Corrigendum: Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

[This corrects the article DOI: 10.3389/fimmu.2018.02558.].

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).

Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters.

Leishmania-activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum) or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.

Clinical and immunopathological findings during long term follow-up in Leishmania infantum experimentally infected dogs.

Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-challenge, subclinically infected dogs presented low CVL clinical scores despite the persistence of Leishmania parasites in the lymph nodes, spleen and skin. Similarities observed among immune profiles in the context of experimental and natural infection seem to suggest that an enduring activation of the host immune response may lead to the control of parasite growth, thereby limiting disease severity.

Epidemiology of canine leishmaniasis in southern Bahia, Brazil.

Leishmaniosis is a zoonosis caused by protozoa of the genus Leishmania. American cutaneous leishmaniosis (ACL) is mainly caused by the species L. amazonensis and L. braziliensis, and American visceral leishmaniosis (AVL) is caused by L. infantum chagasi. In addition to their proven roles as reservoirs of AVL, dogs are also suspected by researchers to be reservoirs of ACL due to reports of this infection in domestic environments and of infected dogs in endemic areas. The aim of this study was to detect Leishmania sp. infection in dogs from Vila Operária, Buerarema, Bahia, using parasitological tests, indirect immunofluorescent assay (IFA) and polymerase chain reaction (PCR). Furthermore, this study also aimed to identify risk factors associated with illness in dogs in this locality by conducting an epidemiological survey. For this purpose, 292 dogs were clinically evaluated for the presence of skin lesions, and the dogs that showed these changes were submitted to scarification injury to enable preparation of slides for microscopic study of amastigotes. Subsequently, the dogs underwent blood sampling for serological (IFA) and molecular (PCR) tests. Additionally, the owners of the dogs answered an epidemiological questionnaire to facilitate the identification of risk factors for exposure of dogs to pathogens of ACL. Of the 292 dogs studied, 13 (4.5%) had lesions suggestive of ACL, but with a negative parasitological examination and 147 (50.3%) were seropositive according to the IFA. Of the 273 dogs studied using PCR test, 10 (3.66%) were positive for L. braziliensis, and all samples were negative for L. infantum chagasi. Wastelands in the peridomicile and the presence of light in the household were risk factors associated with ACL. The results show that Vila Operária has asymptomatic dogs with ACL and that the detection sensitivity of the IFA was higher than that of PCR for the infected dogs.

First report of Trypanosoma cruzi infection in naturally infected dogs from southern Bahia, Brazil.

In order to verify the Trypanosoma cruzi infection in domestic domiciled dogs in a rural endemic area from the south region of the State of Bahia, Polymerase Chain Reaction (PCR) were performed using S35 and S36 primers in 272 dogs living in the district of Vila Operaria, in the municipality of Buerarema. All animals were clinically evaluated; 2.5 mL of blood were collected through venipuncture for the performance of molecular tests. None of these animals showed clinical signs of the illness and only two were identified with the DNA parasite. This result is the first report of natural infection by T. cruzi in domestic dogs in southern Bahia.

First report of Trypanosoma cruzi infection in naturally infected dogs from southern Bahia, Brazil / Primeiro relato de infecção natural por Trypanosoma cruzi em cães do sul da Bahia, Brasil

In order to verify the Trypanosoma cruzi infection in domestic domiciled dogs in a rural endemic area from the south region of the State of Bahia, Polymerase Chain Reaction (PCR) were performed using S35 and S36 primers in 272 dogs living in the district of Vila Operaria, in the municipality of Buerarema. All animals were clinically evaluated; 2.5 mL of blood were collected through venipuncture for the performance of molecular tests. None of these animals showed clinical signs of the illness and only two were identified with the DNA parasite. This result is the first report of natural infection by T. cruzi in domestic dogs in southern Bahia.

Com o objetivo de verificar a infecção por Trypanosoma cruzi em cães domésticos domiciliados em área rural e endêmica do sul da Bahia, foi realizada a Reação em Cadeia da Polimerase (PCR), utilizando-se os iniciadores S35 e S36 em 272 cães domiciliados no distrito da Vila Operária, cidade de Buerarema. Todos os animais foram avaliados clinicamente e, posteriormente, foram coletados 2,5 mL de sangue por punção venosa para realização do diagnóstico molecular. Nenhum dos animais apresentou manifestação clínica da doença e, em apenas dois foram identificados DNA do parasito. Esse resultado é o primeiro relato de infecção natural por T. cruzi em cães domésticos no sul baiano.

Necrotic enteritis in collared (Pecari tajacu) and white-lipped (Tayassu pecari) peccaries.

