Giardia genotypes in pigs in Western Australia: Prevalence and association with diarrhea

Little is known of the prevalence of Giardia species and genotypes in pre- and post-weaned domestic pigs. In the present study, a total of 297 pig faecal samples were screened for the presence of Giardia by PCR and genotyped. An overall prevalence of 31.1% (90/289) (25.8, 36.5 CI) was detected. Giardia was detected in 17% (23/123) (11.8-25.6 CI) of pre-weaned piglet faecal samples and 41% (64/156) (33.3-48.7 CI) post-weaned faecal samples analysed. Sequence analysis identified assemblage A and E in pre- and post-weaned pigs. Assemblage F was identified in one post-weaned pig. Assemblage E was the most prevalent assemblage detected.     

Cryptosporidium suis Infection in Post-Weaned and Adult Pigs in Shaanxi Province,Northwestern China

Cryptosporidium spp., ubiquitous enteric parasitic protozoa of vertebrates, recently emerged as an important cause of economic loss and zoonosis. The present study aimed to determine the distribution and species of Cryptosporidium in post-weaned and adult pigs in Shaanxi province, northwestern China. A total of 1,337 fresh fecal samples of post-weaned and adult pigs were collected by sterile disposable gloves from 8 areas of Shaanxi province. The samples were examined by Sheather’s sugar flotation technique and microscopy at×400 magnification for Cryptosporidium infection, and the species in positive samples was further identified by PCR amplification of the small subunit (SSU) rRNA gene. A total of 44 fecal samples were successfully amplified by the nested PCR of the partial SSU rRNA, with overall prevalence of 3.3%. The average prevalence of Cryptosporidium infection in each pig farms ranged from 0 to 14.4%. Species identification by sequencing of SSU rRNA gene revealed that 42 (3.1%) samples were Cryptosporidium suis and 2 (0.15%) were Cryptosporidium scrofarum. C. suis had the highest prevalence (7.5%) in growers and the lowest in breeding pigs (0.97%). C. suis was the predominant species in pre-weaned and adult pigs, while C. scrofarum infected pigs older than 3 months only. A season-related difference of C. suis was observed in this study, with the highest prevalence in autumn (5.5%) and the lowest (1.7%) in winter. The present study provided basic information for control of Cryptosporidium infection in pigs and assessment of zoonotic transmission of pigs in Shaanxi province, China.     

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.     

Prevalence and Genetic Characterizations of Cryptosporidium spp. in Pre-Weaned and Post-Weaned Piglets in Heilongjiang Province,China

Background

Cryptosporidium spp. are common intestinal protozoa of humans and animals. There have been few studies conducted on the molecular characterizations of pig-derived Cryptosporidium isolates worldwide, especially in China. Thus, the aim of the present study was to understand the prevalence, distribution and genotypes of Cryptosporidium in pigs in Heilongjiang Province, China.

Methodology/Principal Findings

A total of 568 fecal samples from pre-weaned and post-weaned piglets were collected from eight pig farms from four areas of Heilongjiang Province. The average infection rate of Cryptosporidium was 1.6% (9/568) by microscopy. 113 samples were subjected to PCR amplification of the small subunit (SSU) rRNA gene of Cryptosporidium, with 55.8% (63/113) being positive for Cryptosporidium. Cryptosporidium suis (n = 31) and C. scrofarumn (n = 32) were identified by DNA sequencing of the SSU rRNA gene. Three types of C. scrofarumn were found at the SSU rRNA locus, with one novel type being detected. Using species/genotype-specific primers for pig-adapted Cryptosporidium spp., 22 and 23 respectively belonged to C. suis and C. scrofarum mono-infections, with 18 co-infections detected. The infection peaks for C. suis (60%, 24/40) and C. scrofarum (51.2%, 21/41) were respectively found in the piglets of 5 to 8 weeks and more than 8 weeks.

Conclusion/Significance

The detection of C. suis and C. scrofarum in pre-weaned and post-weaned piglets has public health implications, due to the fact that the two species are both zoonotic Cryptosporidium. The novel C. scrofarum type detected may be endemic to China.     

