sand fly feeding and the expression of
salivary gene in wild-collected Ph. papatasi, thevector of ZCL in Iran.
Phlebotomus orientalis salivary proteins and antigens
Iva Rohousova1, Alon Warburg2, Petr Volf1
1Department of Parasitology, Charles University in Prague, Prague, Czech Republic
2Department of Microbiology and Molecular Genetics, The Institute for Medical Research
Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases,
The Hebrew University – Hadassah Medical School, The Hebrew University of Jerusalem,
Jerusalem, Israel
iva.rohousova@natur.cuni.cz
Phlebotomus orientalis is the most important vector of human visceral leishmaniasis
in East Africa. As part of an international collaborative project dedicated to investigating
the ecology and transmission dynamics of visceral leishmaniasis in Ethiopia, we had
a unique opportunity to study salivary proteins of this sand fly species. Sand fly
salivary proteins are an important part of the infective inoculum. They play a key
role in the establishment of Leishmania infection. Furthermore, salivary proteins
can be employed also in ELISA tests to measure exposure to sand fly bites. Some salivary
molecules are highly antigenic and elicit a strong antibody response in repeatedly
exposed hosts. This antibody response can be utilized as a marker of exposure to evaluate
the effectiveness of vector control interventions, to estimate the risk of Leishmania
transmission, or to indicate the feeding preferences of sand flies in search of Leishmania
reservoir hosts. First we studied the transcriptome and proteome of Ph. orientalis
female saliva. Thirteen main protein families were identified in the Ph. orientalis
spitome, including enzymes and antigens known from saliva of other sand fly species
such as apyrases, a hyaluronidase, yellow-related proteins, ParSP25-like proteins,
D7-related proteins, and antigen 5-related proteins. All these Ph. orientalis proteins
showed highest homology with their counterparts in saliva of Ph. perniciosus. Additionally,
Ph. orientalis apyrases showed activity comparable to Ph. perniciosus, while hyaluronidase
activity was the lowest among three Larroussius species tested. Further research was
designed to characterize the main salivary antigens using sera of hosts repeatedly
bitten by Ph. orientalis, either experimentally (murine model) or naturally (domestic
animals from northwest Ethiopia). Individuals belonging to all animal species tested
(cattle, dogs, sheep, goats, donkeys) possessed anti-Ph. orientalis saliva IgG antibodies,
indicating their possible involvement in the transmission of Leishmania donovani as
sources of blood for vector sand flies. The most intensive reactions with murine and
canine sera were detected with the yellow-related proteins, apyrases, D7-related proteins,
and antigen 5-related proteins. The five most auspicious antigens were bacterially-expressed
to determine the possibility of replacing salivary gland homogenate in ELISA tests,
which would enable broader use of this test independent of sand fly colony maintenance.
Out of these five recombinant proteins, yellow-related protein rPorSP24 showed the
most promising features; it achieved high correlation with salivary gland homogenate
(ρ = 0.8) and the highest values of specificity, sensitivity, positive and negative
predictive values in ELISA tests with sera of dogs and sheep naturally-exposed to
Ph. orientalis. Finally, we selected and tested B-cell epitopes from antigenic salivary
proteins to further simplify the serology screening. Two peptides from a yellow-related
protein (PorSP24) and one peptide from a ParSP25-like protein (PorSP65) are hot candidate
antigens to measure anti-Ph. orientalis saliva antibodies in canine sera. Such an
approach may increase the specificity and sensitivity of the reaction and could enable
large-scale epidemiological studies.
Financial support: Bill and Melinda Gates Foundation (OPPGH5336); Czech Science Foundation
(13-05292S).
Parity/nulliparity and sand fly salivary gland-gene expression
Nasibeh Hosseini-Vasoukolaei1,2, Amir Ahmad Akhavan1, Mahmood Jeddi-Tehrani3, Farah
Idali4, Ali Khamesipour5, Mohammad Reza Yaghoobi-Ershadi1, Shaden Kamhawi6, Jesus
G. Valenzuela6
1Department of Medical Entomology and Vector Control, School of Public Health, Tehran
University of Medical Sciences, Tehran, Iran
2Department of Medical Entomology and Vector Control, Health Sciences Research Center,
School of Public Health, Mazandaran University of Medical Sciences, Sari, Iran
3Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran,
Iran
4Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran,
Iran
5Center for Research and Training in Skin Diseases and Leprosy, Tehran University
of Medical Sciences, Tehran, Iran
6Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious
Diseases, National Institute of Health, Rockville, MD 20852, USA
nasibeh.hoseini@gmail.com
In the current study we evaluated the expression of two salivary gland genes, PpSP15
and PpSP44, in both parous and nulliparous wild-collected sand flies. As nulliparous
flies have never oviposited, parity is usually used to determine the age structure
of a population. This study was carried out in Esfahan province, a hyperendemic area
of zoonotic cutaneous leishmaniasis in central Iran. Sand flies were collected using
aspirating tubes and were identified according to morphological characters. Females
of Phlebotomus papatasi were dissected and categorized into two groups, parous and
nulliparous, according to the status of the accessory glands. The expression of PpSP15
and PpSP44 salivary transcripts were assessed in the two groups using Real-Time PCR.
