Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence

Serodiagnosis of Leishmania infantum infection in dogs relies on the detection of antibodies against leishmanial crude extracts or parasitic defined antigens. The expansion of canine leishmaniasis from geographical areas of Brazil in which the infection is endemic to regions in which the disease is emerging is occurring. This fact makes necessary the analysis of the serodiagnostic capabilities of different leishmanial preparations in distinct geographical locations. In this article sera from dogs infected with Leishmania and showing the clinical form of the disease, were collected in three distinct Brazilian States and were tested against soluble leishmanial antigens or seven parasite individual antigens produced as recombinant proteins. We show that the recognition of soluble leishmanial antigens by sera from these animals was influenced by the geographical location of the infected dogs. Efficacy of the diagnosis based on this crude parasite preparation was higher in newly endemic regions when compared with areas of high disease endemicity. We also show that the use of three of the recombinant proteins, namely parasite surface kinetoplastid membrane protein of 11 kDa (KMP-11), and two members of the P protein family (P2a and P0), can improve the degree of sensitivity without adversely affecting the specificity of the diagnostic assays for canine leishmaniasis, independently of the geographical area of residence. In addition, sera from dogs clinically healthy but infected were also assayed with some of the antigen preparations. We demonstrate that the use of these proteins can help to the serodiagnosis of Leishmania infected animals with subclinical infections. Finally, we propose a diagnostic protocol using a combination of KMP-11, P2a y P0, together with total leishmanial extractsThis work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) within the call“CNPq/MS/SCTIE/DECIT N° 32/2014 - Pesquisas sobre Leishmanioses”grant number reference 467389/2014-4. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged. TC received scholarship from Fundação de Amparo a Pesquisa do Estado de Santa Catarina–FAPES

Leishmaniose visceral em Florianópolis: caracterização molecular das cepas de Leishmania infantum isoladas de casos locais e pesquisa vetorial

