High prevalence Giardia duodenalis assemblage B and potentially zoonotic subtypes in sporadic human cases in Western Australia

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Fecal samples from sporadic human clinical cases of giardiasis in Western Australia were analysed at two loci; 18S rRNA and glutamate dehydrogenase (gdh), and G. duodenalis assemblage B isolates were identified in 75% of isolates. Sequence analyses of 124 isolates at the 18S rRNA locus identified 93 isolates as assemblage B and 31 as assemblage A. Analyses of 109 isolates at the gdh locus identified 44 as B3, 38 as B4 and 27 were A2. Infection with Giardia was highest amongst children <5 years of age, with >56% of infections in this age group. The majority of the isolates were from rural areas (91/124) compared with urban areas (33/124). The assemblage A isolates were completely homogenous genetically at the gdh locus, while assemblage B isolates showed variability at the nucleotide but not at the amino acid level at this locus. Some of the assemblage B3 and B4 subtypes identified in humans were previously identified in marsupials in Australia and in a fox, indicating potential zoonotic transmission.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

Genotyping of Giardia duodenalis Isolates from Dogs in Guangdong,China Based on Multi-Locus Sequence

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs'' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.     

Multi-locus analysis of Giardia duodenalis intra-Assemblage B substitution patterns in cloned culture isolates suggests sub-Assemblage B analyses will require multi-locus genotyping with conserved and variable genes

Recent research concerning Giardia duodenalis has focused on resolving possible sub-assemblages within Assemblages A and B to better understand host-specific and zoonotic relationships. In the present study nine cloned, cultured, Assemblage B isolates were used to investigate the intra-Assemblage B substitution patterns of conserved (ssrDNA, ef, h2b, h4) and variable (tpi, gdh, bg) genes to assess their suitability for further application to sub-assemblage analyses. The resolution of each gene was found to be proportional to its substitution rate and for the genetically narrow sample set examined, the variable genes best represented the consensus phylogeny while the conserved genes only established fractions. However it was demonstrated that the spectra of conserved and variable genes were required to ensure accuracy of inferred phylogeny and it was therefore concluded that further research into sub-Assemblage B groups would require a mixture of conserved and variable genes for the multi-locus analyses of this genetically broad assemblage.     

Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, β-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as “AIII”), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or “heterogeneous templates”) in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.     

A natural zoonotic giardiasis: Infection of a child via Giardia cysts in pet chinchilla droppings

Here, we report a case of direct zoonotic transmission of giardiasis between a pet chinchilla and a human. Microscopic and molecular examinations of stool samples from a child and samples of chinchilla droppings revealed cysts/DNA of Giardia intestinalis. The transmission from the chinchilla to the child has been confirmed as coprophagous after the 1-year-old toddler ingested pet chinchilla droppings. Molecular analysis of the gdh gene from both hosts classified the G. intestinalis cysts into the assemblage B genetic group, which has been previously shown to be characteristic of both human and chinchilla giardiasis. Both Giardia sub-assemblages BIII and BIV were present in the chinchilla droppings, whereas only the sub-assemblage BIV was isolated from the child's stool sample. To the best of our knowledge, this is the first report of a true zoonotic transmission of giardiasis, supporting the zoonotic potential of assemblage B.     

Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China

Cryptosporidium and Giardia infections are common causes of diarrhea worldwide. To better understand the transmission of human cryptosporidiosis and giardiasis in Henan, China, 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens were characterized at the species/genotype and subtype levels. Cryptosporidium specimens were analyzed by DNA sequencing of the small subunit rRNA and 60 kDa glycoprotein genes. Among those genotyped, nine belonged to C. hominis and one C. felis, with the former belonging to three subtype families: Ia, Ib, and Id. The three Ib subtypes identified, IbA16G2, IbA19G2, and IbA20G2, were very different from the two common Ib subtypes (IbA9G3 and IbA10G2) found in other areas of the world. The distribution of Giardia duodenalis genotypes and subtypes was assessed by sequence analysis of the triosephosphate isomerase (tpi) gene. The assemblages A (eight belonging to A-I and four A-II) and B (belonging to six new subtypes) were found in 12 and six specimens, respectively. More systematic studies are needed to understand the transmission of Cryptosporidium and G. duodenalis in humans in China.     

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.     

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.     

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.     

