Leishmania infantum (Trypanosomatida: Trypanosomatidae) detection in Nyssomyia neivai (Diptera: Psychodidae) and dogs in Southern Brazil

Leishmania infantum (Trypanosomatida: Trypanosomatidae) detection in Nyssomyia neivai (Diptera: Psychodidae) and dogs in Southern Brazil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Leishmania infantum (Trypanosomatida: Trypanosomatidae) detection in Nyssomyia neivai (Diptera: Psychodidae) and dogs in Southern Brazil Sabrina Fernandes Cardoso, João Victor Costa Guesser, Andressa Alencastre Fuzari Rodrigues, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3961690/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background The sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It has been frequently found in visceral leishmaniasis foci in Santa Catarina (Brazil), a region where Lutzomyia longipalpis , the main vector of Leishmania infantum and the etiological agent of visceral leishmaniasis in the Americas, has not been identified. In the absence of the main Leishmania vector, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and dogs in a region with a recent canine visceral leishmaniasis outbreak in the South of Brazil. Methods We report here a sand fly fauna survey on the Zoonosis Control Center of Tubarão Municipality (Santa Catarina, Brazil). We also conducted molecular testing to detect Leishmania spp. natural infection on captured sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, followed by PCR. Results We collected 3,419 specimens from five sand flies genera. Nyssomyia neivai (85.75%) was the most abundant species, followed by Migonemyia migonei (13.31%), Pintomyia fischeri (0.82%), Evandromyia edwardsi (0.03%), and the genus Brumptomyia spp. (0.09%). Leishmania infantum DNA was detected in two of the 509 analyzed females, yielding a natural infection rate of 0.4%. The L. infantum infected sand flies’ specimens were morphologically and molecularly identified as Ny. neivai . We analyzed 47 dogs’ conjunctival swabs for Leishmania spp. with two positive individuals for L. infantum (infection rate of 4.2%). Conclusions Our results confirm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil. We emphasize the significance of DNA barcoding in supporting the sand flies’ identification, and the conjunctival swab technique proved effective in obtaining enough biological material for detecting L. infantum in dogs. Leishmania infantum Nyssomyia neivai Visceral leishmaniasis Figures Figure 1 Figure 2 Background Leishmaniases are diseases caused by protozoa of the genus Leishmania (Trypanosomatida: Trypanosomatidae), which can affect both humans and other animals, potentially leading to fatal outcomes. The pathogen transmission occurs through a bite of the sand fly vector infected females (Diptera: Psychodidae) [01]. Sand flies constitute a group of approximately 1000 known species worldwide, with 530 species having been identified in the Americas [02]. It is estimated that 98 species may serve as potential natural vectors of Leishmania spp. [03]. In the Americas, Visceral Leishmaniasis (VL), the most severe form of the disease, is caused by Leishmania infantum . This leishmania species is mainly transmitted by its primary vector, the sand flies of the Lutzomyia longipalpis complex [04, 05]. In Brazil, other sand flies’ species have been implicated as potential vectors of L. infantum in areas where the visceral form of the disease occurs and Lu. longipalpis is absent [06, 07]. In the State of Santa Catarina, Brazil, cases of VL affecting both humans and dogs have been reported, yet the conventional vector has not been detected [08]. Nyssomyia neivai , a sand fly species widely distributed throughout Santa Catarina State, is known as the etiologic agent vector of American tegumentary leishmaniasis (ATL) [09]. However, Ny. neivai has been found in abundance in regions with human and canine VL cases within Santa Catarina State [ 10 ]. Based on the VL positive dog identifications in the Zoonosis Control Center (ZCC) of Tubarão municipality, (Santa Catarina, Brazil), the present study aimed to investigate potential vectors implicated in the disease transmission and to identify the presence of new cases of Canine Visceral Leishmaniasis (CVL) in the area. Methods This research was undertaken at the Zoonosis Control Center (ZCC) of Tubarão municipality (28°31′12.64″S, 49°01′03.09″W), located in Southern region of Santa Catarina State, Brazil (Fig. 1 ). The institution facility can house around 60 dogs, and there were reports of CVL outbreaks on the premises approximately three months before the study began. The investigation was carried out from March 2019 to June 2020. Sand fly collections We collected sand flies once a month using CDC light traps (model 512; John W. Hock, Gainesville, FL). These traps were positioned approximately one meter above the ground both inside and outside the dog shelter within ZCC, as well as in the nearby forest area. This arrangement established four distinct collection points, each spaced 30 meters apart. Captures were conducted from 06:00 p.m. to 06:00 a.m. Once the traps were retrieved, the collected sand flies were sorted into males, engorged females and non-engorged females, and the specimens were then preserved in 70% ethyl alcohol and stored at -20ºC until processing. Sand fly taxonomic identification The captured sand flies were identified based on morphological characters, using the taxonomic key proposed by Galati [ 11 ]. Before being mounted on glass slides, all sand flies underwent a previous process of clarification, dehydration and diaphanization of body structures, as described by Fuzari [ 12 ]. The male sand flies were full body assembled on glass slides. Engorged females had the head detached from the rest of the body, while in non-engorged females, the head and the last three abdominal segments were detached from the rest of the body, and then mounted on slides. Remaining fragments of the abdomen and thorax were preserved in 70% ethyl alcohol and stored at -20ºC for subsequent analysis to determine natural infection by Leishmania spp. Molecular identification of Leishmania spp. in sand flies For molecular identification of Leishmania DNA in sand flies, we specifically analyzed non-engorged females. This selection aimed to avoid possible Leishmania detection originated from an undigested blood meal, focusing instead on identifying potential