Fleas (Siphonaptera, Latreille, 1825) from Rio Grande do Sul State, Brazil: Species Diversity, Hosts, and One Health Approach.

Background: Fleas are ectoparasitic insects with holometabolous development. It has a hematophagous habit with mouthparts adapted to sting and suck its hosts. There are about 3000 species in the world, ∼61 in Brazil, and 19 in Rio Grande do Sul state. The objective of the research is to catalog the diversity of fleas recorded in the state, their respective hosts, and endosymbionts. Materials and Methods: To this end, a search was carried out in the scientific literature, from articles, books, to abstracts submitted to congresses. Results: The 19 species of fleas occurring in Rio Grande do Sul are divided into 7 families and 10 genera. These ectoparasites, in addition to being found in the environment, were associated with 10 different families of hosts in Rio Grande do Sul, and on the endosymbiont, agents found associated with fleas, there were 7 different species. The main agents researched in the state are Rickettsia spp. and Bartonella spp. The relationships between parasites, hosts, environment, and etiological agents present different scenarios, whether anthropized or conserved, but unknown. Sometimes, this overlap, a factor that aggravates the possibility of spillovers, either from cosmopolitan fleas in these conserved areas, or from their endosymbionts. Conclusion: Thus, it is important to characterize the environment so that the complexities of each location are known for the adoption of environmental and public health policies in each case. The challenges are extensive, but necessary in view of the One Health perspective.

A cross-sectional study of Leishmania spp. in draft horses from the Distrito Federal, Brazil: Seroprevalence, spatial distribution, and associated factors.

Equine leishmaniasis is a neglected tropical disease caused by the protozoan of the Leishmania genus, and it has been reported in several countries around the world, especially Brazil. Therefore, the present investigation aims to conduct a cross-sectional study to determine the prevalence, spatial distribution, and associated factors with seropositivity for Leishmania spp. in draft horses from the Distrito Federal, Brazil. The serological survey was conducted on 411 animals, employing the Indirect Immunofluorescence Test (IFA) and Enzyme-Linked Immunosorbent Assay (ELISA). The Kappa (κ) and gross agreement indexes evaluated the Leishmania spp. seropositivity by IFA and ELISA test. The statistical analysis was performed using the Chi-square test and logistic regression. The spatial analysis showed the areas with the highest number of seropositive and the Moran autocorrelation analyses between the spatial distribution and the epidemiological model's explanatory variables. A 27.01 % co-positivity was observed with a κ index of 52.64 %. The final model considered the variables: access to water bodies (p-value = 0.008, Odds Ratio (OR) = 2.26, Confidence Interval (CI) = 1.24-4.13), the absence of the use of ectoparasiticide (p-value = 0.008, OR = 1.93 CI = 1.18-3.15) and traveling animal (p-value = 0.059, OR = 1.54, CI = 0.98-2.41). The Kernel map showed hot areas with a high concentration of nine positive animals per area and some lighter areas ranging from five to seven positive animals per area where control measures should be performed. The Moran autocorrelation analysis was significant for the variables: traveling animal (Moran's I = 0.540 and pseudo-p-value = 0.001) and the absence of use ectoparasiticide (Moran's I = 0.259 and pseudo-p-value = 0.005). The current study exposes a high seroprevalence of Leishmania spp. in horses in the Distrito Federal, Brazil. Moreover, it proposes that traveling animal, the access to water bodies and the absence of the use of ectoparasiticide are significantly associated with seropositivity for Leishmania spp. in draft horses, which may contribute to the implementation of prophylactic and controls measures where leishmaniasis is already stalled.

Reactivity of sera from dogs living in a leishmaniasis-endemic area to the COOH-terminal region of cysteine proteinase B.

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n=114), including suspect (n=30) and positive (n=50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n=34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density=0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

Gastrointestinal Parasites in Captive Animals at the Rio de Janeiro Zoo.

