Abstract: Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.
Abstract: Ninety percent of human cryptosporidiosis infections are attributed to two species; the anthroponotic Cryptosporidium hominis and the zoonotic Cryptosporidium parvum. Sequence analysis of the hypervariable gp60 gene, which is used to classify Cryptosporidium to the subtype level, has highlighted extensive intra-species diversity within both C. hominis and C. parvum. The gp60 has also facilitated contamination source tracking and increased understanding of the epidemiology of cryptosporidiosis. Two surface glycoproteins, the gp40 and gp15 are encoded in the gp60 gene; both are exposed to the hosts' immune system and play a pivotal role in the disease initiation process. The extent of genetic diversity observed within the gp60 would support the hypotheses of significant selection pressure placed on the gp40 and gp15. This study used a dual fluorescent terminal-restriction fragment length polymorphism (T-RFLP) analysis to investigate the genetic diversity of Cryptosporidium subtype populations in a single host infection. Terminal-RFLP showed subtype variation within one human Cryptosporidium sample and mouse samples from seven consecutive passages with C. parvum. Furthermore, this was the first study to show that differences in the ratio of subtype populations occur between infections. T-RFLP has provided a novel platform to study infection populations and to begin to investigate the impact of the hosts' immune system on the gp60 gene.
Abstract: Parasites from the Cryptosporidium genus are the most common cause of waterborne disease around the world. Successful management and prevention of this emerging disease requires knowledge of the diversity of species causing human disease and their zoonotic sources. This study employed a spatiotemporal approach to investigate sporadic human cryptosporidiosis in New South Wales, Australia, between January 2008 and December 2010. Analysis of 261 human fecal samples showed that sporadic human cryptosporidiosis is caused by four species; C. hominis, C. parvum, C. andersoni, and C. fayeri. Sequence analysis of the gp60 gene identified 5 subtype families and 31 subtypes. Cryptosporidium hominis IbA10G2 and C. parvum IIaA18G3R1 were the most frequent causes of human cryptosporidiosis in New South Wales, with 59% and 16% of infections, respectively, attributed to them. The results showed that infections were most prevalent in 0- to 4-year-olds. No gender bias or regional segregation was observed between the distribution of C. hominis and C. parvum infections. To determine the role of cattle in sporadic human infections in New South Wales, 205 cattle fecal samples were analyzed. Four Cryptosporidium species were identified, C. hominis, C. parvum, C. bovis, and C. ryanae. C. parvum subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, with 47% of infections attributed to it. C. hominis subtype IbA10G2 was also identified in cattle isolates.
Abstract: Cryptosporidiosis is one of the most common waterborne diseases reported worldwide. Outbreaks of this gastrointestinal disease, which is caused by the Cryptosporidium parasite, are often attributed to public swimming pools and municipal water supplies. Between the months of January and April in 2009, New South Wales, Australia, experienced the largest waterborne cryptosporidiosis outbreak reported in Australia to date. Through the course of the contamination event, 1,141 individuals became infected with Cryptosporidium. Health authorities in New South Wales indicated that public swimming pool use was a contributing factor in the outbreak. To identify the Cryptosporidium species responsible for the outbreak, fecal samples from infected patients were collected from hospitals and pathology companies throughout New South Wales for genetic analyses. Genetic characterization of Cryptosporidium oocysts from the fecal samples identified the anthroponotic Cryptosporidium hominis IbA10G2 subtype as the causative parasite. Equal proportions of infections were found in males and females, and an increased susceptibility was observed in the 0- to 4-year age group. Spatiotemporal analysis indicated that the outbreak was primarily confined to the densely populated coastal cities of Sydney and Newcastle.
Abstract: Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening.
Abstract: The majority of biological data on Cryptosporidium has been collected from humans and domestic animal hosts which creates a bias in knowledge on the biodiversity and evolution of this parasite genus. Further to understanding Cryptosporidium biology are studies encompassing broad hosts that represent diverse taxa sampled across wide geographic ranges. Marsupials represent a group of wildlife hosts from which limited information on Cryptosporidium is available. As marsupial hosts are an ancient mammalian lineage they represent an important group for studying parasite evolution. This review summarises information of the biology, epidemiology and evolution of Cryptosporidium in marsupial hosts, and discusses the importance of further understanding interactions in this parasite-host system.