The fold changes for parous and nulliparous groups of sand flies, respectively, were
2.46 ± 0.14 and 2.04 ± 0.12 for PpSP15 expression and 1.29 ± 0.07 and 1.35 ± 0.11
fold for PpSP44 expression. In this study of salivary gland-gene expression in Ph.
papatasi, only SP15 appeared to be consistently influenced by parity. In contrast,
the modulation of the SP44 expression profile was not statistically significant between
parous and nulliparous flies. A higher expression level of SP15 transcript was observed
in the parous group of flies, which are older compared to nulliparous flies. This
study showed that the status of accessory glands in sand flies is another physiological
factor influencing salivary gene expression profiles.
Different approaches for further application of MALDI-TOF mass spectrometry for species
identification of phlebotomine sand flies
Kristýna Hlavackova1, Vit Dvorak1, Petr Halada2, Petr Volf1
1Department of Parasitology, Charles University, Prague, Czech Republic
2Institute of Microbiology, Academy of Science, Prague, Czech Republic
hlavackova.k@centrum.cz
Traditional morphological approaches to identification of sand fly species rely on
minute characters, which can be difficult to access, especially in females. Moreover,
there is no morphological approach available that would readily enable the identification
of immature stages (larvae, pupae). Nevertheless, conclusive species identification
in endemic areas, where morphologically similar species with different vectorial capacity
and ecology may occur, is critical, emphasizing the importance of developing alternative
molecular methods of species identification. Protein profiling by matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a promising
technique for identification of various organisms including insects. It is simple,
accurate, and requires minimal sample preparation. Extraction of peptides and small
proteins is mixed with a suitable aromatic acid serving as a MALDI matrix. After co-crystallization,
the crystals are irradiated, desorbed and ionized by laser pulses to generate gaseous
peptide/protein ions, which are then measured by mass spectrometer. The obtained mass
spectrum represents a characteristic and unique protein fingerprint, which allows
rapid and effective species identification. We optimized methods of specimen capture,
storage, sample preparation, tested suitability of different body parts of sand fly
for protein identification, and developed a multi-approach protocol that utilizes
a single insect body for several identification methods: morphology-, DNA- and protein-based.
In sand fly females, we studied the influence of blood meal and egg development at
several time intervals after feeding on the quality of protein profile. Moreover,
we tested the possibility of identifying blood-meal origin; sand fly females experimentally
fed on different vertebrate hosts were analyzed at various points in time and protein
profiles of abdomen containing host blood were compared with protein profiles obtained
from host blood only. In larvae (L2 to L4) and pupae of different stages of development,
protein spectra were acquired and compared with profiles of adult sand flies. Impact
of larval diet on the quality of the protein profile was investigated. In summary,
MALDI-TOF protein profiling is a suitable, time- and cost-effective method for species
identification of sand flies including large sets of field-caught sand flies, if these
are collected, stored and analyzed using a standard, optimized protocol.
Financial support: This project is supported by the Czech Science Foundation (GACR
15-04329S) and by VectorNet.
MALDI-TOF protein profiling as a method of choice for high-throughput species identification
of sand flies – an example from the Balkan
Vit Dvorak1, Kristýna Hlavackova1, Petr Halada2, Bulent Alten3, Vladimir Ivovic4,
J. Omeragic5, I. Pajovic6, F. Martinkovic7, O. Mikov8, J. Stefanovska9, Petr Volf1
1Department of Parasitology, Charles University, Prague, Czech Republic
2Institute of Microbiology, Academy of Science, Prague, Czech Republic
3Department of Biology, Hacettepe University, Ankara, Turkey
4Department of Biodiversity, University of Primorska, Koper, Slovenia
5Department of Parasitology, University of Sarajevo, Sarajevo, Bosnia and Herzegovina
6Center of Plant Protection, University of Montenegro, Podgorica, Montenegro
7Department for Parasitology and Parasitic Diseases, University of Zagreb, Zagreb,
Croatia
8Parasitology and Tropical Medicine, National Centre of Infectious and Parasitic Diseases,
Sofia, Bulgaria
9Faculty of Veterinary Medicine, Ss. Cyril and Methodius University, Skopje, Macedonia
vidvorak@natur.cuni.cz
Conclusive species identification of sand flies by morphological analysis remains
a challenging task due to limited availability of robust characters, their intraspecific
variability among different populations and a need of expertise to assess them. According
to proposed scenarios of future climatic trends, sand flies may emerge in regions
with a lack of expertise for their species identification by classical morphological
approach. Therefore, especially for high-throughput assessment of field surveys in
endemic areas, there is a need for complementary methods of species identification
relying on molecular techniques. Protein profiling by matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry emerged as a molecular method recently
used for species identification of various medically important arthropod vectors including
phlebotomine sand flies. With a standardized protocol of specimen capture, storage
and sample preparation and a robust reference database, it provides characteristic
and unique protein fingerprints that allow conclusive species identification. It requires
minimal sample preparation that enables utilization of a single sand fly specimen
for several purposes. Balkan countries are in a transition area in the European part
of the Mediterranean basin where sand fly fauna of the western part meets with eastern
elements and where several species have the limit of their geographical distribution.