BACKGROUND: Gastrointestinal parasites may determine diarrhea, dysentery or even death in captive mammals. These animals tend to be more susceptible to parasitic infections due to confinement and stress. Purpose To increase the information about these etiological agents in captive animals in Brazil, the gastrointestinal parasites of the captive mammals of the Rio de Janeiro Zoo were investigated. METHODS: From 2016 to 2018, 180 fecal samples were collected from animals housed in the Rio de Janeiro Zoo: 63 from animals of the order Primates, 26 of Carnivora, 78 of Artiodactyla, 9 of Perissodactyla and 4 of the order Rheiformes. The feces were processed by direct examination and by the techniques of Faust et al., Sheather, Ritchie, Lutz, and smears were stained with safranin. Immunoenzymatic assays were also performed to investigate antigens of Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica/Entamoeba dispar. RESULTS: Parasite positivity was identified in 68.3% of the fecal samples, with a parasite positivity rate of 68.2% among primates, 65.3% among carnivores, 69.2% among artiodactyls, 33.3% among perissodactyls, and 100% among rheiformes. The most frequently detected parasite was Entamoeba histolytica/E. dispar antigens, which showed a statistically significant positivity rate (33.3%; p = 0.000), particularly in the feces of carnivores (30.7%) and artiodactyls (53.8%). A statistically significant positivity rate of Balantioides coli (11.1%; p = 0.001) was also detected in feces from nonhuman primates, tapirs, collared peccaries and rheas. The positivity of Cryptosporidium sp. antigens in feces of the orders Carnivora, Artiodactyla and Primates was also statistically significant (7.2%, p = 0.010). Oocysts compatible with Cryptosporidium spp. were detected in 6.3% from primates. The helminths most frequently detected were thin-shelled eggs of nematodes (17.7%, p = 0.000), nematode larvae (15.5%, p = 0.000) and Trichuris trichiura eggs (6.1%, p = 0.018). CONCLUSION: The positivity rate for gastrointestinal parasites demonstrates the need for a sanitation management program to be implemented in the zoo, including routine diagnostic parasitology tests followed by specific treatment for each parasitosis.

Prevalência de Campylobacter spp. termofílicos em primatas não humanos da espécie Macaca mulatta provenientes de centro criatório para uso como biomodelos / Prevalence of thermophilic Campylobacter spp. in non-human primates of the Macaca mulatta species from a breeding center for use as biomodels

Objetivo: A campilobacteriose é uma das principais doenças entéricas do mundo. Ocorre não só no homem mas também em primatas não humanos, sendo desta forma importante no monitoramento sanitário de colônias de animais provenientes de criatórios científicos. É causada por bactérias do gênero Campylobacter, cuja detecção em biotérios possibilita não só controlar a doença e prevenir sua disseminação, mas assegurar a qualidade das pesquisas que serão realizadas nestes biomodelos. Com base na importância deste isolamento, nosso objetivo foi a verificação do status sanitário de uma colônia de criação de Macaca mulatta, usando como referência a presença de Campylobacter spp. Métodos: Durante o manejo médico anual coletaram-se 52 swabs fecais de primatas não humanos adultos, o que representou um percentual de 10% da colônia total. Esse material foi submetido ao esquema de semeadura/incubação e identificação de Campylobacter sp. seguindo as recomendações de cultivo microbiológico, incluindo o isolamento, prova de Gram e testes bioquímicos. Todo o processo levou de cinco a sete dias e foi realizado em atmosfera de microaerofilia. Resultados: Em 14 indivíduos foram isoladas bactérias do gênero Campylobacter. Destes, sete eram portadores de Campylobacter coli, seis portadores de Campylobacter jejuni e em um indivíduo não foi possível definir a espécie de Campylobacter isolada. Conclusão: Apesar da baixa prevalência (27%), esses resultados reforçam a necessidade de constante monitoramento microbiológico dos primatas pertencentes à colônia, visando não só a qualidade dos animais fornecidos, mas minimizando o risco de contaminação dentro da colônia e de contágio pelos profissionais que lidam com os animais, já que o Campylobacter possui importante potencial zoonótico.

