Phlebotomine sandflies (Diptera: Psychodidae) from Khandwa and Hoshangabad Districts of Madhya Pradesh, India.

A survey of Phlebotomine sandflies undertaken in April 1985 in Hoshangabad and Khandwa districts of Madhya Pradesh yielded 332 sandflies on 204 sticky traps. Sandflies were collected from feral habitats of termite hills, rodent burrows and tree holes and indoor habitats of human dwellings and cattle sheds. The sandflies belonged to eight species viz., Phlebotomus argentipes, P. papatasi, P. colabaensis, Sergentomyia clydei, S. babu, S. bailyi, S. punjabaensis, and S. indica. Among them P. colabaensis, S. clydei and S. indica were found only in feral habitats, whereas P. argentipes and P. papatasi and other Sergentomyia species collected were found in domestic as well as feral habitats. Termite hills and rodent borrows were the most productive habitats, an observation accounted for by the predilection of sandflies to seek microhabitats offering high humidities and equable temperatures to avoid high summer temperature and low relative humidities of outside environment. Sandfly collection by sticky traps is recommended as an objective method of collection.     

Susceptibility of Phlebotomus argentipes and P. papatasi (Diptera: Psychodidae) to insecticides

Field collected fully fed adults of Phlebotomus argentipes and P. papatasi were screened in the laboratory for susceptibility to DDT, BHC, malathion, deltamethrin, permethrin, lambdacyhalothrin and bendiocarb. Pondichery strain of P. papatasi and P. argentipes showed variations in their susceptibility to insecticides. Both the species were resistant to permethrin but tolerant to DDT and malathion with LD99.99 values of 13.88, 1.92, 1.08 and 34.63, 4.69, 16.32 times more than the deterministic doses respectively. However, they were susceptible to bendiocarb with LD99.99 7.6 and 1.6 times lower than the deterministic doses. While P. papatasi was susceptible to BHC, P. argentipes showed tolerance (1.6 times). The former showed tolerance to deltamethrin and the latter exhibited resistance (34 times). It was reverse in case of lamdacyhalothrin. High susceptibility of the vector sandflies to bendiocarb suggests that this insecticide could be used effectively against OP and pyrethroid resistant populations for Kalaazar control.     

Long-lasting insecticidal nets fail at household level to reduce abundance of sandfly vector Phlebotomus argentipes in treated houses in Bihar (India)

Objective  To determine whether the use of long-lasting insecticidal nets (LLINS) at household level are effective in reducing the abundance of Phlebotomus argentipes , vector of anthroponotic visceral leishmaniasis in India, Nepal and Bangladesh.
Methods  The impact of two long-lasting nets (Olyset and PermaNet) on indoor sandfly abundance was evaluated in selected houses of three endemic hamlets in Bihar (India). It was assumed that most sandflies breed inside the houses and that LLINs would progressively reduce the indoor density during the reproduction season. A campaign of indoor spraying with dichloro-diphenyl-trichloroethane (DDT) interfered with the trial but did not affect the sandfly population.
Results  Only the density of males of P. argentipes was significantly reduced by both the LLINs but not the females.
Conclusions  These findings suggest that most female sandflies are coming from outside and that LLINs do not reduce their entry rate.     

The sandfly fauna in the visceral-leishmaniasis focus of Gedaref,in the Atbara-River area of eastern Sudan

Visceral leishmaniasis (VL) is an acute public-health problem in Sudan. Between 1997 and 2000, four, brief entomological surveys were carried out in Barbar El Fugarra, a village in the state of Gedaref, in the Atbara-River area of eastern Sudan. Between 1996 and 1999, 658 cases of VL occurred among the village's population of about 4000. CDC miniature light-traps set inside and outside human dwellings were used to collect a total of 12,745 sandflies, including five species of the genus Phlebotomus and 19 of Sergentomyia. Phlebotomus papatasi and P. orientalis made up 7% and 5% of the collected sandflies, respectively. Seasonal variation was observed in the numbers of P. orientalis, P. papatasi, S. schwetzi and S. magna caught. Almost all (88%) of the sandflies collected were caught inside houses or granaries and there appeared to be particularly large indoor populations of P. orientalis, P. papatasi, S. schwetzi, S. magna and S. clydei. Phlebotomus orientalis could be responsible for the indoor transmission of the parasites causing the local VL, between humans and between humans and local dogs (which have been found infected by some of the Leishmania zymodemes found in humans). The co-occurrence in this focus of P. papatasi and Arvicanthis niloticus, which are known vectors and reservoir hosts, respectively, of L. major, indicates the possibility that outbreaks of human cutaneous leishmaniasis might occur in the area.     

