Thomas Geurden

Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers

Blastocystis is an enteric protist and one of the most frequently reported parasitic infections in humans and a variety of animal hosts. It has also been reported in numerous parasite surveys of animals in zoological gardens and in particular in non-human primate species. PCR-based methods capable of the direct detection of Blastocystis in faeces were used to detect Blastocystis from various hosts, including non-human primates, Australian native fauna, elephants and giraffes, as well as their keepers from a Western Australian zoo. Additional faecal samples were also collected from elephants and giraffes from four other zoos in Amsterdam (The Netherlands), Antwerp (Belgium), Melbourne and Werribee (Australia). Information regarding the general health and lifestyle of the human volunteers were obtained by questionnaire. Overall, 42% and 63% of animals and zoo-keepers sampled from the Western Australian zoo were positive for Blastocystis, respectively. The occurrence of Blastocystis in elephants and giraffes from other cities was similar. This is the first report of Blastocystis found in the elephant, giraffe, quokka, southern hairy nosed wombat and western grey kangaroo. Three novel and what appear to be highly host-specific subtypes (STs) of Blastocystis in the elephant, giraffe and quokka are also described. These findings indicate that further exploration of the genetic diversity of Blastocystis is crucial. Most zoo-keepers at the Perth Zoo were harbouring Blastocystis. Four of these zoo-keeper isolates were identical to the isolates from the southern hairy nosed wombat and five primate species.

Giardia and other intestinal parasites in different dog populations in Northern Belgium.

The objectives of this study were to obtain data on the prevalence of intestinal parasites in different dog populations in northern Belgium, to estimate the zoonotic risk associated with these infections and to identify potential risk factors. Between 2004 and 2007 a total of 1159 faecal samples were collected from 451 household dogs, 357 dogs from breeding kennels and 351 dogs with gastrointestinal disorders. The samples from dogs with gastrointestinal disorders were sent to the diagnostic Laboratory for Parasitology at Ghent University by veterinary practitioners. In household dogs the prevalence of intestinal parasites was relatively low. Giardia was the most commonly found parasite (9.3%, CI 5.5-13.1), followed by Toxocara canis (4.4%, CI 2.7-6.8). Much higher infection rates were observed in kennel dogs, especially for Giardia spp. (43.9%, CI 37.8-50.0); T. canis (26.3%, CI 21.8-31.2) and Cystoisospora spp. (26.3%, CI 21.8-31.2). Also in dogs with gastrointestinal problems, Giardia spp. (18.1%, CI 13.1-23.1), Cystoisospora spp. (8.8%, CI 6.1-12.3) and T. canis (7.4%, CI 4.9-10.7) were the most frequently detected parasites. In all dog populations pups were more frequently infected with Cystoisospora (P<0.0001 to P<0.05), Giardia (P<0.001 to P<0.05), and T. canis (P<0.0001 to P<0.001) than adult dogs, except for T. canis in household dogs, where this correlation was not significant. A significant association of anthelmintic treatment with T. canis infections was only observed within the household population. Household dogs with a higher number of treatments per year were more frequently infected with T. canis (P<0.05). There was a significant difference between the different breeding kennels for the occurrence of Cystoisospora, Giardia and T. canis (P<0.0001) and large kennels harboured relatively more infected animals than smaller breeding facilities (P<0.05). However, this was not significant for Giardia spp. Breed and gender did not affect the risk of an infection in any of the study populations. Toxocara and Giardia present a zoonotic risk, especially in household dogs, where the majority of Giardia positive samples (80%) belonged to the zoonotic assemblage A. In kennel dogs and clinically affected dogs the host-specific Giardia assemblages C and D were most prevalent (94% and 80%, respectively).

Prevalence and molecular characterisation of Cryptosporidium and Giardia in lambs and goat kids in Belgium

The prevalence of Cryptosporidium and Giardia was studied on 10 intensively reared sheep and goat farms in the province of East Flanders, Belgium. Random faecal samples were collected and examined using the Merifluor((R)) immunofluorescence assay. Cryptosporidium positive samples were withheld for molecular identification using primers targeting the 18S rDNA, 70 kDa heat shock protein and 60 kDa glycoprotein gene. For the molecular identification of Giardia the beta-giardin gene and a recently developed assemblage specific PCR based on the triose phosphate isomerase gene were used. The prevalence of Cryptosporidium in lambs was 13.1% (18/137), on 4 out of 10 farms. In goat kids the Cryptosporidium prevalence was 9.5% (14/148), on 6 out of 10 farms. The molecular characterisation of Cryptosporidium positive isolates indicated that in lambs (n=10) the cervine genotype was predominant, whereas in the goat kids (n=11) only C. parvum was identified, with subgenotypes IIaA15G2R1 and IIdA22G1. The Giardia prevalence was 25.5% (35/137) in lambs with all 10 farms being positive, and 35.8% (53/148) in goat kids with 8 out of the 10 farms being positive. Both in the goat kids and in the lambs the host specific assemblage E was most commonly identified. However, the zoonotic assemblage A was identified in 6 out of 28 goat kids and in 2 out of 8 lambs, based on the beta-giardin sequence alignment. Using the assemblage specific PCR, mixed assemblage A and E infections were additionally identified in 2 lambs and in 5 goat kids. The results of the present study indicate that both Cryptosporidium and Giardia are common parasites on intensively reared sheep and goat farms in the province of East Flanders, Belgium, and that they are a potential source for zoonotic infections.

Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium

Stool samples from Belgian patients suffering from abdominal pain and/or diarrhoea were examined for Cryptosporidium and Giardia. Cryptosporidium-positive samples were genotyped using the 70 kDa heat shock protein and the 60 kDa glycoprotein (GP60) genes: C. hominis was identified in 54.2% and C. parvum in 45.8% of the samples. Sequencing at the GP60 locus indicated that subgenotype IbA10G2 of C. hominis and subgenotype IIaA15G2R1 of C. parvum were the most prevalent, although several other subgenotypes were identified. For Giardia, sequencing at the beta-giardin, triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) genes revealed assemblage B as the most prevalent (74.4%) in human patients. A high degree of heterogeneity was found, especially on the beta-giardin gene, and to a lesser extent on the GDH gene. Furthermore, using a novel species-specific PCR based on the TPI gene, mixed infections with both assemblage A and B were detected in a large number (32.4%) of human patients, which might have important epidemiological implications.

Molecular epidemiology with subtype analysis of Cryptosporidium in calves in Belgium.

The prevalence of Cryptosporidium in calves younger than 10 weeks was estimated in a cross-sectional epidemiological study on 100 dairy (n=499) and 50 beef (n=333) farms in East Flanders (Belgium), using a previously evaluated immunofluorescence assay (Merifluor). The calf prevalence was 37% (95% Probability Interval (PI): 7-70%) in dairy calves and 12% (95% PI: 1-30%) in beef calves. To elucidate the genetic diversity, the Cryptosporidium 18S ribosomal DNA and the 70 kDa heat shock protein gene were targeted. In the majority of the samples C. parvum was present, although C. bovis was also identified, all but one in calves older than 1 month. The porcine-specific C. suis was identified in 1 beef calf. Subtyping of C. parvum positive isolates by sequence analysis of the 60 kDa glycoprotein gene indicated the presence of 4 allele IIa subtypes, along with 1 subtype IIdA22G1. The subtype IIaA15G2R1 was most prevalent, next to subtype IIaA13G2R1 and IIaA16G2R1, and a new subtype IIaA14G2R1. The results of the present study indicate a high prevalence of Cryptosporidium infections in calves in Belgium and confirm that these calves should be considered as a potential zoonotic reservoir for human infections.

Is Giardia a significant pathogen in production animals

Although Giardia duodenalis is recognised worldwide as the most important parasitic cause of gastro-intestinal disorder in human patients, the relevance of infection in production animals is prone to debate. Since the 1980s, clinical disease has been associated with giardiasis in production animals, both in natural conditions and in experimental studies. However, most Giardia research is focussed on the relevance of production animals as a reservoir for zoonotic transmission. In this study, the current knowledge on clinical relevance of giardiasis in production animals is reviewed, along with the diagnosis, treatment and control of infection. Furthermore, future research objectives are discussed.

Molecular characterisation of Giardia duodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms.

Giardia is frequently detected in stools of non-human primates (NHP). However, a molecular identification has been rarely applied to Giardia isolates from NHP, and the distribution of the zoonotic assemblages A and B remains unclear. Moreover, little is known about the genetic variability among the isolates, although this may contribute to the elucidation of the different transmission pathways, including the role of NHP as a reservoir for human giardiasis. Therefore, 258 Giardia samples from 31 NHP species housed in nine zoological gardens and one sanctuary in Belgium and The Netherlands were characterised based on an assemblage-specific PCR targeting the triose phosphate isomerase (tpi) gene to identify both assemblage A and B infections. In addition, a multi-locus sequencing approach based on the glutamate dehydrogenase, the tpi and the beta-giardin genes was used to examine both the genetic variability and the ability to allocate these isolates to different NHP groups. Overall, assemblage B was the most prevalent (78.6%), but mixed assemblage A and B infections occurred in 32.7% of the samples. Sequencing of the isolates revealed the presence of new polymorphisms for both assemblages and at the three loci examined. The majority of the assemblage B isolates could not be grouped into recently described sub-assemblages, particularly at the tpi gene. Isolates could only be allocated to a specific group when polymorphisms of the three loci were combined. The results confirm that NHP are a potential reservoir for zoonotic transmission and advocate the use of assemblage-specific primers in molecular epidemiological surveys, as mixed infections are likely to be underestimated. The high level of heterogeneity within assemblages indicates that a revised nomenclature of these sub-assemblages is needed, but points out the potency of a multi-locus sequencing approach to unravel the complex epidemiology of Giardia duodenalis.