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2018.Leishmanioses são doenças causadas por parasitos do gênero Leishmania sp. (Kinetoplastida: Trypanosomatidae), transmitidas pela da picada de flebotomíneos fêmeas. Três formas clínicas distintas são descritas para as leishmanioses: cutânea, mucocutânea e visceral. A leishmaniose visceral (LV) é a forma mais grave, de elevada mortalidade se não tratada,que possui a espécie Leishmania infantum como agente etiológico nas Américas. O vetor canônico de LV é Lutzomyia longipalpis e o cão doméstico é considerado o principal reservatório. Embora mais de 96% dos casos de LV nas Américas ocorram no Brasil, a região Sul era considerada uma área livre de LV até 2008. Atualmente, a LV canina (LVC) está se espalhando rapidamente em todos os estados do sul, havendo um consequente aumento no número de casos humanos com alta taxa de letalidade. Santa Catarina foi o último estado do país a diagnosticar casos de LV humana (LVH), sendo que o município de Florianópolis possui uma prevalência de 3,4% de LVC. Porém, o principal vetor, Lu. longipalpis, não é encontrado nessa região. Sendo SC uma das áreas endêmicas mais recentes de transmissão de LV no Brasil o objetivo deste trabalho foi estudar a introdução de L. infantum em Florianópolis a partir da análise da variabilidade genética de cepas locais e pesquisar as possíveis espécies de vetores nesta região. Dos casos positivos registrados no município entre 2010 e 2016, foram obtidas amostras de 58 cães e dois casos humanos. A confirmação da espécie L. infantum nessas amostras ocorreu via PCR dos marcadores kDNA e ITS-1. Além disso, foi possível isolar as cepas de 43 dessas amostras. Para tentar identificar a variabilidade genética dos isolados foi realizada a padronização de um painel de marcadores para análise de sequências multilocus (MLSA) de L. infantum. Após amplificação e sequenciamento de 10 desses marcadores de algumas cepas isoladas e a comparação com as sequências de cepas de outros estados do país não foi observado nenhum polimorfismo, indicando baixa variabilidade. Visto que em Florianópolis não há o vetor clássico de LV foram coletados mais de 1.000 flebotomíneos em um dos bairros de Florianópolis (Pantanal) com um grande número de cães diagnosticados com LV. A população de flebotomíneos foi maior nos meses mais quentes do ano e a espécie mais prevalente foi Pintomyia fischeri. Dos indivíduos coletados 365 tiveram o DNA extraído e 31 apresentaram PCR positivo para Leishmania spp., sendo estes das espécies Pi. fischeri, Migonemyia migonei e Nyssomyia neivai.Os resultados sugerem que estas espécies podem participar no ciclo de transmissão dessa parasitose, sendo necessário os estudos de comprovação da competência vetorial. Os dados apontam o rápido espalhamento da doença no município de Florianópolis, sendo que os resultados dos estudos sobre LV nesta região fornecem subsídios para medidas de controle dessa parasitose.Abstract : Leishmaniasis comprises a group of diseases caused by parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae), that are transmitted by bite of infected female phlebotomine. Three major clinical forms of leishmaniasis are found: visceral (VL), cutaneous (CL) and mucocutaneous (ML). Visceral leishmaniasis is the most severe clinical form of the disease, caused by Leishmania infantum in Americas. The canonical vector is Lutzomyia longipalpis and the domestic dog is considered the main reservoir. More than 96% of the human cases of VL (HVL) in the Americas occur in Brazil. However, southern Brazil was a VL-free area up to 2008. Nowadays, canine VL (CVL) is spreading rapidly in all southern States, where the number of human cases is increasing, showing high lethality rates. Santa Catarina is the last state on the country to diagnose autochthonous HVL cases, and nowadays a serological survey in dogs from Florianópolis (SC) revealed a CVL prevalence of 3.4%. As the most recent established endemic area of VL transmission in Brazil, the goal of the present proposal was to study the introduction of L. infantum in Florianópolis, thought the genetic variability comparisons of local strains and determine the parasite vector(s).From positive cases registered in Florianópolis between 2010-2016, samples from 58 CVL cases and 2 HVL cases were obtained. All samples were diagnosed with L. infantum, confirmed by PCR (kDNA and ITS-1) and sequencing. Also, 43 L. infantum strains from VL cases have been isolated. The genetic variability of the strains was performed using the multilocus sequence analysis (MLSA). PCR amplification and sequencing were performed for 10 loci using four local strains. These sequences were compared with sequences from other Brazilian states, however no polymorphic site was found, suggesting low variability. Since Florianópolis is an area without Lu. Longipalpis, the capture of phlebotomine sandflies was initiated, and more than 1,000 individuals were collected in a locality of Florianópolis with CVL cases. The predominant specie was Pintomyia. fischeri and the phlebotomine population had a marked reduction during the winter (June-September). DNA was extracted for 365 female phlebotomines and 31 revealed positive PCR amplification for Leishmania spp., including specimens from Pi. fischeri, Migonemyia migonei e Nyssomyia neivai. The results suggest the possible participation of these three phlebotomine species in the parasite transmission, being necessary the study of their vectorial competence. Our data pointed out to the rapid spreading of VL in Florianópolis, and studies about LV in this region can improve the control strategies for this disease

Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence

Serodiagnosis of Leishmania infantum infection in dogs relies on the detection of antibodies against leishmanial crude extracts or parasitic defined antigens. The expansion of canine leishmaniasis from geographical areas of Brazil in which the infection is endemic to regions in which the disease is emerging is occurring. This fact makes necessary the analysis of the serodiagnostic capabilities of different leishmanial preparations in distinct geographical locations. In this article sera from dogs infected with Leishmania and showing the clinical form of the disease, were collected in three distinct Brazilian States and were tested against soluble leishmanial antigens or seven parasite individual antigens produced as recombinant proteins. We show that the recognition of soluble leishmanial antigens by sera from these animals was influenced by the geographical location of the infected dogs. Efficacy of the diagnosis based on this crude parasite preparation was higher in newly endemic regions when compared with areas of high disease endemicity. We also show that the use of three of the recombinant proteins, namely parasite surface kinetoplastid membrane protein of 11 kDa (KMP-11), and two members of the P protein family (P2a and P0), can improve the degree of sensitivity without adversely affecting the specificity of the diagnostic assays for canine leishmaniasis, independently of the geographical area of residence. In addition, sera from dogs clinically healthy but infected were also assayed with some of the antigen preparations. We demonstrate that the use of these proteins can help to the serodiagnosis of Leishmania infected animals with subclinical infections. Finally, we propose a diagnostic protocol using a combination of KMP-11, P2a y P0, together with total leishmanial extracts.Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) within the call “CNPq/MS/SCTIE/DECIT N° 32/2014 - Pesquisas sobre Leishmanioses” grant number reference 467389/2014-4. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledge