scholarly journals Non-invasive visualisation and identification of fluorescent Leishmania tarentolae in infected sand flies

2018 ◽  
Vol 3 ◽  
pp. 160 ◽  
Author(s):  
Hector M. Diaz-Albiter ◽  
Clément Regnault ◽  
Edubiel A. Alpizar-Sosa ◽  
Dagmara McGuinness ◽  
Michael Barrett ◽  
...  
Keyword(s):  
Post Infection ◽  
Parasite Population ◽  
Sand Fly ◽  
Neglected Diseases ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Leishmania Tarentolae ◽  
Green Fluorescent ◽  
Parasite Populations ◽  
Non Destructive

Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods: Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results: Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites (L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion: These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies.

scholarly journals Leishmania infantum Infection Modulates the Jak-STAT Pathway in Lutzomyia longipalpis LL5 Embryonic Cells and Adult Females, and Affects Parasite Growth in the Sand Fly

2021 ◽  
Vol 2 ◽  
Author(s):  
Erich Loza Telleria ◽  
Daisy Aline Azevedo-Brito ◽  
Barbora Kykalová ◽  
Bruno Tinoco-Nunes ◽  
André Nóbrega Pitaluga ◽  
...  
Keyword(s):  
Post Infection ◽  
Leishmania Infantum ◽  
Janus Kinase ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Embryonic Cells ◽  
Adult Females ◽  
Parasite Survival ◽  
Stat Pathway

Phlebotomine sand flies (Diptera, Psychodidae) belonging to the Lutzomyia genus transmit zoonoses in the New World. Lutzomyia longipalpis is the main vector of Leishmania infantum, which is the causative agent of visceral leishmaniasis in Brazil. To identify key molecular aspects involved in the interaction between vector and pathogens and contribute to developing disease transmission controls, we investigated the sand fly innate immunity mediated by the Janus kinase/signal transducer and activator of transcription (Jak-STAT) pathway in response to L. infantum infection. We used two study models: L. longipalpis LL5 embryonic cells co-cultured with L. infantum and sand fly females artificially infected with the parasite. We used qPCR to follow the L. longipalpis gene expression of molecules involved in the Jak-STAT pathway. Also, we modulated the Jak-STAT mediated immune response to understand its role in Leishmania parasite infection. For that, we used RNAi to silence the pathway regulators, protein inhibitor of activated STATs (PIAS) in LL5 cells, and STAT in adult females. In addition, the pathway suppression effect on parasite development within the vector was assessed by light microscopy in late-phase infection. The silencing of the repressor PIAS in LL5 cells led to a moderate increase in a protein tyrosine phosphatase 61F (PTP61F) expression. It suggests a compensatory regulation between these two repressors. L. infantum co-culture with LL5 cells upregulated repressors PIAS, suppressor of cytokine signaling (SOCS), and PTP61F. It also downmodulated virus-induced RNA-1 (VIR-1), a pathway effector, indicating that the parasite could repress the Jak-STAT pathway in LL5 cells. In Leishmania-infected L. longipalpis females, STAT and the antimicrobial peptide attacin were downregulated on the third day post-infection, suggesting a correlation that favors the parasite survival at the end of blood digestion in the sand fly. The antibiotic treatment of infected females showed that the reduction of gut bacteria had little effect on the Jak-STAT pathway regulation. STAT gene silencing mediated by RNAi reduced the expression of inducible nitric oxide synthase (iNOS) and favored Leishmania growth in sand flies on the first day post-infection. These results indicate that STAT participated in the iNOS regulation with subsequent effect on parasite survival.

scholarly journals Odour of domestic dogs infected with Leishmania infantum is attractive to female but not male sand flies: Evidence for parasite manipulation

2021 ◽  
Vol 17 (3) ◽  
pp. e1009354
Author(s):  
Monica E. Staniek ◽  
James G. C. Hamilton
Keyword(s):  
Leishmania Infantum ◽  
Etiologic Agent ◽  
Human Infection ◽  
T Test ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Control Female ◽  
Behavioural Experiments ◽  
Paired T Test

