Insecticide-impregnated dog collars reduce infantile clinical visceral leishmaniasis under operational conditions in NW Iran: A community-wide cluster randomised trial.

OBJECTIVE: To assess the effectiveness of community-wide deployment of insecticide-impregnated collars for dogs- the reservoir of Leishmania infantum-to reduce infantile clinical visceral leishmaniasis (VL). METHODS: A pair matched-cluster randomised controlled trial involving 40 collared and 40 uncollared control villages (161 [95% C.L.s: 136, 187] children per cluster), was designed to detect a 55% reduction in 48 month confirmed VL case incidence. The intervention study was designed by the authors, but implemented by the Leishmaniasis Control Program in NW Iran, from 2002 to 2006. RESULTS: The collars provided 50% (95% C.I. 17·8%-70·0%) protection against infantile VL incidence (0·95/1000/yr compared to 1·75/1000/yr). Reductions in incidence were observed across 76% (22/29) of collared villages compared to pair-matched control villages, with 31 fewer cases by the end of the trial period. In 11 paired villages, no further cases were recorded post-intervention, whereas in 7 collared villages there were 9 new clinical cases relative to controls. Over the trial period, 6,835 collars were fitted at the beginning of the 4 month sand fly season, of which 6.9% (95% C.I. 6.25%, 7.56%) were lost but rapidly replaced. Collar coverage (percent dogs collared) per village varied between 66% and 100%, with a mean annual coverage of 87% (95% C.I. 84·2, 89·0%). The variation in post-intervention clinical VL incidence was not associated with collar coverage, dog population size, implementation logistics, dog owner compliance, or other demographic variables tested. Larger reductions and greater persistence in incident case numbers (indicative of transmission) were observed in villages with higher pre-existing VL case incidence. CONCLUSION: Community-wide deployment of collars can provide a significant level of protection against infantile clinical VL, achieved in this study by the local VL Control Program, demonstrating attributes desirable of a sustainable public health program. The effectiveness is not dissimilar to the community-level protection provided against human and canine infection with L. infantum

Co-infection of Phlebotomus papatasi (Diptera: Psychodidae) gut bacteria with Leishmania major exacerbates the pathological responses of BALB/c mice

Clinical features and severity of the leishmaniasis is extremely intricate and depend on several factors, especially sand fly-derived products. Bacteria in the sand fly’s gut are a perpetual companion of Leishmania parasites. However, consequences of the concomitance of these bacteria and Leishmania parasite outside the midgut environment have not been investigated in the infection process. Herein, a needle infection model was designed to mimic transmission by sand flies, to examine differences in the onset and progression of L. major infection initiated by inoculation with “low” or “high” doses of Enterobacter cloacae and Bacillus subtilis bacteria. The results showed an alteration in the local expression of pro- and anti-inflammatory cytokines in mice receiving different inoculations of bacteria. Simultaneous injection of two bacteria with Leishmania parasites in the low-dose group caused greater thickness of ear pinna and enhanced tissue chronic inflammatory cells, as well as resulted in multifold increase in the expression of IL-4 and IL-1β and a decrease in the iNOS expression, without changing the L. major burden. Despite advances in scientific breakthroughs, scant survey has investigated the interaction between micro and macro levels of organization of leishmaniasis that ranges from the cellular to macro ecosystem levels, giving rise to the spread and persistence of the disease in a region. Our findings provide new insight into using the potential of the vector-derived microbiota in modulating the vertebrate immune system for the benefit of the host or recommend the use of appropriate antibiotics along with antileishmanial medicines

Simultaneous Morphological and Molecular Characterization of Tatera indica in Southwestern Iran

Background: Interest in Tatera indica rodent arises mostly because it is believed that this species is survived among four subspecies reported from Iran, two of which exist in Khuzestan Province. In addition, it might has a role as res­ervoir hosts of zoonotic cutaneous leishmaniasis in the transmission of Leishmania major in some of the widespread Asian foci including southwestern Iran. Methods: Diagnostic morphological and molecular markers for T. indica were sought by characterizing from indi­vidual specimens, such as some taxonomic features and mitochondrial cytochrome b gene that had previously proven useful for the taxonomy of rodents. Wild rodents were caught using live wooden and wire traps. The specimens were identified morphologically using external criteria and molecularly by sequencing of Cyt b gene and phylogenetic analyses. Results: Forty one T. indica were collected and identified morphologically in Khuzestan Province, Iran. Two morphotypes of T. indica were found and classified but sequencing and phylogenetic analyses of mitochondrial Cyt b gene did not support any subspecies between two morphotypes of T. indica.  Because all 21 sequences of both morphotypes of T. indica had no variation with only one common and novel haplotype (GenBank accession No KP001566). Conclusion: This is the first time that T. indica was characterized molecularly in Iran. There is no molecular evi­dence for T. indica morphotypes or subspecies, and so a population genetics approach using several polymorphic genes might be employed using species-specific molecular markers. In addition, more specimens of T. indica species in large geographical locations should be tested.

