Cryptosporidium spp. in quails (Coturnix coturnix japonica) in Henan, China: molecular characterization and public health significance

The prevalence of Cryptosporidium spp. was investigated in scale quail (Coturnix coturnix japonica) farms in Henan Province, China between September 2006 and August 2007. One thousand eight hundred and eighteen fecal samples from 47 quail farms in five areas were collected for the examination of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 13.1% (95% CI 13.1±1.6%) (29 of 47 farms), with 72-100-day-old quails having the highest prevalence (23.6%, 95% CI 23.6±2.6%) (χ(2)=64.91; ρ<0.01). The highest prevalence was observed in autumn (21.8%, 95% CI 21.8±3.1%) and the lowest in winter (χ(2)=74.83; ρ<0.01). Two hundred and thirty-nine Cryptosporidium-positive samples were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 42 were further analyzed by DNA sequencing of the PCR products. Two Cryptosporidium species were identified, Cryptosporidium baileyi in 237 birds on 29 farms, and potentially zoonotic Cryptosporidium meleagridis in only two birds on two farms. These findings may suggest that quails are not a major source of zoonotic Cryptosporidium in the study area.     

Prevalence of Cryptosporidium spp. in pigs in Shanghai, China

A survey on prevalence of Cryptosporidium spp. in pigs at 12 farms in 8 suburban districts and 1 county of Shanghai was conducted under Sheather's sucrose flotation protocol and modified acid-fast stain methods from 2006 to 2009. A total of 2323 faecal samples were collected and Cryptosporidium spp. oocysts were detected in 800 samples (34.4%). Cryptosporidium was found in all 12 pig farms. Significant variations of prevalence were observed in different farms ranging from 14.1% to 90.6%. A follow-up survey on a positive pig farm for 13 consecutive months revealed that the most serious infection of Cryptosporidium spp. in pigs happened in winter and spring, and the lowest infection season was summer. Cryptosporidium spp. infection was mainly found in piglets within 2 months and no infection was found among those pigs of 90-180 days of age. The genotype analyses were carried out through PCR-RFLP and partial sequences analysis of small subunit ribosomal RNA (SSU rRNA) in some of the positive samples. Cryptosporidium suis (57/69, 82.6%), Cryptosporidium pig genotype II (6/69, 8.7%) and mixed infection of above two species/genotype (6/69, 8.7%) were found to be the main species/genotype in pigs in Shanghai area.     

Real-timeqPCR检测鸡胚中贝氏隐孢子虫方法研究

为检测鸡胚中贝氏隐孢子虫,本研究根据GenBank公布的贝氏隐孢子虫和原鸡18SrRNA基因序列设计针对贝氏隐孢子虫的特异性引物,并以贝氏隐孢子虫卵囊基因组DNA为模板,建立检测贝氏隐孢子虫SYBRGreen实时荧光定量PCR（Real—timeqPCR）方法,同时对贝氏隐孢子虫在鸡胚中培养7d后尿囊膜和尿囊液样品中虫体DNA进行检测。结果表明,建立的SYBRGreenreal—timeqPCR方法可特异性检测到贝氏隐孢子虫,建立的标准曲线线性关系良好（R2=0．998）,卵囊基因组DNA检测阈值为10个卵囊,重复检测结果显示试验重现性较好。该方法成功检测到尿囊膜和尿囊液中虫体DNA。本研究建立了快速、特异的定量检测鸡胚中贝氏隐孢子虫real—timeqPCR方法,可用于对贝氏隐孢子虫在鸡胚中繁殖规律分析和抗隐孢子虫药物筛选等研究。     