Globally visceral leishmaniasis (VL) causes thousands of human deaths every year. In South America, the etiologic agent, Leishmania infantum, is transmitted from an infected canine reservoir to human hosts by the bite of the sand fly vector; predominantly Lutzomyia longipalpis. Previous evidence from model rodent systems have suggested that the odour of infected hosts is altered by the parasite making them more attractive to the vector leading to an increased biting rate and improved transmission prospects for the pathogen. However, there has been no assessment of the effect of Le infantum infection on the attractiveness of dogs, which are the natural reservoirs for human infection. Hair collected from infected and uninfected dogs residing in a VL endemic city in Brazil was entrained to collect the volatile chemical odours present in the headspace. Female and male Lu. longipalpis sand flies were offered a choice of odour entrained from infected and uninfected dogs in a series of behavioural experiments. Odour of uninfected dogs was equally attractive to male or female Lu. longipalpis when compared to a solvent control. Female Lu. longipalpis were significantly more attracted to infected dog odour than uninfected dog odour in all 15 experimental replicates (average 45.7±0.87 females attracted to infected odour; 23.9±0.82 to uninfected odour; paired T-test, P = 0.000). Male Lu. longipalpis did not significantly prefer either infected or uninfected odour (average 36.1±0.4 males to infected odour; 35.7±0.6 to uninfected odour; paired T-test, P = 0.722). A significantly greater proportion of females chose the infected dog odour compared to the males (paired T-test, P = 0.000). The results showed that the odour of dogs infected with Le. infantum was significantly more attractive to blood-seeking female sand flies than it was to male sand flies. This is strong evidence for parasite manipulation of the host odour in a natural transmission system and indicates that infected dogs may have a disproportionate significance in maintaining infection in the canine and human population.

Entomological Studies in Itaúna, Brazil, an Area With Visceral Leishmaniasis Transmission: Fauna Survey, Natural Leishmania Infection, and Molecular Characterization of the Species Circulating in Phlebotomine Sand Flies (Diptera: Psychodidae)

2019 ◽  
Vol 56 (5) ◽  
pp. 1368-1376 ◽  
Author(s):  
Josiane V Lopes ◽  
Erika M Michalsky ◽  
Nathalia C L Pereira ◽  
Adão J V de Paula ◽  
Fabiana O Lara-Silva ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Natural Infection ◽  
Local Population ◽  
Influential Factor ◽  
Sand Fly ◽  
Health Concern ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Phlebotomine Sand Flies

Abstract Among neglected tropical diseases, visceral leishmaniasis (VL) shows great relevance in global terms and is a serious public health concern due to the possibility of severe and lethal forms in humans. In this study, we evaluate entomological factors such as diversity and abundance of phlebotomine sand flies (Diptera:Psychodidae) and the Leishmania species circulating in these species in possible association with VL transmission in the Brazilian town Itaúna. The entomological collections were performed during three consecutive nights, always in the third week of each month, within a period of 12 mo. A total of 1,786 sand fly specimens were collected, from which 20% were collected inside houses. The influence of three local climatic variables (temperature, rainfall, relative humidity) on the population sizes of these insects was evaluated. Temperature was the most influential factor, with a significant positive correlation with the local population size of phlebotomine sand flies collected per month. Lutzomyia longipalpis (Lutz & Neiva, 1912) was the predominant species in the study area. Leishmania DNA was detected in nine out of 133 pools of sand fly females, using nested/PCR, which resulted in a minimal natural infection rate of 2.91%. DNA from Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida), was detected in Evandromyia cortelezzii (Bréthes, 1923), Ev. evandroi (Costa, Lima & Antunes, 1936), Ev. lenti (Mangabeira, 1938), and Ev. termitophila (Martins, Falcão & Silva, 1964), besides Lu. longipalpis. Our study indicates favorable conditions for VL spreading in Itaúna due to the presence of Lu. longipalpis and Le. infantum-infected phlebotomine sand flies.

scholarly journals Detection of Leishmania infantum DNA in Pintomyia evansi and Lutzomyia longipalpis in Honduras

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Wilfredo Sosa-Ochoa ◽  
Javier Varela Amador ◽  
Yokomi Lozano-Sardaneta ◽  
Gabriela Rodriguez Segura ◽  
Concepcion Zúniga Valeriano ◽  
...  
Keyword(s):  
Pacific Coast ◽  
First Record ◽  
Abundant Species ◽  
Central American ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Endemic Region ◽  
Transmission Cycle ◽  
The Pacific

Abstract Background The two most abundant sand fly species on the Honduran Pacific coast are Lutzomyia (Lutzomyia) longipalpis and Pintomyia (Pifanomyia) evansi. Both species are known vectors of Leishmania (Leishmania) infantum, the etiological agent of visceral leishmaniasis (VL) in the Americas. Although VL and non-ulcerative cutaneous leishmaniasis (NUCL) are endemic on the Pacific versant of the Central American Pacific, the latter is the most frequent manifestation of leishmaniasis there. We evaluated the circulation of Leishmania spp. in the sand fly species on El Tigre Island, an endemic area of NUCL. Results We collected 222 specimens of six sand fly species. Lu. longipalpis (180 specimens; 81%) and Pif. (Pi.) evansi (35 specimens; 16%) were the most abundant species. L. (L.) infantum DNA was detected in nine of the 96 specimens analyzed; seven of these specimens were identified as Lu. longipalpis, and the remaining two were Pi.evansi, with an infection rate of 9.4% and 2.7%, respectively. Conclusion We present the first record of L. (L.) infantum DNA in Pi.evansi from a NUCL endemic region of Central America. Our results suggest that Pi. evansi could be a secondary vector of L. (L.) infantum in the transmission cycle of leishmaniasis. The detection of natural infections of L. (L.) infantum in sand flies in this region contributes to an understanding of the epidemiology of leishmaniasis in Honduras.

