Identification of a Novel Cryptosporidium Genotype in Pigs

Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp. by microscopy. Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium. Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001). Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status.     

Prevalence of Cryptosporidium genotypes in pre and post-weaned pigs in Australia

A total of 289 pig faecal samples were collected from pre-weaned and post-weaned piglets and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia and screened at the 18S rRNA locus for the presence of Cryptosporidium. An overall prevalence of 22.1% (64/289) was identified. Cryptosporidium was more prevalent in post-weaned animals (p < 0.05); 32.7% (51/156) versus 10.6% (13/123) for pre-weaned animals. Sequence analysis identified Cryptosporidium suis in all pre-weaned isolates genotyped (7/13). In post-weaned pigs that were genotyped (n = 38), the non-zoonotic Cryptosporidium species, pig genotype II was identified in 32 samples and C. suis in 6 samples. The zoonotic species Cryptosporidium parvum was not detected, suggesting that domestic pigs do not pose a significant public health risk. Pig genotype II was significantly (p < 0.05) associated with ‘normal’ stools, indicating an asymptomatic nature in the porcine host.     

Cryptosporidium pig genotype II diagnosed in pigs from the state of Rio De Janeiro, Brazil

Pigs may represent a source of Cryptosporidium sp. infection to humans. The objective of this study was to identify the Cryptosporidium species present in pigs from the State of Rio de Janeiro, Brazil, and verify what risks pigs represent in the transmission of human cryptosporidiosis, because there is no such information to date in Brazil. Ninety-one samples of pig feces were collected from 10 piggeries in 2 municipalities located in the north and northwest regions of the State of Rio de Janeiro, Brazil. A nested polymerase chain reaction (PCR) protocol to amplify an 830-bp fragment of the small subunit rDNA (SSU rRNA) gene was followed by sequencing of all positive PCR samples. Two samples (2.2%) were Cryptosporidium sp. positive and were identified as pig genotype type II (PGII). This genotype has been observed in an immunocompetent person, in cattle without pigs nearby, and from a potential human source. Its potential for zoonotic transmission is little known and should be rigorously studied.     

Cryptosporidium species: preliminary descriptions of the prevalence and genotype distribution among school children and hospital patients in the Venda region, Limpopo Province, South Africa

In the present study, the prevalence and species distribution of Cryptosporidium among school children and hospital patients in the Venda region of South Africa was determined. Real time PCR (qPCR) was used for initial screening to detect positive samples while a nested PCR followed by restriction fragment length polymorphism was used to determine the species genotype. From a total of 244 stool samples tested, 44 (18%) had Cryptosporidium with no significant difference (chi(2)=0.04; P=0.841) between samples collected from patients attending hospitals 36/197 (18%) and the samples from primary schools 8/47 (17%). The age groups most affected were those from 2 to 5 years old (28.6%) and 50 to 59 years old (50.0%). Cryptosporidium was detected in 4 (12.5%) of the 31 HIV positive individuals. Fifty-seven percent of the Cryptosporidium positive samples were diarrheic and 26 (59.1%) had elevated lactoferrin content. C. hominis (82%) was more common than C. parvum (18%). This study has demonstrated the high prevalence of Cryptosporidium infections in the Venda region and its implications in causing diarrhea and inflammation.     

Cryptosporidium spp. from small mammals in the New York City watershed

The objective of this study was to assess the potential role that wildlife plays in environmental degradation of watersheds through the contamination of the water supply with zoonotic genotypes of Cryptosporidium. Cryptosporidium isolates recovered from wildlife in the New York City (NYC) watershed were examined to determine genotype using a polymerase chain reaction protocol targeting the 18-Small Subunit (SSU) rRNA locus. Seventy-seven DNA samples recovered from 12 wildlife host species captured in the NYC watershed were amplified and sequenced. Data on risk factors associated with the perpetuation of these genotypes also were collected and analyzed. Although many genotypes appeared to be host-specific, 38% of the samples examined were identified as Cryptosporidium parvum, indicating the presence of zoonotic Cryptosporidium. Adult animals were more likely to shed the zoonotic strains of Cryptosporidium spp. Animals captured in the fall and winter were more likely to be infected with C. parvum than those captured in spring and summer.     