natural infections. Consequently, we individually extracted DNA of non-engorged female sandflies body fragments using the protocol established by Jowett [ 13 ]. We assembled sample pools, each containing genetic material from up to ten individuals for initial molecular screening. A polymerase chain reaction (PCR) was conducted targeting the ITS1 region of Leishmania spp. rDNA, using the primers and amplification conditions described by El-Tai et al. [ 14 ] and Schönian et al. [ 15 ]. This region amplifies a sequence of 300 to 350 base pairs (bp), depending on the Leishmania species. Following a positive pool identification, the original 10 female samples were submitted to an individual analysis through a second PCR reaction under the same conditions. Therefore, we were able to identify the real number of Leishmania infected sand flies, thus accurately determining the natural infection rate and vector identification. The amplified fragments resulting from the individual sample PCR reactions were purified using the QIAquick® PCR purification kit (Qiagen), according to the manufacturer's instructions. The purified fragments were submitted to a Sanger sequencing at Fiocruz Sequencing Unit (RPT1A, Rio de Janeiro, Brazil) for Leishmania spp. identification. The sequences were aligned using the Geneious Prime program to obtain consensus sequences followed by BLASTN analysis. The identified sequences were submitted to GenBank ( www.ncbi.nlm.nih.gov ) under accession numbers: OP718537 and OP718538. Sand fly molecular identification In addition to morphological characterization, Leishmania spp. positive sandflies were submitted to a molecular identification through DNA barcoding. The primers used, and the reaction conditions were performed as described by Pinto et al. [ 16 ]. Purified PCR products were sequenced and analyzed following the procedure outlined previously. The resulting sequences have been deposited in GenBank under the following accession numbers: OP719771, OP719772, OP719773 and OP719774. Dogs’ sample collection We collected biological samples from 47 dogs sheltered at ZCC using the non-invasive conjunctival swab technique [ 17 ]. During the procedure, the lower eyelid was slightly separated so that the cotton swab could be rubbed on the ocular conjunctiva to collect cells from the right eye of each dog. Only the swabs tips were transferred to sterile tubes containing 200µl of extraction buffer-SQ solution (Tris HCL 10 mM; EDTA 2 mM; Triton absolute 0.2%) and stored at -20°C until analysis. Dogs molecular diagnosis of Leishmania spp. To extract DNA from the swab, the cotton was macerated with the SQ-buffer, and then 2µl of Proteinase K (20mg/ml) was added. The solution was incubated at 37ºC for one hour in a dry bath, then heated to 100ºC for 20 minutes to denature the Proteinase K. Individual samples were submitted to a PCR reaction followed by amplicons sequencing to identify positive dogs for Leishmania spp., as detailed previously. The resulting sequences were submitted to GenBank, under the accession numbers: OP724554 and OP724555. Results and discussion 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. neiva i (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. Conclusions In this study, we provide the first evidence of L. infantum infecting both Ny. neivai and dogs within the same area in Brazil. Considering the absence of Lu. Longipalpis in Santa Catarina State thus far, our data suggests that Ny. neivai might act as a VL vector transmitting L. infantum in this region. Furthermore, we emphasize the importance of using DNA barcoding as a tool to support fast and numerous sand fly’s identification. Additionally, our study confirms the conjunctival swab technique efficiency in successfully detecting L. infantum in dogs. Abbreviations PCR: Polymerase chain reaction; VL: Visceral leishmaniasis; ATL: American tegumentary leishmaniasis; ZCC: Zoonosis Control Center; CVL: Canine visceral leishmaniasis; bp: Base pairs; μl: Microliter; HCL: Hydrochloric acid; EDTA: Ethylenediaminetetraacetic acid; SC: Santa Catarina. Declarations Acknowledgements The authors are indebted PDTIS-FIOCRUZ for the use of its DNA sequencing facility, and LAMEB-UFSC for helping with the photos. Funding The work was funded by the National Council for Scientific and Technological Development MCTI/CNPq/CAPES/FAPs no 16/2014 (to ANP and LDPR), and FIOCRUZ (ANP). Availability of data and materials The datasets generated and/or analyzed during the current study are available in the GenBank database under the accession numbers: OP718537, OP718538, OP719771, OP719772, OP719773, OP719774, OP724554, OP724555. Authors’ contributions SFC, ANP, and LDRP participated in data generation and analysis; SFC, JVCG, AAFR, RPB carried sand fly collections and morphological identification; SFC drafted the manuscript; ANP and LDPR helped in the paper drafting by critically reading the original manuscript; ANP and LDPR were the principal investigators, participated in its design and coordination. All authors read and approved the final manuscript. Ethics approval and consent to participate This study was reviewed and approved by the Ethics in Animal Use Committee of the Federal University of Santa Catarina, Florianópolis, Brazil (process number: 8644190221, ID 001784). Formal written and oral consent was obtained from the Zoonoses Control Center, where the study was carried out. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. This paper is part of the Master thesis of Sabrina Fernandes Cardoso from Cell and Developmental Biology (PPGBCD) Graduation Program from Biological Science Center (CCB) at Federal University of Santa Catarina (UFSC). References Sherlock IRA. Importância médico-veterinária: a importância dos flebotomíneos. In: Rangel EF, Ralph L, (Org). Flebotomíneos do Brasil. Fiocruz. 2003;15-22. Shimabukuro PHF, de Andrade AJ, Galati EAB. Checklist of American sand flies (Diptera, Psychodidae, Phlebotominae): genera, species, and their distribution. Zookeys. 2017;(660):67-106. Galati EAB. Morfologia e taxonomia: classificação de Phlebotominae. In: Rangel EF, Lainson R (eds). Flebotomíneos do Brasil. Fiocruz. 2003;23-51. OPAS. