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Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

ABSTRACT: BACKGROUND:Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS:Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE:Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

SUBMITTER: Sima M

PROVIDER: S-EPMC4795800 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Project description:Sand fly saliva plays an important role in blood feeding and Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.A cDNA library from salivary glands of P. arabicus females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic.This transcriptomic analysis of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.

Project description:BackgroundSticky traps are generally viewed as interceptive sand fly sampling methods; although no previous experimental evidence has supported this assumption. In this study, we tested this assumption experimentally for Phlebotomus orientalis, the principal vector of visceral leishmaniasis in East Africa, and propose an explanation for the highly male-biased collection of sticky traps.MethodsA number of field experiments were carried out in March-June 2016-2019, in Gedarif state, eastern Sudan. In the first experiment, we compared numbers of P. orientalis caught on sticky traps made of black, red, transparent, white, yellow, green and blue A4 size papers set simultaneously at different lunar light conditions. In the second and third experiments, we compared numbers of P. orientalis captured on sticky traps placed side-by-side horizontally or vertically on the ground, or horizontally on a 15 cm height stool. We also witnessed mating behaviour of sand flies following their landing on un-sticky papers placed on the ground.ResultsPhlebotomus orientalis showed significant attraction to white, yellow and transparent traps, with negligible numbers caught on the black and the red traps. Similarly, significantly higher numbers of P. orientalis were attracted to the horizontal traps, resulting in an 8-fold increase in sand fly trapping efficacy as compared to the vertical traps. Placing the traps on the stools resulted in significant reduction in this attraction. In contrast to the sticky traps that captured only very few females; we found that when male sand flies land on un-sticky white paper they successfully lure females and copulate with them.ConclusionsWe demonstrate that, for P. orientalis, sticky traps are more attractant-based than interception-based sampling tools. Further, our findings support the notion that males of this sand fly species likely utilize the bright surface of the trap papers to perform mating rituals that attract the females for copulation. However, pre-mature death in the sticky oil hampers the completion of these rituals, and thus results in failure to attract the females. These findings inform our understanding of P. orientalis behaviour and have important implications for optimization of sticky trap design for vector surveillance purposes.

Project description:BackgroundIn East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands.Methodology/principal findingsTwo cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase.ConclusionsThis is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition.

Project description:BackgroundVisceral leishmaniasis (VL) also known as kala-azar is a growing health problem in Ethiopia with an estimated annual VL incidence between 3700 and 7400. The disease is mainly endemic in northwestern parts of the country. The aim of the current study was to determine the sand fly fauna and ecology of Phlebotomus orientalis in two endemic and ecologically distinct areas of northwestern Ethiopia.MethodsSand flies were collected using CDC light traps, sticky traps and pyrethrum spray catches from peri-domestic, mixed forest, farm field and indoor habitats from both Libo-Kemkem (May 2011-April 2012) and Metema (October 2012-September 2013) districts.ResultsA total of 51,411 sand fly specimens were collected and identified (10,776 from highland and 40, 635 from the lowland areas). Seven species were found in the highland area: two Phlebotomus spp. (P. orientalis and P. rodhaini) and five Sergentomyia species. Whereas 19 species were found in the lowland area: six Phlebotomus (P. orientalis, P. rodhaini, P. bergeroti, P. duboscqi, P. papatasi and P. martini) and 13 Sergentomyia species. Of the Phlebotomus spp., P. orientalis was the predominant species in both the highland (99.9%) and lowland (93.7%) areas. Indoor collections using pyrethrum spray catches and sticky traps indicated that P. orientalis has a strong exophilic and exophagic behaviors in both districts. In both areas, this species showed seasonal occurrence and showing abundance during the dry months (March-May/June) of the year and increasing in numbers till the rain season, when numbers dropped dramatically. Mean density of P. orientalis in the two areas had positive and significant correlation with mean temperature in light trap collections (P<0.05). However, mean density of P. orientalis in the two areas in sticky trap collections had positive and insignificant association with the temperature (P>0.05). Regarding the rainfall pattern, density of P. orientalis had negative and statistically insignificant correlation (for light trap collections for both areas) and significant correlation (for sticky trap collections for lowland area).ConclusionsThe current study indicated the variation in sand fly fauna between the highland and lowland districts, wherein, P. orientalis was found to be the most abundant Phlebotomus species. The study also determined that P. orientalis exhibits distinct seasonality, where its abundance increases during the dry season and disappears when the rainy period starts in both study areas. This entomological observation on the bionomics of P. orientalis provides significant evidence for considering vector control or preventive measures in the areas studied.

Project description:BackgroundThe sand fly Phlebotomus papatasi is an Old World vector of Leishmania major, the etiologic agent of zoonotic cutaneous leishmaniasis. This study describes the courtship behaviour of P. papatasi and compares it with that of Lutzomyia longipalpis, the New World vector of visceral leishmaniasis. Understanding the details of courtship behaviour in P. papatasi may help us to understand the role of sex pheromones in this important vector.ResultsP. papatasi courtship was found to start with the female touching the male, leading him to begin abdomen bending and wing flapping. Following a period of leg rubbing and facing, the male flaps his wings while approaching the female. The female then briefly flaps her wings in response, to indicate that she is willing to mate, thereby signaling the male to begin copulation. Male P. papatasi did not engage in parading behaviour, which is performed by male L. longipalpis to mark out individual territories during lekking (the establishment and maintenance of mating aggregations), or wing-flap during copulation, believed to function in the production of audio signals important to mate recognition. In P. papatasi the only predictor of mating success for males was previous copulation attempts and for females stationary wing-flapping. By contrast, male L. longipalpis mating success is predicted by male approach-flapping and semi-circling behaviour and for females stationary wing-flapping.ConclusionsThe results show that there are important differences between the mating behaviours of P. papatasi and L. longipalpis. Abdomen bending, which does not occur in L. longipalpis, may act in the release of sex pheromone from an as yet unidentified site in the male abdomen. In male L. longipalpis wing-flapping is believed to be associated with distribution of male pheromone. These different behaviours are likely to signify significant differences in how pheromone is used, an observation that is consistent with field and laboratory observations.

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Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

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