Abstract: Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.
Abstract: Management and control of cryptosporidiosis in human requires knowledge of Cryptosporidium species contributing to human disease. Markers that are able to provide information below the species level have become important tools for source tracking. Using the hypervariable surface antigen, glycoprotein 60 (GP60), C. hominis (n=37) and C. parvum (n=32) isolates from cryptosporidiosis cases in New South Wales, Australia, were characterised. Extensive variation was observed within this locus and the isolates could be divided into 8 families and 24 different subtypes. The subtypes identified have global distributions and indicate that anthroponotic and zoonotic transmission routes contribute to sporadic human cryptosporidiosis in NSW.
Abstract: The highly polymorphic 60 kDa glycoprotein (GP60) of Cryptosporidium is an important tool for investigating the epidemiology of this parasite. Characterization of the GP60 gene has only been performed for 3 of the 20 known Cryptosporidium species, and has already enabled sub-typing and source tracking of species with human significance. We have characterised a fourth species, Cryptosporidium fayeri, at the GP60 locus using isolates (n=26) from different marsupial hosts to assess the diversity of GP60 within this species. The analysis demonstrated that C. fayeri isolates could be assigned to 6 subtypes which were associated with host species and locality. The intra-species diversity for the host-adapted C. fayeri was less than the diversity for human pathogenic species suggesting that the GP60 locus is under less selective pressure in these than host-adapted species.
Abstract: Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E.trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line.
Abstract: Cryptosporidium is the most common non-viral cause of diarrhea worldwide. Of the 5 described species that contribute to the majority of human infections, C. parvum is of major interest due to its zoonotic potential. A species-specific fluorescence in situ hybridisation probe was designed to the variable region in the small subunit of the 18S rRNA of C. parvum and labeled with Cy3. Probe specificity was validated against a panel of 7 other Cryptosporidium spp. before it was applied to 33 human faecal samples positive for cryptosporidiosis which were obtained during the period from 2006-2007. Results were compared to PCR-RFLP targeting the 18S rDNA. FISH results revealed that 19 of the 33 isolates analysed were identified as C. parvum. Correlation of PCR-RFLP and FISH was statistically significant (P<0.05), resulting in a calculated correlation coefficient of 0.994. In this study, species identification by FISH and PCR-RFLP provided preliminary evidence to support both anthroponotic and zoonotic transmission of sporadic cases of cryptosporidiosis in the Sydney basin. In conclusion, FISH using a C. parvum-specific probe provided an alternative tool for accurate identification of zoonotic Cryptosporidium which will be applied in the future to both epidemiological and outbreak investigations.
Abstract: To investigate the possible role of common brushtail possums (Trichosurus vulpecula) in the transmission of Toxoplasma gondii within a zoo environment, a serological survey of a free-ranging population resident within Taronga Zoo, Sydney, Australia was undertaken using the modified agglutination test (MAT). For comparison, the seroprevalence of T. gondii antibodies was also assessed in a possum population inhabiting a felid-free, non-urban woodland habitat. Six of 126 possums (4.8%) from the zoo population had antibodies to T. gondii with a MAT titre of 25 or higher, while in contrast, all of the 17 possums from woodland were seronegative. These observations suggest that possums were at a higher risk of exposure to the parasite as a consequence of co-existing with domestic, stray and captive felids associated with urbanisation. Screening of captive felids at the zoo indicated 16 of 23 individuals (67%) and all 6 species were seropositive for T. gondii, implicating them as a possible source of the parasite within the zoo setting. In addition captive, non-felid carnivores including the chimpanzee (Pan troglodytes), saltwater crocodile (Crocodylus porosus), dingo (Canis lupis) and leopard seal (Hydrurga leptonyx) were tested for the presence of T. gondii antibodies as these species predate and are a leading cause of death amongst zoo possums. In total, 5 of 23 individuals (22%) were seropositive, representing 2 of the 4 carnivorous species; the dingo and chimpanzee. These data suggest that carnivory was not a highly efficient pathway for the transmission of T. gondii and the free-ranging possum population posed minimal threat to the health of zoo animals.