Screening sandflies for natural infection with Leishmania donovani, using a non-radioactive probe based on the total DNA of the parasite

Digoxigenin-labelled total, Leishmania donovani DNA was used as a probe to detect the parasite in Indian Phlebotomus argentipes. The probe was quite sensitive, detecting as little as 0.3 pg parasite DNA, equivalent to approximately 100 parasites. Positive signals could be detected in 12 (32%) of the 38 small (two- to 30-fly) pools of the wild-caught P. argentipes investigated and in a pool of 10 laboratory-bred P. argentipes fed on a patient with post-kala-azar dermal leishmaniasis. None of the wild-caught P. papatasi, P. sergenti or Sergentomyia sp. or of the newly emerged, laboratory-bred P. argentipes investigated were positive by this probe. The results indicate that such a probe may be very useful in screening sandflies for L. donovani, and so detecting the main vectors and transmission pathways of this parasite, in India and elsewhere.     

Identification of bloodmeals of phlebotomine sandflies using the agarose gel diffusion method.

The source of blood meal of wild caught Phlebotomid sandflies collected in areas around Pondicherry was identified using an agarose gel diffusion method. A total of 497 blood samples obtained from freshly engorged Phlebotomid sandflies were tested. Of these 66.8% were positive to human antisera, 29.2% to bovine antisera and the remaining 4.0% to avain antisera. The human blood indices of P. papatasi and P. argentipes were 0.91 and 0.04, respectively.     

The sandflies (Diptera: Psychodidae) in the Turkish province of Hatay: some possible vectors of the parasites causing human cutaneous leishmaniasis

The province of Hatay, which lies on the Mediterranean coast in south-eastern Turkey, forms an important endemic area for human cutaneous leishmaniasis (HCL). Between the June and November of 2001, the sandflies in eight areas of the province, Erzin, Dortyol, Iskenderun, Kirikhan, Antakya, Samandag, Altinozu and Yayladagi, were surveyed, using sticky-paper traps placed in goat, sheep, cow or chicken sheds close to houses. Although 217 of the 998 sandflies caught belonged to the genus Sergentomyia, the rest were of Phlebotomus species, including some species that, elsewhere, are known to act as vectors of the parasites causing HCL. The Sergentomyia caught were either S. theodori (59.9%) or S. dentata (40.1%). Most of the Phlebotomus were P. sergenti (60.8%) but P. papatasi (18.8%), P. syriacus (9.3%), P. neglectus (5.1%), P. tobbi (2.7%), P. simici (2.2%), P. alexandri (0.9%) and P. kyreniae (0.1%) were also identified (the latter three species for the first time in Hatay province). The identification of the main vector species of sandfly in the province is complicated because it is still uncertain which Leishmania species cause(s) the local HCL. It seems likely, however, that P. sergenti is the main vector in Antakya, Iskenderun, Dortyol, Erzin and Kirikhan. In areas such as Altinozu and Yayladagi, where, although P. sergenti is very rare or absent, HCL cases still occur each year, other species, such as the relatively abundant P. papatasi and P. syriacus, may be the main vectors.     

Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia

From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize.     

