The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada

The objective of this study was to compare the occurrence and the genotypes and species of Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in the Regional Municipality of Waterloo, Ontario, in an effort to determine the potential for zoonotic transmission from these animals. Pooled manure samples were collected from 45 dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporidium oocysts was determined by immunofluorescence microscopy, while nested-PCR and DNA sequencing were used to determine genotypes and species. The overall farm prevalence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattle farms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However, on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6% of total pooled pen manure samples, respectively, with the occurrence of both parasites being generally higher in calves than in older animals. Most Giardia isolates were identified as either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assemblage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequently identified species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanae and Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the total pooled pen manure samples were positive for Giardia and Cryptosporidium, respectively, with no obvious correlation with age. All Giardia isolates in beef cattle were identified as G. duodenalis Assemblage E, while all Cryptosporidium isolates were identified by sequence analysis as C. andersoni, although microscopic analyses, and subsequent restriction fragment length polymorphism analyses, indicated that other Cryptosporidium species were also present. The results of this study indicate that although Giardia and Cryptosporidium were identified in a higher overall percentage of the pooled beef cattle manure samples than in dairy cattle, firmly established zoonotic genotypes and species were much more common in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairy calves, may, therefore, pose a greater risk of infection to humans than beef cattle. However, these results may also provide evidence of potential zooanthroponotic transmission (human to animal).     

Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates

The prevalence of Cryptosporidium, Giardia, and Enterocytozoon bieneusi in cats from Bogota (Colombia) was determined from fecal specimens and scrapings of duodenal and ileal mucosa screened by PCR. All PCR-positive specimens were sequenced to determine the genotype(s) present. Of 46 cats, 6 (13%) were positive for Cryptosporidium, 5 (11%) were infected with C. felis and one (2%) with C. muris. Three (6.5%) cats were infected with Giardia duodenalis Assemblage F. Eight (17%) cats were infected with four genotypes of E. bieneusi: genotype D-like (9%), K (4%), Peru 10 (2%), and Peru 5 (2%). This is the first report on the presence of zoonotic species/genotypes of Cryptosporidium and E. bieneusi in cats in Colombia.     

Genotype characterisation of Giardia duodenalis isolates from domestic and farm animals by SSU-rRNA gene sequencing

In order to investigate the genotypes of Giardia duodenalis from domestic and farm animals in Italy, 21 Giardia isolates, 17 from dogs, 1 from cat and 3 from dairy calves, were genetically characterised by SSU-rRNA gene sequencing. Among dogs, 76.5% of isolates showed the dog-specific genotypes (Assemblages C, D and C/D mixed Assemblage) and 23.5% exhibit potential zoonotic genotypes (Assemblage A and A/C mixed Assemblages). The cat isolate belonged to assemblage A, whereas the sequences among the isolates from calves were found to correspond to hoofed-livestock genotype, namely Assemblage E. These findings suggest that infection of humans by zoonotic genotypes from domestic animals could be of low epidemiological significance, although possible. The present study represents the first contribute to the knowledge of G. duodenalis genotypes in domestic and farm animals from Italy.     

Presence and molecular characterisation of Giardia and Cryptosporidium in alpacas (Vicugna pacos) from Peru

The presence of Giardia and Cryptosporidium was investigated in 274 faecal samples of alpacas (Vicugna pacos) from 12 herds from Peru by immunofluorescence microscopy and PCR amplification and sequencing of fragments of the ssu-rRNA and β-giardin genes from Giardia spp., as well as the ssu-rRNA gene from Cryptosporidium spp. A total of 137 samples (50.0%) were positive for Giardia spp., and 12 samples (4.4%) for Cryptosporidium spp. In ten samples (3.6%), co-infection by both pathogens was found. Herd prevalence was found to be 91.7% (11/12 herds) for Giardia and 58.3% (7/12 herds) for Cryptosporidium. Regarding the age of the animals, although Giardia was detected in animals as young as 1 week, the prevalence increased with age, reaching 80% by 8 weeks. Similarly, the highest percentage of Cryptosporidium detection (20%) was also found in the 8 week-old group. By PCR, 92 of the 274 analysed samples were positive for Giardia. Sequencing of the amplicons showed the existence of Giardia duodenalis assemblage A in 67 samples; G. duodenalis assemblage E in 24 samples; and inconsistent results between the two molecular markers used in a further sample. Cryptosporidium was only detected by PCR in 3 of the 274 samples; Cryptosporidium parvum was identified in two samples and Cryptosporidium ubiquitum in one sample. This study is the first performing molecular characterisation of both parasites in Peruvian alpacas, and the first report of C. ubiquitum in this host. The identification of G. duodenalis assemblage A, C. parvum and C. ubiquitum, suggests that zoonotic transmission of these enteropathogens between alpacas and humans is possible.     

