An evaluation of techniques to diagnose Dioctophyme renale in dogs.

Dioctophyme renale is a nematode with zoonotic potential that affects the kidneys of carnivorous, wild, and domestic mammals. In this study, we sought to evaluate the indirect ELISA method against routine methods used to diagnose dioctophimosis. Hence, 38 dogs parasitized by D. renale, as confirmed by surgery, were selected. The dogs were evaluated by abdominal ultrasound and urinalysis, and their sera were tested by indirect ELISA using D. renale adult secretion and excretion antigen (DES). Five dogs were followed up with serum collections on day 0 (day of surgery) and 30, 60, and 90 days after surgery to evaluate antibody kinetics. Abdominal ultrasound and indirect ELISA successfully diagnosed 37 dogs parasitized by D. renale, while urinalysis diagnosed 29 animals. The positive animals were parasitized with 1-7 parasites; 17 dogs were infected by male and female parasites, 15 only by female parasites, and six were parasitized only by male parasites. When assessing specificity and sensitivity, all techniques showed 100% specificity and 81.6%, 97.4%, and 97.4% sensitivity for urinalysis, ultrasound, and ELISA, respectively (p < 0.001). The five positive dogs that were followed up after surgery showed a progressive decrease in mean absorbances in indirect ELISA (0.644, 0.516, 0.511, and 0.440, respectively). This study demonstrated that the indirect ELISA using the DE antigen could diagnose dioctophimosis regardless of the number, sex, and location of the parasites, with the potential to be used in epidemiological research and implementing immunological and molecular studies, opening new lines of research on D. renale.

Dioctophyme renale é um nematódeo que possui potencial zoonótico e acomete o rim de mamíferos carnívoros, silvestres e domésticos. Neste estudo busca-se avaliar a técnica de ELISA indireto frente metodologias de rotina utilizadas no diagnóstico da dioctofimose. Para isto, 38 cães participaram do estudo, sendo todos parasitados por D. renale, confirmados por cirurgia. Esses cães foram avaliados por ultrassom abdominal, urinálise e seus soros testado por ELISA indireto utilizando antígeno de excreção e secreção (DES) de adultos de D. renale. Destes, cinco cães foram acompanhados com coletas de soro, no dia zero (dia da cirurgia) e após 30, 60 e 90 dias da cirurgia para avaliar a cinética dos anticorpos. O ultrassom abdominal e ELISA indireto apresentaram sucesso no diagnóstico de 37 cães parasitados por D. renale, enquanto que a urinálise diagnosticou 29 animais. Os animais positivos possuíam de um a sete parasitos, 17 cães apresentaram infecções por macho e fêmeas, 15 somente por fêmeas e seis foram parasitados apenas por machos. Na avaliação da especificidade e sensibilidade, todas as técnicas apresentaram 100% de especificidade e 81,6%, 97,4%, 97,4% de sensibilidade para urinálise, ultrassom e ELISA, respectivamente (p < 0,001). Os cinco cães positivos que foram acompanhados após a cirurgia apresentaram diminuição progressiva nas médias de absorbâncias no ELISA indireto (0,644, 0,516, 0,511 e 0,440, respectivamente). O estudo demonstrou que o ELISA indireto utilizando o antígeno DES poderia diagnosticar dioctofimose, independentemente do número, sexo e localização dos parasitos, com potencial para ser utilizada em estudos epidemiológicos e na implementação de estudos imunológicos e moleculares, abrindo novas linhas de pesquisa sobre Dioctophyme renale.

Antigenicity assessment of the Theileria equi merozoite antigen (EMA-2) expressed in Pichia pastoris in mice and horses.

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.