[Show abstract][Hide abstract] ABSTRACT: There are very few studies on the diversity and public health significance of Cryptosporidium species in zebu cattle and water buffaloes in developing countries. In this study, PCR-restriction fragment length polymorphism and DNA sequence analyses of the small-subunit (SSU) rRNA gene were used to genotype Cryptosporidium specimens from 12 zebu cattle calves, 16 water buffalo calves, and four swamp deer (Cervus duvaucelii) collected from the buffer zone of the Chitwan National Park, Nepal. All Cryptosporidium specimens from cattle and buffaloes belonged to Cryptosporidium ryanae, whereas those from deer belonged to Cryptosporidium ubiquitum. Comparison of the SSU rRNA gene sequences obtained with those from earlier studies has identified a nucleotide substitution unique to all C. ryanae isolates from Nepal, in addition to some sequence heterogeneity among different copies of the gene. The finding of the dominance of a unique C. ryanae variant in both zebu cattle and water buffaloes in Nepal indicates that there is unique cryptosporidiosis transmission in bovine animals in the study area, and cross-species transmission of some Cryptosporidium spp. can occur between related animal species sharing the same habitats.
[Show abstract][Hide abstract] ABSTRACT: Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.
[Show abstract][Hide abstract] ABSTRACT: To assess the prevalence and public health significance of rabbit cryptosporidiosis, a total of 1,081 fecal specimens were collected between October 2007 and April 2008 from rabbits on eight farms in five different areas in Henan Province, China, and were examined by microscopy after Sheather's sucrose flotation and modified acid-fast staining. The average infection rate of Cryptosporidium was 3.4% (37/1,081 samples). There was a significant association between the prevalence of Cryptosporidium and the age of animals (chi(2) = 57.13; P < 0.01); the prevalence of cryptosporidiosis in 1- to 3-month-old rabbits was the highest (10.9%). The Cryptosporidium species in microscopy-positive specimens were genotyped by sequence analyses of the 18S rRNA, 70-kDa heat shock protein (HSP70), oocyst wall protein (COWP), and actin genes and were subtyped by sequence analysis of the 60-kDa glycoprotein (gp60) gene. Only the Cryptosporidium rabbit genotype was identified, with 100% sequence identity to published sequences of the 18S rRNA, HSP70, COWP, and actin genes, and the strains belonged to three gp60 subtypes (VbA36, VbA35, and VbA29). In view of the recent finding of the Cryptosporidium rabbit genotype in human outbreak and sporadic cases, the role of rabbits in the transmission of human cryptosporidiosis should be reassessed.
[Show abstract][Hide abstract] ABSTRACT: To date, little is known about the prevalence, genotypes and zoonotic potential of Cryptosporidium spp. affecting horses, especially in North America. A cross-sectional study was conducted in New York, USA between February 25th and May 1st 2009. Fecal samples were collected from three hundred and forty nine 1-10-week-old foals and their dams on 14 different broodmare farms. All fecal samples were screened for Cryptosporidium spp. using a direct immunofluorescence assay (DFA). DNA extraction and PCR-RFLP analysis of the small-subunit (SSU) rRNA gene were performed on all the foal samples. PCR-positive samples were subtyped by DNA sequencing of the 60-kDa glycoprotein (gp60) gene. On DFA, 13/175 (7.4%) foal samples and 3/174 (1.7%) mare samples were designated positive for Cryptosporidium spp., whereas on SSU rRNA-based PCR, 9/175 (5.1%) foal samples were positive. Cryptosporidium PCR-positive foals were significantly older (13-40 days, median age of 28 days) compared with negative foals (4-67 days, median 18 days, p=0.02). The number of foals with diarrhea or soft feces was not significantly different between positive and negative foals (p=0.09). PCR-RFLP analysis of the SSU rRNA gene and DNA sequencing of the gp60 gene identified the parasite as subtype VIaA14G2 of the horse genotype. This is the first report of a group of foals affected with the Cryptosporidium horse genotype, which has recently been detected in humans. As other contemporary molecular studies have identified C. parvum in foals, it seems that equine cryptosporidiosis should be considered a zoonosis.
[Show abstract][Hide abstract] ABSTRACT: Subtyping was conducted in late 2007 on 57 Cryptosporidium specimens from sporadic cases in Colorado, Idaho, New Mexico, and Iowa. One previously rare Cryptosporidium hominis subtype was identified in 40 cases (70%) from all four states, and the Cryptosporidium horse genotype was identified in a pet shop employee with severe clinical symptoms.
[Show abstract][Hide abstract] ABSTRACT: Small-subunit (SSU) rRNA-based methods have been commonly used in the differentiation of Cryptosporidium species or genotypes. In order to develop a new tool for confirming the genotypes of Cryptosporidium species, parts of the 90-kDa heat shock protein (Hsp90) genes of seven Cryptosporidium species and genotypes known to infect humans (C. hominis, C. parvum, C. meleagridis, C. canis, C. muris, C. suis, and the cervine genotype), together with one from cattle (C. andersoni), were sequenced and analyzed. With the exception of C. felis from cats and C. baileyi from birds, the Hsp90 genes of all tested Cryptosporidium species were amplified. Phylogenetic analysis of the hsp90 sequences from all these species is congruent with previous studies in which the SSU rRNA, 70-kDa heat shock protein, oocyst wall protein, and actin genes were analyzed and showed that gastric and intestinal parasites segregate into two distinct clades. In this study, the secondary products of hsp90 produced after PCR-restriction fragment length digestion with StyI and HphI or with BbsI showed that parasites within the intestinal or gastric clade could be differentiated from each other. These data confirm the utility of the Hsp90 gene as a sensitive, specific, and robust molecular tool for differentiating species and/or genotypes of Cryptosporidium in clinical specimens.
[Show abstract][Hide abstract] ABSTRACT: To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from
five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each
associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and
wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected.
The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very
few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed
(9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant
risk to human health.