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Mar, 2024 Reviews received at journal 05 Mar, 2024 Reviews received at journal 02 Mar, 2024 Reviewers agreed at journal 19 Feb, 2024 Reviewers agreed at journal 19 Feb, 2024 Reviewers agreed at journal 17 Feb, 2024 Reviewers invited by journal 17 Feb, 2024 Editor assigned by journal 17 Feb, 2024 Submission checks completed at journal 17 Feb, 2024 First submitted to journal 16 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3961690","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":273730880,"identity":"0a8046f7-fe58-49f3-aeef-8bea56da64f0","order_by":0,"name":"Sabrina Fernandes Cardoso","email":"","orcid":"","institution":"Federal University of Santa Catarina (UFSC)","correspondingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"Fernandes","lastName":"Cardoso","suffix":""},{"id":273730882,"identity":"4db81ee9-ee3d-44e5-be52-36850c5e4e67","order_by":1,"name":"João Victor Costa Guesser","email":"","orcid":"","institution":"Federal University of Santa Catarina (UFSC)","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Victor Costa","lastName":"Guesser","suffix":""},{"id":273730883,"identity":"19f99bee-f327-4822-884f-b704fb02268e","order_by":2,"name":"Andressa Alencastre Fuzari Rodrigues","email":"","orcid":"","institution":"Oswaldo Cruz Institute (IOC), FIOCRUZ","correspondingAuthor":false,"prefix":"","firstName":"Andressa","middleName":"Alencastre Fuzari","lastName":"Rodrigues","suffix":""},{"id":273730888,"identity":"5d65f876-ef6a-45b4-b13c-ce5c7e3ae458","order_by":3,"name":"Reginaldo Peçanha Brazil","email":"","orcid":"","institution":"Oswaldo Cruz Institute (IOC), FIOCRUZ","correspondingAuthor":false,"prefix":"","firstName":"Reginaldo","middleName":"Peçanha","lastName":"Brazil","suffix":""},{"id":273730890,"identity":"f43d6df6-5967-4c7e-8382-5b91f1b6e9fd","order_by":4,"name":"Luísa Damazio Pitaluga Rona","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPgbGBiiT+QBDBZAyIKSFDaGFLYHhDHFa4IDHgEgtEsnNH34w3JMzZz/zTeLAHzsGc+kDhLQktkn2MBQbW/bkbpM42JbMYNmXQFgLAw9DQuKGA7nbpD82HGAwOEPQYYnNH/+AtJx/8wzoMOK0NEiDbbmRwyZxgI0YLTwP26RlDBKMDW48M7YA+oXHsoeAFn729Mcf31QkyBmcT354AxhicuY8BLRAAFJcEKdhFIyCUTAKRgF+AAA68D80rWLZ0QAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Santa Catarina (UFSC)","correspondingAuthor":true,"prefix":"","firstName":"Luísa","middleName":"Damazio Pitaluga","lastName":"Rona","suffix":""},{"id":273730893,"identity":"11036065-5f60-48cd-975d-e116ca3ba6e3","order_by":5,"name":"André Nóbrega Pitaluga","email":"","orcid":"","institution":"Oswaldo Cruz Institute (IOC), FIOCRUZ","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Nóbrega","lastName":"Pitaluga","suffix":""}],"badges":[],"createdAt":"2024-02-16 15:37:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3961690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3961690/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51390336,"identity":"fd8761da-5dc5-4f1b-90c8-8689fcc0cf14","added_by":"auto","created_at":"2024-02-20 18:19:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4415690,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the Zoonosis Control Center of the municipality of Tubarão, located in the South of Santa Catarina State, Brazil, and images of the sampling points: \u003cstrong\u003eA-\u003c/strong\u003e Trap inside the dogs' stall; \u003cstrong\u003eB-\u003c/strong\u003eTrap in front of the dogs' stall; \u003cstrong\u003eC-\u003c/strong\u003e Traps installed in a forest area; \u003cstrong\u003eD-\u003c/strong\u003eTrap next to the dogs' stall.\u003c/p\u003e","description":"","filename":"20240106Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3961690/v1/d711f51b57373b9f06d44e76.png"},{"id":51390337,"identity":"20a69708-b536-4fbc-bf00-e1873497d113","added_by":"auto","created_at":"2024-02-20 18:19:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":569934,"visible":true,"origin":"","legend":"\u003cp\u003eDashboard displaying a summary of survey results:\u003cstrong\u003eA-\u003c/strong\u003e Adult non-engorged female sand fly. \u003cstrong\u003eB-\u003c/strong\u003e Polymerase chain reaction (PCR) for detecting natural \u003cem\u003eLeishmania\u003c/em\u003e spp. infection, using primers targeting the ITS1 region of rDNA, which amplifies within the range of 300 to 350 base pairs (bp), depending on the \u003cem\u003eLeishmania \u003c/em\u003especies. Line M: Molecular marker (100 bp), lines F1-F4: female sand flies (\u003cem\u003eNy. neivai\u003c/em\u003e) positively identified for trypanosomatids, lines F1 and F3: \u003cem\u003eNy. neivai\u003c/em\u003e females positive for \u003cem\u003eL. infantum\u003c/em\u003e, Line PC: Positive control using \u003cem\u003eL. infantum\u003c/em\u003e DNA, line NC: negative control, without DNA, employing water instead. \u003cstrong\u003eC\u003c/strong\u003e- Female sand fly specimens mounted on glass slide, showcase morphological features of \u003cem\u003eNy. neivai\u003c/em\u003e spermatheca. \u003cstrong\u003eD\u003c/strong\u003e- PCR conducted for molecular identification of \u003cem\u003eNy. neivai\u003c/em\u003e using DNA barcoding. Line M: Molecular marker (100 bp), lines M1-M4: female \u003cem\u003eNy. Neivai \u003c/em\u003esand flies, Line PC: Positive control using \u003cem\u003eNy. neivai \u003c/em\u003eDNA, line NC: negative control, without DNA, using water instead. \u003cstrong\u003eE-\u003c/strong\u003eCollection of biological material from dogs for \u003cem\u003eLeishmania\u003c/em\u003e spp. infection detection, employing the non-invasive conjunctival swab technique.\u003c/p\u003e","description":"","filename":"20240106Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3961690/v1/7a8ebc6e3add7f1bede1ee8a.png"},{"id":51391667,"identity":"8f41c010-bdcb-4fc9-9442-4476b71e5deb","added_by":"auto","created_at":"2024-02-20 18:27:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2064920,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3961690/v1/fdffa01a-f65f-4ddb-b48b-4afe3a98c8e9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Leishmania infantum (Trypanosomatida: Trypanosomatidae) detection in Nyssomyia neivai (Diptera: Psychodidae) and dogs in Southern Brazil","fulltext":[{"header":"Background","content":"\u003cp\u003eLeishmaniases are diseases caused by protozoa of the genus \u003cem\u003eLeishmania\u003c/em\u003e (Trypanosomatida: Trypanosomatidae), which can affect both humans and other animals, potentially leading to fatal outcomes. The pathogen transmission occurs through a bite of the sand fly vector infected females (Diptera: Psychodidae) [01].