Field trial of efficacy of the Leish-tec® vaccine against canine leishmaniasis caused by Leishmania infantum in an endemic area with high transmission rates.

BACKGROUND: Because domestic dogs are reservoir hosts for visceral leishmaniasis (VL) in Brazil, one of the approaches used to reduce human disease incidence is to cull infected dogs. However, the results of controlled intervention trials based on serological screening of dogs and killing of seropositive animals are equivocal. A prophylactic vaccine to protect dogs from being infectious to the sand fly vector could be an effective strategy to provide sustained control. Here, we investigated whether a currently licensed commercial subunit rA2 protein-saponin vaccine (Leish-tec®) had an additional effect to dog culling on reducing the canine infectious populations. METHODOLOGY/PRINCIPAL FINDINGS: This prospective study was conducted in an L. infantum highly endemic area of southeast Brazil. At the onset of the intervention, all of the eligible dogs received through subcutaneous route a three-dose vaccine course at 21-day intervals and a booster on month 12. For the purpose of comparison, newly recruited healthy dogs were included as the exposed control group. To ascertain vaccine-induced protection, dogs were screened on clinical and serological criteria every 6 months for a 2-year follow-up period. Antibody-based tests and histopathological examination of post-mortem tissue specimens from euthanized animals were used as a marker of infection. The standardized vaccine regime, apart from being safe, was immunogenic as immunized animals responded with a pronounced production of anti-A2-specific IgG antibodies. It should be noted the mean seroconversion time for infection obtained among immunized exposed dogs (~ 18 months), which was twice as high as that for unvaccinated ones (~ 9 months). After two transmission cycles completed, the cumulative incidence of infection did differ significantly (P = 0.016) between the vaccinated (27%) and unvaccinated (42%) dogs. However, the expected efficacy for the vaccine in inducing clinical protection was not evident since 43% of vaccine recipients developed disease over time. Our estimates also indicated that immunoprophylaxis by Leish-tec® vaccine in addition to dog culling might not have an impact on bringing down the incidence of canine infection with L. infantum in areas of high transmission rates. CONCLUSIONS/SIGNIFICANCE: Leish-tec® as a prophylactic vaccine showed promise but needs to be further optimized to be effective in dogs under field conditions, and thereby positively impacts human incidence.

CANINE VISCERAL LEISHMANIASIS CASE INVESTIGATION IN THE JACARE REGION OF NITEROI, RIO DE JANEIRO, BRAZIL.

American visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpis was not detected. Our results are indicative of an ongoing transmission in the area.

Canine viscerall leishmaniasis case investigation in the Jacare region of Niteroi, Rio de Janeiro, Brazil / Investigação de caso de leishmaniose visceral canina na região do Jacaré, município de Niterói, Rio de Janeiro, Brasil

SUMMARYAmerican visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpiswas not detected. Our results are indicative of an ongoing transmission in the area.

RESUMOA leishmaniose visceral americana é zoonose em expansão no Brasil com transmissão vetorial, onde o cão é o principal reservatório urbano. Partindo de um caso canino da doença no Jacaré, Niterói/RJ, realizouse inquérito epidemiológico canino e levantamento entomológico para avaliar a extensão da enfermidade na localidade. As amostras foram coletadas ao redor do caso e os cães identificados por testes sorológicos (teste rápido de imunocromatografia em dupla plataforma, ensaio imunoenzimático/ELISA, reação de imunofluorescência indireta/RIFI). O diagnóstico parasitológico foi realizado nos animais reagentes em ao menos um desses exames. O levantamento entomológico foi feito com armadilhas luminosas e coleta manual. As associações entre variáveis caninas coletadas e o desfecho (ELISA e RIFI reagentes) foram avaliadas pelo teste qui-quadrado e ajustadas pela regressão logística multivariada para variáveis com p< 0,1 na análise bivariada. Foram detectados 17 casos de LVA entre os 110 cães avaliados (prevalência de 15,5%). Presença de ectoparasitas (OR 6,5; 95% IC 1,1-37,4), animais com sinais clínicos (OR 9,5; 95% IC 1,2-76,6) e casos prévios de LVA canina na residência (OR 17,9; 95% IC 2,2-147,1) foram associados ao desfecho. Não foi detectada Lutzomyia longipalpis. Nossos resultados indicam situação de transmissão contínua na área.