Fecal shedding of Cryptosporidium oocysts in healthy alpaca crias and their dams

Objective-To determine the apparent prevalence of shedding of Cryptosporidium spp in healthy alpaca crias and their dams on 14 farms in New York and 1 farm in Pennsylvania. Design-Cross-sectional study. Animals-110 alpaca crias and their 110 dams. Procedures-Fecal samples were obtained from 220 alpacas at 14 alpaca farms in New York and 1 farm in Pennsylvania. For each animal, age, sex, and health condition were recorded. A fecal score (1 = normally formed; 2 = soft or loose; 3 = diarrhetic) was recorded for each cria. Cryptosporidium oocysts were identified in fecal samples by a direct immunofluorescence assay. Results-Apparent prevalence of fecal shedding of Cryptosporidium oocysts was 8% (95% confidence interval, 4% to 15%) in dams and was 7% (95% confidence interval, 3% to 13%) in crias. There was no significant difference in age between dams with positive fecal test results for Cryptosporidium oocysts (median age, 4 years; range, 3 to 8 years) and dams with negative results (median age, 4 years; range, 2.5 to 19 years). No significant difference was found in age between crias with positive fecal test results (median age, 20 days; range, 7 to 53 days) and those with negative results (median, 36 days; range, 2 to 111 days). No significant difference in fecal scores was found between crias with positive versus negative fecal test results. Conclusions and Clinical Relevance-A higher than previously reported apparent prevalence of fecal shedding of Cryptosporidium oocysts in healthy alpacas was found. A zoonotic risk should be considered, especially for Cryptosporidium parvum.     

The prevalence of Cryptosporidium, and identification of the Cryptosporidium horse genotype in foals in New York State

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.     

Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves

Fifteen dairy farms in seven states on the east coast of the US were each visited on two consecutive years to determinate the prevalence of Cryptosporidium species in pre-weaned (5 days to 2 months) and post-weaned calves (3-11 months), respectively. After each of 971 fecal specimens collected directly from each calf was sieved and subjected to density gradient centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy, and polymerase chain reaction (PCR). For all PCR-positive specimens the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified from all farms. Types of housing appeared to have no influence with regard to prevalence of infection. Of 971 calves, 345 were infected with Cryptosporidium (35.5%), but more pre-weaned calves (253 of 503; 50.3%) than post-weaned calves (92 of 468; 19.7%) were found to be infected. A total of 278 PCR-positive specimens characterized by gene sequencing revealed Cryptosporidium parvum, Cryptosporidium andersoni, and two unnamed Cryptosporidium genotypes Bovine B (AY120911) and deer-like genotype (AY120910). The prevalence of these Cryptosporidium species and genotypes appeared to be age related between pre- and post-weaned calves. C. parvum, the only zoonotic species/genotype, constituted 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves. These findings clearly demonstrate that earlier reports on the presence and prevalence of C. parvum in post-weaned cattle that were based solely on oocyst morphology must be reassessed using molecular methods to validate species and genotype. This finding also indicates that persons handling or otherwise exposed to calves under 2 months of age are at greater risk of zoonotic infection from Cryptosporidium than the risk of infection from exposure to older calves.     

Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States

The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.     

A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age

Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1-8 weeks of age) (45.8%) than in post-weaned calves (3-12 months of age) (18.5%) and heifers (12-24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in pre-weaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1.     

灌南县域肉羊源人兽共患隐孢子虫的流行病学调查及其类型分析

以隐孢子虫(Cryptosporidium spp.)18S rRNA为靶基因,通过巢氏PCR检测发现在采集的101份新鲜粪便样品中18份样本为阳性。不同地区羊场的隐孢子虫感染率分别为：李集镇36%(18/50),新安镇0%(0/30)、堆沟港镇0%(0/21)未检出隐孢子虫。但通过对4羊场的分析发现,有2个羊场(50%)为隐孢子虫感染阳性,且不同的羊场感染率差异显著,因此单纯的以地区来评价隐孢子虫的感染率,是值得商榷的。山羊隐孢子虫的感染率为33.3%,湖羊隐孢子虫的感染率为2%。2～6月龄的育肥羊隐孢子虫的感染率为36%,6～10月龄的育成羊(0%)。对检测为阳性的样品进行了隐孢子虫18S rRNA基因片段序列分析,发现18个样品全部为肖氏隐孢子虫(Cryptosporidium xiaoi),不存在泛在隐孢子虫(Cryptosporidium ubiquitum)。在检测隐孢子虫感染阳性的1个山羊场和1个羊湖羊场均存在肖氏隐孢子虫感染,不存在泛在隐孢子虫,更未发现肖氏隐孢子虫和泛在隐孢子虫的混合感染。目前的数据提示肖氏隐孢子虫对2～6月龄山羊(33.3%)和湖羊(2%)具有更高的...     