An outbreak of necrotic enteritis caused by Clostridium perfringens type C was diagnosed in captive collared (Pecari tajacu) and white-lipped (Tayassu pecari) peccaries housed in the Laboratory of Applied Ethology of Universidade Estadual de Santa Cruz located in Ilhéus, State of Bahia, Brazil. Four collared peccaries and three white-lipped peccaries, all juveniles (25-105 days old), were affected. For all affected animals, lethargy and inappetance were followed by sudden death within 24 hours. Histopathology of intestinal wall, culture of C. perfringens type C, and the identification of beta-toxin from intestinal content confirmed the diagnosis.

Frequency of species of the genus Eimeria in naturally infected cattle in Southern Bahia, Northeast Brazil.

The aim of this study was to determine the presence of species of the genus Eimeria species in naturally infected bovines in Southern Bahia, Northeast Brazil. The study population comprised 117 Zebu crossbred cattle that belonged to 10 dairy herds with extensive or semi-extensive production systems. The modified Gordon and Whitlock technique was used to determine positive samples and number of oocysts per gram of feces. Statistical analyses were performed using the chi-square test with Yates correction and a 95% confidence interval. Thirty-nine cattle (33.33%) were positive, and ten different species were identified in infected animals: E. bovis (24.79%); E. canadensis (8.55%); E. zuernii (6.83%); E. ellipsoidalis (5.99%); E. cylindrica (3.42%); E. auburnensis (3.42%); E. brasiliensis (2.56%); E. bukidnonensis (1.71%); E. alabamensis (0.85%), and E. subspherica (0.85%). Higher parasitism was observed in animals up to one year of age (p = 0.005), but no animal presented clinical signs of the disease. As the presence of clinical eimeriosis was not evidenced and all animals were Zebu crossbred cattle from extensive or semi-extensive production systems, further studies should be conducted to investigate the effects of these factors on disease development.

Frequency of species of the Genus Eimeria in naturally infected cattle in Southern Bahia, Northeast Brazil / Frequência de espécies do gênero Eimeria em bovinos naturalmente infectados no Sudeste da Bahia, Nordeste do Brasil

The aim of this study was to determine the presence of species of the genus Eimeria species in naturally infected bovines in Southern Bahia, Northeast Brazil. The study population comprised 117 Zebu crossbred cattle that belonged to 10 dairy herds with extensive or semi-extensive production systems. The modified Gordon and Whitlock technique was used to determine positive samples and number of oocysts per gram of feces. Statistical analyses were performed using the chi-square test with Yates correction and a 95 percent confidence interval. Thirty-nine cattle (33.33 percent) were positive, and ten different species were identified in infected animals: E. bovis (24.79 percent); E. canadensis (8.55 percent); E. zuernii (6.83 percent); E. ellipsoidalis (5.99 percent); E. cylindrica (3.42 percent); E. auburnensis (3.42 percent); E. brasiliensis (2.56 percent); E. bukidnonensis (1.71 percent); E. alabamensis (0.85 percent), and E. subspherica (0.85 percent). Higher parasitism was observed in animals up to one year of age (p = 0.005), but no animal presented clinical signs of the disease. As the presence of clinical eimeriosis was not evidenced and all animals were Zebu crossbred cattle from extensive or semi-extensive production systems, further studies should be conducted to investigate the effects of these factors on disease development.

O objetivo deste estudo foi determinar a presença de espécies do gênero Eimeria em bovinos naturalmente infectados, na região Sudeste da Bahia, Nordeste do Brasil. A população do estudo incluiu 117 bovinos mestiços de raças Zebuínas que pertenciam a 10 fazendas leiteiras com sistemas de produção extensivo ou semiextensivo. A técnica de Gordon e Whitlock modificada foi utilizada para determinar as amostras positivas e o número de oocistos por grama de fezes. A análise estatística foi realizada utilizando o teste do qui-quadrado com correção de Yates e intervalo de confiança de 95 por cento. Trinta e nove animais (33,33 por cento) foram positivos, e dez diferentes espécies foram identificadas nos animais infectados: E. bovis (24,79 por cento), E. canadensis (8,55 por cento), E. zuernii (6,83 por cento), E. ellipsoidalis (5,99 por cento ), E. cylindrica (3,42 por cento), E. auburnensis (3,42 por cento), E. brasiliensis (2,56 por cento), E. bukidnonensis (1,71 por cento), E. alabamensis (0,85 por cento) e E. subspherica (0,85 por cento). Maior parasitismo foi observado em animais com até um ano de idade (p = 0,005), mas nenhum animal apresentou sinais clínicos que fossem compatíveis com a parasitose. Como não foi observado presença de eimeriose clínica e como todos os animais eram mestiços zebuínos e pertencentes ao sistema de criação extensivo ou semiextensivo, novos estudos devem ser conduzidos para comprovar a influência desses fatores no surgimento da doença.