Serological survey of cases of canine visceral leishmaniasis and evaluation of phlebotomine fauna on Marambaia Island, municipality of Mangaratiba, Rio de Janeiro state, Brazil* / Levantamento sorológico de casos de leishmaniose visceral canina e avaliação da fauna de flebotomíneos na Ilha da Marambaia, município de Mangaratiba, estado do Rio de Janeiro, Brasil

The state of Rio de Janeiro is epidemiologically classified as a region of low incidence for American visceral leishmaniasis (AVL); however, endemic areas have expanded dramatically in recent years. In 2009 and 2010, autochthonous cases of AVL were reported in the municipality of Maricá and in the neighborhood of Laranjeiras in the city of Rio de Janeiro, respectively. In 2006, samples isolated in infected animals in the coastal area of the municipality of Mangaratiba were characterized as L. (L.) chagasi. In 2002, on Marambaia Island, located in Mangaratiba, only cases of American cutaneous leishmaniasis (ACL) were recorded. This study aimed to reassess the prevalence of CVL and identify the phlebotomine fauna on Marambaia Island, Mangaratiba, in 2012. To this end, a canine serological survey was carried out using IFA, ELISA and DPP techniques. In addition, phlebotomi were captured with the help of light traps, HP type. The census totaled 116 dogs, and 17 animals tested positive with prevalence of 14.6%. From April to November 2012, 2,524 sandfly specimens of nine different species were captured. We observed the maintenance of CVL prevalence in the region, as well as the presence of the Lutzomyia longipalpis vector.

O estado do Rio de Janeiro é classificado epidemiologicamente como uma região de baixa incidência de leishmaniose visceral americana (LVA), no entanto, áreas endêmicas têm se expandido drasticamente nos últimos anos. Em 2009 e 2010, foram notificados casos autóctones de LVA no município de Maricá e no bairro de Laranjeiras, na cidade de Rio de Janeiro, respectivamente. Em 2006, em amostras de animais infectados no litoral do município de Mangaratiba foram isoladas e caracterizadas L. (L.) chagasi. Em 2002, na Ilha da Marambaia, localizada em Mangaratiba, foram registrados apenas os casos de leishmaniose tegumentar americana (LTA). Este estudo teve como objetivo avaliar a prevalência da LVC e identificar a fauna de flebotomíneos na Ilha da Marambaia, Mangaratiba, em 2012. Para este fim, um inquérito sorológico canino foi realizado utilizando as técnicas IFA, ELISA e DPP. Além disso, foram capturados flebótomos com a ajuda de armadilhas luminosas, tipo HP. O censo totalizou 116 cães e 17 animais foram positivos com a prevalência de 14,6%. Entre abril e novembro de 2012, foram capturados, nove espécies diferentes flebotomíneos, um total de 2.524 espécimes. Observou-se a manutenção da LVC prevalência na região, bem como a presença do vetor Lutzomyia longipalpis.