\u003c/p\u003e \u003cp\u003eSand flies constitute a group of approximately 1000 known species worldwide, with 530 species having been identified in the Americas [02]. It is estimated that 98 species may serve as potential natural vectors of \u003cem\u003eLeishmania\u003c/em\u003e spp. [03].\u003c/p\u003e \u003cp\u003eIn the Americas, Visceral Leishmaniasis (VL), the most severe form of the disease, is caused by \u003cem\u003eLeishmania infantum\u003c/em\u003e. This leishmania species is mainly transmitted by its primary vector, the sand flies of the \u003cem\u003eLutzomyia longipalpis\u003c/em\u003e complex [04, 05]. In Brazil, other sand flies\u0026rsquo; species have been implicated as potential vectors of \u003cem\u003eL. infantum\u003c/em\u003e in areas where the visceral form of the disease occurs and \u003cem\u003eLu. longipalpis\u003c/em\u003e is absent [06, 07]. In the State of Santa Catarina, Brazil, cases of VL affecting both humans and dogs have been reported, yet the conventional vector has not been detected [08].\u003c/p\u003e \u003cp\u003e \u003cem\u003eNyssomyia neivai\u003c/em\u003e, a sand fly species widely distributed throughout Santa Catarina State, is known as the etiologic agent vector of American tegumentary leishmaniasis (ATL) [09]. However, \u003cem\u003eNy. neivai\u003c/em\u003e has been found in abundance in regions with human and canine VL cases within Santa Catarina State [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the VL positive dog identifications in the Zoonosis Control Center (ZCC) of Tubar\u0026atilde;o municipality, (Santa Catarina, Brazil), the present study aimed to investigate potential vectors implicated in the disease transmission and to identify the presence of new cases of Canine Visceral Leishmaniasis (CVL) in the area.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis research was undertaken at the Zoonosis Control Center (ZCC) of Tubar\u0026atilde;o municipality (28\u0026deg;31\u0026prime;12.64\u0026Prime;S, 49\u0026deg;01\u0026prime;03.09\u0026Prime;W), located in Southern region of Santa Catarina State, Brazil (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The institution facility can house around 60 dogs, and there were reports of CVL outbreaks on the premises approximately three months before the study began. The investigation was carried out from March 2019 to June 2020.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSand fly collections\u003c/h2\u003e\n \u003cp\u003eWe collected sand flies once a month using CDC light traps (model 512; John W. Hock, Gainesville, FL). These traps were positioned approximately one meter above the ground both inside and outside the dog shelter within ZCC, as well as in the nearby forest area. This arrangement established four distinct collection points, each spaced 30 meters apart. Captures were conducted from 06:00 p.m. to 06:00 a.m. Once the traps were retrieved, the collected sand flies were sorted into males, engorged females and non-engorged females, and the specimens were then preserved in 70% ethyl alcohol and stored at -20\u0026ordm;C until processing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eSand fly taxonomic identification\u003c/h2\u003e\n \u003cp\u003eThe captured sand flies were identified based on morphological characters, using the taxonomic key proposed by Galati [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Before being mounted on glass slides, all sand flies underwent a previous process of clarification, dehydration and diaphanization of body structures, as described by Fuzari [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. The male sand flies were full body assembled on glass slides. Engorged females had the head detached from the rest of the body, while in non-engorged females, the head and the last three abdominal segments were detached from the rest of the body, and then mounted on slides. Remaining fragments of the abdomen and thorax were preserved in 70% ethyl alcohol and stored at -20\u0026ordm;C for subsequent analysis to determine natural infection by \u003cem\u003eLeishmania\u003c/em\u003e spp.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular identification of\u003c/strong\u003e \u003cstrong\u003eLeishmania\u003c/strong\u003e \u003cstrong\u003espp. in sand flies\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFor molecular identification of \u003cem\u003eLeishmania\u003c/em\u003e DNA in sand flies, we specifically analyzed non-engorged females. This selection aimed to avoid possible \u003cem\u003eLeishmania\u003c/em\u003e detection originated from an undigested blood meal, focusing instead on identifying potential natural infections. Consequently, we individually extracted DNA of non-engorged female sandflies body fragments using the protocol established by Jowett [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eWe assembled sample pools, each containing genetic material from up to ten individuals for initial molecular screening. A polymerase chain reaction (PCR) was conducted targeting the ITS1 region of \u003cem\u003eLeishmania\u003c/em\u003e spp. rDNA, using the primers and amplification conditions described by El-Tai et al. [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e] and Sch\u0026ouml;nian et al. [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. This region amplifies a sequence of 300 to 350 base pairs (bp), depending on the \u003cem\u003eLeishmania\u003c/em\u003e species.\u003c/p\u003e\n \u003cp\u003eFollowing a positive pool identification, the original 10 female samples were submitted to an individual analysis through a second PCR reaction under the same conditions. Therefore, we were able to identify the real number of \u003cem\u003eLeishmania\u003c/em\u003e infected sand flies, thus accurately determining the natural infection rate and vector identification.\u003c/p\u003e\n \u003cp\u003eThe amplified fragments resulting from the individual sample PCR reactions were purified using the QIAquick\u0026reg; PCR purification kit (Qiagen), according to the manufacturer\u0026apos;s instructions. The purified fragments were submitted to a Sanger sequencing at Fiocruz Sequencing Unit (RPT1A, Rio de Janeiro, Brazil) for \u003cem\u003eLeishmania\u003c/em\u003e spp. identification. The sequences were aligned using the Geneious Prime program to obtain consensus sequences followed by BLASTN analysis. The identified sequences were submitted to GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e) under accession numbers: OP718537 and OP718538.