Sand fly collection
Field sampling was carried out at four different sites of the ZCC facility in Tubarão municipality (Santa Catarina State). The sand flies were collected monthly, from March 2019 to June 2020, yielding a total of 3,419 sand flies captured. Across all sampling points, the number of males (2,478) exceed that of females (941) resulting in a male/female ratio of 2,63 (Table 1), a similar proportion to several sand flies’ studies [18, 19, 20].
Table 1
Number of sand flies collected and species.
Sand flies species | | | | | | Total | % |
Males | Females |
(n) | (%) | Engorged | Non-engorged | (n) | (%) |
Nyssomyia neivai | 2209 | 89.14 | 286 | 437 | 723 | 76.83 | 2932 | 85.75 |
Migonemyia migonei | 251 | 10.13 | 136 | 68 | 204 | 21.68 | 455 | 13.31 |
Pintomyia fischeri | 15 | 0.61 | 9 | 4 | 13 | 1.38 | 28 | 0.82 |
Brumptomyia spp. | 3 | 0.12 | 0 | 0 | 0 | 0.00 | 3 | 0.09 |
Evandromyia edwardsi | 0 | 0 | 1 | 0 | 1 | 0.11 | 1 | 0.03 |
Total | 2478 | 100 | 432 | 509 | 941 | 100 | 3419 | 100 |
The knowledge of regional sand fly fauna is crucial, since many species are leishmaniasis vectors, leading to a more effective and sustainable disease control measures [04]. In this study, five genera of sand flies and four species were identified based on their morphology. The most abundant sand fly was Ny. neivai (85.75%), followed by Migonemyia migonei (13.31%), Pintomyia fischeri (0.82%), Brumptomyia spp. (0.09%) and Evandromyia edwardsi (0.03%) (Table 1). These results align with a previous study that identify the presence of these sand fly species in Santa Catarina State [18] and contribute to a deeper knowledge of the State sand flies fauna and canine leishmania transmission, which are limited.
Nyssomyia neivai was the predominant species found in this study, which is consistent with its identification in studies of sand flies’ fauna in southern Brazil [21, 22, 23]. This species is prevalent in colder and drier Brazilian regions, thriving in South, Southeast, and Midwest Brazil [24]. It is an important ATL vector, and its infection with L. infantum was previously documented in Santa Catarina and Minas Gerais States [18, 25].
Leishmania DNA detection in sand flies
A total of 509 non-engorged females were tested for Leishmania spp. infection, accounting for 54.1% of collected females (Fig. 2-A). Among the 53 analyzed pools, four (7.5%) were positive, presenting a PCR banding pattern with the expected size range (300 bp). After performing a second PCR, and closely examining the DNA individually from each insect of the four positive pools, we identify four specimens positive for trypanosomatids DNA (Fig. 02-B).
The positive sand flies sequencing data identified L. infantum as the cause of natural infection in 50% of positive samples (Fig. 02-B: F1 and F3), resulting in a 0.4% natural infection rate. This infection rate is in alignment with previous reports that found Leishmania infected sand flies ranging from 0.2–9.1% [26]. The remaining positive samples (Fig. 02-B: F2 and F4) yielded inconclusive species identification, which may be due to the low coverage (< 35%) compared to GenBank sequences, suggesting a putative close related trypanosomatid specie not yet available at GenBank. The inconclusive samples showed PCR fragments size different of expected for L. infantum (Fig. 02-B: F2 and F4), suggesting a broader spectrum for trypanosomatids detection using ITS1 primers [14, 15].
The four sand flies naturally infected with trypanosomatids were identified as Ny. neivai, confirmed through both morphological and molecular analyses (Fig. 02: C and D).
Our findings present the first DNA barcode sequences of Ny. neivai originating from Brazil, ranging in length from 554 to 672 base pairs. This is an important step towards a precise leishmania vector sand fly species identification in South America since GenBank currently holds shorter sequences only from Argentina samples (543 bp) (access numbers: MN857519 up MN857540) [27]. Indeed, Ny. neivai has been found naturally infected by both i) the etiologic agents of ATL [22, 28, 29, 30, 31], as well as ii) L. infantum, the causative agent of VL in Brazil [18, 25].
Reports from the Epidemiological Surveillance Directorate of Santa Catarina State (DIVE/SC) indicated a significant prevalence of Ny. neivai in areas with CVL and human VL foci in Florianópolis - SC. Notably, Lu. Longipalpis, the primary vector of L. infantum, is absent in these areas [32, 33]. Our study revealed a similar entomological profile, including natural infection of Ny. neivai by L. infantum. This discovery strongly suggests that Ny. neivai might be a vector in this region, which is supported by i) the insect’s high abundance in areas with CVL cases, ii) the absence of Lu. Longipalpis, and iii) the natural infection of Ny. neivai by L. infantum. Nonetheless, additional research is needed to confirm the species’ role in the VL cycle.
Leishmania DNA detection in dogs
A total of 47 dogs from Tubarão Municipality ZCC were tested for Leishmania spp. infection using a non-invasive method for sample collection - the conjunctival swab (Fig. 02-E). This simple and efficacy technique, combined with ITS1 PCR amplification, in diagnosing CVL in asymptomatic animals was previously demonstrated by Leite et al. [34] as Leishmania amastigote forms are frequently found in the ocular conjunctiva [35]. The dogs examined were mixed breed, and asymptomatic for leishmaniasis throughout the sampling period. Leishmania PCR amplification was evident in four samples, with the band profile aligning with the expected ITS1 targeting. Within this subset, two samples were positive for L. infantum, resulting in an CVL infection rate of 4.2%. The remaining two positive dogs samples lead to an inconclusive outcomes regarding the trypanosomatids species, which may be due to the low sequence coverage compared to GenBank sequences or due a non-identified trypanosomatid species.
Here we identified a high prevalence of Ny. neivai combined with L. infantum natural infection, also detected in infected dogs within the studied area. The L. infantum ITS1 sequence profile was identical in all four samples, two positive sand flies and two positive dogs. This suggests that Ny. neivai is attracted to feed on dogs and supports the parasite’s infection after blood digestion, potentially enabling transmission through subsequent sand fly bites. Nonetheless, further investigations into the vectorial competence of the species will be necessary. Nevertheless, the dogs from different origins gathering in Zoonosis Control Centers (CCZ) should follow Leishmania diagnostics on a regular basis since the parasite vector are widely disperse in Brazil. Also, periodically vectorial surveillance for Leishmania natural infection should be implemented as a preventive measurement to avoid CVL transmission.