Prevalence of Giardia duodenalis and Cryptosporidium spp. in dogs from different living conditions in Uberlândia, Brazil

Infection rates with Giardia duodenalis and Cryptosporidium spp. were compared among dogs living under different conditions. Stool samples (n = 433) collected from dogs of different ages, gender, living conditions and origin were analyzed using three techniques, i.e., centrifugal flotation in zinc sulfate solution, centrifugal flotation in sucrose solution, and methylene blue gram safranin staining. Eighty-nine of the samples were from stay dogs living in shelters run by animal protection societies, 199 were from kennels and 122 from households. A total of 119 (29.0%) had G. duodenalis cysts and six (1.4%) were positive for Cryptosporidium spp. oocysts. Dogs from kennels were most frequently affected by G. duodenalis (49.7%) while those from shelters showed a higher prevalence of Cryptosporidium spp. (2.2%). A significant difference (p < or = 0.05) was observed between immature dogs and adults only with respect to Giardia infection. There was no significant difference between the gender with regard to the presence of either protozoan.     

Prevalence of Giardia duodenalis assemblages among dairy herds in the New York City Watershed

A longitudinal herd-level study was carried out to determine the cumulative incidence of Giardia duodenalis infections in dairy cattle in the New York City Watershed. We also sought to assess the changes in infection pattern of animals diagnosed as shedding Giardia over time, determine risk factors that may be associated with G. duodenalis infections, and identify potentially zoonotic infections. A total of 2109 fecal samples were randomly collected from dairy cattle at 34 farms in the New York City Watershed on a seasonal basis. A total of 504 Giardia-positive samples were identified by zinc sulfate flotation. The overall cumulative incidence of G. duodenalis based on flotation results was 23.9% with 73.8% of all infections occurring in animals under 180 days of age (372/504). The intensity of infection ranged from 2 to 563,200 cysts/gram of feces. Cattle shedding Cryptosporidium spp. oocysts were twice as likely to shed G. duodenalis cysts in comparison to the animals that did not shed oocysts (1.81 95% CI 1.26-2.60 p=0.0012). In the multivariate analysis, only the age of the animal and the presence of dogs on the farm were significantly associated with the likelihood of shedding G. duodenalis. DNA was extracted from positive samples and analyzed by polymerase chain reaction (PCR) of the beta-giardin and triosephosphate isomerase genes of Giardia spp. 304 samples were analyzed by PCR of which 131 were sequenced. 22.1% of sequenced samples were identified as assemblage A and 77.9% were identified as assemblage E. Interestingly, 100% of specimens identified as assemblage A were from calves under 84 days of age indicating that younger cattle are important reservoirs for potentially zoonotic assemblages of G. duodenalis.     

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.     

Comparison of conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections in dogs and cats

Faecal samples of 270 dogs and 100 cats from 3 animal shelters in Germany were comparatively examined using conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections. Giardia cysts were found in 9.5% and 0% of the faecal samples in dogs and cats, respectively, examined once using the ZnCl2-NaCl flotation. However, the Giardia coproantigen ELISA (ProSpecT Giardia Microplate Assay) was positive in 29.5% and 22.4% of the samples from dogs and cats, respectively. Direct faecal smears stained with carbol fuchsin showed Cryptosporidium oocysts in one dog (0.4%) and one cat (1%). In contrast, the Cryptosporidium coproantigen ELISA (ProSpecT Cryptosporidium Microplate Assay) reacted positively in 23% of the samples from dogs and 30% of the samples from cats. Both coproantigen ELISAs were more often positive in coproscopically Giardia-negative canine faecal samples that contained Isospora burrowsi/ohioensis oocysts than in faecal samples without any parasite stage. Possible reasons for these observations are discussed.     