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eSand fly molecular identification\u003c/h2\u003e\n \u003cp\u003eIn addition to morphological characterization, \u003cem\u003eLeishmania\u003c/em\u003e spp. positive sandflies were submitted to a molecular identification through DNA barcoding. The primers used, and the reaction conditions were performed as described by Pinto et al. [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Purified PCR products were sequenced and analyzed following the procedure outlined previously. The resulting sequences have been deposited in GenBank under the following accession numbers: OP719771, OP719772, OP719773 and OP719774.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eDogs\u0026rsquo; sample collection\u003c/h2\u003e\n \u003cp\u003eWe collected biological samples from 47 dogs sheltered at ZCC using the non-invasive conjunctival swab technique [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. During the procedure, the lower eyelid was slightly separated so that the cotton swab could be rubbed on the ocular conjunctiva to collect cells from the right eye of each dog. Only the swabs tips were transferred to sterile tubes containing 200\u0026micro;l of extraction buffer-SQ solution (Tris HCL 10 mM; EDTA 2 mM; Triton absolute 0.2%) and stored at -20\u0026deg;C until analysis.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDogs molecular diagnosis of\u003c/strong\u003e \u003cstrong\u003eLeishmania\u003c/strong\u003e \u003cstrong\u003espp.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo extract DNA from the swab, the cotton was macerated with the SQ-buffer, and then 2\u0026micro;l of Proteinase K (20mg/ml) was added. The solution was incubated at 37\u0026ordm;C for one hour in a dry bath, then heated to 100\u0026ordm;C for 20 minutes to denature the Proteinase K. Individual samples were submitted to a PCR reaction followed by amplicons sequencing to identify positive dogs for \u003cem\u003eLeishmania\u003c/em\u003e spp., as detailed previously. The resulting sequences were submitted to GenBank, under the accession numbers: OP724554 and OP724555.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSand fly collection\u003c/h2\u003e \u003cp\u003eField sampling was carried out at four different sites of the ZCC facility in Tubar\u0026atilde;o 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a similar proportion to several sand flies\u0026rsquo; studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of sand flies collected and species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSand flies species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMales\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eFemales\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEngorged\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNon-engorged\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNyssomyia neivai\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e85.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMigonemyia migonei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePintomyia fischeri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBrumptomyia spp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEvandromyia edwardsi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eNy. neiva\u003c/em\u003ei (85.75%), followed by \u003cem\u003eMigonemyia migonei\u003c/em\u003e (13.31%), \u003cem\u003ePintomyia fischeri\u003c/em\u003e (0.82%), \u003cem\u003eBrumptomyia\u003c/em\u003e spp. (0.09%) and \u003cem\u003eEvandromyia edwardsi\u003c/em\u003e (0.03%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results align with a previous study that identify the presence of these sand fly species in Santa Catarina State [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and contribute to a deeper knowledge of the State sand flies fauna and canine leishmania transmission, which are limited.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNyssomyia neivai\u003c/em\u003e was the predominant species found in this study, which is consistent with its identification in studies of sand flies\u0026rsquo; fauna in southern Brazil [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This species is prevalent in colder and drier Brazilian regions, thriving in South, Southeast, and Midwest Brazil [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It is an important ATL vector, and its infection with \u003cem\u003eL. infantum\u003c/em\u003e was previously documented in Santa Catarina and Minas Gerais States [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003eDNA detection in sand flies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 509 non-engorged females were tested for \u003cem\u003eLeishmania\u003c/em\u003e spp. infection, accounting for 54.1% of collected females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe positive sand flies sequencing data identified \u003cem\u003eL. infantum\u003c/em\u003e as the cause of natural infection in 50% of positive samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e-B: F1 and F3), resulting in a 0.4% natural infection rate. This infection rate is in alignment with previous reports that found \u003cem\u003eLeishmania\u003c/em\u003e infected sand flies ranging from 0.2\u0026ndash;9.1% [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The remaining positive samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e-B: F2 and F4) yielded inconclusive species identification, which may be due to the low coverage (\u0026lt;\u0026thinsp;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 \u003cem\u003eL. infantum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e-B: F2 and F4), suggesting a broader spectrum for trypanosomatids detection using ITS1 primers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe four sand flies naturally infected with trypanosomatids were identified as \u003cem\u003eNy. neivai\u003c/em\u003e, confirmed through both morphological and molecular analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e: C and D).