Distribution of Giardia duodenalis Assemblages A and B among Children Living in a Remote Indigenous Community of the Northern Territory,Australia

Giardiasis is a communicable gastrointestinal disease caused by Giardia duodenalis and two genetic assemblages, A and B, cause human infection. In remote Indigenous communities of Australia, giardiasis is highly prevalent among children but disease transmission is poorly understood. This study investigated the prevalence of Giardia and genetic subtypes contributing to human disease in a remote Indigenous community, in the Northern Territory of Australia. Eighty-seven faecal samples were collected from 74 children (<15 years) over an 18 month period, and the distribution of positive cases relative to participant age and gender were examined. Screening by microscopy and 18S rRNA PCR amplification showed 66.7% (58/87) of faecal samples were positive for Giardia. Both males and females were equally affected and high detection rates were obtained for participants aged 0–<5 years and 5–<10 years (66.0 and 60.0% respectively). For 58.6% of the positive samples, Giardia was only detected by 18S rRNA PCR. Approximately 75% of cases were assemblage B, and subassemblage analyses using terminal restriction fragment length polymorphism of the glutamate dehydrogenase gene demonstrated that a variety of genetic variants were present. The high proportion of positive cases that were not detectable by microscopy, and dominance of assemblage B cases highlights the need for further research in this community, to assess the contribution of Giardia to chronic gastrointestinal disease among children, and to understand conditions conductive to assemblage B transmission.     

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.     

Biological and Genetic Characterization of Cryptosporidium spp. and Giardia duodenalis Isolates from Five Hydrographical Basins in Northern Portugal

To understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and β-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.     

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.     

Prevalence of Cryptosporidium and Giardia species in animals in irrigation catchments in the southwest of Australia

Screening of 445 animal faecal samples in irrigation catchments in Western Australia (WA) was conducted to identify the prevalence of Cryptosporidium and Giardia species. Of the samples positive for Giardia duodenalis, 30.7% (12/36) were the zoonotic Assemblage A, while approximately 13% (4/30) of Cryptosporidium positives were zoonotic. This is the first finding of Giardia Assemblage A in native marsupials and birds and indicates that marsupials and possibly birds may potentially be a reservoir of zoonotic Giardia.     

Multilocus genotyping of Giardia duodenalis from pigs in Korea

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is an important zoonotic parasite infecting livestock (including pigs) through ingesting cysts in contaminated food or water. This parasite has been classified into eight different genetic assemblages, A to H. Here, we examined the individual-level prevalence of G. duodenalis in domestic pig farms and confirmed host specificity by genotype comparisons. Samples were collected from southern and central Korea, between May 2017 and January 2019. DNA directly extracted from 745 pig fecal specimens were tested by PCR for G. duodenalis small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), and β-giardin gene sequences. Based on ssu rRNA PCR, 110 (14.8%) were positive for G. duodenalis. Infection risk was the highest in the fattener group (31/139, 22.3%) and during the autumn season (52/245, 21.2%: p < .001). No statistically significant differences in risk for infection were observed between fecal types (normal versus diarrheal). Fifty ssu rRNA samples, three gdh samples, and five β-giardin samples were successfully sequenced and genotyped. Ssu rRNA assemblage sequence analysis identified E (40.0%, 20/50), D (34.0%, 17/50), C (24.0%, 12/50), and A (2.0%, 1/50). The gdh locus identified three samples as assemblage E, and the β-giardin locus identified four samples as assemblage E and one as assemblage C. Assemblage A sequences obtained (ssu rRNA; MK430919) had 100% identity with Giardia sequences isolated from a Korean individual (AJ293301), indicating the potential of zoonotic transmission. Continuous management and monitoring for prevention of transmission and protection of animal and human health are essential.     

Non-archetypal Type II-like and atypical strains of Toxoplasma gondii infecting marsupials of Australia

Australia is geographically isolated and possesses a remarkable diversity of wildlife species. Marsupials are highly susceptible to infection with the cosmopolitan parasite Toxoplasma gondii. Of 46 marsupials screened for T. gondii by multilocus PCR-DNA sequencing at polymorphic genes (B1, SAG3, GRA6, GRA7), 12 were PCR-positive; the majority (67%; 9/12) were infected by non-archetypal Type II-like or atypical strains. Six novel alleles were detected at B1, indicating greater diversity of genotypes than previously envisaged. Two isolates lethal to marsupials, were avirulent to mice. The data support the conclusion that Australia’s isolation may have favoured the persistence of non-archetypal ancestral genotypes.