Molecular identification of Cryptosporidium parvum and Giardia duodenalis in the Italian water buffalo (Bubalus bubalis)

Livestock are commonly infected with protozoan parasites of the genera Cryptosporidium and Giardia, and some of the species and genotypes found in these animals have zoonotic significance. We characterized isolates of both parasites recovered from the Italian water buffalo (Bubalus bubalis), an economically important species whose milk is used for the production of "buffalo mozzarella" fresh cheese. Molecular analysis of the Cryptosporidium small subunit ribosomal DNA gene and of the Giardia beta-giardin gene shows the presence of both zoonotic parasites (Cryptosporidium parvum and Giardia duodenalis assemblage A) and host-specific parasites (G. duodenalis assemblage E), suggesting that water buffaloes can contribute to environmental contamination with oocysts and cysts potentially infectious to humans if their faeces are improperly disposed of. On the other hand, mozzarella cheese is probably a safe product, given that its production involves the treatment of cheese curd at 85-95 degrees C, which is likely to kill or inactivate the parasites.     

Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans).

Feces and duodenal scrapings were collected from 22 coyotes (Canis latrans) killed in managed hunts in northeastern Pennsylvania. Polymerase chain reaction (PCR) methods were used to detect Giardia and Cryptosporidium spp. PCR-amplified fragments of Giardia and Cryptosporidium spp. SSU-rRNA genes were subjected to DNA sequence analysis for species/genotype determination. Seven coyotes (32%) were positive for G. duodenalis: three assemblage C, three assemblage D, and one assemblage B. Six coyotes (27%) were positive for Cryptosporidium spp. One isolate shared 99.7% homology with C. muris, whereas five others (23%) shared 100% homology with C. canis, coyote genotype. This is the first report on multiple genotypes of Giardia spp. in coyotes and on the prevalence of Cryptosporidium spp. genotypes in coyotes.     

Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland

In the United Kingdom and Australia sheep have been implicated as sources of Cryptosporidium and Giardia that infect humans, but no such studies have been conducted in North America. Therefore, a study was undertaken to investigate the prevalence of these parasites in sheep on a farm in Maryland. Feces were collected from 32 pregnant ewes 1, 2, and 3 days after parturition and from each of their lambs 7, 14, and 21 days after birth. The presence of Cryptosporidium oocysts and Giardia cysts was determined by both immunofluorescence microscopy and PCR/gene sequence analysis. PCR was consistently more sensitive than microscopy. The prevalence, by PCR, of Cryptosporidium in ewes and lambs was 25 and 77.4%, respectively. Three species/genotypes of Cryptosporidium were identified: C. parvum, a novel C. bovis-like genotype, and Cryptosporidium cervine genotype. Cryptosporidium parvum and the cervine genotype have been reported worldwide in human infections. The novel C. bovis-like genotype is reported here for the first time. The prevalence of Giardia in ewes and lambs was 12 and 4%, respectively. Most infections were Assemblage E which is not zoonotic; however, one ewe was infected with zoonotic Assemblage A. The identification of only two lambs infected with C. parvum and one ewe infected with G. duodenalis Assemblage A suggests a low prevalence of these zoonoses. However, the high prevalence of the zoonotic cervine genotype indicates that sheep should be considered a potential environmental source of this human pathogen.     

Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm.

Prevalences of Cryptosporidium spp. and Giardia duodenalis in relation to age and season were investigated on a dairy farm in The Netherlands over the course of 1year. The whole herd was sampled five times, whereas calves younger than about 2 months were sampled every 2-3 weeks. Associations between diarrhoea and presence of one or more pathogens (Cryptosporidium spp., G. duodenalis, rotavirus) were investigated. Potential transmission routes of Cryptosporidium spp. were evaluated and positive samples of Cryptosporidium spp. and G. duodenalis were identified to genotype level by PCR microsatellite identification and fingerprinting. Shedding of Cryptosporidium spp. was found in all age categories but peaked in calves 1-3 weeks old (39.1%). Herd prevalence of shedding for Cryptosporidium spp. varied from 2.4% in June to 22.2% in December. Shedding of G. duodenalis was found in all age categories but peaked in animals 4-5 months old (54.5%). Herd prevalence of shedding for G. duodenalis varied from 0.8% in June to 15.5% in February. Cryptosporidium spp. and rotavirus appeared to be significantly associated with diarrhoea in calves. Microsatellite analysis showed two different subtypes (C3 and C1) of Cryptosporidium parvum calf strains. Two genotypes of G. duodenalis were found, one positive by A lineage specific PCR and thus closely related to human genotypes and one genotype, which was negative by A and B lineage specific PCR. The results indicate that cow-to-calf and indirect calf-to-calf transmission both are important routes for acquiring infection with Cryptosporidium spp.     

