First record of Cryptosporidium infection in a raccoon dog (Nyctereutes procyonoides viverrinus)

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.     

Cryptosporidium and host resistance: historical perspective and some novel approaches

Cryptosporidium parvum is recognized as a major cause of diarrheal disease in neonatal bovine calves. In addition, this protozoan parasite has emerged as an important cause of disease in both immunocompromised and immunocompetent humans. Despite years of research, no consistently effective means of prevention or treatment are readily available for cryptosporidiosis in any species. Infection through ingestion of contaminated water has been widely documented; C. parvum was reported to be responsible for the largest waterborne outbreak of infectious disease in US history. In addition to its role as a primary disease agent, C. parvum has potential to initiate or exacerbate other gastrointestinal disorders, such as inflammatory bowel disease. Thus, control of C. parvum infection in both animals and humans remains an important objective. Research in our laboratory has focused on understanding mechanisms of resistance to C. parvum. We have demonstrated that acquisition of intestinal flora increases resistance to C. parvum. Substances present in the intestinal mucosa of adult animals can transfer resistance when fed to susceptible infants. Both expression of intestinal enzymes and rate of proliferation of epithelial cells may be altered following C. parvum infection. These and other changes may have profound effects on host resistance to C. parvum.     

Biology of Cryptosporidium parvum in pigs: from weaning to market

Cryptosporidium parvum is commonly identified as infecting domestic livestock and humans. Prevalence of C. parvum in pigs has been reported, however, the duration and infection pattern of naturally acquired Cryptosporidium infections in pigs has not been reported. This study was undertaken to investigate the age of oocyst shedding and duration of natural Cryptosporidium parvum infections in pigs from weaning to market weight. Fecal samples were collected from weaned Yorkshire-Landrace piglets (n=33) twice per week until Cryptosporidium oocysts were detected. Upon oocyst detection, fecal samples were collected three times per week and pigs were monitored throughout the study for diarrhea and examined after concentration and immunofluroescent staining. Cryptosporidium isolates were genotyped by polymerase chain reaction to amplify the HSP70 gene which was subsequently sequence analyzed. All 33 pigs shed oocysts some time during the study. The mean age of initial oocyst detection was 45.2 days post-weaning with the mean duration of infection 28.7 days. Mean number of Cryptosporidium oocysts was low and declined to zero prior to study completion. Episodes of diarrhea were not associated with oocyst excretion. Genetic sequences were obtained for 10 of the pigs. All of the 10 isolates aligned as the Cryptosporidium parvum 'pig' genotype. This study demonstrates that the age and duration of oocyst shedding in pigs infected with C. parvum porcine genotype is different from other livestock species.     

Identification of genotypes of Cryptosporidium parvum isolates from a patient and a dog in Japan

Cryptosporidium parvum (C. parvum) is recognized as a significant pathogen in humans and animals, primarily as a cause of diarrheal illness. Recent genetic and biological studies indicate that C. parvum is not a single species but composed of genetically distinct multiple genotypes. Thus, it is valuable to distinguish between genotypes in the epidemiology of Cryptosporidium infection in humans and animals. Although C. parvum has been detected in humans and animals in Japan, the genotype of isolates remains unclear because identification has been performed only by conventional microscopy. We report herein the genotypes of C. parvum isolates distinguished by the polymerase chain reaction (PCR)-based diagnostic method. C. parvum isolates, originally obtained from a patient and a pet dog, were found to have cattle and dog genotypes, respectively.     

Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic

To study the diversity of Cryptosporidium spp. in various hosts, we used the variability of the small-subunit rRNA gene and the Cryptosporidium oocyst wall protein genes. Oocysts from humans, cattle, horses, dogs, field mice, chickens, reptiles, deer, goat, cat, antelope and from a sample of water reservoir were assayed. The zoonotic C. parvum bovine genotype sequence was found to be present in the most of isolates. This study shows a complex epidemiology pattern for C. parvum bovine genotype infections. The identification of cattle, horse, and deer isolates emphasizes a transmission route for C. parvum via these hosts, and identifies a potential source for human infection in the Czech Republic. Furthermore, C. andersoni from a cow, C. baileyi from a chicken, C. felis from a cat, C. meleagridis from a dog, and C. saurophilum and C. serpentis from reptiles were also identified in the isolates from the Czech Republic.     

