Camargo Vinícius da Silva, Santana Bruna Nicoleti, Ferrari Elis Domingos, Nakamura Alex Akira, Nagata Walter Bertequini, Nardi Ana Rita Moraes, Meireles Marcelo Vasconcelos
Faculdade de Medicina Veterinária, Universidade Estadual Paulista - UNESP, Araçatuba, SP, Brasil.
Curso de Medicina Veterinária, Faculdades Adamantinenses Integradas, Adamantina, SP, Brasil.
Rev Bras Parasitol Vet. 2018 Jan-Mar;27(1):61-66. doi: 10.1590/S1984-296120180010. Epub 2018 Mar 1.
This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.
本研究采用多种诊断方法，对巴西南部和东南部圈养金丝雀（Serinus canaria）中隐孢子虫属的发生情况进行检测，并对其进行分子特征分析。总共498份粪便样本通过使用谢弗氏溶液的离心浮选法进行纯化。隐孢子虫属的诊断采用三种诊断方法：孔雀石绿负染色法、靶向18S rRNA基因的巢式PCR，随后对扩增片段进行测序，以及靶向检测鸡隐孢子虫和鸟类基因型III特异性18S rRNA的双重实时PCR。通过显微镜分析、巢式PCR和双重实时PCR方案结果得出，隐孢子虫属的总体阳性率（至少在一种检测方案中呈阳性的总样本数）为13.3%（66/498）。通过显微镜检查和巢式PCR检测隐孢子虫属的阳性率分别为2.0%（10/498）和4.6%（23/498）。对通过巢式PCR扩增的20个样本进行测序，鉴定出鸡隐孢子虫（3.0%；15/498）、鸟类基因型I隐孢子虫（0.8%；4/498）和鸟隐孢子虫（0.2%；1/498）。双重实时PCR显示，鸡隐孢子虫的阳性率为7.8%（39/498），鸟类基因型III的阳性率为2.4%（12/498）。在检测隐孢子虫属方面，孔雀石绿负染色法与巢式PCR有显著差异。在检测金丝雀胃内隐孢子虫时，双重实时PCR比巢式PCR/测序更敏感。