Production and Characterization of IgY against Canine IgG: Prospect of a New Tool for the Immunodiagnostic of Canine Diseases

This study describes the production of a new avian polyclonal antibody (IgY) against canine IgG, as another tool for the immunodiagnostic using IgY technology. The immunization protocol caused neither deaths nor pathologies, and no decline in egg laying capacity was detected. The total concentration of isolated IgY was constant, without significant difference (p> 0.05), with average of 97.55 mg of IgY/yolk. The IgY revealed a strong sensitivity and specificity in recognition against canine IgG by ELISA. After the immunization, there was a significant increase in the production of IgY specific from the first to the second month (p <0.05), reaching a stable peak without decrease in the production by the end of the analysis period (p > 0.05). The IgY demonstrated a suitable specificity in Western blot against the purified and serum canine IgG, not enabling recognition of canine IgM or IgG of other animal species. The specific IgY in the egg yolks of immunized hens proved to be a molecule with an appropriate purity and desired specificity against the immunizing antigen. Moreover, its constant production in large quantities during the four months analyzed indicated that IgY antibody production technology could be considered as an excellent alternative to the standard methods.

Seroepidemiological aspects of Leishmania spp. in dogs in the Itaguai micro-region, Rio de Janeiro, Brazil.

This study evaluated factors associated with the frequency of Leishmania spp. antibodies in dogs residing in the Itaguai micro-region, State of Rio de Janeiro, Brazil. Blood samples were collected from 524 dogs. The serum samples were submitted to indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for Leishmania spp. The frequency of seropositive dogs was 28.24% (n = 148) in the micro-region, and among the three municipalities within that region, the highest frequency (p < 0.05) was observed in Seropedica (59.46%), followed by Itaguai (29.05%) and Mangaratiba (11.49%). Regarding factors associated with the host, mongrel dogs and those over the age of two presented higher frequency of antibodies to Leishmania spp. (p < 0.05). Concerning factors related to the environment and habits of the animal, dogs residing in rural areas (FR = 1.67, p = 0.0002), living outside the residence (FR = 1.42, p = 0.0197), with access to forest, streams and pastures (FR = 2.81, p = 0.0007), remaining loose (FR = 1.66, p = 0.0073), and those that had no shelter (FR = 2.16, p < 0.0001) were more likely to be seropositive. Canine leishmaniasis is a disease with high occurrence in the Itaguai micro-region, and aspects such as the definition of breed, age, habits and care by owners showed significant association in this micro-region.

Seroepidemiological aspects of Leishmania spp in dogs in the Itaguai micro-region, Rio de Janeiro, Brazil / Aspectos soroepidemiológicos de Leishmania spp em cães na microrregião de Itaguaí, Rio de Janeiro, Brasil

This study evaluated factors associated with the frequency ofLeishmania spp. antibodies in dogs residing in the Itaguai micro-region, State of Rio de Janeiro, Brazil. Blood samples were collected from 524 dogs. The serum samples were submitted to indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) forLeishmania spp. The frequency of seropositive dogs was 28.24% (n = 148) in the micro-region, and among the three municipalities within that region, the highest frequency (p < 0.05) was observed in Seropedica (59.46%), followed by Itaguai (29.05%) and Mangaratiba (11.49%). Regarding factors associated with the host, mongrel dogs and those over the age of two presented higher frequency of antibodies to Leishmaniaspp. (p < 0.05). Concerning factors related to the environment and habits of the animal, dogs residing in rural areas (FR = 1.67, p = 0.0002), living outside the residence (FR = 1.42, p = 0.0197), with access to forest, streams and pastures (FR = 2.81, p = 0.0007), remaining loose (FR = 1.66, p = 0.0073), and those that had no shelter (FR = 2.16, p < 0.0001) were more likely to be seropositive. Canine leishmaniasis is a disease with high occurrence in the Itaguai micro-region, and aspects such as the definition of breed, age, habits and care by owners showed significant association in this micro-region.