野生动物隐孢子虫的种类和基因型

隐孢子虫病（Cryptosporidiosis）是一种全球性的人兽共患原虫病,具有广泛的宿主范围,可以感染鸟类、哺乳类、鱼类、爬行类、两栖类以及人在内的240多种动物。野生动物隐孢子虫做为人隐孢子虫病感染的重要传播来源,对其进行生物学研究具有重要的公共卫生意义。本文分别叙述了野生动物隐孢子虫的种类及基因型,为进一步研究野生动物的隐孢子虫病提供了有效的参考。     

Factors associated with shedding of Cryptosporidium parvum versus Cryptosporidium bovis among dairy cattle in New York State

OBJECTIVE: To isolate and speciate Cryptosporidium DNA from fecal samples obtained from dairy cattle in New York State and identify factors associated with whether cattle were shedding Cryptosporidium parvum versus Cryptosporidium bovis. DESIGN: Cross-sectional study. SAMPLE POPULATION: 115 fecal samples positive for DNA coding for the Cryptosporidium 18S rRNA gene from dairy cattle in New York State. PROCEDURES: A PCR assay was used to amplify DNA from fecal samples; amplification products were submitted for bidirectional DNA sequencing. Logistic regression was used to test for associations between various host factors and Cryptosporidium spp. RESULTS: 70 of the 115 (61%) fecal samples were found to have C parvum DNA, 42 (37%) were determined to have C bovis DNA, and 3 (3%) were found to have C parvum deer-type DNA. The presence of diarrhea at the time of fecal sample collection, oocyst count, and breed were associated with whether cattle were infected with C parvum or C bovis, with animals more likely to be infected with C parvum if they had diarrhea, had a high oocyst count, or were Holsteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that C parvum and C bovis can be isolated from dairy cattle in New York State and that various factors affect whether cattle infected with Cryptosporidium spp are infected with C parvum or C bovis. Findings also lend credence to the theory that C bovis may be more host adapted and thus less pathogenic to dairy cattle than C parvum.     

Molecular epidemiology of cryptosporidium and giardia in humans on prince edward island, Canada: evidence of zoonotic transmission from cattle

To determine the zoonotic potential of Cryptosporidium and Giardia in Prince Edward Island (PEI), Canada, 658 human faecal specimens were screened that were submitted to the Queen Elizabeth Hospital diagnostic laboratory. Overall, 143 (22%) samples were Cryptosporidium positive, while three (0.5%) were positive for Giardia. Successful genotyping of 25 Cryptosporidium isolates by sequence analysis of the HSP70 gene revealed that 28 and 72% were C. hominis and C. parvum, respectively. Cryptosporidium isolates from humans and previously genotyped C. parvum from beef cattle were subtyped by sequence analysis of the GP60 gene. Subtyping identified three subtypes belonging to the family IIa. All three subtypes IIaA16G2RI (55%), IIaA16G3RI (22%) and IIaA15G2RI (22%) were found in the animal isolates, while two of the subtypes found in the animals, IIaA16G2RI (80%) and IIaA15G2RI (20%), were also identified in the human isolates. Cryptosporidium infection in humans peaked in April-June. Molecular epidemiological analysis of the human data showed a C. parvum peak in the spring and a relatively smaller peak for C. hominis in July-September. The majority (57%) of human Cryptosporidium isolates were found in children between 5 and 10 years of age. All three Giardia isolates were identified as G. duodenalis assemblage A. The overall Cryptosporidium prevalence in our human samples was high relative to other studies, but because the samples were submitted to a hospital diagnostic laboratory, the results may not be representative of the general population. Further, the presence of the same zoonotic C. parvum subtypes in cattle and human isolates implies that transmission is largely zoonotic and cattle may be a source of sporadic human infections on PEI. The presence of Giardia in people on PEI is rare, and the assemblage A found in humans might originate from humans, livestock or other domestic or wild animals.     