Seasonal and nocturnal landing/biting behaviour of Phlebotomus argentipes (Diptera: Psychodidae)

The nocturnal activity of Phlebotomus argentipes (Diptera: Psychodidae), the main vector of Leishmania donovani in India, was studied throughout a year, with monthly collections, between 18.00 and 06.00 hours, of the sandflies landing on 15 humans and 15 cows in the village of Bahapur, Patna district. The cattle appeared to be better as bait, since more than five female P. argentipes were caught on them for each one caught on the human bait. Overall, although P. argentipes were caught during each collection hour from 18.00--06.00 hours, the numbers caught landing/biting peaked at 23.00--24.00 hours. There were, however, slight seasonal variations in the timing of this peak in activity. The numbers of P. argentipes caught/collection night also varied with season, being significantly higher during the summer than during the rainy season or winter (P < 0.01 for each), and apparently inversely correlated with rainfall. Male P. argentipes were caught in much higher numbers than the females of this species, with female:male ratios of 1:8 for the flies caught on human bait and of 1:13 for those collected from cattle. If the probability of a female P. argentipes being infected with L. donovani does not vary with season, peak transmission of this parasite to humans probably occurs between February and May, at the middle of night.     

Seasonal variation of cave-dwelling phlebotomine sandflies (Diptera:Psychodidae) in Phra Phothisat Cave, Saraburi Province, Thailand

Phlebotomine sandflies are widely distributed in Thailand. In view of recent occurrence of indigenous cases of leishmaniasis in Thailand, a bionomic study of sandflies was undertaken in Phra Phothisat cave, Saraburi Province, Thailand from August 2005 to July 2006. The insects were collected monthly by CDC light traps between 06:00 PM and 06:00 AM. They were preserved in 80% alcohol and mounted with Hoyer's medium for species identification. A total of 5,514 sandflies were collected with a female:male ratio of 2.5:1. The collected sandflies belong to 13 species in the genera Phlebotomus and Sergentomyia, namely S. silvatica (43.5%), S. anodontis (31.5%), S. dentata (15.3%), S. barraudi (3.3%), P. argentipes (2.0%), P. philippinensis gouldi (1.0%), P. stantoni (0.5%), S. gemmea (0.5%), P. major major (0.1%), S. perturbans (0.1%), S. iyengari (0.1%), S. bailyi (0.1%), and P. teshi (0.1%). The results revealed seasonal variation in sandflies with the highest peak in July (436 sandflies/trap-night). Some of the sandflies could not be identified and were assumed to be new species. Soil samples inside the cave were analyzed for chemical characteristics. The soil was characterized by moderate acidity (pH 5.8) with various amount of chemicals and nutrients.     

First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi (Diptera; Psychodidae) in Fars province, southern Iran

Zoonotoc cutaneous leishmaniasis is endemic in several parts of Iran. Jahrom district is one of the most important endemic foci of leishmaniasis located in Fars province, southern Iran. To identify the vectors of leishmaniasis in this area, a total of 349 sandflies were collected during May to August 2009. They were caught from outdoors in five regions of Jahrom district including villages of Mousavieh, Ghotb-Abad, Heydar-Abad, Fath-Abad and Jahrom County. Eleven species of Phlebotomine (three Phlebotomus spp. and eight Sergentomyia spp.) were detected. To determine the sandflies naturally infected by Leishmania spp., 122 female sandflies were dissected and evaluated microscopically using Giemsa-stained slides. Natural infection of 2 out of 38 (5.26%) P. papatasi and 1 out of 8 (12.5%) P. salehi to Leishmania major was confirmed in the region. Sequencing and nested polymerase chain reaction-based detection of Leishmania were carried out to confirm the microscopic findings. Five (13.16%) P. papatasi and two (25%) P. salehi were positive in nested polymerase chain reaction assay. All positive samples were shown 72-76% similarity with L. major Friedlin. On the basis of our knowledge, this is the first molecular detection of L. major within naturally infected P. salehi in this region in southern Iran.     

Phlebotomine sandflies (Diptera: Psychodidae) of Marrakech city, Morocco

The sandflies at 17 sites within or near the Moroccan city of Marrakech were surveyed between 2002 and 2006. Overall 2310 specimens were collected and five species were identified. Phlebotomus (Phlebotomus) papatasi was the most prevalent species (47.4%), followed by Sergentomyia (Sergentomyia) minuta (16.6%), S. (S.) fallax (16.1%), P. (Paraphlebotomus) sergenti (12.8%) and P. (Larroussius) longicuspis (7.1%). Phlebotomus sergenti appeared to be the most endophilic species. When the collection sites were categorized into five types of habitat, only P. papatasi and S. minuta were found to be common in every habitat type. Urbanization seemed to have a significant effect on the numbers and species of sandfly. In the year after the demolition and redevelopment of the Akioud neighbourhood, there were far fewer sandflies and species of sandfly than observed before the redevelopment. Two years later, however, the sandfly population was found to be much larger than that seen before the neighbourhood was demolished.     

