Rabbits as reservoirs: An updated perspective of the zoonotic risk from Cryptosporidium and Giardia.

Rabbits are highly abundant in many countries and can serve as reservoirs of diseases for a diversity of pathogens including the enteric protozoan parasites, Cryptosporidium and Giardia. Both parasites shed environmentally robust environmental stages (oo/cysts) and have been responsible for numerous waterborne outbreaks of diseases. Cryptosporidium hominis and C. parvum are responsible for most infections in humans, while Giardia duodenalis assemblages A and B, cause most human cases of giardiasis. Cryptosporidium cuniculus, the dominant species infecting rabbits, is the only spceies other than C. hominis and C. parvum to have caused a waterborne outbreak of gastritis, which occurred in the United Kingdom in 2008. This review examines the prevalence of Cryptosporidium and Giardia species in rabbits to better understand the public health risks of contamination of water sources with Cryptosporidium and Giardia oo/cysts from rabbits. Despite the abundance of C. cuniculus in rabbits, reports in humans are relatively rare, with the exception of the United Kingdom and New Zealand, and reports of C. cuniculus in humans from the United Kingdom have declined substantially since the 2008 outbreak. Subtyping of C. cuniculus has supported the potential for zoonotic transmission. Relatively few studies have been conducted on Giardia, but assemblage B dominates. However, improved typing methods are required to better understand the transmission dynamics of Giardia assemblages in rabbits. Similarly, it is not well understood if pet rabbits or contaminated water are the main source of C. cuniculus infections in humans. Well-planned studies using high-resolution typing tools are required to understand the transmission dynamics better and quantify the public health risk of Cryptosporidium and Giardia from rabbits.

Critters and contamination: Zoonotic protozoans in urban rodents and water quality.

Rodents represent the single largest group within mammals and host a diverse array of zoonotic pathogens. Urbanisation impacts wild mammals, including rodents, leading to habitat loss but also providing new resources. Urban-adapted (synanthropic) rodents, such as the brown rat (R. norvegicus), black rat (R. rattus), and house mouse (Mus musculus), have long successfully adapted to living close to humans and are known carriers of zoonotic pathogens. Two important enteric, zoonotic protozoan parasites, carried by rodents, include Cryptosporidium and Giardia. Their environmental stages (oocysts/cysts), released in faeces, can contaminate surface and wastewaters, are resistant to common drinking water disinfectants and can cause water-borne related gastritis outbreaks. At least 48 species of Cryptosporidium have been described, with C. hominis and C. parvum responsible for the majority of human infections, while Giardia duodenalis assemblages A and B are the main human-infectious assemblages. Molecular characterisation is crucial to assess the public health risk linked to rodent-related water contamination due to morphological overlap between species. This review explores the global molecular diversity of these parasites in rodents, with a focus on evaluating the zoonotic risk from contamination of water and wasterwater with Cryptosporidium and Giardia oocysts/cysts from synanthropic rodents. Analysis indicates that while zoonotic Cryptosporidium and Giardia are prevalent in farmed and pet rodents, host-specific Cryptosporidium and Giardia species dominate in urban adapted rodents, and therefore the risks posed by these rodents in the transmission of zoonotic Cryptosporidium and Giardia are relatively low. Many knowledge gaps remain however, and therefore understanding the intricate dynamics of these parasites in rodent populations is essential for managing their impact on human health and water quality. This knowledge can inform strategies to reduce disease transmission and ensure safe drinking water in urban and peri­urban areas.

The risk of wild birds contaminating source water with zoonotic Cryptosporidium and Giardia is probably overestimated.