Profiles of airborne fungi in buildings and outdoor environments in the United States

We examined 12,026 fungal air samples (9,619 indoor samples and 2,407 outdoor samples) from 1,717 buildings located across the United States; these samples were collected during indoor air quality investigations performed from 1996 to 1998. For all buildings, both indoor and outdoor air samples were collected with an Andersen N6 sampler. The culturable airborne fungal concentrations in indoor air were lower than those in outdoor air. The fungal levels were highest in the fall and summer and lowest in the winter and spring. Geographically, the highest fungal levels were found in the Southwest, Far West, and Southeast. The most common culturable airborne fungi, both indoors and outdoors and in all seasons and regions, were Cladosporium, Penicillium, nonsporulating fungi, and Aspergillus. Stachybotrys chartarum was identified in the indoor air in 6% of the buildings studied and in the outdoor air of 1% of the buildings studied. This study provides industrial hygienists, allergists, and other public health practitioners with comparative information on common culturable airborne fungi in the United States. This is the largest study of airborne indoor and outdoor fungal species and concentrations conducted with a standardized protocol to date.     

Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.     

Molecular identification and prevalence of Isospora sp. in pigs in Western Australia using a PCR-RFLP assay

Little is known about the prevalence of Isospora in domestic pigs in Western Australia. A total of 289 pig faecal samples were collected from pre- and post-weaned pigs and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia. Faecal samples were screened using a PCR-RFLP assay based on the ITS-1 rDNA locus. An overall prevalence of 10.4% (30/289) was identified. Isospora was detected in 16.3% (20/123) of pre-weaned animals and 6.4% (10/156) of post-weaned animals. PCR-RFLP analysis confirmed the presence of Isospora suis in 86.7% of the positive Isospora isolates. Isospora was significantly associated with diarrhea and the findings of this study suggest that management factors such as cleaning practices, flooring types and stocking densities need to be investigated in the porcine host to find new and improved measures for control.     

Detection of Cryptosporidium oocysts in water: effect of the number of samples and analytic replicates on test results

Due to the small number of Cryptosporidium oocysts in water, the number of samples taken and the analyses performed can affect the results of detection. In this study, 42 water samples were collected from one watershed during 20 storm events over 1 year, including duplicate or quadruplicate samples from 16 storm events. Ten samples from four events had three to eight subsamples. They were processed by EPA method 1623, and Cryptosporidium oocysts present were detected by immunofluorescent microscopy or PCR. Altogether, 24 of 39 samples (47 of 67 samples and subsamples) analyzed by microscopy were positive for Cryptosporidium. In contrast, 36 of 42 samples (62 of 76 samples and subsamples) were positive by PCR, including 10 microscopy-negative samples (13 microscopy-negative samples and subsamples). Six of the 24 microscopy-positive samples were negative by PCR, and all samples had one or less oocyst in a 0.5-ml packed pellet volume calculated. Discordant results were obtained by microscopy and PCR from six and three of the storm events, respectively, with multiple samples. Discordant microscopy or PCR results were also obtained among subsamples. Most of the 14 Cryptosporidium genotypes were found over a brief period. Cryptosporidium-positive samples had a mean of 1.9 genotypes per sample, with 39 of the 62 positive samples/subsamples having more than one genotype. Samples/subsamples with more than one genotype had an overall PCR-positive rate of 73%, compared to 34% for those with one genotype. The PCR amplification rate of samples was affected by the volume of DNA used in PCR.     

Profiles of Airborne Fungi in Buildings and Outdoor Environments in the United States

We examined 12,026 fungal air samples (9,619 indoor samples and 2,407 outdoor samples) from 1,717 buildings located across the United States; these samples were collected during indoor air quality investigations performed from 1996 to 1998. For all buildings, both indoor and outdoor air samples were collected with an Andersen N6 sampler. The culturable airborne fungal concentrations in indoor air were lower than those in outdoor air. The fungal levels were highest in the fall and summer and lowest in the winter and spring. Geographically, the highest fungal levels were found in the Southwest, Far West, and Southeast. The most common culturable airborne fungi, both indoors and outdoors and in all seasons and regions, were Cladosporium, Penicillium, nonsporulating fungi, and Aspergillus. Stachybotrys chartarum was identified in the indoor air in 6% of the buildings studied and in the outdoor air of 1% of the buildings studied. This study provides industrial hygienists, allergists, and other public health practitioners with comparative information on common culturable airborne fungi in the United States. This is the largest study of airborne indoor and outdoor fungal species and concentrations conducted with a standardized protocol to date.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Detection of methicillin-resistant Staphylococcus aureus in Iberian pigs

