Phlebotomus argentipes is a predominant vector of Leishmania donovani, the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes, sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti-P. argentipes saliva IgG responses. The anti-rPagSP06 and anti-P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti-P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area.

• Klíčová slova
• Bangladesh, IgG antibodies, Leishmania donovani, Marker of exposure, Phlebotomus argentipes, Salivary glands,
• MeSH
• hmyzí proteiny * imunologie   MeSH
• kousnutí a bodnutí epidemiologie   MeSH
• Leishmania donovani MeSH
• lidé MeSH
• Phlebotomus * MeSH
• slinné proteiny a peptidy * imunologie   MeSH
• sliny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Bangladéš epidemiologie   MeSH
• Názvy látek
• hmyzí proteiny * MeSH
• slinné proteiny a peptidy * MeSH

Leishmania parasites alternate in their life cycle between promastigote stages that develop in the gut of phlebotomine sand flies and amastigotes residing inside phagocytic cells of vertebrate hosts. For experimental infections of sand flies, promastigotes are frequently used as this way of infection is technically easier although ingestion of promastigotes by sand flies is unnatural. Here we aimed to answer a critical question, to what extent do promastigote-initiated experimental infections differ from those initiated with intracellular amastigotes. We performed side-by-side comparison of Leishmania development in Phlebotomus argentipes females infected alternatively with promastigotes from log-phase cultures or amastigotes grown ex vivo in macrophages. Early stage infections showed substantial differences in parasite load and representation of morphological forms. The differences disappeared along the maturation of infections; both groups developed heavy late-stage infections with colonization of the stomodeal valve, uniform representation of infective metacyclics and equal efficiency of transmission. The results showed that studies focusing on early phase of Leishmania development in sand flies should be initiated with intracellular amastigotes. However, the use of promastigote stages for sand fly infections does not alter significantly the final outcome of Leishmania donovani development in P. argentipes and their transmissibility to the vertebrate host.

• Klíčová slova
• Leishmania donovani, Phlebotomus, amastigotes, promastigotes, transmission,
• MeSH
• hmyz - vektory parazitologie   MeSH
• interakce hostitele a parazita MeSH
• Leishmania donovani růst a vývoj   MeSH
• leishmanióza viscerální parazitologie   přenos   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• Phlebotomus parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Visceral leishmaniasis (VL), the most severe form of the disease, is caused by Leishmania donovani in the Indian sub-continent (ISC). Whole genome sequencing studies revealed that two parasite populations exist in the ISC: a main population named the Core Group (CG) found mostly in the lowlands, and a new, genetically different subpopulation called ISC1. Parasites belonging to the CG were shown to be responsible for the recent epidemics, while the ISC1 variant was originally identified in hilly districts of Nepal and was later on increasingly found in the lowlands. Importantly, the ISC1 and CG isolates differ in their drug susceptibility and virulence signatures, suggesting that ISC1 constitutes an emerging and functionally different variant of L. donovani. In present study we aimed to address the potential of ISC1 transmission by the natural vector of L. donovani in the lowlands, Phlebotomus argentipes. By experimental infection of sand flies with parasites of the different genotypes, we demonstrate that ISC1 and CG strains are developing similarly in P. argentipes, suggesting that P. argentipes is a fully competent vector for ISC1 parasites. Integration of previous and current findings shows thus that ISC1 is a new and different variant of L. donovani, fully adapted to spread in the ISC through the main vector. This information is directly useful for managers of the elimination program. Furthermore, integration of our successive studies (genotyping, phenotyping and vector competence) demonstrates the relevance of molecular surveillance and should be of interest for scientists working on vector borne diseases and control managers.

• Klíčová slova
• Experimental development, Leishmania donovani, Sand fly,
• MeSH
• fylogeneze MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania donovani klasifikace   genetika   izolace a purifikace   MeSH
• leishmanióza viscerální přenos   MeSH
• lidé MeSH
• Phlebotomus parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Indie MeSH