Evaluation of a dot-immunoblot assay for detecting leishmanial antigen in naturally infected Phlebotomus argentipes (Diptera: Psychodidae)

A simple and highly reproducible dot-immunoblot assay was developed to detect leishmanial antigen in Phlebotomus argentipes that were naturally infected with Leishmania donovani. The test was sensitive to as little as 10 ng of antigenic protein (equivalent to the gut content of one laboratory-infected sandfly) and also appeared to be specific, in that it gave a positive result with some P. argentipes (the primary vector of L. donovani in India) and L. donovani but not with P. papatasi or other pathogens.When used to investigate a large number of sandflies collected from two areas of the Indian state of Bihar where visceral leishmaniasis is endemic, the assay appeared sufficiently sensitive and specific to detect the naturally infected insects. The simplicity, reproducibility, high sensitivity and high specificity of the assay should make it useful for field studies, particularly in determining the prevalence of sandfly infection, the local level of transmission, and the impact of vector-control programmes.     

Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan.

Phlebotomine vectors can in some instances transmit only certain species of Leishmania. Comparison of a large number of vector/parasite pairs revealed that species-specific differences in vectorial competence were in every case directly correlated with the ability of promastigotes to attach to the sand-fly midgut, the variable outcomes of which were controlled by structural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. The ability of Phlebotomus papatasi to transmit only Leishmania major could be attributed to the unique, highly substituted nature of L. major LPG that provides for multiple terminally exposed beta-linked galactose residues for binding. While the relatively unsubstituted LPGs of other Leishmania species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of Phlebotomus argentipes, which was found to be a potentially competent vector for every Leishmania species examined. The data suggest that at least some phlebotomine vectors differ with respect to the parasite recognition sites which they express and that midgut adhesion is a sufficiently critical component of vectorial competence as to provide the evolutionary drive for LPG structural polymorphisms.     

Entomological survey of sandflies (Diptera: Psychodidae) in a new focus of zoonotic cutaneous leishmaniosis in Iran.

A total of 22,982 sandflies representing six species were collected using sticky (oiled) paper traps in Badrood rural district, Natanz county from May 1995 to May 1996. Phlebotomus papatasi was the predominant species. The average indoor resting density of P. papatasi ranged from 11.8 (May 1995) to 135.8 per 10 traps (September 1995). The average indoor resting density of this species varied between 39.7-701.3 per 10 traps in May and September, respectively in 1995. The activity of P. papatasi starts from late April or early May and extends to mid October with two peaks, one in mid or late June and the second one in early or mid September. Natural leptomonad infection was found in P. papatasi and Sergentomyia sintoni. P. papatasi could be a vector of humans and gerbils and it is susceptible to DDT in this area.     

Zooprophylaxis: impact of breeding rabbits around houses on reducing the indoor abundance of Phlebotomus papatasi

Zooprophylaxis is the use of animals to deviate vectors from humans. The indoor abundance of Phlebotomus papatasi in houses with rabbit holes in the peridomestic areas are significantly lower than the indoor abundance in houses without rabbit holes in their peridomestic areas. Introduction of rabbits in artificial underground holes in peridomestic areas reduced significantly the indoor abundance of P. papatasi. Cleaning rabbit holes in peridomestic area by removing all rabbit feces induced a significant increase in the abundance of P. papatasi inside bedrooms. The ecologic niche made around houses in endemic areas by creating active rabbit holes is a major source of attractiveness of P. papatasi, and therefore it may deviate the vector from humans to rabbits. Although rabbit holes are breeding sites for P. papatasi, rabbits are not competent reservoirs for Leishmania major. Our overall findings strongly suggest that zooprophylaxis could be effective in reducing the indoor abundance of P. papatasi and subsequently may be used to control the transmission L. major in rural areas.     

