<i>Cryptosporidium</i> spp. and <i>Giardia</i> spp. in feces and water and the associated exposure factors on dairy farms

The aims of this study were to verify the prevalence of Cryptosporidium spp. and Giardia spp. in animal feces and drinking water on dairy farms and to identify a possible relation between the exposure factors and the presence of these parasites. Fecal samples from cattle and humans and water samples were collected on dairy farms in Paraná, Brazil. Analysis of (oo)cysts in the feces was performed by the modified Ziehl-Neelsen staining and centrifugal flotation in zinc sulfate. Test-positive samples were subjected to nested PCR amplification of the 18SSU ribosomal RNA gene for identification of Cryptosporidium and Giardia and of the gp60 gene for subtyping of Cryptosporidium. Microbiological analysis of water was carried out by the multiple-tube method and by means of a chromogenic substrate, and parasitological analysis was performed on 31 samples by direct immunofluorescence and nested PCR of the genes mentioned above. Identification of the species of Cryptosporidium was performed by sequencing and PCR with analysis of restriction fragment length polymorphisms. The prevalence of Giardia and Cryptosporidium was higher in calves than in adults. Among the samples of cattle feces, Cryptosporidium parvum was identified in 41 (64%), C. ryanae in eight (12.5%), C. bovis in four (6.3%), C. andersoni in five (7.8%), and a mixed infection in 20 samples (31.3%). These parasites were not identified in the samples of human feces. Thermotolerant coliform bacteria were identified in 25 samples of water (45.5%). Giardia duodenalis and C. parvum were identified in three water samples. The gp60 gene analysis of C. parvum isolates revealed the presence of two strains (IIaA20G1R1 and IIaA17G2R2) in the fecal samples and one (IIaA17G2R1) in the water samples. The presence of coliforms was associated with the water source, structure and degradation of springs, rain, and turbidity. The prevalence of protozoa was higher in calves up to six months of age. C. parvum and G. duodenalis were identified in the water of dairy farms, as were thermotolerant coliforms; these findings point to the need for guidance on handling of animals, preservation of water sources, and water treatment.本研究旨在明确巴西巴拉那州乳制品农场的动物粪便与饮用水中隐孢子虫属（Cryptosporidium spp.）和贾第鞭毛虫属（Giardia spp.）的流行率，并探究暴露因素与这类寄生虫检出情况之间的潜在关联。研究团队在该地区的乳制品农场采集了牛与人类的粪便样本以及水样。粪便中的（卵）囊检测采用改良齐尔-尼尔森染色法与硫酸锌离心浮聚法完成。对于检测呈阳性的样本，通过嵌套PCR扩增18SSU核糖体RNA基因以鉴定隐孢子虫与贾第鞭毛虫，并扩增gp60基因以实现隐孢子虫的亚型分型。水质微生物学分析采用多管发酵法与显色底物法；同时针对31份水样，通过直接免疫荧光法以及上述基因的嵌套PCR开展寄生虫学分析。隐孢子虫的物种鉴定通过测序以及限制性片段长度多态性（Restriction Fragment Length Polymorphism, RFLP）分析完成。犊牛粪便中贾第鞭毛虫与隐孢子虫的流行率显著高于成年牛。在牛粪便样本中，共鉴定出微小隐孢子虫（Cryptosporidium parvum）41份（64%）、瑞氏隐孢子虫（C. ryanae）8份（12.5%）、牛隐孢子虫（C. bovis）4份（6.3%）、安德森隐孢子虫（C. andersoni）5份（7.8%），另有20份样本（31.3%）存在混合感染。人类粪便样本中未检出上述寄生虫。25份水样（45.5%）检出耐热大肠菌群。3份水样中检出十二指肠贾第鞭毛虫（Giardia duodenalis）与微小隐孢子虫。对微小隐孢子虫分离株的gp60基因分析显示，粪便样本中存在IIaA20G1R1与IIaA17G2R2两种亚型，水样中检出IIaA17G2R1亚型。大肠菌群的检出与水源类型、泉眼的结构与退化程度、降雨情况以及浊度显著相关。6月龄以内犊牛的原虫流行率更高。本研究在乳制品农场的水体中检出了微小隐孢子虫、十二指肠贾第鞭毛虫与耐热大肠菌群，上述结果提示需制定针对动物操作管理、水源保护以及水处理的相关指南。