BACKGROUND: Species belonging to the subgenus Sauroleishmania are parasites of reptiles, and traditionally considered to be non-pathogenic to mammals. Knowledge of the development of these parasites in sand flies and their mechanism of transmission is currently lacking. The main aim of this study was to test the susceptibility of various sand fly species to infection by two Sauroleishmania species, focusing on the localization of parasites in the sand fly intestinal tract. METHODS: The development of Leishmania (Sauroleishmania [S.]) adleri and Leishmania (S.) hoogstraali was studied in six sand fly species (Phlebotomus orientalis, P. argentipes, P. sergenti, P. papatasi, P. duboscqi, Sergentomyia schwetzi). Sand flies were fed through a chick-skin membrane on blood containing Sauroleishmania promastigotes, and they were dissected at various time intervals post blood meal (PBM). Guts were examined microscopically for the presence of parasites, and the intensity and localizations of infections were recorded. Morphological forms of both Sauroleishmania species developing in P. orientalis were analyzed. Experimental infections of geckos using sand fly-derived promastigotes were also performed, and the reptiles were repeatedly examined for Sauroleishmania infection by xenodiagnosis and PCR analysis. RESULTS: High infection rates for both Sauroleishmania species were observed in P. orientalis and P. argentipes, with the parasites migrating anteriorly and undergoing a peripylarian type of development, including colonization of the stomodeal valve. Conversely, the development of L. (S.) adleri in P. sergenti, P. papatasi and Se. schwetzi was restricted to the sand fly hindgut (hypopylarian type of development). Five morphological forms were distinguished for both Sauroleishmania species developing in P. orientalis. All experimentally infected geckos scored negative for Sauroleishmania based on xenodiagnosis and molecular analysis. CONCLUSIONS: The results showed that Sauroleishmania promastigotes can undergo either a peripylarian or hypopylarian type of development in the sand fly intestinal tract, depending on the sand fly species infected. We demonstrated that P. argentipes and P. orientalis, two sand fly species known as permissive vectors for mammalian parasites of subgenus Leishmania, are also highly susceptible to Sauroleishmania as the parasites developed mature late-stage infections, including colonization of the sand fly stomodeal valve. Thus, the role of Phlebotomus sand flies in transmission of Sauroleishmania should be reconsidered and further investigated.

• Klíčová slova
• Geckos, Leishmaniasis, Phlebotomus, Sand flies, Sauroleishmania, Sergentomyia,
• MeSH
• ještěři * MeSH
• Leishmania * MeSH
• Phlebotomus * parazitologie   MeSH
• Psychodidae * parazitologie   MeSH
• savci MeSH
• xenodiagnóza MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: During blood feeding, sand flies inoculate salivary proteins that interact with the host haemostatic system. The blocking of biogenic amines such as serotonin and histamine helps to limit vasodilatation and clot formation, and thus enables the insect to finish the blood-feeding process. In sand flies, an amine-binding ability is known only for the yellow-related proteins of Phlebotomus and Lutzomyia vectors, but not yet for members of the genus Sergentomyia. METHODS: The ability of Phlebotomus argentipes and Sergentomyia schwetzi recombinant yellow-related salivary proteins to bind histamine and serotonin was measured by microscale thermophoresis. Both sand fly species were also fed through a chicken-skin membrane on blood mixed with histamine or serotonin in order to check the effects of biogenic amines on sand fly fitness. Additionally, fecundity and mortality were compared in two groups of P. argentipes females fed on repeatedly-bitten and naive hamsters, respectively. RESULTS: The P. argentipes recombinant yellow-related protein PagSP04 showed high binding affinity to serotonin and low affinity to histamine. No binding activity was detected for two yellow-related proteins of S. schwetzi. Elevated concentrations of serotonin significantly reduced the amount of eggs laid by P. argentipes when compared to the control. The fecundity of S. schwetzi and the mortality of both sand fly species were not impaired after the experimental membrane feeding. Additionally, there were no differences in oviposition or mortality between P. argentipes females fed on immunized or naive hamsters. CONCLUSIONS: Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or anti-saliva antibodies without any influence on their fitness. The serotonin binding by salivary yellow-related proteins may play an important role in Phlebotomus species feeding on mammalian hosts, but not in S. schwetzi, which is adapted to reptiles.

• Klíčová slova
• Anti-saliva antibodies, Histamine, Mortality, Oviposition, Phlebotomus argentipes, Sergentomyia schwetzi, Serotonin, Yellow-related proteins,
• MeSH
• biogenní aminy * krev   farmakologie   MeSH
• fertilita účinky léků   MeSH
• histamin krev   MeSH
• hmyzí proteiny chemie   metabolismus   MeSH
• kousnutí a bodnutí hmyzem imunologie   MeSH
• křečci praví MeSH
• krev metabolismus   MeSH
• molekulární evoluce MeSH
• mortalita MeSH
• Phlebotomus metabolismus   MeSH
• plazi MeSH
• protilátky MeSH
• Psychodidae metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• savci MeSH
• serotonin krev   MeSH
• slinné proteiny a peptidy * chemie   metabolismus   MeSH
• sliny imunologie   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biogenní aminy * MeSH
• histamin MeSH
• hmyzí proteiny MeSH
• protilátky MeSH
• rekombinantní proteiny MeSH
• serotonin MeSH
• slinné proteiny a peptidy * MeSH