A Bayesian evaluation of three diagnostic assays for the detection of Giardia duodenalis in symptomatic and asymptomatic dogs.

A Bayesian approach was used to evaluate three commonly used diagnostic assays for the detection of Giardia duodenalis in dogs: microscopical examination (ME), a commercial immunofluorescence assay (IFA: MerifluorGiardia test) and a commercial immunochromatographic assay (SNAP: Idexx SNAPGiardia test). These assays were evaluated for use in two different settings: in a cross-sectional epidemiological survey in household dogs and in a clinical survey, both conducted in the northern part of Belgium. A total of 272 faecal samples from household dogs and 141 faecal samples from clinically affected dogs were examined using these three diagnostic assays. The Bayesian analysis indicated that all tests were highly specific (specificity above 90%), and that the IFA is more sensitive than SNAP and ME, both in an epidemiological and in a clinical setting. For all three tests, the estimated sensitivity values were higher in the clinical compared to the epidemiological survey, whereas the specificity values were comparable in both studies. The results of the present study indicate that IFA is a highly specific and sensitive technique for the detection of G. duodenalis cysts, both for use in an epidemiological or clinical survey. The SNAP is a specific and fairly sensitive technique for the diagnosis of Giardia in clinically affected dogs. Overall, the ME was found to be a specific diagnostic technique, although lacking sensitivity.

Prevalence, molecular typing and risk factor analysis for Giardia duodenalis infections in dogs in a central London rescue shelter

A cross-sectional survey to investigate canine infections with Giardia duodenalis was undertaken at a central London rescue shelter between October 2006 and March 2007. The objectives of the study were to (i) estimate the prevalence of infection in dogs admitted to a London dog shelter using a commercially available ELISA-based test kit; (ii) identify the relative importance of potential dog level risk factors for infection; and (iii) identify the occurrence of different G. duodenalis assemblages present in this population in order to identify presence of any potentially zoonotic assemblages. A faecal sample was collected from each dog entering the shelter within 1 day of arrival. Each sample was tested for the presence of parasite cyst wall protein using the Giardia SNAP test kit (Idexx Laboratories). Samples were graded for faecal consistency on a standard scale and data on age, breed, categorized breed group, sex and neutered status were collected for each dog. Associations between infection status and each dog level variable were investigated using univariable and multivariable logistic regression. Selected G. duodenalis-positive samples were genotyped using previously described primers targeting the 18S rDNA gene and the beta-giardin gene. Samples from a total of 878 dogs were collected and the true prevalence found to be 21.0% (95% CI 16.7-25.4%). In the present study, the odds of infection decreased with increasing age (adjusted odds ratio 0.66, 95% CI 0.54-0.80, p<0.0001) and was increased for Rottweilers (adjusted odds ratio 2.12, 95% CI 1.03-4.34, p=0.04). Of the 51 samples selected for genotyping, 41 samples yielded a good amplification at one or both of the targeted genes, demonstrating the occurrence of mainly dog-specific assemblages C and D. The potentially zoonotic assemblage A and a mixed template C/D were found in two individual dogs. The results of the present study illustrate the high prevalence of G. duodenalis in shelter dogs. Although predominantly infected with dog-specific assemblages, the identification of assemblage A suggests that appropriate precautions should be taken to minimize the risk of transmission to staff.

Parasitic infections in dairy cattle around Hanoi, northern Vietnam

In northern Vietnam, dairy cattle are mainly managed in small-scale farms, where animals are kept confined and feeding occurs by cut and carry methods. In the present study the occurrence of parasitic infections was examined in five provinces around Hanoi. A total of 201 farms were visited, and 334 stool and 239 blood samples were collected from calves younger than 3 months, animals between 3 and 24 months and adult cows. Furthermore, 254 milk samples were collected from lactating animals. Coproscopical examination indicated a high prevalence of nematode eggs (Cooperia spp., Haemonchus and Oesophagostomum spp.) in animals (n=176) between 3 and 24 months (66%) and in adult cows (n=90; 54%). In these age groups the prevalence of Fasciola was 28% and 39%, respectively, and for Paramphistomum the prevalence was 78% and 82%, respectively. Fifty percent of the calves younger than 3 months (n=68) were positive for Giardia, and none for Cryptosporidium. Most Giardia isolates were identified as the non-zoonotic G. duodenalis assemblage E on the beta-giardin gene. The blood samples were examined with commercially available Svanovir((R))Elisas for the presence of Anaplasma marginale and Babesia bigemina specific antibodies, and a prevalence of 28% and 54% was found, respectively. In the milk samples Neospora caninum specific antibodies (Svanovir((R))Elisa) were detected in 30% of the lactating animals. The present study demonstrates that parasitic infections occur frequently in dairy cattle around Hanoi although animals are mainly kept confined, and indicates that further research on the economic impact of these infections is needed.