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts

Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll(?) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. Results: (a) Diagnostic methods: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(?) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.     

Cryptosporidium and Giardia as foodborne zoonoses

Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.     

Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing

Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptosporidium DNA in 36 of 107 cattle (33.64%), in 26 of 85 sheep (30.5%) and in 17 of 78 horses (21.79%). The HSP LAMP specific to C. muris and C. andersoni, amplified Cryptosporidium DNA in one cow (0.9%), five sheep (5.8%) and seven horses (8.9%). The gp60 LAMP assay, shown to detect C. parvum produced no amplified Cryptosporidium DNA, likely due to low sample DNA concentrations. The specificity of LAMP assays was confirmed by sequencing of the LAMP products generated in positive samples. Sequence products from the three LAMP assays showed high identity to the target gene sequences confirming the specificity of LAMP. In this study, the LAMP procedure was clearly superior to nested PCR in the detection of Cryptosporidium species DNA. Use of LAMP is proposed as an efficient and effective tool for epidemiologic survey studies including screening of healthy animals in which Cryptosporidium oocyst shedding is characteristically low and likely below the detection limit of PCR in conventional sample concentrates.     

水源中隐孢子虫种类和基因型鉴定技术研究进展

目前，隐孢子虫有效种有20个。隐孢子虫的分子流行病学研究表明，隐孢子虫具有宿主特异性。目前，感染人的隐孢子虫种和基因型有C．horninis、C.parvum、C．meleagridis、C.felis、C.canis、C.nuris、C．suis genotype和C．cervine genotype等。水源性隐孢子虫暴发，具有重要的公共卫生意义。分子鉴定技术已广泛应用于检测水中隐孢子虫，水中检测到的隐孢子虫有9个。通过分析其分子组成特性表明，成年牛可能是水污染的主要根源，除了居民用水和农业污染外，鹿、麝鼠、田鼠、鸟和其它野生动物可能也与水污染有关。隐孢子虫分子基因型鉴定工具可能为水源保护性研究提供一个更有力的溯源追踪工具。     

Cryptosporidiosis in People: It's Not Just About the Cows

Cryptosporidiosis is one of the most common causes of infectious diarrhea in people. Although dairy calves are high-risk hosts, the role of other livestock, pets, and humans in the disease should not be underestimated. Some Cryptosporidium species and strains are specific to people, others are specific to animals while some are zoonotic pathogens. Cryptosporidium hominis is the species responsible for the majority of human cases in the United States, Sub-Saharan Africa, and Asia, while Cryptosporidium parvum accounts for more human cases in Europe and particularly in the United Kingdom. A deeper understanding of Cryptosporidium host range, reservoirs, and transmission is needed to develop preventive strategies to protect the general public.     

Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed

OBJECTIVE: To determine the risk posed by Cryptosporidium parvum and Cryptosporidium hominis from dairy cattle in the New York City watershed (NYCW). SAMPLE POPULATION: Samples from cattle at risk for shedding Cryptosporidium organisms on randomly selected dairy farms in the NYCW. PROCEDURE: Feces were collected for 4 years from calves at risk for infection on 37 dairies. Oocysts were detected by use of centrifugation concentration-flotation microscopy. The DNA was directly isolated from fecal samples and used to amplify fragments of the small subunit ribosomal RNA and thrombospondin-related adhesion protein C-2 genes by use of nested polymerase chain reaction assays. Small subunit ribosomal RNA fragments were restriction digested by the enzyme Vspl and thrombospondin-related adhesion protein C-2 fragments were digested by Eco91l to distinguish between C hominis (formerly known as genotype 1) and C parvum (formerly known as genotype 2). RESULTS: Of 437 fecal samples examined, 214 contained oocysts. Amplicons were generated for 200 samples. We can be certain, with 95% confidence, that cattle in the NYCW did not harbor C hominis. CONCLUSIONS AND CLINICAL RELEVANCE: Cryptosporidium infections in cattle are under examination because of the potential contamination of public waters by manure. Although cattle may be the source of zoonotic infection via C parvum, they pose little risk for C hominis (the strain commonly isolated from humans in waterborne outbreaks of disease). Other sources of oocysts should be considered when investigating outbreaks attributable to contaminated urban drinking water because cattle pose only a small risk via shedding of C hominis.     