\u003c/p\u003e \u003cp\u003eOur findings present the first DNA barcode sequences of \u003cem\u003eNy. neivai\u003c/em\u003e 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) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Indeed, \u003cem\u003eNy\u003c/em\u003e. \u003cem\u003eneivai\u003c/em\u003e has been found naturally infected by both i) the etiologic agents of ATL [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], as well as ii) \u003cem\u003eL. infantum\u003c/em\u003e, the causative agent of VL in Brazil [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReports from the Epidemiological Surveillance Directorate of Santa Catarina State (DIVE/SC) indicated a significant prevalence of \u003cem\u003eNy. neivai\u003c/em\u003e in areas with CVL and human VL foci in Florian\u0026oacute;polis - SC. Notably, \u003cem\u003eLu. Longipalpis\u003c/em\u003e, the primary vector of \u003cem\u003eL. infantum\u003c/em\u003e, is absent in these areas [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study revealed a similar entomological profile, including natural infection of \u003cem\u003eNy. neivai\u003c/em\u003e by \u003cem\u003eL. infantum\u003c/em\u003e. This discovery strongly suggests that \u003cem\u003eNy. neivai\u003c/em\u003e might be a vector in this region, which is supported by i) the insect\u0026rsquo;s high abundance in areas with CVL cases, ii) the absence of \u003cem\u003eLu. Longipalpis\u003c/em\u003e, and iii) the natural infection of \u003cem\u003eNy. neivai\u003c/em\u003e by \u003cem\u003eL. infantum\u003c/em\u003e. Nonetheless, additional research is needed to confirm the species\u0026rsquo; role in the VL cycle.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003eDNA detection in dogs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 47 dogs from Tubar\u0026atilde;o Municipality ZCC were tested for \u003cem\u003eLeishmania\u003c/em\u003e spp. infection using a non-invasive method for sample collection - the conjunctival swab (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e02\u003c/span\u003e-E). This simple and efficacy technique, combined with ITS1 PCR amplification, in diagnosing CVL in asymptomatic animals was previously demonstrated by Leite et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] as \u003cem\u003eLeishmania\u003c/em\u003e amastigote forms are frequently found in the ocular conjunctiva [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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 \u003cem\u003eL. infantum\u003c/em\u003e, 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.\u003c/p\u003e \u003cp\u003eHere we identified a high prevalence of \u003cem\u003eNy. neivai\u003c/em\u003e combined with \u003cem\u003eL. infantum\u003c/em\u003e natural infection, also detected in infected dogs within the studied area. The \u003cem\u003eL. infantum\u003c/em\u003e ITS1 sequence profile was identical in all four samples, two positive sand flies and two positive dogs. This suggests that \u003cem\u003eNy. neivai\u003c/em\u003e is attracted to feed on dogs and supports the parasite\u0026rsquo;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 \u003cem\u003eLeishmania\u003c/em\u003e diagnostics on a regular basis since the parasite vector are widely disperse in Brazil. Also, periodically vectorial surveillance for \u003cem\u003eLeishmania\u003c/em\u003e natural infection should be implemented as a preventive measurement to avoid CVL transmission.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we provide the first evidence of \u003cem\u003eL. infantum\u003c/em\u003e infecting both \u003cem\u003eNy. neivai\u003c/em\u003e and dogs within the same area in Brazil. Considering the absence of \u003cem\u003eLu. Longipalpis\u003c/em\u003e in Santa Catarina State thus far, our data suggests that \u003cem\u003eNy. neivai\u003c/em\u003e might act as a VL vector transmitting \u003cem\u003eL. infantum\u003c/em\u003e in this region. Furthermore, we emphasize the importance of using DNA barcoding as a tool to support fast and numerous sand fly\u0026rsquo;s identification. Additionally, our study confirms the conjunctival swab technique efficiency in successfully detecting \u003cem\u003eL. infantum\u003c/em\u003e in dogs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCR: Polymerase chain reaction; VL: Visceral leishmaniasis; ATL: American tegumentary leishmaniasis; ZCC: Zoonosis Control Center; CVL: Canine visceral leishmaniasis; bp: Base pairs; \u0026mu;l: Microliter; HCL: Hydrochloric acid; EDTA: Ethylenediaminetetraacetic acid; SC: Santa Catarina.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are indebted PDTIS-FIOCRUZ for the use of its DNA sequencing facility, and LAMEB-UFSC for helping with the photos.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was funded by the National Council for Scientific and Technological Development MCTI/CNPq/CAPES/FAPs no 16/2014 (to ANP and LDPR), and FIOCRUZ (ANP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the GenBank database under the accession numbers:\u0026nbsp;OP718537, OP718538, OP719771, OP719772, OP719773, OP719774, OP724554, OP724555.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSFC, ANP, and LDRP participated in data generation and analysis; SFC, JVCG, AAFR, RPB carried sand fly collections and morphological identification; SFC drafted the manuscript; ANP and LDPR helped in the paper drafting by critically reading the original manuscript; ANP and LDPR were the principal investigators, participated in its design and coordination. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Ethics in Animal Use Committee of the Federal University of Santa Catarina, Florian\u0026oacute;polis, Brazil (process number: 8644190221, ID 001784). Formal written and oral consent was obtained from the Zoonoses Control Center, where the study was carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eThis paper is part of the Master thesis of Sabrina Fernandes Cardoso from Cell and Developmental Biology (PPGBCD) Graduation Program from Biological Science Center (CCB) at Federal University of Santa Catarina (UFSC).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSherlock IRA. Import\u0026acirc;ncia m\u0026eacute;dico-veterin\u0026aacute;ria: a import\u0026acirc;ncia dos flebotom\u0026iacute;neos. In: Rangel EF, Ralph L, (Org). Flebotom\u0026iacute;neos do Brasil. 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Br Med Bull. 1972;28(1):44-8.\u003c/li\u003e\n\u003cli\u003eR\u0026ecirc;go FD, Souza GD, Miranda JB, Peixoto LV, Andrade-Filho JD. Potential vectors of leishmania parasites in a recent focus of visceral leishmaniasis in neighborhoods of Porto Alegre, State of Rio Grande do Sul, Brazil. J Med Entomol. 2020;57(4):1286-1292.