Detection of a New Strain of Wolbachia Pipientis in Phlebotomus Perfiliewi Transcaucasicus, a Potential Vector of Visceral Leishmaniasis in North West of Iran, by Targeting the Major Surface Protein Gene

Background: Wolbachia pipientis is maternally inherited endoparasitic bacterium belonging to the α-proteobacteria, infecting 20–75% of all insect species including sand flies. The Wolbachia surface protein (wsp) was employed as an appropriate marker for strain typing. The objective of our research was to find the possibility of detection of W. pipientis in Phlebotomus perfiliewi transcaucasicus.Methods: Individual sand flies were screened for the presence of W. pipientis. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes.Results: Two haplotypes of W. pipientis were found in P. perfiliewi transcaucasicus. The common haplotype of W. pipientis was found to be identical to the sequences of those submitted in GenBank. New strain (haplotype) of W. pipientis was found novel. The sequence of new strain of W. pipientis occurs in P. perfiliewi transcaucasicus is very different from those already submitted in GenBank.Conclusion: Finding one genetically modified new strain of W. pipientis in P. perfiliewi transcaucasicus, now we can conclude that further documents and studies need to reach the role of cytoplasmic incompatibility of W. pipientis through wild sand fly populations to drive a deleterious gene into and to reduce the density of natural populations of sand flie

Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis

Individual, naturally occurring Phlebotomus mongolensis and Phlebotomus caucasicus from Iran were screened for infections with the maternally inherited intracellular Rickettsia-like bacterium Wolbachia pipientis via targeting a major surface protein gene (wsp). The main objective of this study was to determine if W. pipientis could be detected in these species. The sandflies were screened using polymerase chain reaction to amplify a fragment of the Wolbachia surface protein gene. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes. Two strains of Wolbachia were found. Strain Turk 54 (accession EU780683) is widespread and has previously been reported in Phlebotomus papatasi and other insects. Strain Turk 07 (accession KC576916) is a novel strain, found for first time in the two sister species. A-group strains of W. pipientis occur throughout much of the habitat of these sandflies. It is possible that Wolbachia is transferred via horizontal transmission. Horizontal transfer could shed light on sandfly control because Wolbachia is believed to drive a deleterious gene into sandflies that reduces their natural population density. With regard to our findings in this study, we can conclude that one species of sandfly can be infected with different Wolbachia strains and that different species of sandflies can be infected with a common strain

Molecular characterization of leishmania infection from naturally infected sand flies caught in a focus of cutaneous leishmaniasis (eastern iran).

Background: Cutaneous leishmaniasis due to Leishmania major is a serious and increasing problem affecting manyrural areas of 17 out of 31 provinces in Iran. Little is known about sand fly fauna and leishmaniases in Eastern Iran and no study has been carried out in Sarbisheh County. The aim of this study was to determine sand flies composi- tion and probable Leishmania infection to find the probable vectors of leishmaniasis in Sarbisheh district. Methods: Sand flies were caught using both sticky papers and CDC light traps in August 2010. They were identified morphologically and analyzed for Leishmania infection by amplification of ITS-rDNA. Results: Totally, 842 specimens were caught and 8 species recorded. They belonged to the genera Phlebotomus and Sergentomyia: P. (Phlebotomus) papatasi, P. (Paraphlebotomus) sergenti, P. (Pa.) caucasicus, P. (Pa.) mongolensis, P. (Pa.) jacusieli, S. (Sergentomyia) dentata, S. (Se.) sintoni and S. (Sintonius) clydei. All collected females were processed for Leishmania DNA detection by PCR amplifying of Internal Transcribed Spacer1 (partial sequence),5.8S (complete sequence) and ITS2 (partial sequence) fragments. Thirteen females were positive for Leishmania DNA. The sequencing of the 430 bp amplicons indicated that 9  P. papatasi and 3 females belonging to the Caucasicus group carried L. major DNA whereas one P. sergenti carried L. tropica DNA. Conclusion: Phlebotomus papatasi and P. sergenti are, like in several places, the probable vectors of cutaneous leishmaniases in this emerging or unknown focus of cutaneous leishmaniases

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies