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The leishmaniases are multifactorial zoonotic diseases requiring a multidisciplinary One Health approach for diagnosis and control. For leishmaniasis diagnosis, here we describe production of a new recombinant protein based on a kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. Culturing was carried out, and the secreted recombinant protein with a C-terminal histidine tag purified using nickel affinity chromatography on the culture supernatant, yielding a final product at 0.4 mg/mL. An indirect enzyme linked immunosorbent assay (ELISA) was standardised using sera from 74 Brazilian patients with cutaneous leishmaniasis and 11 with visceral leishmaniasis. Optimal ELISA conditions were established for the Lbk39 antigen in comparison with a crude extract from L. braziliensis. The sensitivity, specificity analysis and receiver operating characteristic (ROC) curve were determined with a significance level of 5%. The ROC curve showed a good accuracy with an area under curve (AUC) = 0.967, p < 0.001 (0.941-0.993) for CL patients and an AUC = 100 (100-100) for VL patients. The values of sensitivity and specificity were 88 and 98% for CL and 100 and 100% for VL, respectively. The study showed good production and expression of the target protein and has generated a potential new antigen for the diagnosis of leishmaniasis.

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BACKGROUND: Despite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date. METHODOLOGY/PRINCIPAL FINDINGS: Females from colonies of the midges Culicoides nubeculosus Meigen and C. sonorensis Wirth & Jones and the sand fly Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae) were experimentally infected with two different species of Leishmania, originating from Australia (Leishmania sp. AM-2004) and Brazil (Leishmania enriettii). In addition, the infectivity of L. enriettii infections generated in guinea pigs and golden hamsters for Lu. longipalpis and C. sonorensis was tested by xenodiagnosis. Development of L. enriettii in Lu. longipalpis was relatively poor compared to other Leishmania species in this permissive vector. Culicoides nubeculosus was not susceptible to infection by parasites from the L. enriettii complex. In contrast, C. sonorensis developed late stage infections with colonization of the thoracic midgut and the stomodeal valve. In hamsters, experimental infection with L. enriettii led only to mild symptoms, while in guinea pigs L. enriettii grew aggressively, producing large, ulcerated, tumour-like lesions. A high proportion of C. sonorensis (up to 80%) feeding on the ears and nose of these guinea pigs became infected. CONCLUSIONS/SIGNIFICANCE: We demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".

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We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10³) and high (105) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis.

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