Development of a real-time PCR assay for detection of Cryptosporidium canis in dog fecal samples.

Cryptosporidiosis is an emerging zoonotic disease caused by the worldwide distributed parasitic protozoa Cryptosporidium spp. The host-adapted species Cryptosporidium canis is most frequently found in dogs, although human infections with this species have been described. This study aimed to develop a real-time PCR targeting the HSP70 protein gene for C. canis DNA detection in dog fecal samples collected from two municipalities in the state of São Paulo, Brazil. Furthermore, the occurrence of Cryptosporidium spp. and. C. canis was also determined by nested PCR. Fecal samples from 367 dogs (21 puppies and 346 adults) were purified by water-ether sedimentation. A real-time PCR protocol targeting the HSP70 gene for the species-specific detection of C. canis was developed and compared with nested PCR results. Real-time PCR identified C. canis in 15.3% (58/367) samples. Nested PCR revealed that 10.4% (38/367) of samples were positive for Cryptosporidium spp. All sequenced 18S rRNA amplicons were C. canis. There was a higher prevalence of Cryptosporidium spp. and C. canis in puppies compared to adult dogs. No non-specific amplification was observed in C. canis specific real-time PCR assay.

The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR.

Infection by Cryptosporidium serpentis occurs in reptiles, particularly in snakes. This disease is characterized by chronic infection with the presence of hypertrophic gastritis. The objectives of this study were to use real-time polymerase chain reaction (PCR) targeting the heat shock protein 70 (Hsp70) gene for the detection of C. serpentis in fecal samples from snakes and to determine the analytical and epidemiological specificity and sensitivity of this approach relative to the gold standard of nested PCR for the amplification of a fragment of the 18S subunit of the ribosomal RNA (18S rRNA) gene followed by the sequencing of amplified fragments (nPCR/S). Individual fecal samples were collected on a single occasion from 503 asymptomatic adult snakes housed in the serpentarium of the Butantan Institute in São Paulo, Brazil. The nested PCR revealed that 60 samples (11.98%) were positive for Cryptosporidium sp. The sequencing of amplified fragments, which was possible for 38 samples, resulted in the identification of Cryptosporidium tyzzeri (7), Cryptosporidium muris (4), Cryptosporidium varanii (12) and C. serpentis (15) in fecal samples from several snake species. The real-time PCR approach indicated that 17 samples (3.37%) were positive for C. serpentis, whereas the nPCR/S indicated that 15 samples (2.98%) were positive for C. serpentis. The epidemiological sensitivity and specificity of real-time PCR were 93.8% and 99.5%, respectively. Thus, we conclude that real-time PCR targeting the Hsp70 gene is a sensitive and specific method for the detection of C. serpentis in snake fecal samples.

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.