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.     

Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal

Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin-ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and beta-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.     

Risk factors associated with Cryptosporidium parvum infection in cattle

A 2-year, cross-sectional study was conducted to identify risk factors for Cryptosporidium sp. infection in bovine farms in central Italy. Faecal samples were collected on 248 farms, from 2024 calves and analysed using ELISA and immunofluorescent assay (IFA) commercial kits. In all 101 samples confirmed to be positive with IFA, the aetiological agent was identified as Cryptosporidium parvumand a large genetic variability was detected by subtype analysis. The prevalence of farm infection ranged from 3.4% to 35.6%. Univariate analysis showed a number of putative risk factors, including the type of farm, stalling of calves, late supply of colostrum, number of heads and contact between calves and adults. However, multivariate analysis confirmed that the higher risk for calves was associated with housing calves separately from their dams, a characteristic practice of dairy herd, whereas calves being nursed by their dams, a characteristic of cow-calf herd resulted as a protective factor.     

Viability and infectivity of two Cryptosporidium parvum bovine isolates from different geographical location

The viability of two Cryptosporidium parvum bovine isolates from Spain and Colombia was evaluated by in vitro excystation, inclusion/exclusion of two fluorogenic vital dyes (DAPI and PI) and infectivity assay in a suckling murine model. Excystation percentages were similar for both Spain and Colombia isolates (83% and 87%, respectively). The total viability of the Spain isolate, measured by inclusion/exclusion of two fluorogenic vital dyes, was 71% in comparison with that detected for oocysts of the Colombia isolate, 32.3%. The bovine C. parvum oocysts of both isolates were viable and infectious for suckling Swiss CD-1 mice. However, infectivity percentage and the mean intensity of infection were consistently higher in the Spain isolate than those from Colombia isolate. It was not possible to obtain a good correlation between in vitro excystation, inclusion/exclusion of vital dyes and in vivo infectivity for the Colombia isolate, while data obtained with the Spain isolate indicated that there was an apparent strong correlation between excystation efficiency, total viability and the infectivity. Although a comparative analysis of genetic variation among these isolates from different geographical location is necessary, variations observed between the both isolates seemed to be a result of parasite adaptation to environmental stresses such as temperature which appears to have a direct effect on the permeability of the oocysts.     

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 study and genotyping of Cryptosporidium baileyi and Cryptosporidium parvum from free-range and commercial broiler chickens in Guilan province,Iran

Cryptosporidiosis acutely impacts the digestive and/or respiratory tract of the birds in many species of various orders. More importantly, it is also well known as a significant zoonotic disease, which can lead to diarrhea in humans and livestock. Regarding increasing demand for free-range products and increasing the number of free-range poultry farms, the present paper evaluated histopathological and molecular detection of Cryptosporidium baileyi and Cryptosporidium parvum in free-range and commercial broiler chickens in the north part of Iran. For this purpose, 100 fecal and tissue samples of the chickens in Guilan province were collected. After microscopic examination using Ziehl-Neelsen staining, molecular analyses of the fecal samples were processed by Nested-PCR targeting the 18S rRNA gene followed by sequencing of the amplicons and phylogenetic analyses. Eventually, the tissue samples were studied for histological lesions. Findings demonstrated the presence of Cryptosporidium baileyi and Cryptosporidium parvum in 6 % and 2 % of fecal samples, respectively. This is the first identification of C.parvum in avian hosts in Iran, and for the first time, C.baileyi and C.parvum are shown in native free-range chickens in Iran. All of the PCR positive birds with clinical symptoms showed gross lesions of respiratory infections. There was no significant difference between infection rate in free-range and commercial broiler chickens; however, the infection rate was significantly higher in chickens <25 days old. To conclude, we present here a notable Cryptosporidium infection rate in the free-range chicks in Iran, which notify the role of this host as a reservoir and should be more noted due to the economic and zoonotic importance.     