Aims: Iberian pigs are bred in Spain for the production of high‐value dry‐cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin‐resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. Methods and Results: From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. Conclusions: Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. Significance and Impact of the Study: To our knowledge, this is the first detection of MRSA in food animals raised in free‐range conditions.     

The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.     

Cryptosporidiosis in an urban community.

Over three months Cryptosporidium oocysts were identified in faecal samples from 43 (5%) of 867 patients presenting to their general practitioners with gastrointestinal symptoms. Cryptosporidium was the second most common enteric pathogen identified. Of the 867 patients, 329 were children aged under 5, of whom 24 (7%) excreted Cryptosporidium. A characteristic clinical presentation of infection with Cryptosporidium was recognised--namely, mild gastroenteritis with four to six watery, mucoid, and offensive motions a day, which lasted for one to two weeks. The source of infection was not identified, but direct contact with farm animals was not a feature and no association with a common water supply could be established.     

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.     

Prevalence of Cryptosporidium infection in cattle in Isfahan, Iran

Cryptosporidium are commonly identified as intestinal pathogens in humans and animals. Fecal samples from 480 cattle randomly selected from 30 regions in Isfahan, Iran, were examined to investigate the prevalence of Cryptosporidium infection. Cryptosporidium oocysts were identified by using sheather's concentration and the Ziehl-Neelsen modified staining technique in 30 of 480 cattle ranging from less than 6 mo of age to older than 1 yr of age. Infected animals were found in 86.6% (26/30) of regions investigated. Overall prevalence of infection was 6.25%, but higher in cattle less than 6 mo of age (10.8%) and this was statistically significant (P<0.05). Both sexes of cattle were infected with Cryptosporidium parasites, but prevalences were higher in diarrheic (56.7%) than in non-diarrheic (39%) cattle. Cryptosporidium appears to be prevalent in cattle in Isfahan.     

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.     

Cryptosporidium Species and Genotypes in HIV-Positive Patients in Lima, Peru

ABSTRACT: Cryptosporidium parasites from a cross-sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to deteimine the diversity of Cryptosporidium spp. in HIV-positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium - positive specimens from 300 patients using a small subunit rRNA-based PCR-RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV-positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.     

Genotypes of Cryptosporidium from Sydney water catchment areas

AIMS: Currently cryptosporidiosis represents the major public health concern of water utilities in developed nations and increasingly, new species and genotypes of Cryptosporidium are being identified in which the infectivity for humans is not clear. The complicated epidemiology of Cryptosporidium and the fact that the majority of species and genotypes of Cryptosporidium cannot be distinguished morphologically makes the assessment of public health risk difficult if oocysts are detected in the raw water supplies. The aim of this study was to use molecular tools to identify sources of Cryptosporidium from the Warragamba catchment area of Sydney, Australia. METHODS AND RESULTS: Both faecal and water samples from the catchment area were collected and screened using immunomagnetic separation (IMS) and immunofluorescence microscopy. Samples that contained Cryptosporidium oocysts were genotyped using sequence and phylogenetic analysis of the 18S rDNA, and the heat-shock (HSP-70) gene. Analysis identified five Cryptosporidium species/genotypes including C. parvum (cattle genotype), C. suis, pig genotype II, the cervid genotype and a novel goat genotype. CONCLUSIONS: Monitoring and characterization of the sources of oocyst contamination in watersheds will aid in the development and implementation of the most appropriate watershed management policies to protect the public from the risks of waterborne Cryptosporidium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that quantification by IMS analysis can be combined with the specificity of genotyping to provide an extremely valuable tool for assessing the human health risks from land use activities in drinking water catchments.