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.     

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.     

Giardia duodenalis and Cryptosporidium spp. in a veterinary college bovine teaching herd

In a preliminary study, we commonly identified Giardia duodenalis in adult dairy cattle from a veterinary college teaching herd. Therefore, the present study was carried out in order to better understand the potential of adult cattle to act as a source for G. duodenalis infections for students and staff at the veterinary college. Fecal samples were collected bi-weekly from this herd of adult cattle (n=30) over an 8-month period to determine the prevalence of G. duodenalis and Cryptosporidium spp. within the herd. Nested PCR followed by DNA sequencing was then performed on a subset of positive samples in order to better understand the zoonotic potential of these infections. Every cow was sampled between 11 and 18 times, depending on the date the animal joined the teaching herd. In total, 507 fecal samples were collected from 30 different cows and examined for cysts and oocysts using epifluorescence microscopy. G. duodenalis prevalence during the course of the study ranged from 37% (11/30) to 64% (18/28), with a mean of 49%. Cumulative G. duodenalis prevalence was 73% (22/30). Zoonotic G. duodenalis assemblage A genotype was identified in 43% (6/14) of the G. duodenalis-positive samples on which PCR and genetic sequencing were successfully performed. G. duodenalis assemblage E was identified in 57% (8/14) of these samples. Cryptosporidium spp. oocysts were not detected in the feces of any cows during the study period. The presence of the zoonotic G. duodenalis assemblage A in 43% of the sequenced samples indicates that there is a potential risk of infection for students and staff at this research and teaching facility, although the roles of cows as sources of giardiasis in humans remain uncertain. Furthermore, due to the large amount of feces they produce, adult cattle may serve as important sources for G. duodenalis infections in young cattle, or other animals in the facility, despite relatively low numbers of cysts excreted per gram of feces. In contrast, the results of this study indicate that this herd posed a negligible risk of transmitting Cryptosporidium parvum infections to humans.     

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-acre, commercial sheep farms in southern Western Australia, on two separate occasions (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old), were individually identified using ear tags (a numbered tag and a radio-frequency tag). A total of 1155 faecal samples were collected only from these individually identified lambs on five separate sampling occasions. All samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). The overall prevalences (lambs positive for a parasite on at least one of the five samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia prevalences at individual samplings ranged between 18.5 and 42.6% in lambs and were <10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium ubiquitum was most commonly detected in younger lambs and Cryptosporidium parvum was detected in lambs at all five samplings, typically in older lambs and as part of a mixed species infection with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most common genotype detected at all five samplings, with greater proportions of assemblage A and mixed assemblage A and E infections identified in older lambs. This longitudinal study identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed extensively on pastures, while reinforcing that sampling a random selection of animals from a flock/herd on one occasion (point prevalence), underestimates the overall prevalence of these parasites in the flock/herd across an extended time period. Based on these findings, grazing lambs were identified as a low risk source of zoonotic Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five samplings in some lambs, indicating that these individuals were either unable to clear the naturally acquired protozoan infections or were repeatedly re-infected from their environment or other flock members.     

Genotype identification and prevalence of Giardia duodenalis in pet dogs of Guangzhou, Southern China

Giardia duodenalis is a flagellated parasite and is considered one of the most common causes of protozoal diarrhea in both humans and animals worldwide. This paper represents the first study of the prevalence of G. duodenalis in pet dogs in Guangzhou, China. Faecal samples (209 specimens) were obtained from young (<6 months old), adult (6 months to 3 years) and elder dogs (>3 years old). 8.61% (18/209) faecal samples were recorded positive using microscopy examination, and 11.00% (23/209) using PCR. The prevalence was significantly higher in diarrheic dogs (26.31%) compared with non-diarrheic dogs (5.10%), while it was higher in young (25.58%) than both adult (7.37%) and elder (7.04%) dogs and the difference was statistically significant (P<0.05). The prevalence in male dogs 11.30% (13/115) was higher than females 10.87% (10/92), and in suburban dogs (12.15%) higher than urban 9.80%, but the difference was not statistically significant (P>0.05). Sequence analysis of the 23 PCR-positive samples revealed the presence of Assemblage D (18/23), and zoonotic Assemblage A (5/23). The present investigation reported a high infection rate of G. duodenalis in pet dogs, especially in young dogs. Genotypic characterization demonstrated that the zoonotic Assemblage A was found, a fact that poses a potential risk of G. duodenalis transmission from pet dogs to humans. It is suggested that pet owners should take appropriate hygiene measures to prevent and control giardiasis in this region.     