Abstract: Cryptosporidium macropodum n. sp is described. Oocysts of C. macropodum from the feces of kangaroos (Macropus spp.) are morphologically indistinguishable from other mammalian Cryptosporidium species, including C. parvum, C. hominis, C. suis, and C. canis. The oocysts are fully sporulated on excretion, lack sporocysts, and have an average width of 4.9 microm (4.5-6.0), a length of 5.4 microm (5.0-6.0), and a length:width ratio of 1.1. Phylogenetic analyses of the 18S ribosomal RNA, actin, and heat shock protein 70 (HSP70) loci demonstrate that C. macropodum is genetically distinct from all described Cryptosporidium species, including others found in marsupials. The parasite seems to be highly host-specific, because it has been found only in marsupials to date. Therefore, based on biological and molecular data, we consider C. macropodum a new species.
Abstract: The common brushtail possum (Trichosurus vulpecula) is one of the most abundant native marsupials in urban Australia, having successfully adapted to utilize anthropogenic resources. The habituation of possums to food and shelter available in human settlements has facilitated interaction with people, pets, and zoo animals, increasing the potential for transmission of zoonotic Cryptosporidium pathogens. This study sought to examine the identity and prevalence of Cryptosporidium species occurring in possums adapted to urban settings compared to possums inhabiting remote woodlands far from urban areas and to characterize the health of the host in response to oocyst shedding. Findings indicated that both populations were shedding oocysts of the same genotype (brushtail possum 1 [BTP1]) that were genetically and morphologically distinct from zoonotic species and genotypes and most closely related to Cryptosporidium species from marsupials. The urban population was shedding an additional five Cryptosporidium isolates that were genetically distinct from BTP1 and formed a sister clade with Cryptosporidium parvum and Cryptosporidium hominis. Possums that were shedding oocysts showed no evidence of pathogenic changes, including elevated levels of white blood cells, diminished body condition (body mass divided by skeletal body length), or reduced nutritional state, suggesting a stable host-parasite relationship typical of Cryptosporidium species that are adapted to the host. Overall, Cryptosporidium occurred with a higher prevalence in possums from urban habitat (11.3%) than in possums from woodland habitat (5.6%); however, the host-specific nature of the genotypes may limit spillover infection in the urban setting. This study determined that the coexistence of possums with sympatric populations of humans, pets, and zoo animals in the urban Australian environment is unlikely to present a threat to public health safety.
Abstract: Current DNA extraction methods for parasites are labour-intensive and usually involve several steps, increasing the potential for cross-contamination. We describe here a closed-tube DNA extraction procedure based upon the use of a thermostable proteinase that enabled sensitive amplification of target loci from parasites from diverse lineages including Apicomplexa, Sarcomastgophora and Nematoda. Moreover, this procedure is not subject to cross-contamination and is readily adaptable to automation.
Abstract: A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.
Abstract: The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.
Abstract: Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality.
Abstract: Molecular characterisation of Cryptosporidium oocysts isolated from faeces collected from eastern grey kangaroos Macropus giganteus inhabiting an Australian water catchment revealed that this host was susceptible to three types of Cryptosporidium. Nucleotide sequence analysis of the 18S rDNA, Cryptosporidium oocyst wall protein (COWP) and a 70kDa heat shock protein (HSP70) identified an isolate identical to the described Cryptosporidium 'marsupial' genotype. A second isolate had less than 0.5% variation, compared to the described Cryptosporidium 'marsupial' genotype, within the sequences of the 18S rDNA, COWP and HSP70 and 10% variation in the internal transcribed spacer 1 (ITS1). Multilocus analysis of the third Cryptosporidium revealed a novel genotype that had a degree of genetic variation, at the four loci characterised, which was greater than or equivalent to that used to discriminate between currently recognised Cryptosporidium species. These findings have increased our current understanding on the molecular epidemiology of Cryptosporidium in Australian wildlife and have provided information on the types of Cryptosporidium marsupials may shed into the environment.