High prevalence of Cryptosporidium infection caused by C. scrofarum and C. suis among pigs in Thailand

Cryptosporidium spp. is an important intestinal protozoan causing diarrhea among both healthy and immunocompromised patients especially those with HIV/AIDS. Cryptosporidium spp. can be transmitted via foodborne, waterborne and person-to-person routes. In addition, several Cryptosporidium species are zoonotic. This study aimed to determine the prevalence of Cryptosporidium infection among pigs raised in both smallholder (<50 heads/farm) and large scale farms (50–500 heads/farm) in Chonburi Province, eastern Thailand using nested PCR amplifying the small subunit of the ribosomal RNA (SSU-rRNA) gene. DNA sequencing was also performed to identify the species of Cryptosporidium. A total of 245 fecal samples were collected from 11 pig farms. The overall prevalence of Cryptosporidium infection was 20.8% (51/245) which were found in both smallholder and small large scale pig farms. The prevalence of Cryptosporidium infection among pigs aged ≤6 months was significantly higher than those aged >6 months (p < .001). Among 51 Cryptosporidium positive samples, Cryptosporidium scrofarum (42/51, 82.4%) and Cryptosporidium suis (9/51, 17.6%) were identified. The prevalence of C. scrofarum infection observed among pigs aged ≤6 months was significantly higher when compared with those aged >6 months (20.7% and 2.1%, respectively, p < .001). The high prevalence of C. scrofarum and C. suis infections among pigs could be a potential source of infection to humans.     

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.     

Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008

Cryptosporidium is a gastrointestinal parasite that is recognised as a significant cause of non-viral diarrhea in both developing and industrialised countries. In the present study, a longitudinal analysis of 248 faecal specimens from Australian humans with gastrointestinal symptoms from 2005 to 2008 was conducted. Sequence analysis of the 18S rRNA gene locus and the 60 kDa glycoprotein (gp60) gene locus revealed that 195 (78.6%) of the cases were due to infection with Cryptosporidium hominis, 49 (19.8%) with Cryptosporidium parvum and four (1.6%) with Cryptosporidium meleagridis. A total of eight gp60 subtype families were identified; five C. hominis subtype families (Ib, Id, Ie, If and Ig), and two C. parvum subtype families (IIa and IId). The Id subtype family was the most common C. hominis subtype family identified in 45.7% of isolates, followed by the Ig subtype family (30.3%) and the Ib subtype family (20%). The most common C. parvum subtype was IIaA18G3R1, identified in 65.3% of isolates. The more rare zoonotic IId A15G1 subtype was identified in one isolate. Statistical analysis showed that the Id subtype was associated with abdominal pain (p < 0.05) and that in sporadic cryptosporidiosis, children aged 5 and below were 1.91 times and 1.88 times more likely to be infected with subtype Id (RR 1.91; 95% CI, 1.7-2.89; p < 0.05) and Ig (RR 1.88; 95% CI, 1.10-3.24; p < 0.05) compared to children aged 5 and above. A subset of isolates were also analysed at the variable CP47 and MSC6-7 gene loci. Findings from this study suggest that anthroponotic transmission of Cryptosporidium plays a major role in the epidemiology of cryptosporidiosis in Western Australian humans.     

Concentrations,Viability, and Distribution of Cryptosporidium Genotypes in Lagoons of Swine Facilities in the Southern Piedmont and in Coastal Plain Watersheds of Georgia