猴源人隐孢子虫的分离与鉴定

为了解感染不同灵长类动物的隐孢子虫种类以及与感染人的人隐孢子虫(C.hominis)之间的遗传差异,本研究采用形态学和分子生物学方法对猴源隐孢子虫进行分离和鉴定。利用常规方法分离猴粪便中的隐孢子虫卵囊,通过改良抗酸染色和荧光显微镜观察对其进行形态学鉴定;并采用PCR方法扩增其卵囊壁蛋白(COWP)基因和18SrRNA基因,扩增产物克隆至pMD-18-T载体中,对阳性克隆进行测序并作进化树分析。结果表明:抗酸染色和荧光检查结果与所报道的人隐孢子虫的结果一致。PCR扩增产物经电泳检测,明显地出现554bp和370bp大小的片段,与预期结果一致;两种基因的序列分析结果显示该猴源隐孢子虫与C.hominis的相似性均为100%。由此可认为本次分离的隐孢子虫为C.hominis。     

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.     

Prevalence and characterization of multidrug resistance and variant Salmonella genomic island 1 in Salmonella isolates from cattle,poultry and humans in Iran

Salmonella enterica is a common food‐borne pathogen with occasional multidrug resistance (MDR). Salmonella genomic island (SGI1) is a horizontally transmissible genomic island, containing an MDR gene cluster. All Salmonella serotypes are public health concern, although there is an additional concern associated with those that harbour SGI1. In Iran, there are no data on the presence of SGI1 variants in Salmonella isolates. The present study was conducted to identify MDR‐ and SGI1‐carrying Salmonella strains isolated from various sources and to compare their genetic relatedness between human and animal sources. In total, 242 Salmonella isolates collected from chicken, cattle, and humans from 2008 through 2014 were studied. The isolates were tested for resistance to 14 antimicrobials via the disc diffusion method. They were also tested for the presence of SGI1 variants via PCR, and genetic relatedness was evaluated based on pulsed‐field gel electrophoresis (PFGE). Resistance to at least one antimicrobial agent was observed in 132 (54%) Salmonella isolates (n = 242), while more than 40% of the isolates showed MDR. Based on PCR analysis, eight variants of SGI1, including SGI1, SGI1‐B, SGI1‐C, SGI1‐D, SGI1‐F, SGI1‐I, SGI1‐J and SGI1‐O, were found in both human and animal isolates. Statistical analysis revealed no significant difference in the prevalence of SGI1 variants between human and animal isolates (p > 0.05). Macrorestriction PFGE analysis of the isolates with the same SGI1 variant and resistance patterns revealed genetic relatedness ranging from 70% to 100% among human and animal isolates. According to our review, this is the first documentation of SGI1 in Salmonella isolates in Iran. The presence of similar SGI1 variants in both humans and animals, along with their related PFGE patterns, suggests that food‐producing animals may be a source of MDR Salmonella isolates in Iran.     

Infection with Cryptosporidium spp. in humans and cattle in Manitoba.

Between October 1, 1983 and October 31, 1984, fecal specimens from 3656 persons with enteritis and 182 calves, representing 148 herds having a neonatal diarrhea problem, were examined for oocysts of Cryptosporidium spp. Oocysts were found in 1% of human and 25% of bovine specimens. All infected persons were immunocompetent. Children under five years of age had an infection rate of 25/100,000 compared to 1.4/100,000 in older people (p less than 0.005). Rates in northern communities were four to seven times as high as those in southern Manitoba. Human infections occurred most commonly in late summer and fall. In beef calves infection occurred in winter and spring, the calving season in Manitoba. Epidemiological association between the infection in people and in cattle could not be established.     

Genotyping of Cryptosporidium parvum isolates in bovine population in Kolkata and characterization of new bovine genotypes

Cryptosporidium infection may have adverse effect in health and production potential of cattle herd. The exact profile of Cryptosporidium infection in bovine population of India in general, particularly from Kolkata is scarce. We here report systematic investigation of clinical and genetic profiling of promiscuous Cryptosporidium infection in selected representative cattle farms from Kolkata as well as some surrounding local areas. The current study was conducted in the period of October to September, 2000–2001 with 149 diarrhoeic and non-diarrhoeic cattle of different age groups from two Government cattle farms, Harringhata Cattle Unit and Kalyani State Livestock Farm and animals raised by local farmers. Among these 149 samples, diarrhoea was recorded in 79 cases (53%) and non-diarrhoeic in 70 (46.9%). Out of 149 faecal samples screened microscopically, 32.9% from diarrhoeic faecal samples and 7.1% from healthy faecal samples revealed the presence of oocysts. Cryptosporidium genus was confirmed by DNA typing with nested PCR. The PCR-RFLP analysis was carried out for genotype identification. In course of PCR-RFLP, unique band patterns were obtained in two of our samples. The unusual RFLP products were characterized by DNA sequencing and homology analysis with other reported variants. This is the first report of identification and characterization of such a variant from the area of present investigation. Further study will be required to understand the phylogenetic origin and functional significance in virulence and morbidity of this genotype.     