鸭源贝氏隐孢子虫对鸡的致病性

设计3个不同剂量的鸭源隐孢子虫试验感染2日龄雏鸡,通过临床症状、增重、排卵囊规律和法氏囊指数及寄生器官组织扫描电镜观察,证实贝氏隐孢子虫160万个卵囊感染即可引起明显的呼吸道症状,发病鸡增重显著降低,法氏囊严重萎缩。贝氏隐孢子虫致病程度和排卵囊规律与感染剂量相关,主要病变表现呼吸道病变和法氏囊炎症。     

Age-related and housing-dependence of Cryptosporidium infection of calves from dairy and beef herds in South Bohemia, Czech Republic

Data of the prevalence, age-related and housing-dependence of naturally acquired cryptosporidiosis on 11 dairy and 11 beef farms in South Bohemia (Czech Republic) were collected. The farms were visited over four consecutive years (from 2002 to 2005). The prevalence of Cryptosporidium in pre-weaned (animals until second month of age) and post-weaned (animals from the third month of age) calves was determined. A total of 7001 faecal samples were collected, concentrated by Sheather's floatation method and stained by aniline-carbol-methyl violet. All samples were examined by light microscopy. Cryptosporidium parvum and C. andersoni oocysts were differentiated on morphological criteria. Of the 7021 specimens, 1814 (25.8%) were positive for Cryptosporidium oocysts; 561 samples (8%) for C. parvum and 1253 (17.8%) for C. andersoni. Pre-weaned dairy calves had higher infection levels of C. parvum than pre-weaned beef calves. The prevalence of C. parvum ranged from 1.4 to 56.5% on dairy farms. Only three cases of C. parvum oocysts shedding in pre-weaned calves on beef farms were found. Only one case of C. andersoni infection in pre-weaned calves was detected and no infections of C. parvum in post-weaned calves were found. The prevalence of C. andersoni reached 35.5% on dairy farms and 61.7% on beef farms. Calves that were on pasture all year long, had a lower probability of C. andersoni infection than those calves kept in a cowshed during the winter season.     

Molecular characterization of Cryptosporidium in Brazilian sheep

Feces were collected from 125 sheep between January and December 2007, on ten farms in the State of Rio de Janeiro, Brazil, and examined for the presence of Cryptosporidium. Ninety samples were collected from lambs 2 to 6 months of age, and 35 were from sheep over 12 months of age. All samples were subjected to molecular analysis by polymerase chain reaction (nested PCR) in two steps of the SSU rRNA. Two samples (1.6%) from the lambs were positive, and after sequencing were identified as Cryptosporidium ubiquitum. This species has been reported worldwide and it is considered a zoonotic pathogen since it has been found and in several animal species and humans. However, because of the low frequency of C. ubiquitum found, the risk for public health in this region may not be high.     

伊犁河谷新疆褐牛隐孢子虫感染情况调查

为了解伊犁河谷区域新疆褐牛隐孢子虫病流行情况,从该地区的伊宁市和察布查尔锡伯自治县的4个养殖场采集356份粪便样品,提取基因组DNA,以隐孢子虫小亚基核糖体DNA(SSU rDNA)为靶基因,进行套式PCR扩增,并对阳性样品进行序列分析。结果显示:伊宁市和察布查尔锡伯自治县各有1个养殖场的新疆褐牛发现隐孢子虫感染,总的感染率为3.37%,其中伊宁市褐牛的感染率为4.10%,察布查尔锡伯自治县褐牛的感染率为2.48%;序列比对结果显示,存在2种隐孢子虫感染,分别为安氏隐孢子虫(C.andersoni)和瑞氏隐孢子虫(C.ryanae),其中C.andersoni是优势种(91.67%);成年牛的感染率最高(5.59%),而断奶前犊牛未发现隐孢子虫感染;冬季感染率(5.68%)显著高于春季(1.11%)(P<0.05)。本研究首次对伊犁河谷区域新疆褐牛感染的隐孢子虫进行了分子鉴定,发现褐牛的隐孢子虫感染率显著低于我国其他品种牛的感染率。该研究结果为深入了解新疆褐牛隐孢子虫的流行情况,制定有效的防控措施提供了数据支持。     

Giardia and Cryptosporidium in dairy calves in British Columbia.

A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive.     

First report of Cryptosporidium deer-like genotype in Malaysian cattle

Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.     

Molecular characterisation of Cryptosporidium isolates from pre-weaned calves in Romania: is there an actual risk of zoonotic infections?