\u003c/li\u003e\n\u003cli\u003eGuimar\u0026atilde;es VCFV, Pruzinova K, Sadlova J, Volfova V, Myskova J, Filho SPB, Volf P. Lutzomyia migonei is a permissive vector competent for Leishmania infantum. Parasit Vectors. 2016;9:159.\u003c/li\u003e\n\u003cli\u003eDIVE. Boletim epidemiol\u0026oacute;gico das leishmanioses. Florian\u0026oacute;polis: DIVE, 2021. \u003c/li\u003e\n\u003cli\u003eMarcondes CB, Concei\u0026ccedil;\u0026atilde;o MB, Portes MG, Sim\u0026atilde;o BP. Phlebotomine sandflies in a focus of dermal leishmaniasis in the eastern region of the Brazilian State of Santa Catarina: preliminary results (Diptera: Psychodidae). Rev Soc Bras Med Trop. 2005;38(4):353-5.\u003c/li\u003e\n\u003cli\u003eDIVE. Relat\u0026oacute;rio de pesquisa entomol\u0026oacute;gica. Florian\u0026oacute;polis: DIVE, 2017. \u003c/li\u003e\n\u003cli\u003eGalati, EAB. Phlebotominae (Diptera, Psychodidae): classification, morphology and terminology of adults and identification of American Taxa. In Rangel EF, Shaw JJ. (eds). Brazilian Sand Flies. Springer. 2018;9-212.\u003c/li\u003e\n\u003cli\u003eFuzari AA. Avalia\u0026ccedil;\u0026atilde;o do papel epidemiol\u0026oacute;gico das leishmanioses em comunidades inseridas em \u0026aacute;rea de preserva\u0026ccedil;\u0026atilde;o ambiental, Parque Estadual da Serra da Tiririca, Niter\u0026oacute;i e Maric\u0026aacute;, RJ, Brasil. (Tese). Instituto Oswaldo Cruz: P\u0026oacute;s-gradua\u0026ccedil;\u0026atilde;o em Medicina Tropical, 2016.\u003c/li\u003e\n\u003cli\u003eJowett T. Preparation of nucleic acids. In: Roberts DB, editor. Drosophila: a practical approach. Oxford: IRL Press. 1998;275\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eEl Tai NO, Osman OF, El Fari M, Presber W, Sch\u0026ouml;nian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans. R. Soc. Trop. Med. Hyg. 2000;94:575\u0026ndash;579. \u003c/li\u003e\n\u003cli\u003eSch\u0026ouml;nian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003;47(1):349-58.\u003c/li\u003e\n\u003cli\u003ePinto IS, Chagas BD, Rodrigues AAF, Ferreira AL, Rezende HR, Bruno RV, et al. DNA barcoding of neotropical sand flies (Diptera, Psychodidae, Phlebotominae): species identification and discovery within Brazil. PLoS One. 2015;10(10):e0140636.\u003c/li\u003e\n\u003cli\u003eGao CH, Ding D, Wang JY, Steverding D, Wang X, Yang YT, Shi F. Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples. Parasit Vectors. 2015 Jul 15;8:370. doi: 10.1186/s13071-015-0991-2. PMID: 26169060; PMCID: PMC4501202.\u003c/li\u003e\n\u003cli\u003eDias ES, Michalsky EM, Nascimento JC, Ferreira EC, Lopes JV, Fortes-Dias CL. Detection of \u003cem\u003eLeishmania infantum\u003c/em\u003e, the etiological agent of visceral leishmaniasis, in \u003cem\u003eLutzomyia neivai\u003c/em\u003e, a putative vector of cutaneous leishmaniasis. J Vector Ecol. 2013;38(1):193-6.\u003c/li\u003e\n\u003cli\u003eFigueiredo FB, Madeira MF. Os Parasitos e a quest\u0026atilde;o da infec\u0026ccedil;\u0026atilde;o em animais Dom\u0026eacute;sticos e Domiciliados. In: Concei\u0026ccedil;\u0026atilde;o SF; Alves CR (org). Leishmanioses do continente americano. 22. ed. Rio de Janeiro: Fiocruz. 2014;259-273.\u003c/li\u003e\n\u003cli\u003eQuiroga C, Cevallos V, Morales D, Baldeon M, Cardenas P, Rojas-Silva P, et al. Molecular identification of \u003cem\u003eleishmania\u003c/em\u003e spp. in sand flies (Diptera: Psychodidae, Phlebotominae) from Ecuador. J Med Entomol. 2017;54(6):1704-1711.\u003c/li\u003e\n\u003cli\u003eMarcondes CB, Concei\u0026ccedil;\u0026atilde;o MB, Portes MGT, Sim\u0026atilde;o BP. Phlebotomine sandflies in a focus of dermal leishmaniasis in the eastern region of the Brazilian State of Santa Catarina: preliminary results (Diptera: Psychodidae). Rev Soc Bras Med Trop. 2005;38(4):353-5.\u003c/li\u003e\n\u003cli\u003eMarcondes CB, Bittencourt IA, Stoco PH, Eger I, Grisard EC, Steindel M. Natural infection of \u003cem\u003eNyssomyia neivai\u003c/em\u003e (Pinto, 1926) (Diptera: Psychodidae, Phlebotominae) by \u003cem\u003eLeishmania \u003c/em\u003e(\u003cem\u003eViannia\u003c/em\u003e) spp. in Brazil. Trans R Soc Trop Med Hyg. 2009;103(11):1093-1097.\u003c/li\u003e\n\u003cli\u003eSilva AM, Camargo NJ, Santos DR, Massafera R, Ferreira AC, Postai C, et al. Diversidade, Distribui\u0026ccedil;\u0026atilde;o e Abund\u0026acirc;ncia de Flebotom\u0026iacute;neos (Diptera: Psychodidae) no Paran\u0026aacute;. Neotrop Entomol. 2008;37(2):209-225. \u003c/li\u003e\n\u003cli\u003eMcIntyre S, Rangel EF, Ready PD, Carvalho BM. Species-specific ecological niche modelling predicts different range contractions for \u003cem\u003eLutzomyia intermedia\u003c/em\u003e and a related vector of \u003cem\u003eLeishmania braziliensis\u003c/em\u003e following climate change in South America. Parasit Vectors. 2017;10(1):157.\u003c/li\u003e\n\u003cli\u003eSaraiva L, Carvalho GML, Gontijo CMF, Quaresma PF, Lima ACVMR. Falc\u0026atilde;o AL, et al. Natural infection of \u003cem\u003eLutzomyia neivai\u003c/em\u003e and \u003cem\u003eLutzomyia sallesi\u003c/em\u003e (Diptera:Psychodidae) by \u003cem\u003eLeishmania infantum chagasi\u003c/em\u003e in Brazil. J. Med. Entomol. 2009;46(5):1159\u0026ndash;1163 \u003c/li\u003e\n\u003cli\u003eBritto CFPC, Pereira DP. Diagn\u0026oacute;stico molecular de \u003cem\u003eLeishmania\u003c/em\u003e spp. em fleb\u0026oacute;tomos provenientes de \u0026aacute;reas de ocorr\u0026ecirc;ncia de leishmanioses. In: Concei\u0026ccedil;\u0026atilde;o SF; Alves CR (org). Leishmanioses do continente americano. 22. ed. Rio de Janeiro: Fiocruz, 2014;217-231. \u003c/li\u003e\n\u003cli\u003eMoya SL, Pech-May A, Quintana MG, Manteca-Acosta M, Salom\u0026oacute;n OD. Phylogenetic relationships of closely-related phlebotomine sand flies (Diptera: Psychodidae) of \u003cem\u003eNyssomyia\u003c/em\u003e genus and \u003cem\u003eLutzomyia\u003c/em\u003e subgenus. Mem Inst Oswaldo Cruz. 2020;14;115:e200220. \u003c/li\u003e\n\u003cli\u003eC\u0026oacute;rdoba-Lan\u0026uacute;s E, Grosso ML, Pi\u0026ntilde;ero JE, Valladares B, Salom\u0026oacute;n OD. Natural infection of \u003cem\u003eLutzomyia neivai\u003c/em\u003e with \u003cem\u003eLeishmania\u003c/em\u003e spp. in northwestern Argentina. Acta Trop. 2006;98(1):1-5\u003c/li\u003e\n\u003cli\u003eNeitzke-Abreu HC, Reinhold-Castro KR, Venazzi MS, Scodro RBL, Dias Ade C, Silveira TGV. Detection of \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) in \u003cem\u003eNyssomyia neivai\u003c/em\u003e and \u003cem\u003eNyssomyia whitmani\u003c/em\u003e by multiplex polymerase chain reaction, in Southern Brazil. Rev Inst Med Trop S\u0026atilde;o Paulo. 