Incidence of Cryptosporidium parvum in the dairy cattle population in a New York City Watershed

A longitudinal study of 2-year duration was conducted to determine the risk, as measured by incidence rate, of Cryptosporidium parvum infection among dairy cattle in the Catskill/Delaware Watershed of New York City (NYC), and the factors that predispose animals to the likelihood of infection. A proportional sampling scheme with follow up at quarterly farm visits was employed for heifers and cows. Additionally, all calves born on the 39 study farms were sampled once during the first four weeks of life and at least once more before weaning. Samples were analyzed for the presence of C. parvum using a quantitative centrifugation concentration flotation technique and a C. parvum-specific enzyme-linked immunosorbent assay (ELISA). Of the 9914 fecal samples collected, 747 were found to contain C. parvum. The average number of oocysts detected was 1.3x10(5)/g (range: 1.0/g--8.2x10(6)/g). The average age at time of first detection of the organism was 15.0 days with a standard deviation of 6.59 days. The age range of animals infected with C. parvum in the study population was 3--60 days (inclusive). The unadjusted (crude) incidence rate of C. parvum among the entire study population was 2.05 per 1000 animal-days. The unadjusted incidence rate among pre-weaned calves was 15.55 per 1000 animal-days. After controlling for age and prior protozoal risk level, no seasonal impact on the incidence of C. parvum was detected among animals less than 61 days by negative binomial regression. A seasonal impact was identified among the oocyst counts of infected animals after controlling for age and prior protozoal risk level.     

The homologous and interspecies transmission of Cryptosporidium parvum and Cryptosporidium meleagridis

Experimental infections have been performed in several mammals and birds to determine the host's specificity for Cryptosporidium parvum (of human and bovine origin) and for Cryptosporidium meleagridis (isolated from broiler chicken). The C. parvum infection (of human origin) was established also in calves (Bos taurus), lambs (Ovis aries), mice (Mus musculus), and rats (Ratus norvegicus), but not in broiler chickens (Gallus domestica). The C. parvum infection (of bovine origin), was achieved in calves, lambs, dogs (Canis familiaris), cats (Felis domesticus), rabbits (Orytolagus cuniculus), mice, rats, and guinea-pigs (Cavia porcelus) but not in broiler chickens. We have demonstrated that C. meleagridis can produce infection in broiler chickens and in 5 mammalian species (calves, pigs, rabbits, rats, mice) but not in guinea-pigs.     

Genetic analysis of Akabane virus isolates from cattle in Korea

Bayesian Inference (BI) and Neighbor Joining (NJ) analyses of the phylogenetic relationships between the nucleotide sequences of the N gene of Akabane virus revealed an unclear topology among genogroups I–III, which was probably caused by genetic reassortment or recombination between these genogroups. In contrast, nucleotide and amino acid phylogenetic tree analyses of the M RNA segment agreed with the topologies obtained by using the BI and NJ methods. Therefore, distinct genogrouping of Akabane virus isolates should be performed using the M RNA segment. Four Korea isolates were classified into genogroup II together with Akabane virus strains isolated from all areas of Japan, including Okinawa Island. However, more nationwide isolates and more clinical data from Korean cattle farms will be required in the future to confirm the precise relationships between genotypes and pathogenicity.     

Cryptosporidium parvum in cattle, sheep and pigs in Galicia (N.W. Spain).

Infection by Cryptosporidium parvum has been detected for the first time in cattle and swine in Galicia (N.W. Spain). The organisms were also found in one of 69 sheep examined. Although in most cases the parasite was found in young diarrhoeic animals, its presence in asymptomatic mature cattle and piglets was also detected.     

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.