Padronização de ELISA para detecção de anticorpos anti-Toxoplasma gondii e Neospora caninum em soro e fezes de cervídeos
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Data
2021-12-01
Autores
Baldini, Maria Helena Mazzoni [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A maior parte dos dados descritos na literatura sobre patógenos de cervídeos neotropicais são provenientes de levantamentos sorológicos, informações sobre vías de transmissão e avaliações clínicas ainda são escasos. O presente trabalho teve como objetivos: a) estimar a prevalência de cervídeos positivos para Toxoplasma gondii e Neospora canium em 92 animais mantidos em cativeiro; b) Avaliar a probabilidade de diferentes vias de transmissão do T. gondii e N. caninum; c) avaliar a sintomatologia clínica e resposta imune humoral de machos de veados-catingueiros (Mazama gouazoubira) infectados experimentalmente com oocistos da cepa ME49 (genotipo tipo II) de T. gondii e d) padronizar a técnica de ELISA indireta para detecção de anticorpos fecais específicos para T. gondii e N. caninum. Inicialmente, foram colhidas amostras dos animais cervídeos pertencentes a dois criadouro conservacionistas e comparados os resultados sorológicos obtidos nos testes de imunofluorescência indireta (RIFI) e ELISA indireto pelo teste de concordância Kappa de Cohen. Em seguida, foram desenhadas árvores genealógicas dos animais de cada espécie para estimativa da taxa de transmissão congênita de cada doença. A prevalência estimada para T. gondii foi de 20,73% pela tecnica de RIFI e 25,60% por ELISA, apresenando uma concordância rasoável (ϰ = 0,277) entre as duas técnicas sorológicas utilizadas. Já para N. caninum a prevalência estimada foi de 40,24% por RIFI e 39,02% por ELISA com uma correlação quase perfeita (ϰ = 0,83) entre as técnicas. A taxa de transmissão congênita, estimada com base em números de filhos positivos de mães positivas entre o número total de filhos de mães positivas, diferiu bastante entre as doenças, sendo de 0% para toxoplasmose e 81,25% para neosporose. No capítulo 3, para a infecção experimental dos animais, quatro machos da espécie Mazama gouazoubira foram infectados experimentalmente com 5000 oocistos esporulados de T. gondii da cepa ME49, enquanto um animal soronegativo foi usado como controle. Os animais foram monitorados quanto aos parâmetros clínicos de -7 a 40 dias após a infecção (dpi). Amostras de sangue foram coletadas semanalmente até 49 dpi para detecção de anticorpos pelo teste de ELISA. A cada 15 dias os animais foram submetidos à contenção química e coleta de sêmen por eletroejaculação e o material genético das amostras de semen foi extraído para posterior análise por PCR para T. gondii. Apenas dois dos quatro animais infectados desenvolveram resposta de anticorpos detectável no teste de ELISA, e mesmo sem nenhum animal ter manifestado sinais clínicos da infecção, o material genético do parasita foi encontrado no sêmen dos animais soropositivos aos 35 e 49 dpi. A terceira etapa do projeto visou a padronização da técnica de ELISA para detecção de imunoglobulinas fecais contra os dois patógenos. Amostras de fezes frescas foram coletadas, extraídas e armazenadas em nitrogênio líquido ou processadas imediatamente. Um pool de amostras de animais soropositivos foi utilizado como controle positivo para N. caninum e um pool de amostras de animais experimentalmente infectados foi usado como controle para T. gondii. Mesmo com diversas alterações de protocolos não foi possível contornar o problema da baixa especificidade do teste, constatada pelos altos ruídos nas leituras das placas. Foi possível concluir que as técnicas sorológicas provaram ser muito úteis para estudos epidemiológicos em animais de cativeiro, tendo diversas aplicações além da simples estimativa da prevalência, porém sem a padronização de um método não invasivo de detecção de anticorpos, estudos epidemiológicos em populações de vida-livre ainda são um grande desafio. Nesses estudos foi possível inferir que a principal via de transmissão do N. caninum em cervídeos é a via congênita, de forma semelhante ao que se observa em bovinos , enquanto que o T. gondii parece utilizar da via horizontal para a transmissão na maioria dos casos. Além disso , a infecção experimental com 5000 oocistos esporulados de T. gondii não causou sinais clínicos nos animais estudados, sugerindo que a espécie apresenta certa resistência à doença, por outro lado, a detecção do DNA do parasito no sêmen dos animais sugere que a transmissão sexual é uma possibilidade nessas espécies, o que deve ser levado em consideração por criadouros conservacionistas.