Effect of the irrigation and reclamation transformation of the Karshi Steppe on burrow sandflies of the genus Phlebotomus--vectors of the causative agent of zoonotic cutaneous leishmaniasis

Studies of land irrigation effect on Phlebotomus sandflies, carriers of Leishmania major (a zoonotic cutaneous leishmaniasis causative agent)--were performed for 15 field work periods, from 1967 to 1981, at 18 sites in various natural areas of the Karshi Steppe (Uzbek SSR). Over 43500 sandfly specimen were caught and identified. Regularities in sandfly number changes, first of all, that of Ph. papatasi, after irrigation were determined, with respect to various proximity of great gerbils' settlements, where sandflies breed in the irrigated fields. Well-drained sites situated higher than irrigated fields are practically unaffected by irrigation, sandflies number and specific composition being practically unchanged. At the sites situated either on the same level with irrigated fields or 3-5 m lower the number of sandflies changes usually in 2 stages: 1) short-term increase in Ph. papatasi number at the very beginning of the irrigation period; 2) formation of stably high or low Phlebotomus numbers due to stabilization of hydrothermal regime in the holes of great gerbils.     

Prevalence of sand flies and Leishmania donovani infection in a natural population of female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.     

Molecular Evidence Indicates That Phlebotomus major sensu lato (Diptera: Psychodidae) Is the Vector Species of the Recently-Identified Sandfly Fever Sicilian Virus Variant: Sandfly Fever Turkey Virus

Abstract Sandfly fever turkey virus (SFTV) is a recently-discovered sandfly fever Sicilian virus (SFSV) variant (family Bunyaviridae, genus Phlebovirus), characterized during retrospective evaluation of febrile disease outbreaks in Turkey. In addition to causing sandfly fever, SFTV was observed to induce elevation of liver enzymes, and to cause thrombocytopenia in affected individuals. This study was conducted to identify vectors for phleboviruses including SFTV in Ankara province, Turkey, where evidence indicates ongoing circulation of SFTV, as well as Toscana virus. Sandfly sampling was performed in Ankara province in the vicinity or in animal housing facilities in 15 peri-domestic sites. Male sandflies were identified morphologically, whereas females were evaluated individually for Phlebovirus RNA via a nested-PCR assay with consensus primers. Selected individuals and PCR-positive sandflies were subjected to barcoding via cytochrome c oxidase sequence analyses. The source of blood meals in virus-infected sandflies was investigated using a multiplexed PCR targeting the mitochondrial cytochrome b gene of various vertebrates. A total of 667 sandflies were captured in 11 locations. Morphological identification of males (n=226) revealed Phlebotomus major sensu lato as the most abundant species (38.9%), followed by Phlebotomus sergenti (20.4%), Phlebotomus halepensis (17.7%), Phlebotomus papatasi (10.2%), Phlebotomus simici (3.98%), Larrousius spp. (3.53%), Phlebotomus tobbi (1.32%), Phlebotomus perfiliewi perfiliewi (1.32%), and others. Virus sequences were detected in 3 (3/441) sandflies, two of which were characterized as P. major s.l. via barcoding. The detected sequences in sandflies were identified as SFTV, and were identical or similar to sequences from patients from the same area and the prototype SFTV strain. Bovine and human blood meals were demonstrated in SFTV-infected sandflies. P. major s.l. has been identified as the vector species for SFTV. Bovidae need to be evaluated as probable amplifying hosts for SFTV.     

First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA

Two PCR methods were compared for their sensitivity in detecting cultured Leishmania major, before being used to estimate infection rates in female sandflies (Phlebotomus papatasi) collected from peridomestic animal shelters and the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in Isfahan province, central Iran. A semi-nested PCR was used to amplify a fragment of minicircle kinetoplast (k) DNA with a length and sequence diagnostic for L. major, and a nested PCR was developed to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) with a sequence diagnostic for L. major. The semi-nested PCR was less sensitive than the nested PCR when using DNA extracted from cultured promastigotes of L. major, but it was more sensitive for detecting L. major in wild-caught sandflies. At the edges of two Isfahan villages, infection rates were significantly higher in P.papatasi collected outside gerbil burrows (14/28) compared with those from peridomestic animal shelters (2/21). This is the first record of L. major detected in P.papatasi from peridomestic sites in Isfahan province.