BACKGROUND: Sand fly females require a blood meal to develop eggs. The size of the blood meal is crucial for fecundity and affects the dose of pathogens acquired by females when feeding on infected hosts or during experimental membrane-feeding. METHODS: Under standard laboratory conditions, we compared blood meal volumes taken by females of ten sand fly species from four genera: Phlebotomus, Lutzomyia, Migonomyia, and Sergentomyia. The amount of ingested blood was determined using a haemoglobin assay. Additionally, we weighed unfed sand flies to calculate the ratio between body weight and blood meal weight. RESULTS: The mean blood meal volume ingested by sand fly females ranged from 0.47 to 1.01 µl. Five species, Phlebotomus papatasi, P. duboscqi, Lutzomyia longipalpis, Sergentomyia minuta, and S. schwetzi, consumed about double the blood meal size compared to Migonomyia migonei. The mean body weight of females ranged from 0.183 mg in S. minuta to 0.369 mg in P. duboscqi. In males, the mean body weight ranged from 0.106 mg in M. migonei to 0.242 mg in P. duboscqi. Males were always lighter than females, with the male-to-female weight ratio ranging from 75% (in Phlebotomus argentipes) to 52% (in Phlebotomus tobbi). CONCLUSIONS: Females of most species took a blood meal 2.25-3.05 times their body weight. Notably, the relatively tiny females of P. argentipes consumed blood meals 3.34 times their body weight. The highest (Mbl/Mf) ratios were found in both Sergentomyia species studied; females of S. minuta and S. schwetzi took blood meals 4.5-5 times their body weight. This parameter is substantially higher than that reported for mosquitoes and biting midges.

• Klíčová slova
• Lutzomyia, Phlebotomus, Sergentomyia, Blood meal, Haemoglobin, Prediuresis,
• MeSH
• krev MeSH
• Phlebotomus fyziologie   MeSH
• Psychodidae * fyziologie   MeSH
• stravovací zvyklosti * MeSH
• tělesná hmotnost * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Lectins which agglutinate Leishmania promastigotes were demonstrated in gut lysates from laboratory colonies of five Phlebotomus and two Lutzomyia species. In general, the highest agglutination titres were found in P. halepensis and Lu. longipalpis (Jacobina). Marked differences were found in the agglutination of promastigotes of various Leishmania species and strains and high agglutination titres were observed in some natural vector-parasite combinations, such as Phlebotomus argentipes and Le. donovani. Intraspecific variability, in agglutination of Le. major strains, could be related to the varying infectivity of the strains to laboratory animals. Similar carbohydrates, of which the most effective were D-mannosamine and N-acetyl-D-glucosamine, inhibited the agglutination of Le. major and Le. donovani promastigotes by midgut extracts from P. papatasi and Lu. longipalpis. D-Mannosamine and N-acetyl-D-glucosamine inhibited agglutination of promastigotes in all vector-parasite combinations. The results of the carbohydrate-inhibition tests indicate that the lectin specificities in Phlebotomus are similar to those in Lutzomyia.

• MeSH
• acetylglukosamin fyziologie   MeSH
• aglutinační testy MeSH
• hexosaminy fyziologie   MeSH
• Leishmania účinky léků   MeSH
• lektiny analýza   antagonisté a inhibitory   fyziologie   MeSH
• Phlebotomus chemie   MeSH
• Psychodidae chemie   MeSH
• sacharidy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylglukosamin MeSH
• hexosaminy MeSH
• lektiny MeSH
• mannosamine MeSH Prohlížeč
• sacharidy MeSH

BACKGROUND: Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results. METHODS: We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6-72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite. RESULTS: In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation. CONCLUSIONS: We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania.

• Klíčová slova
• Blood meal digestion, Leishmania donovani, Leishmania major, Phlebotomus, Proteases, Sand fly, Sergentomyia,
• MeSH
• gastrointestinální trakt enzymologie   parazitologie   MeSH
• králíci MeSH
• krev metabolismus   MeSH
• Leishmania donovani fyziologie   MeSH
• Leishmania major fyziologie   MeSH
• Phlebotomus parazitologie   MeSH
• proteasy metabolismus   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteasy MeSH

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

• MeSH
• genomika MeSH
• křížení genetické MeSH
• Leishmania donovani * genetika   MeSH
• leishmanióza kožní * MeSH
• leishmanióza viscerální * diagnóza   prevence a kontrola   epidemiologie   MeSH
• Phlebotomus * genetika   MeSH
• Psychodidae * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector. METHODS: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites. RESULTS: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector. CONCLUSIONS: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature.

• MeSH
• křečci praví MeSH
• Leishmania donovani * MeSH
• Leishmania infantum * MeSH
• paromomycin farmakologie   MeSH
• Phlebotomus * MeSH
• Psychodidae * MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• paromomycin MeSH