Natural infection by Cryptosporidium SPP. in production animals: first description of subtype IIaa15G2R1 in goat kids and piglets in Brazil

Cryptosporidiosis is a severe enteric disease, with varied clinical manifestations. In young animals the infection is more common and may be more severe. In this study the polymerase chain reaction (PCR) was used to detect Cryptosporidium parasites in goat kids, calves, lambs, piglets and colts sharing the same environment. Fecal samples were collected directly from the rectum of 192 goat kids, 184 calves, 44 lambs, 47 piglets and 26 colts aged up to twelve months, males and females, of different breeds, from the Brazilian states of Goiás, Mato Grosso do Sul, Minas Gerais and São Paulo. PCR was used for amplifying a fragment of 18S rRNA gene and the gene encoding the surface glycoprotein GP60. Positive PCR amplification was observed in 16.7% (32/192) goat kids, 6.5% (12/184) calves and 2.1% (1/47) piglets. Based on the sequencing of 18S rRNA PCR products, all samples from goat kids were identified as C. parvum. Among calves, C. parvum was identified in 41.7% (5/12), C. andersoni in 16.7% (2/12), C. ryanae in 16.7% (2/12) and C. bovis in 25% (3/12) of the animals. All GP60 sequences were classified as genotype IIaA15G2R1 and were found in goat kids, calves and piglets sharing the same environment. This is the first description of the molecular identification and genotyping of Cryptosporidium in goat kids and piglets in Brazil. We conclude that Cryptosporidium species and C. parvum GP60 subtypes that infect livestock in Brazil, may act as sources of zoonotic infection for other animals and humans.

Diagnóstico de criptosporidiose em amostras fecais de bezerros por imunofluorescência direta e microscopia de contraste de fase / Diagnosis of cryptosporidiosis in fecal samples of calves using direct immunofluorescence and phase contrast microscopy

O presente estudo teve como objetivo comparar as técnicas de imunofluorescência direta (IFD) e a microscopia de contraste de fase em solução de Sheather (MCF), para detecção de oocistos de Cryptosporidium spp. em amostras fecais de bezerros. A determinação dos limiares detecção da IFD e da MCF foi realizada utilizando cinco alíquotas de uma amostra fecal de bezerro, comprovadamente negativa para Cryptosporidium spp., adicionadas com diferentes quantidades de oocistos de Cryptosporidium parvum. Ao exame das 5 alíquotas, a IFD e a MCF apresentaram, respectivamente, limiares de detecção de 3,3x104 (duas alíquotas positivas) e 3,3x105 oocistos (1 alíquota positiva) por grama de fezes. Foram também realizadas a comparação entre a positividade obtida e uma análise semiquantitativa do número de oocistos observados por campo de microscopia, em ambos os métodos, em 300 amostras fecais de bezerros. Entre as 300 amostras, 19,7 por cento (59/300) foram positivas pela IFD, com diferença estatisticamente significante (P=0,0098) quando comparada com a positividade obtida pela MCF, que foi de 11,7 por cento (35/300). As amostras positivas foram submetidas à reação em cadeia da polimerase para amplificação de fragmentos da subunidade 18S do rRNA, com posterior sequenciamento dos fragmentos amplificados, o que permitiu a identificação de Cryptosporidium andersoni em 11,9 por cento (7/59) e de C.parvum em 88,1 por cento (52/59) das amostras. Os resultados observados comprovam que a IFD foi mais eficiente que a MCF para detecção de oocistos de Cryptosporidium spp. em amostras fecais de bezerros.

This study aimed to compare the direct immunofluorescence assay (DIF) and the phase contrast microscopy in Sheather solution (PCM) for detection of Cryptosporidium oocysts in fecal samples from calves. The determination of the thresholds of detection of DIF and PCM was performed using five aliquots of a fecal sample from a calf negative for Cryptosporidium spp. oocysts, spiked with different amounts of Cryptosporidium parvum oocysts. The screening of the five aliquots revealed that the DIF and MCF showed respectively, detection thresholds of 3.3x104 (2 positive aliquots) and 3.3x105 (1 positive aliquot) oocysts per gram of fecal sample. Further analyses were accomplished in order to compare the positivity results and to determine semi-quantitatively the number of oocysts per field of microscopy, in both methods, in 300 fecal samples from calves. Among the 300 samples, 19.7 percent (59/300) were positive by DIF, result that was statistically significant (P=0.0098) when compared with the positivity obtained by the PCM, which was 11.7 percent (35/300). The positive samples were submitted to the nested-PCR assay for amplification of fragments of the 18S subunit of rRNA, following sequencing of amplified fragments, allowing the identification of Cryptosporidium andersoni in 11.9 percent (7/59) and C. parvum in 88.1 percent (52/59) of the samples. The present results indicate that the DIF was more effective than PCM in the detection of Cryptosporidium in fecal samples from calves.

Evaluation of reduction in egg shedding of gastrointestinal nematodes in cattle following administration of anthelmintics / Avaliação na redução de ovos de nematódeos gastrintestinais em bovinos após a administração de anti-helmínticos

To evaluate the necessary time and to identify the differences among anthelmintics for the reduction of parasite eggs shed in feces, five groups of cattle were treated with moxidectin, ivermectin, levamisole phosphate, and albendazole sulphoxide. Levamisole promoted a reduction in egg count per gram (EPG-R) of 97.4 percent after 24 hours, moxidectin 98.3 percent after 36 hours, and albendazole sulphoxide 95.9 percent after 36 hours post- treatment. Cooperia spp. and Haemonchus spp. showed resistance to ivermectin. The results demonstrated that the diagnosis of parasite resistance in cattle can be based on a count of eggs per gram (EPG) carried out as soon as seven days after the treatment.

No intuito de avaliar o tempo necessário para redução dos valores de OPG visando identificar o início de atuação dos anti-helmínticos, cinco grupos de novilhos, naturalmente infectados por nematódeos gastrintestinais foram tratados com moxidectina, ivermectina, fosfato de levamisol e sulfóxido de albendazol. O levamisol promoveu redução no número de ovos de nematódeos eliminados nas fezes (R-OPG) de 97,4 por cento 24 horas após a aplicação, a moxidectina de 98,3 por cento após 36 horas, e o sulfóxido de albendazol de 95,9 por cento após 36 horas. Foi registrada a presença de Cooperia spp. e Haemonchus spp. com resistência a ivermectina. A contagem de OPG realizada aos sete dias pós-tratamento apresentou resultados similares aos obtidos nas contagens realizadas 10 e 14 dias após a aplicação dos anti-helmínticos avaliados, demonstrando que o intervalo adequado entre o tratamento anti-helmíntico e o exame para verificar a redução do OPG pode ser de 7 dias.