Abstract: A combined immunomagnetic separation (IMS) and flow cytometry (FC) technique was developed for the sensitive detection of Cryptosporidium in faecal samples. The IMS/FC technique was found to be approximately 50-fold more sensitive than formol-ether concentration, which is commonly used for Cryptosporidium epidemiological investigations. Of 31 faecal samples from captive animals 16 were found to contain Cryptosporidium oocysts when analysed using the IMS/FC compared to four when using formol-ether concentration (FEC). In a wild population of eastern grey kangaroos Macropus giganteus 66.3% of infected animals were shedding <500oocysts/gfaeces when analysed using IMS/FC. This is below the detection limit for the FEC method. The dispersal of Cryptosporidium in host populations is aggregated, with many individuals shedding low numbers of oocysts and few individuals shedding numbers of oocysts sufficiently high to be detected by FEC. This research demonstrates that the prevalence and oocyst shedding intensity of Cryptosporidium in animal populations will be significantly underestimated using standard detection methods.
Abstract: Resistance, especially to the anthelmintic benzimidazoles (BZ), has been reported in horse cyathostomes world-wide. Diagnosis of resistance has traditionally been made by faecal egg count reduction (FECR) trials, however, this technique has limitations. Some of the shortcomings may be resolved by refining the test or by using an in vitro test. FECR tests and the larval development assay (LDA) were performed on adult horses held on 15 different horse properties across a wide geographical area of NSW, Australia. FECR were measured before and 10-14 after days treatment with oxibendazole (OBZ), morantel (MOR) or ivermectin (IVM) at recommended dose rates. Eight properties were rejected following low pre-treatment egg counts, leaving seven in the study. On these, the majority of larvae recovered from faecal cultures were cyathostomes. Using a definition of resistance as a FECR of <90%, resistance to OBZ was present on six properties and to MOR on two properties. Resistance to IVM was not detected. An alternative method of calculating FECR based on individual horse egg counts pre- and post-treatment was developed and results from the same properties compared with the results of the LDA. For example, for the BZ, correlation coefficients of values of lethal concentration to kill 50% of population (LC50) on LDA and FECR percentages were -0.536 before and -0.704 after OBZ treatment. We conclude that the LDA has the potential to be a single visit test for detection of anthelmintic resistance in horse cyathostomes but requires further investigation and standardisation.
Abstract: Three Streptomyces isolates were identified as producing macrolide antibiotics of the bafilomycin or leucanicidin types during an evaluation of Australian actinomyces for the production of inhibitors of larval development in the parasitic nematode, Haemonchus contortus. Bafilomycins A1, B1, C1, and D were obtained from culture A239 and the 2-O-methyl-L-rhamnosyl derivative of bafilomycin A1, leucanicidin, from cultures A223 and A240. All these 'bafilolides' gave similar patterns of inhibition typified by an initial paralysis of newly hatched L1 larvae and a lethal toxicity within 24 h. LD50 values for inhibition of larval development of McMaster H. contortus ranged from 0.23 micrograms ml-1 for leucanicidin to 2.5 micrograms ml-1 for bafilomycin D. The bafilolides had broad spectrum nematocidal activity, being equi-potent as inhibitors of H. contortus, Trichostrongylus colubriformis and Ostertagia circumcincta larval development. Further, all bafilolides caused some inhibition of H. contortus L3 motility, with the semi-synthetic analogue, bafilomycin B2, the most potent inhibitor (LP50 against McMaster H. contortus 1.9 microgram ml-1). Nematode strains resistant to the known benzimidazole, levamisole and avermectin anthelmintics showed no cross resistance to the bafilolides, supporting the hypothesis that the bafilolides act by an independent mechanism.