Waste lagoons of swine operations are a source of Cryptosporidium oocysts. Few studies, however, have reported on oocyst concentrations in swine waste lagoons; none have reported on oocyst viability status, nor has there been a systematic assessment of species/genotype distributions across different types of swine facilities. Ten swine waste lagoons associated with farrowing, nursery, finishing, and gestation operations were each sampled once a month for a year. Oocysts were extracted from triplicate 900-ml effluent samples, enumerated by microscopy, and assessed for viability by dye exclusion/vital stain assay. DNA was extracted from processed samples, and 18S ribosomal DNA (rDNA) genes were amplified by PCR and sequenced for species and genotype identification. Oocysts were observed at each sampling time at each lagoon. Annual mean concentrations of total oocysts and viable oocysts ranged between 24 and 51 and between 0.6 and 12 oocysts ml⁻¹ effluent, respectively. The species and genotype distributions were dominated (95 to 100%) by Cryptosporidium suis and Cryptosporidium pig genotype II, the latter of which was found at eight of the lagoons. The lagoon at the gestation facility was dominated by Cryptosporidium muris (90%), and one farrowing facility showed a mix of pig genotypes, Cryptosporidium muris, and various genotypes of C. parvum. The zoonotic C. parvum bovine genotype was observed five times out of 407 18S rDNA sequences analyzed. Our results indicate that pigs can have mixed Cryptosporidium infections, but infection with C. suis is likely to be dominant.Over the last few decades, pork production in North America has undergone significant growth and centralization into large concentrated swine (Sus scrofa) operations with more animals on fewer farms (18). A consequence of the increase in numbers of swine per facility is a concomitant increased concentration of swine waste. Present housing facilities for swine are designed to collect feces and urine in wastewater lagoons, in which the waste undergoes anaerobic transformations. One of several public health concerns over swine lagoons is the potential presence of infectious bacteria, viruses, and protozoa (4). Because of the notoriety given to swine waste lagoon spills in the coastal flood plain of North Carolina that were associated with a series of hurricanes in 1998 and 1999 (21), large-scale swine operations have become a focus of environmental and public health concerns.The cause of the massive outbreak of cryptosporidiosis in Milwaukee, WI, in 1993 was afterwards determined to be Cryptosporidium hominis, the human genotype of C. parvum and an obligate parasite of humans (33, 44). At the time, however, it was thought to be caused by C. parvum (22). Because of this initial misidentification of the cryptosporidial source of the outbreak, the connection between C. parvum and large-scale confined livestock operations has become a focused area of research. Although manure-associated outbreaks of C. parvum have implicated bovine sources, a Canadian study found that the prevalence of Cryptosporidium in swine lagoons was greater than that in dairy liquid manure (9). Olson et al. (24) also reported the presence of Cryptosporidium oocysts of undetermined genotype at four of six hog operations in Canada. Atwill et al. (2) observed C. parvum oocysts in feces of feral pigs. Hutchison et al. (13) observed C. parvum oocysts of undetermined genotype in 5 and 13% of fresh and stored fecal samples, respectively, from pigs of undeclared age. Guselle et al. (10) followed the course of a naturally occurring C. parvum infection in 33 weaned pigs. Following the protocol of the genetic analysis of Morgan et al. (23), Guselle et al. (10) identified this C. parvum genotype as being adapted to pigs. At the time, the zoonotic potential of this C. parvum pig-adapted genotype was considered uncertain (23).Recently, two genotypes of Cryptosporidium have been recognized as host adapted to swine: Cryptosporidium suis (formerly Cryptosporidium pig genotype I) and Cryptosporidium pig genotype II (28, 29). Xiao et al. (37) reported on an immunocompromised person who was infected with a Cryptosporidium pig genotype and thus implicated Cryptosporidium from swine as potentially zoonotic and a public health concern. Before molecular methods were developed to differentiate pig genotypes of Cryptosporidium from other species, C. parvum was thought to infect 152 species of mammals and consist of several cryptic species (6). An extensive survey of swine effluent from swine finishing operations in Ireland indicated a prevalence of both C. suis and Cryptosporidium pig genotype II (39). Hamnes et al. (11) reported prevalence of both C. suis and Cryptosporidium pig genotype II in feces of suckling pigs across Norway and thus implicated farrowing operations as sources of this parasite.Other than the prevalence of Cryptosporidium in feces of young pigs and effluent lagoons of older pigs in finishing operations, little comprehensive data on oocyst concentrations, viability of oocysts, and distributions of Cryptosporidium species and genotypes have been reported. No systematic study of swine lagoon effluents from large-scale facilities has been reported for the four separate stages of swine development, (i) breeding and gestation, (ii) farrowing (parturition), (iii) nursery (in which weaned piglets are kept until 8 to 9 weeks of age), and (iv) finishing (in which 8- to 9-week-old pigs are kept to market weight). The objective of this investigation was to determine for 1 year the frequencies, concentrations, viability statuses, and distributions of Cryptosporidium species and genotypes in lagoons associated with the four types of swine operations in the Southern Piedmont and in coastal plain watersheds of Georgia.     

Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China

Neonatal diarrhea is one of the most important syndromes in dairy cattle. Among enteropathogens, Cryptosporidium spp. are primary causes of diarrhea, but outbreaks due to cryptosporidiosis are rarely reported in cattle. From January to April in 2016, severe diarrhea was observed in over 400 neonatal dairy calves on a large dairy farm in Jiangsu Province of East China. Approximately 360 calves died due to watery diarrhea despite antibiotic therapy. In this study, 18 fecal specimens were collected from seriously ill calves on this farm during the diarrhea outbreak, and analysed for common enteropathogens by enzymatic immunoassay (EIA). In a post-outbreak investigation, 418 and 1372 specimens collected from animals of various age groups were further analysed for rotavirus and Cryptosporidium spp. by EIA and PCR, respectively, to assess their roles in the occurrence of diarrhea on the farm. Cryptosporidium spp. were genotyped using established techniques. Initial EIA tests showed that 15/18 seriously ill calves during the outbreak were positive for Cryptosporidium parvum, while 8/18 were positive for rotavirus. The overall infection rate of Cryptosporidium in pre-weaned calves on the farm was 22.7%, with odds of the Cryptosporidium infection during the outbreak 4.4–23.5 times higher than after the outbreak. Four Cryptosporidium spp. were identified after the outbreak including C. parvum (n = 79), Cryptosporidium ryanae (n = 48), Cryptosporidium bovis (n = 31), and Cryptosporidium andersoni (n = 3), with co-infections of multiple species being detected in 34 animals. Infection with C. parvum (73/79) was found in the majority of calves aged ≤3 weeks, consistent with the age of ill calves during the outbreak. All C. parvum isolates were identified as subtype IIdA19G1. In the post-outbreak investigation, C. parvum infection was associated with the occurrence of watery diarrhea in pre-weaned calves, C. ryanae infection was associated with moderate diarrhea in both pre- and post-weaned calves, while no association was identified between rotavirus infection and the occurrence of diarrhea. Results of logistic regression analysis further suggested that C. bovis infection might also be a risk factor for moderate diarrhea in calves. Thus, we believe this is the first report of a major outbreak of severe diarrhea caused by C. parvum IIdA19G1 in dairy calves. More attention should be directed toward preventing the dissemination of this virulent subtype in China.     

Prevalence and genotypes of Cryptosporidium in livestock in Hualien Country,Eastern Taiwan

Cryptosporidium spp. is a group of protozoans that cause diarrheal disease in both humans and animals. In Taiwan, very little information is available about the epidemiology of Cryptosporidium spp. in domesticated animals, especially in Eastern Taiwan where agriculture is one of the main industries. Therefore, this study aimed to investigate the occurrence of Cryptosporidium spp. in livestock in Hualien Country of Eastern Taiwan and identify their genotypes. Excrements from dogs (n = 81), cattle (n = 156), and pigs (n = 142) were randomly collected from different pastures or farm in Hualien Country. Microscopic examination and nested PCR were performed on all samples and both showed identical results, with 4.94% (4/81) of dogs, 24.36% (38/156) of cattle, and 16.20% (23/142) of pigs being infected with Cryptosporidium species. Positive samples were then sequenced and analyzed. DNA sequencing revealed that all four positive samples isolated from dogs were Cryptosporidium canis (C. canis); 38 positive samples from cattle were identified as C. bovis (8/38), C. canis (1/38), C. ryanae (4/38), and C. scrofarum (25/38); and 22 positive samples isolated from pigs were identified as C. scrofarum while one was identified as C. suis. In addition, the infective rates of animals from indoor farms (57.14% of all positive samples) are much higher than the rates from pastures. This study provided evidence of the occurrence of Cryptosporidium spp. in Hualien country, and farming conditions largely affect their infection rates. Therefore, precautions should be made to control Cryptosporidium spp. transmission.     

Prevalence of and management factors contributing to Cryptosporidium sp. infection in pre-weaned and post-weaned calves in Johor, Malaysia

A cross-sectional study was carried out to identify species and determine the prevalence of Cryptosporidium sp. shedding in pre-weaned and post-weaned dairy calves and to identify management factors that may be contributing to disease. A total of 240 calf faecal samples were collected from 16 farms in two districts in Johor, Malaysia, and screened by PCR. The overall Cryptosporidium prevalence was 27.1%. The prevalence of Cryptosporidium species in pre-weaned calves was 32.4% for C. parvum, 26.5% for C. bovis, followed by C. andersoni (20.6%), C. ryanae (11.8%) and mixed sp. (8.8%). The prevalence of Cryptosporidium species in post-weaned calves was 35% for C. bovis followed by C. andersoni and C. ryanae (30% each) and mixed sp. (5%). Subtyping analysis of 8 of the 11 C. parvum isolates at the gp60 locus identified five isolates as IIdA15G1, one as IIa18A3R1 and two isolates as IIa17G2R1. Management factors that increased the risk of Cryptosporidium infection included having other cattle farms close by, feeding calves with saleable milk, keeping pre-weaned calves in pens with slatted floors and keeping post-weaned calves in pens with a sand floor.     