Este estudo avaliou os fatores associados à frequência de anticorpos específicos para Leishmania spp. em cães domiciliados na microrregião de Itaguaí, Rio de Janeiro. Foram colhidas amostras de sangue de 524 cães. As amostras de soro foram submetidas a reação de imunofluorescência indireta (RIFI) e ensaio imunoenzimático indireto (ELISA-teste) para Leishmania spp. A frequência de cães soropositivos foi de 28,24% (n = 148) na microrregião e, entre os três municípios avaliados, a maior frequência (p < 0,05) foi observada em Seropédica (59,46%), seguida de Itaguaí (29,05%) e Mangaratiba (11,49%). Em relação aos fatores associados ao hospedeiro, observou-se que cães sem raça definida e aqueles com idade acima de dois anos apresentaram maior frequência de anticorpos para Leishmania spp. (p < 0,05). Em relação aos fatores relacionados ao ambiente e ao hábito do animal, os cães residentes em áreas rurais (RF = 1,67, p = 0,0002), animais que vivem fora da residência (RF = 1,42, p = 0,0197), com acesso à mata, córregos e pastagens (FR = 2,81, p = 0,0007), que permanecem soltos (RF = 1,66, p = 0,0073), e aqueles que não possuem abrigo (RF = 2,16, p < 0,0001) apresentaram maior chance de serem soropositivos. A leishmaniose canina é uma enfermidade com elevada ocorrência na microrregião de Itaguai, e aspectos como definição racial, idade, hábitos e cuidados estabelecidos pelo proprietário mostraram associação significativa nessa microrregião.

Implementação da tecnologia de IgY para o diagnóstico da leishmaniose visceral canina / Implementation of IgY technology for the diagnosis of canine visceral leishmaniasis

Os métodos de diagnóstico sorológico são ainda as ferramentas mais realistas e aplicáveis para identificação de cães com leishmaniose visceral. A pesquisa por métodos sorológicos mais eficientes e de produção simplificada é importante para a identificação do reservatório canino, ação primordial para o controle da doença. Este estudo teve por objetivo o desenvolvimento e a avaliação do desempenho de um ELISA para leishmaniose visceral canina, utilizando como conjugado enzimático uma IgY anti-IgG canina. Para isto, galinhas poedeiras foram imunizadas com IgG canina purificada e a IgY anti-IgG foi isolada das gemas dos ovos por precipitação com polietilenoglicol e purificada por meio de adsorção tiofílica. A especificidade frente ao antígeno imunizante foi verificada por imunodifusão radial dupla, ELISA e western-blot. O ELISA foi desenvolvido utilizando IgY conjugada a enzima peroxidase, tendo seus parâmetros de acurácia avaliados e comparados com o ELISA utilizando IgG de mamíferos conjugada a peroxidase. A concentração total de IgY isolada nas gemas foi constante, sem diferença significativa nos meses analisados (...), com média de 97,55 mg de IgY/ gema. A IgY produzida reconheceu a IgG canina, demonstrando uma forte sensibilidade e especificidade no ELISA, porém na imunodifusão não foi detectada ligação da IgY produzida a IgG canina. Após a imunização, houve um aumento significante da produção de IgY específica do primeiro para o segundo mês (...) onde ocorreu um pico estável sem queda de produção até o final do período analisado (...). A IgY demonstrou ainda, forte reatividade no western-Blot frente a IgG canina purificada e a presente no soro de cão clinicamente saudável, não sendo capaz de reconhecer IgG de outras espécies animais. A conjugação da IgY a enzima peroxidase ocorreu sem danos à sua estrutura, com discriminação entre as amostras positivas e negativas em todas as diluições do conjugado e concentrações do antígeno testadas. O ELISA utilizando IgY conjugada a peroxidase apresentou 100 por cento de sensibilidade e 100 por cento de especificidade. A comparação dos parâmetros de acurácia do ELISA utilizando IgY com o ELISA utilizando IgG de mamíferos demonstraram valores idênticos. A repetibilidade do ensaio de ELISA IgY foi superior a encontrada com o ELISA IgG, com coeficiente de correlação intraclasse (ICC) estatisticamente significante (...). Um maior agrupamento das amostras negativas e positivas, também verificada quando a correlação das duplicatas nas duas observações foram analisadas (...). Diante de todos esses resultados a IgY ligada à peroxidase demonstrou ser um conjugado enzimático de excelente qualidade, revelando um melhor desempenho quando comparado ao conjugado produzido por mamíferos.

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine.

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.