scholarly journals Reduced Tissue Parasitic Load and Infectivity to Sand Flies in Dogs Naturally Infected by Leishmania (Leishmania) chagasi following Treatment with a Liposome Formulation of Meglumine Antimoniate

2008 ◽  
Vol 52 (7) ◽  
pp. 2564-2572 ◽  
Author(s):  
Raul R. Ribeiro ◽  
Eliane P. Moura ◽  
Vitor M. Pimentel ◽  
Weverton M. Sampaio ◽  
Sydnei M. Silva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Nodes ◽  
The Other ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Cervical Lymph Nodes ◽  
Meglumine Antimoniate ◽  
Liposome Formulation ◽  
Group I

ABSTRACT The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

scholarly journals Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

2018 ◽  
Author(s):  
Tom Beneke ◽  
François Demay ◽  
Edward Hookway ◽  
Nicole Ashman ◽  
Heather Jeffery ◽  
...  
Keyword(s):  
Life Cycle ◽  
Large Scale ◽  
Gene Knockout ◽  
Sand Fly ◽  
Relative Fitness ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Label Free ◽  
Flagellar Proteins

AbstractThe protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.Author SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2020 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Developmental Cycle ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Time Points

Abstract Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

scholarly journals Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis

2020 ◽  
Author(s):  
Karina Mondragon-Shem ◽  
Katherine Wongtrakul-Kish ◽  
Radoslaw P. Kozak ◽  
Shi Yan ◽  
Iain Wilson ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Immune Responses ◽  
Sand Fly ◽  
Salivary Proteins ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Detailed Structural Analysis ◽  
Highly Sensitive ◽  
Sds Page ◽  
Before And After

AbstractDuring Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host’s immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

Evaluation of a dinotefuran-permethrin-pyriorixyfen insect repellent in dogs exposed to experimental infestations of sand flies (Lutzomyia longipalpis)

2018 ◽  
Vol XXIII (136) ◽  
pp. 58-73
Author(s):  
Bruna Cerruti de Godoi ◽  
Sandra Fernandez ◽  
Paula Bandiera Leite ◽  
Sidimar Sossai ◽  
Leonardo Pinto Brandão ◽  
...  
Keyword(s):  
Urban Areas ◽  
Statistical Significance ◽  
Sand Fly ◽  
Canine Leishmaniasis ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Major Player ◽  
High Incidence ◽  
Receive Treatment ◽  
And Control

Visceral leishmaniasis is a serious zoonosis, and has a high incidence in Brazil. The ubiquitous presence of sand flies is a major player in the high incidence of leishmaniasis in urban areas of Brazil. Prevention and control or canine leishmaniasis is important to decrease the incidence of the zoonosis. Control is done with topical use of products with antiparasitic, repellent and insecticide action. This study evaluated the repellent efficacy of line-on application of a topical permethrin-dinotefuran-pyriproxyfen formulation in dogs. Six dogs were treated with the product and six did not receive treatment. All dogs were sedated and exposed to sand flies of the species Lutzomyia longipalpis weekly for 28 days. The result showed that the product had an efficacy of 90.4% in repelling sand flies. There was statistical significance (p ≤ 0,05) difference in the number (mean) of live engorged sand fly females between the two groups in all evaluations. The product showed repellent efficacy against Lutzomyia longipalpis for 28 days.

scholarly journals Assessment of sand fly (Diptera, Psychodidae) control using cypermethrin in an endemic area for visceral leishmaniasis, Montes Claros, Minas Gerais State, Brazil

2011 ◽  
Vol 27 (11) ◽  
pp. 2117-2123 ◽  
Author(s):  
Ricardo Andrade Barata ◽  
Erika Monteiro Michalsky ◽  
Ricardo Toshio Fujiwara ◽  
João Carlos França-Silva ◽  
Marília Fonseca Rocha ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Endemic Area ◽  
Residual Effect ◽  
Minas Gerais ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Light Traps ◽  
Minas Gerais State ◽  
Transmission Area

Montes Claros in Minas Gerais State, Brazil, was considered an intense transmission area for visceral leishmaniasis. This study evaluated sand fly fauna after insecticide application. Captures were performed in 10 districts from September 2005 to August 2006 with CDC light traps inside and outside each residence. Cypermethrin was sprayed in two cycles during November/2005 and May/2006. The 636 specimens collected, belonging to 10 species, were predominantly Lutzomyia longipalpis (79%), and most frequently males (70%). The highest percentage of specimens were captured in areas surrounding domiciles (85.8%). The main species were observed to be sensitive to treatment with the insecticide. The results showed a reduction in the number of sand flies collected after use of cypermethrin in homes and annexes, and with residual effect lasting from two to four months.

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