2014;56(5):391-5. \u003c/li\u003e\n\u003cli\u003eOliveira DM, Reinhold-Castro KR, Bernal MVZ, Legriffon CMO, Lonardoni MVC, Teodoro U, et al. Natural infection of \u003cem\u003eNyssomyia neivai\u003c/em\u003e by \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia\u003c/em\u003e) spp. in the state of Paran\u0026aacute;, Southern Brazil, detected by multiplex polymerase chain reaction. Vector Borne Zoonotic Dis. 2011;11(2):137-43.\u003c/li\u003e\n\u003cli\u003ePita-Pereira D, Souza GD, Zwetsch A, Alves CR, Britto C, Rangel EF. First report of \u003cem\u003eLutzomyia \u003c/em\u003e(\u003cem\u003eNyssomyia\u003c/em\u003e) \u003cem\u003eneivai\u003c/em\u003e (Diptera: Psychodidae: Phlebotominae) naturally infected by \u003cem\u003eLeishmania\u003c/em\u003e (\u003cem\u003eViannia) braziliensis\u003c/em\u003e in a peri urban area of south Brazil using a multiplex polymerase chain reaction assay. Am J Trop Med Hyg. 2009;80(4):593-5.\u003c/li\u003e\n\u003cli\u003eDIVE. Relat\u0026oacute;rio de pesquisa entomol\u0026oacute;gica em \u0026aacute;rea com focos de Leishmaniose Visceral Canina e Humana. Florian\u0026oacute;polis: DIVE, 2019. \u003c/li\u003e\n\u003cli\u003eDIVE. Relat\u0026oacute;rio de pesquisa entomol\u0026oacute;gica em \u0026aacute;rea de foco de Leishmaniose Visceral Humana. Florian\u0026oacute;polis: DIVE, 2017. \u003c/li\u003e\n\u003cli\u003eLeite RS, Ferreira SA, Ituassu LT, de Melo MN, de Andrade ASR. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples. Vet Parasitol. 2010;170(3-4):201-6.\u003c/li\u003e\n\u003cli\u003eBarbosa VT, Silva MAG, Sousa MG, Gering AP, Santos HD, Laus JL. Detec\u0026ccedil;\u0026atilde;o de formas amastigotas em exame parasitol\u0026oacute;gico de esfrega\u0026ccedil;o obtido a partir de suabe conjuntival de c\u0026atilde;es com leishmaniose visceral. Arq. Bras. Med. Vet. Zootec. 2012;64(6):1465-1479.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Leishmania infantum, Nyssomyia neivai, Visceral leishmaniasis","lastPublishedDoi":"10.21203/rs.3.rs-3961690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3961690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe sand fly \u003cem\u003eNyssomyia neivai\u003c/em\u003e is one of the most abundant species in Southern Brazil. It has been frequently found in visceral leishmaniasis foci in Santa Catarina (Brazil), a region where \u003cem\u003eLutzomyia longipalpis\u003c/em\u003e, the main vector of \u003cem\u003eLeishmania infantum\u003c/em\u003e and the etiological agent of visceral leishmaniasis in the Americas, has not been identified. In the absence of the main \u003cem\u003eLeishmania\u003c/em\u003e vector, this study aimed to identify the sand fly fauna and diagnose any potential \u003cem\u003eLeishmania\u003c/em\u003e spp. infection in sand flies and dogs in a region with a recent canine visceral leishmaniasis outbreak in the South of Brazil.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe report here a sand fly fauna survey on the Zoonosis Control Center of Tubar\u0026atilde;o Municipality (Santa Catarina, Brazil). We also conducted molecular testing to detect \u003cem\u003eLeishmania\u003c/em\u003e spp. natural infection on captured sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of \u003cem\u003eLeishmania\u003c/em\u003e spp. using a non-invasive technique for the collection of biological material, followed by PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe collected 3,419 specimens from five sand flies genera. \u003cem\u003eNyssomyia neivai\u003c/em\u003e (85.75%) was the most abundant species, followed by \u003cem\u003eMigonemyia migonei\u003c/em\u003e (13.31%), \u003cem\u003ePintomyia fischeri\u003c/em\u003e (0.82%), \u003cem\u003eEvandromyia edwardsi\u003c/em\u003e (0.03%), and the genus \u003cem\u003eBrumptomyia\u003c/em\u003e spp. (0.09%). \u003cem\u003eLeishmania infantum\u003c/em\u003e DNA was detected in two of the 509 analyzed females, yielding a natural infection rate of 0.4%. The \u003cem\u003eL. infantum\u003c/em\u003e infected sand flies\u0026rsquo; specimens were morphologically and molecularly identified as \u003cem\u003eNy. neivai\u003c/em\u003e. We analyzed 47 dogs\u0026rsquo; conjunctival swabs for \u003cem\u003eLeishmania\u003c/em\u003e spp. with two positive individuals for \u003cem\u003eL. infantum\u003c/em\u003e (infection rate of 4.2%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results confirm the presence of \u003cem\u003eNy. neivai\u003c/em\u003e naturally infected with \u003cem\u003eL. infantum\u003c/em\u003e in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil. We emphasize the significance of DNA barcoding in supporting the sand flies\u0026rsquo; identification, and the conjunctival swab technique proved effective in obtaining enough biological material for detecting \u003cem\u003eL. infantum\u003c/em\u003e in dogs.\u003c/p\u003e","manuscriptTitle":"Leishmania infantum (Trypanosomatida: Trypanosomatidae) detection in Nyssomyia neivai (Diptera: Psychodidae) and dogs in Southern Brazil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 18:19:43","doi":"10.21203/rs.3.rs-3961690/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-10T10:31:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-05T20:14:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-03T01:48:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94b29e3e-8779-4f3b-ab3d-de685db61ce5","date":"2024-02-19T12:25:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"883b3cfd-9450-426f-9bf8-03f2edf47bf3","date":"2024-02-19T09:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e0a2276f-196e-4fc2-a0a6-17f792d55779","date":"2024-02-17T19:22:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-17T17:50:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-17T09:18:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-17T09:15:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-02-16T15:31:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aae3dbfe-2be8-446d-a255-0de816cc2228","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-28T13:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-20 18:19:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3961690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3961690","identity":"rs-3961690","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}