Effects of isobutyrate supplementation in pre- and post-weaned dairy calves diet on growth performance,rumen development,blood metabolites and hormone secretion

Isobutyrate supplements could improve rumen development by increasing ruminal fermentation products, especially butyrate, and then promote the growth performance of calves. The objective of this study was to evaluate the effects of isobutyrate supplementation on growth performance, rumen development, blood metabolites and hormone secretion in pre- and post-weaned dairy calves. In total, 56 Chinese Holstein male calves with 30 days of age and 72.9±1.43 kg of BW, blocked by days of age and BW, were assigned to four groups in a randomized block design. The treatments were as follows: control, low-isobutyrate, moderate-isobutyrate and high-isobutyrate with 0, 0.03, 0.06 and 0.09 g isobutyrate/kg BW per calf per day, respectively. Supplemental isobutyrate was hand-mixed into milk of pre-weaned calves and the concentrate portion of post-weaned calves. The study consisted of 10 days of an adaptation period and a 50-day sampling period. Calves were weaned at 60 days of age. Seven calves were chosen from each treatment at random and slaughtered at 45 and 90 days of age. BW, dry matter (DM) intake and stomach weight were measured, samples of ruminal tissues and blood were determined. For pre- and post-weaned calves, DM intake and average daily gain increased linearly (P<0.05), but feed conversion ratio decreased linearly (P<0.05) with increasing isobutyrate supplementation. Total stomach weight and the ratio of rumen weight to total stomach weight tended to increase (P=0.073) for pre-weaned calves and increased linearly (P=0.021) for post-weaned calves, whereas the ratio of abomasum weight to total stomach weight was not affected for pre-weaned calves and decreased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. Both length and width of rumen papillae tended to increase linearly for pre-weaned calves, but increased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. The relative expression of messenger RNA for growth hormone (GH) receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly (P<0.05) for pre- and post-weaned calves with increasing isobutyrate supplementation. Blood concentrations of glucose, acetoacetate, β-hydroxybutyrate, GH and IGF-1 increased linearly (P<0.05) for pre- and post-weaned calves, whereas blood concentration of insulin decreased linearly with increasing isobutyrate supplementation. The present results indicated that isobutyrate promoted growth of calves by improving rumen development and its ketogenesis in a dose-dependent manner.     

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.     

Prevalence and Identity of Cryptosporidium spp. in Pig Slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

Prevalence of the Cryptosporidium pig genotype II in pigs from the Yangtze River Delta, China

Background

Cryptosporidium spp. is prevalent globally, pigs are an important Cryptosporidium reservoir. In China, little data regarding rates of Cryptosporidium infections in pigs are available. The present study was therefore aimed at characterizing the distribution of Cryptosporidium species in pigs from two different cities, Shaoxing and Shanghai, from the Yangtze River delta.

Methodology/Principal Findings

Nested PCR to amplify the 18S rRNA locus on DNA extracted from fecal samples (n = 94) revealed the positive rate of Cryptosporidium in pigs from two cities was approximately 17.0%. The positive rates in Shanghai and Shaoxing were 14.3% and 25.0% respectively. Amplified sequences were verified by sequencing. The identified strain belonged to the C. pig genotype II using BLAST analysis in the NCBI database.

Conclusion/Significance

Our finding of Cryptosporidium pig genotype II in pigs in the Yangtze River delta area suggests that pig farms in this region must be considered a public health threat and proper control measures be introduced.     

Molecular epidemiology of Cryptosporidium spp. in an agricultural area of northern Vietnam: A community survey

The purpose of this study was to investigate the occurrence of Cryptosporidium infection and the potential for transmission of Cryptosporidium spp. between animals and humans in northern Vietnam. A total of 2715 samples (2120 human diarrheal samples, 471 human non-diarrheal samples, and 124 animal stool samples) were collected through our community survey in an agricultural area. All samples were tested for Cryptosporidium spp. by direct immunofluorescence assay (DFA) using a fluorescent microscope. DNA extraction, PCR amplification of three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp. Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were identified as C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample), C. hominis (from four human diarrheal samples), and C. suis (from two pig samples) by sequencing the amplified COWP and/or SSU-rRNA genes. In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples. Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. While C. hominis and C. suis were only detected in human and pig samples, respectively, C. canis was detected in samples from both dogs and humans. We suspect that C. canis infections in humans at this study site may be due to environmental contamination with animal and human feces.     

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 10⁶ oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.     

Genetic Diversity of Cryptosporidium spp. in Captive Reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.     

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60 × 3.99 μm (n = 50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.