Most of the data on neotropical deer pathogens come from serological surveys, little is known about transmission routes and clinical evaluations.The present study aimed to: a) estimate the prevalence of positive cervids for Toxoplasma gondii and Neospora canium in animals kept in captivity; b) Assess different routes of infection of T. gondii and N.caninum on these deer populations; c) Evaluate the clinical symptomatology and humoral immune response of male brocket deer (Mazama gouazoubira) experimentally infected with T. gondii (ME49 strain) oocysts; and d) Standardize an indirect ELISA technique for non-invasive detection of specific fecal antibodies to T. gondii and N. caninum. Initially, deer serum samples collected from animals from two conservation breeding centers were tested by both techniques making it possible to compare the results obtained in the indirect immunofluorescence tests (IFAT) and indirect ELISA by the Cohen's Kappa concordance test. Furthermore, family trees were drawn for each species to estimate the congenital transmission rate of both diseases. The T. gondii prevalence were 20,73% by IFAT and 25,60 by ELISA, with , with an fair agreement of ϰ = 0.277 between the two serological techniques. For N. caninum, the antibody prevalence was 40,24% by IFAT and 39,02% by ELISA, with a almost perfect correlation of ϰ = 0.83 between techniques. The congenital transmission rate, estimated by the number of positive offspring from positive mothers by the total of positive mother offspring, differed significantly between diseases, resulting in 0% for Toxoplasmosis and 81,25% for Neosporosis. In Chapter 3, four Mazama gouazoubira males were inoculated with 5000 sporulated T. gondii oocysts (ME49), while one seronegative animal was used as a control. Animals were monitored for clinical parameters from -7 to 40 days post-infection (dpi). Blood samples were collected weekly up to 49 dpi for antibody detection by the previously standardized ELISA test. Once every 15 days, the animals were chemical restraint for semen collection by electroejaculation. DNA of the semen samples was extracted for T. gondii PCR analysis. Only two from four infected animals developed an antibody response detectable in the ELISA test, and despite the animals didn’t manifest any clinical signs, the parasite's genetic material was found in the semen of the seropositive animals at 35 and 49 dpi. In the third step, the standardization of ELISA technique for detection of fecal immunoglobulins against the two pathogens was carried out, aiming to apply the methodology to epidemiological studies in free-living animals. Fresh stool samples were collected, extracted and stored in liquid nitrogen or processed immediately. A pool of samples from seropositive animals was used as a positive control for N. caninum and a pool of samples from experimentally infected animals was used as a control for T. gondii. Even with several protocol changes, it was not possible to overcome the problem of the low specificity evidenced by the high background at plate readings. Finally, it was possible to conclude that serological techniques have proved very useful for epidemiological studies in captive animals, having several applications beyond the simple estimation of prevalence, but without the standardization of a noninvasive method of detection of antibodies, epidemiological studies in free-living populations are still challenging. Whith these studies it was possible to infer tha the congenital transmission is the major route of infection os N. caninum in neotropical deer, in the other hand, horizontal transmission seem to be more important to T. gondii epidemiology in these species. Furthermore, the experimental infection with 5000 T. gondii oocysts did not caused clinical signs in Mazama gouazoubira, implying that the specie present some resistance to the disease, still, the detection of the parasite in semen samples indicate the possibility of sexual transmission of T. gondii in cervids, vi and breeding centers should take this into account for better reproductive performance of the animals.
Most of the data on neotropical deer pathogens come from serological surveys, little is known about transmission routes and clinical evaluations.The present study aimed to: a) estimate the prevalence of positive cervids for Toxoplasma gondii and Neospora canium in animals kept in captivity; b) Assess different routes of infection of T. gondii and N.caninum on these deer populations; c) Evaluate the clinical symptomatology and humoral immune response of male brocket deer (Mazama gouazoubira) experimentally infected with T. gondii (ME49 strain) oocysts; and d) Standardize an indirect ELISA technique for non-invasive detection of specific fecal antibodies to T. gondii and N. caninum. Initially, deer serum samples collected from animals from two conservation breeding centers were tested by both techniques making it possible to compare the results obtained in the indirect immunofluorescence tests (IFAT) and indirect ELISA by the Cohen's Kappa concordance test. Furthermore, family trees were drawn for each species to estimate the congenital transmission rate of both diseases. The T. gondii prevalence were 20,73% by IFAT and 25,60 by ELISA, with , with an fair agreement of ϰ = 0.277 between the two serological techniques. For N. caninum, the antibody prevalence was 40,24% by IFAT and 39,02% by ELISA, with a almost perfect correlation of ϰ = 0.83 between techniques. The congenital transmission rate, estimated by the number of positive offspring from positive mothers by the total of positive mother offspring, differed significantly between diseases, resulting in 0% for Toxoplasmosis and 81,25% for Neosporosis. In Chapter 3, four Mazama gouazoubira males were inoculated with 5000 sporulated T. gondii oocysts (ME49), while one seronegative animal was used as a control. Animals were monitored for clinical parameters from -7 to 40 days post-infection (dpi). Blood samples were collected weekly up to 49 dpi for antibody detection by the previously standardized ELISA test. Once every 15 days, the animals were chemical restraint for semen collection by electroejaculation. DNA of the semen samples was extracted for T. gondii PCR analysis. Only two from four infected animals developed an antibody response detectable in the ELISA test, and despite the animals didn’t manifest any clinical signs, the parasite's genetic material was found in the semen of the seropositive animals at 35 and 49 dpi. In the third step, the standardization of ELISA technique for detection of fecal immunoglobulins against the two pathogens was carried out, aiming to apply the methodology to epidemiological studies in free-living animals. Fresh stool samples were collected, extracted and stored in liquid nitrogen or processed immediately. A pool of samples from seropositive animals was used as a positive control for N. caninum and a pool of samples from experimentally infected animals was used as a control for T. gondii. Even with several protocol changes, it was not possible to overcome the problem of the low specificity evidenced by the high background at plate readings. Finally, it was possible to conclude that serological techniques have proved very useful for epidemiological studies in captive animals, having several applications beyond the simple estimation of prevalence, but without the standardization of a noninvasive method of detection of antibodies, epidemiological studies in free-living populations are still challenging. Whith these studies it was possible to infer tha the congenital transmission is the major route of infection os N. caninum in neotropical deer, in the other hand, horizontal transmission seem to be more important to T. gondii epidemiology in these species. Furthermore, the experimental infection with 5000 T. gondii oocysts did not caused clinical signs in Mazama gouazoubira, implying that the specie present some resistance to the disease, still, the detection of the parasite in semen samples indicate the possibility of sexual transmission of T. gondii in cervids, vi and breeding centers should take this into account for better reproductive performance of the animals.
Descrição
Palavras-chave
Patologia Veterinária, Toxoplasmose, Neospora, Transmissão, Animais silvestres