At the same time, data on sand fly presence and species composition are scarce due
to recent upheavals. Due to two field surveys in the Balkan area and adjacent countries
(Bosnia and Hercegovina, Bulgaria, Croatia, Hungary, Macedonia, Montenegro, Serbia
and Slovenia) in summer of 2015, approx. 700 sand fly specimens of a total of 8000
captured were analyzed by protein profiling. The sand flies were trapped by CDC traps
placed in domestic, peridomestic and sylvatic sites and processed according to standardized
protocol. An in-house reference spectrum database was used as reference comprising
16 sand fly species based on specimens from both laboratory colonies as well as field-collected
specimens whose species identity was conclusively confirmed by morphological analysis
and DNA sequencing. Analyzed specimens belonged to species of four subgenera of the
genus Phlebotomus: Adlerius (Ph. balcanicus and Ph. simici), Larroussius (Ph. neglectus,
Ph. perfiliewi, Ph. tobbi), Paraphlebotomus (Ph. sergenti) and Phlebotomus (Ph. papatasi).
Moreover, two species of the genus Sergentomyia were also recorded (Se. minuta and
Se. dentata). The method proved very suitable for identification of female specimens
of the subgenus Larroussius (492 analyzed specimens) where species-specific diagnostic
markers were characterized beside the overall protein spectra. All dubious specimens
where morphological and protein-based identification did not agree were further characterized
by a sequencing analysis (cytochrome oxidase 1 gene) which confirmed the identification
by MALDI-TOF MS in all cases. MALDI-TOF protein profiling proved to be a time- and
cost-effective method of choice for species identification of large sets of field-caught
sand flies if these are collected, stored and analyzed using a standard, optimized
protocol.
Financial support: This project is supported by the Czech Science Foundation (GA15-04329S)
and by VectorNet.
New generation sequencing (NGS) as a tool for identification of pooled sand flies
Nazli Ayhan1,2, Vit Dvorak3, Cigdem Alkan1,2, Petr Volf3, Rémi N. Charrel1,2
1UMR “Émergence des Pathologies Virales” (EPV: Aix-Marseille Universite – IRD 190
– Inserm 1207 – EHESP), Marseille, France
2Institut hospitalo-universitaire Méditerranée infection, APHM Public Hospitals of
Marseille, Marseille, France
3Department of Parasitology, Charles University, Prague, Czech Republic
remi.charrel@univ-amu.fr
The recent renewed interest in viruses transmitted by sand flies has raised technical
questions which should not be neglected to achieve multidisicplinary project and to
share the entomological material collected from field work in a manner that can be
beneficial for all bodies involved in the project. Identification of sand flies at
the species level remains a difficult task and should be conducted at the individual
level which is time and labor-consuming regardless the technique used (morphologic,
sequencing, mass spectrometry). Manipulations of sand flies lead to a degradation
of live viruses and of their RNA; therefore, the requirements of virologists is to
keep the insects “as untouched as possible”. When a virus is isolated, and sequenced,
the next question is “what is the vector?”. Frequently facing this paradoxical situation,
we developed a method that allows to identify the different species of sand flies
contained in a pool. This method takes advantage (i) of PCR using generic primers
to amplify sequences that can discriminate the species of sand flies, (ii) of NGS
capability to generate large numbers of reads that can be statistically analysed.
This technique has been initially used “experimentally” from field-collected pools
of sandflies. In view of the promising results, we have decided to evaluate this method
in a more “scientific” and “systematic” manner based on previously identified sandflies.
The results of both stages will be presented and discussed. This method cannot replace
gold standard techniques for identification; however, it may provide direct information
concerning the species of sand flies acting as vectors of viruses.