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.     

Prevalence and risk factors of Giardia duodenalis in dogs from Romania

The protozoan Giardia duodenalis is a mammalian-infecting parasite that produces diarrhoea and malabsorption in its hosts. A survey to investigate canine infections with G. duodenalis in Romania was undertaken between June 2008 and December 2009. The objectives of the study were to (i) estimate the prevalence of infection in different dog populations (kennels, shelters, shepherd, household) using microscopy and a commercially available enzyme-linked immunosorbent assay (ELISA) test kit; (ii) to establish the level of agreement and characteristics of the tests; and (iii) to identify risk factors for infection by multivariate logistic regression models. Faecal samples were collected from 614 dogs aged from 1 month to 16 years (mean ± SD=2.88 ± 2.86 years). Each sample was tested for the presence of cysts using a flotation method with saturated sodium chloride solution and 416 out of 614 stool samples were further examined for the presence of G. duodenalis specific antigens using Giardia Microwell ELISA (SafePath? Laboratories). Giardia cysts were identified in 8.5% of total dogs (52/614) and statistical significantly more frequently in dogs living in communities. The cysts prevalence according with dog populations was as follows: 7.2%(9/125) in kennel dogs; 16.5%(27/164 in shelter dogs; 4.3%(2/46) in shepherd dogs; 4.8%(4/84) in household dogs from urban areas; and 5.1%(10/195) in household dogs from rural areas. The overall prevalence of Giardia infection by ELISA was 34.6% (144/416). The prevalence was significantly higher in kennel dogs (50%; 13/26), shelter dogs (47.7%; 74/155) and shepherd dogs (40.5%; 17/42) than in household dogs from urban areas (34.1%; 15/44) and household dogs from rural areas (16.8%; 25/149). It was noticed poor agreement between microscopy and ELISA (k=0.19). The microscopy performed best, with an Youden Index of 0.74, a Se of 73.68% and a Sp of 100%. ELISA had 100% Sp, but only 19.44% Se. Young dogs (up to 12 months age) and living in communities were identified as risk factors for infection by multivariate logistic regression analysis. 71.2% (37/52) Giardia cysts positive dogs presented co-infections with other intestinal parasites: Toxocara canis (14/52; 26.9%), Isospora ohioensis (12/52; 23.1%), Ancylostoma caninum (9/52; 17.3%), Uncinaria stenocephala (7/52; 13.5%), Trichocephalus vulpis (6/52; 11.5%), Hammondia heydorni/Neospora caninum (5/52; 9.6%), Sarcocystis spp. (5/52; 9.6%), Isospora canis (4/52; 7.7%), Capillaria aerophila (3/52; 5.8%), Strongyloides stercoralis (2/52; 93.8%), Dipylidium caninum (1/52; 1.9%) and Toxascaris leonina (1/52; 1.9%).     

Occurrence of Giardia and Cryptosporidium in Norwegian red foxes (Vulpes vulpes)

Faecal samples from 269 Norwegian wild red foxes (Vulpes vulpes) shot during the hunting season (October-April) in 2002-2004 were examined for the presence of Giardia and Cryptosporidium. Cryptosporidium oocysts were detected in samples from 6 (2.2%) of the foxes, and Giardia cysts in 13 (4.8%) of the foxes. The prevalence of Giardia infection was significantly higher in juvenile male foxes than in adult male foxes, but no other significant differences between age and sex were found. No significant differences in prevalence related to geographical origin of animals were found. Insufficient nucleated Cryptosporidium oocysts were isolated for successful PCR, but genotyping of Giardia duodenalis isolates from seven foxes demonstrated a high degree of heterogeneity amongst them, with all isolates belonging to the zoonotic Assemblages A and B.