Prevalence of Cryptosporidium parvum infection and pattern of oocyst shedding in calves in Japan

Cryptosporidium parvum infection and the pattern of oocyst shedding were observed in calves. A total of 480 fecal samples were collected from 30 calves (age, < or =30 days) over a period of 10 months from June 1998 to March 1999. A sucrose centrifugal flotation technique revealed 28/30 (93%) calves were passing Cryptosporidium oocysts. Oocyst shedding was first detected on the sixth day after birth, with 8% of the calves testing positive. This rate increased day by day and reached approximately 80% by day 15. Oocyst shedding varied from 1 to 13 days, with a mean of 7 days. Calves infected with C. parvum had a significantly higher rate of diarrhea (33%) than non-infected calves (8%) (P<0.05), suggesting C. parvum infection as the likely cause. The mean number of oocysts excreted by calves < or =30 days old was approximately 6x10(7) per gram of feces. These results indicated that one calf would excrete some 6x10(11) oocysts in the first month after birth, taking both the quantity of feces in a day and the period of excretion into consideration. Accordingly, it is clear that calves are important in the spread of cryptosporidiosis to calves and humans.     

Abomasal cryptosporidiosis in cattle

A 6-week-old calf and nine feedlot steers shed oocysts similar to Cryptosporidium muris-like oocysts. There were massive populations of this Cryptosporidium in the peptic glands of most of these animals. The oocysts were larger and more oval than the frequently reported type which is generated in the intestines of many animal species and thought to be similar to Cryptosporidium parvum. The pattern of shedding of this newly discovered Cryptosporidium in the steers was continuous over a period of months whereas the C. parvum-like oocysts cease to be shed 1 to several weeks after onset. The nature of the host-parasite interaction in abomasal cryptosporidiosis is yet to be determined. Morphologic changes that resulted from the interaction were an approximate 10% increase in abomasal mucosal thickness, widening of gland lumens in the middle region, and atrophy of epithelium in the same region.     

Antibody responses following administration of a Cryptosporidium parvum rCP15/60 vaccine to pregnant cattle

Cryptosporidium parvum is a zoonotic Apicomplexa-protozoan pathogen that causes gastroenteritis and diarrhea in mammals worldwide. Globally, C. parvum is ubiquitous on dairy operations and is the pathogen most commonly diagnosed in association with calf diarrhea. Here, we describe the antibody response in 20 pregnant cows to a recombinant C. parvum oocyst surface protein (rCP15/60) vaccine compared with 20 controls, and the antibody response in 19 calves fed the rCP15/60-immune colostrum from these vaccinated cows compared with 20 control calves. Cows vaccinated with rCP15/60 produced a significantly greater antibody response compared to controls (p<0.0001) and this response was strongly associated with the subsequent level of colostral antibody (r=0.82, p<0.0001). Calves fed rCP15/60-immune colostrum showed a dose-dependent absorption of antibody, also associated with colostral antibody levels (r=0.83, p<0.0001). Currently, drug therapy against cryptosporidiosis is limited making development of an effective vaccine attractive. This report describes the first stages in development of a C. parvum rCP15/60 vaccine designed to confer passive protection to calves against cryptosporidiosis.     

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido,Japan

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.