Diarrheic fecal samples from 258 pre-weaned calves (1-30 day-old) from 9 dairy farms located in Banat region, Romania, were microscopically examined for the presence of Cryptosporidium oocysts. Overall, 65 (25%) samples were found positive. A higher percent of infection was recorded in calves aged between 8 and 14 days compared with other age categories (1-7, 8-14, 15-21 and 22-30 days; p<0.05). Genetic characterization was carried out on all Cryptosporidium-positive samples. After DNA extraction, Cryptosporidium species were determined by a nested PCR of the small subunit rRNA gene (18S) followed by RFLP analysis with SspI, VspI and MboII restriction enzymes. The restriction patterns showed that animals were infected with Cryptosporidium parvum. Subsequently, subtyping of 13 C. parvum isolates, based on sequence analysis of the 60 kDa glycoprotein (GP60) gene, showed 2 subtypes (IIaA15G2R1 and IIaA16G1R1) belonging to the subtype family IIa. This is the first molecular study of bovine Cryptosporidium infection in Romania.     

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil

A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of S?o Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C. parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C. parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife.     

High prevalence of Cryptosporidium bovis and the deer-like genotype in calves compared to mature cows in beef cow-calf operations

Recent studies have identified the novel, host adapted Cryptosporidium bovis and the deer-like genotype in dairy cattle from farms in the United States, China, India and Europe. This novel species and genotype appear to be more prevalent in older, post-weaned dairy cattle than previously thought. However, little information is available on their prevalence in beef cow-calf operations. In the present study, we determined the prevalence of Cryptosporidium species in 98 calves (6-8 months old) and 114 cows (>2 years old) in seven beef cow-calf herds in western North Dakota. DNA was extracted from fecal samples and Cryptosporidium spp. were identified by amplification of the 18S rRNA gene followed by sequencing or RFLP analysis. All seven herds tested positive for Cryptosporidium. Overall, 43/212 (20.3%) animals were positive. Only five of these positives were from cows. C. bovis, the deer-like genotype and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. C. parvum was not identified in any of the positive samples. C. bovis, the deer-like genotype and C. andersoni were detected in 6/7, 5/7 and 2/7 herds, respectively. C. bovis and the deer-like genotype were primarily detected in calves, while C. andersoni was only detected in cows. Six isolates could not be typed. These results show a relatively high prevalence of C. bovis and the deer-like genotype in 6-8-month-old beef calves compared to cows older than 2 years in the seven herds studied.     

Genotyping of Cryptosporidium spp. from free-living wild birds from Brazil

In wild and domestic birds, cryptosporidiosis is often associated with infections by Cryptosporidium galli, Cryptosporidium baileyi and Cryptosporidium meleagridis. In addition to these species, a number of avian Cryptosporidium species yet to be fully characterized are commonly found among exotic and wild avian isolates. The present study aimed to detect and identify samples of Cryptosporidium spp. from free-living wild birds, in order to contribute to the knowledge of the variability of this parasite in the free-living population of Brazil. Stool samples were collected from 242 birds, with the following proportions of individuals: 50 Emberizidae (20.7%), 112 Psittacidae (46.3%), 44 Cardinalidae (18.2%), 12 Turdidae (5.0%), eight Ramphastidae (3.3%), seven Icteridae (2.9%), three Estrilididae (1.2%), two Contigidae (0.8%), two Thraupidae (0.8%) and two Fringilidae (0.8%). Among the 242 fecal samples from wild birds, 16 (6.6%) were positive for the presence of oocysts of Cryptosporidium. Molecular characterization of the 16 samples of Cryptosporidium, were performed with phylogenetic reconstructions employing 292 positions of 18S rDNA. None of the samples of birds was characterized as C. meleagridis. C. galli was identified in one rufous-bellied thrush (Turdus rufiventris), five green-winged saltators (Saltator similis), one slate-coloured seedeater (Sporophila schistacea), one goldfinch (Carduelis carduelis) and three saffron finches (Sicalis flaveola). One goldfinch isolate, one buffy-fronted seedeater (Sporophila frontalis), one red-cowled cardinal (Paroaria dominicana) and one other saffron finch (S. flaveola) were identified as C. baileyi. Avian genotype II was found in an isolate from a white-eyed parakeet (Aratinga leucophthalma). Clinical symptoms of cryptosporidiosis in birds have already been described and the number of wild birds which were shedding parasites was high. Therefore, further epidemiological research and disease surveillance of birds in the wild is warranted.