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 marsupials in Australia

A total of 421 fecal samples from a variety of captive and wild marsupial hosts in Western Australia, Victoria and South Australia were screened for the presence of Giardia species/genotypes using PCR and sequence analysis of a fragment of the 18S rRNA gene. Giardia spp. were identified in 13.4% (28/209) of samples from captive marsupials and 13.7% (29/212) of samples from wild marsupials. Sequence analysis at the 18S locus identified the zoonotic Giardia duodenalis Genotypes A and B in both captive and wild marsupials. Eight isolates were typed as genotype B3 and B4 at the gdh locus, although 7/8 were typed as genotype A at the 18S rRNA locus. The possible reasons for this discordance are discussed. This is the first report of genotype B and only the second report of genotype A in marsupials. As some of the genotype B isolates were identical to human-derived Giardia gdh sequences, these results suggest that marsupials in catchments may pose a public health risk and therefore warrant further investigation.     

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.     

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.     

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.     

Cryptosporidium and Giardia: Treatment options and prospects for new drugs

Cryptosporidium species and Giardia intestinalis are the most common enteric protozoan pathogens affecting humans worldwide. In recent years, nitazoxanide has been licensed in the United States for the treatment of cryptosporidiosis in non-immunodeficient children and adults, becoming the first drug approved for treating this disease. There is a need for a highly effective treatment for cryptosporidiosis in immunodeficient patients, but the quest for such a drug has proven to be elusive. While not effective against Cryptosporidium, nitroimidazoles such as metronidazole or tinidazole are effective treatments for giardiasis and can be administered as a single dose. Albendazole and nitazoxanide are effective against giardiasis but require multiple doses. Nitazoxanide is the first new drug developed for treating giardiasis in more than 20 years. New potentially promising drug targets in Cryptosporidium and Giardia have been identified, but there appears to be little activity toward clinical development of new drugs.     

Evidence supporting zoonotic transmission of Cryptosporidium in rural New South Wales

Cryptosporidium hominis, which has an anthroponotic transmission cycle and Cryptosporidium parvum, which is zoonotic, are the primary species of Cryptosporidium that infect humans. The present study identified the species/genotypes and subgenotypes of Cryptosporidium in 7 human and 15 cattle cases of sporadic cryptosporidiosis in rural western NSW during the period from November 2005 to January 2006. The species/genotype of isolates was determined by PCR sequence analysis of the 18S rRNA and C. parvum and C. hominis isolates were subgenotyped by sequence analysis of the GP60 gene. Fourteen of 15 cattle-derived isolates were identified as C. parvum and 1 as a C. bovis/C. parvum mixture. Of the human isolates, 4 were C. parvum and 3 were C. hominis. Two different subgenotypes were identified with the human C. hominis isolates and six different subgenotypes were identified within the C. parvum species from humans and cattle. All four of the C. parvum subtypes found in humans were also found in the cattle, indicating that zoonotic transmission may be an important contributor to sporadic human cases cryptosporidiosis in rural NSW.     

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite. Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is, however, a large amount of future work needed to fully understand the proteome of this parasite.     

Involvement of 14-3-3 protein post-translational modifications in Giardiaduodenalis encystation

14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia.     

Giardia/giardiasis — A perspective on diagnostic and analytical tools

Giardiasis is a gastrointestinal disease of humans and other animals caused by species of parasitic protists of the genus Giardia. This disease is transmitted mainly via the faecal–oral route (e.g., in water or food) and is of socioeconomic importance worldwide. The accurate detection and genetic characterisation of the different species and population variants (usually referred to as assemblages and/or sub-assemblages) of Giardia are central to understanding their transmission patterns and host spectra. The present article provides a background on Giardia and giardiasis, and reviews some key techniques employed for the identification and genetic characterisation of Giardia in biological samples, the diagnosis of infection and the analysis of genetic variation within and among species of Giardia. Advances in molecular techniques provide a solid basis for investigating the systematics, population genetics, ecology and epidemiology of Giardia species and genotypes as well as the prevention and control of giardiasis.     

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.     

Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Cryptosporidium in birds, fish and amphibians

Whilst considerable information is available for avian cryptosporidiosis, scant information is available for Cryptosporidium infections in fish and amphibians. The present review details recent studies in avian cryptosporidiosis and our current knowledge of piscine and amphibian infections.     

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.     

Molecular characterisation of Cryptosporidium outbreaks in Western and South Australia

Molecular typing at the 18S rRNA and Gp60 loci was conducted on Cryptosporidium-positive stool samples from cases collected during 2007 Western Australian and South Australian outbreaks of cryptosporidiosis. Analysis of 48 Western Australian samples identified that all isolates were C. hominis and were from five different Gp60C. hominis subtype families. The IbA10G2 subtype was most common across all age groups (37/48). In South Australia, analysis of 24 outbreak samples, identified 21 C. hominis isolates, two C. parvum isolates and one sample with both C. hominis and C. parvum. All C. hominis isolates were identified as the IbA10G2 subtype.     

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.     

Developmental stages and molecular phylogeny of Hepatozoon tuatarae, a parasite infecting the New Zealand tuatara, Sphenodon punctatus and the tick, Amblyomma sphenodonti

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 × 228 μm. Partial sequence data of the parasite’s small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.     

Occurrence and genotypes of Giardia isolated from lambs in Spain

Three hundred and eighty six faecal specimens were randomly collected from 1- to 3-month-old lambs from 16 farms in Spain to investigate the presence of different genotypes of Giardia duodenalis. Individual specimens were examined by IFA (Immunofluorescence assay) and β-giardin PCR polymerase chain reaction. Cysts of G. duodenalis were shed by lambs in every flock analyzed, showing a prevalence by farms of 100%. The average prevalence of G. duodenalis for the 386 specimens was 42%, ranging from 8.3 to 80% depending on the farm. β-giardin PCR positive samples were sequenced to determine the genotypes present at each farm and seven new subtypes of β-giardin Assemblage E are reported in this study. In each farm, one to six different β-giardin subtypes were found, showing the high variability of the target. Also, one flock had the zoonotic Assemblage A. This is the first report of Giardia subgenotype A-1 in sheep in Spain.     

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.