Cryptosporidium and Giardia are important waterborne protozoan parasites that are resistant to disinfectants commonly used for drinking water. Wild birds, especially wild migratory birds, are often implicated in the contamination of source and wastewater with zoonotic diseases, due to their abundance near water and in urban areas and their ability to spread enteric pathogens over long distances. This review summarises the diversity of Cryptosporidium and Giardia in birds, with a focus on zoonotic species, particularly in wild and migratory birds, which is critical for understanding zoonotic risks. The analysis revealed that both avian-adapted and zoonotic Cryptosporidium species have been identified in birds but that avian-adapted Cryptosporidium species dominate in wild migratory birds. Few studies have examined Giardia species and assemblages in birds, but the non-zoonotic Giardia psittaci and Giardia ardeae are the most commonly reported species. The identification of zoonotic Cryptosporidium and Giardia in birds, particularly C. parvum and G. duodenalis assemblages A and B in wild migratory birds, is likely due to mechanical carriage or spillback from birds co-grazing pastures contaminated with C. parvum from livestock. Therefore, the role of wild migratory birds in the transmission of zoonotic Cryptosporidium and Giardia to source water is likely overestimated. To address knowledge gaps, it is important to conduct more extensive studies on the prevalence of Cryptosporidium and Giardia in a broader range of migratory wild birds. There is also a need to investigate the extent to which zoonotic infections with C. hominis/C. parvum and G. duodenalis assemblages A and B are mechanical and/or transient, and to assess the load and viability of zoonotic oo/cysts shed in avian faeces. Understanding the contribution of birds to zoonoses is essential for effective disease surveillance, prevention, and control.

Cryptosporidium and Giardia in cats and dogs: What is the real zoonotic risk?

Due to the close bond between humans and companion animals, a thorough understanding of the diversity of Cryptosporidium species and Giardia assemblages in cats and dogs is essential to determine the potential zoonotic risks. Analysis of molecular studies shows that C. felis and C. canis are the main species infecting cats and dogs, respectively. These species are largely host-specific, as despite intense association with humans, prevalence of C. felis and C. canis in humans is low and predominantly in immunocompromised individuals and low-income countries. There have been reports of C. parvum in cats and dogs and two reports of C. hominis in dogs. In most studies conducted to date, however, the prevalence of zoonotic species was low and may be associated with coprophagy and or/spillback, but this remains to be determined. Results of subtyping studies suggest that for C. felis and C. canis, some zoonotic transmission may occur but host-adapted subtypes also exist. Giardia duodenalis assemblages C and D are commonly reported in dogs, with assemblages F and A most common in cats. Assemblages C, D and F are largely host-specific as there are only a handful of reports of them in humans. Reports of assemblage A and B in cats and dogs may be due to coprophagy or spillback from owners. Despite the extent of pet ownership and the close contact between humans and companion animals worldwide, the overall risk of zoonotic transmission from cats and dogs to humans is uncertain but thought to be low due to C. canis, C. felis and G. duodenalis assemblages C, D and F being predominantly host-specific, the relatively low prevalence of C. parvum (and C. hominis) in cats and dogs (which may be due to mechanical carriage), and low oo/cyst shedding. Carefully designed epidemiological studies of cats and dogs and their owners using subtyping tools are essential to better quantify the extent of spillover and spillback of Cryptosporidium and Giardia between pets and their owners.

Detection of unusual Cryptosporidium parvum subtype in patients with gastrointestinal cancer in Egypt.

While the importance of cryptosporidiosis in immunocompromised persons is well known, the prevalence of Cryptosporidium spp. in cancer patients is not clear. The current study was designed to assess the occurrence and genetic characteristics of Cryptosporidium spp. in patients with gastrointestinal (GI) cancer in Egypt. Stool samples were collected from 100 patients with GI malignancies and 20 healthy individuals without any GI manifestations (control group). They were screened by microscopy and the immunochromatographic RIDA®QUICK Cryptosporidium kit. Subtyping of Cryptosporidium spp. was conducted by sequence analysis of the glycoprotein 60 (gp60) locus. Sociodemographic, environmental data and information on GI symptoms, cancer types, and clinical treatment were obtained via a questionnaire. By microscopy and RIDA®QUICK, only 7% (7/100) of GI cancer patients were positive for Cryptosporidium, compared with 40% (40/100) by gp60 nPCR. No positives were obtained from the control group. Male sex (P = 0.02) and younger age (P = 0.004) were major Cryptosporidium risk factors for infection. The occurrence of Cryptosporidium was also significantly more frequent (P = 0.003) in watery stool samples. Sequence analysis of the gp60 amplicons (~ 400 bp) identified a novel C. parvum subtype with nine TCA repeats and eleven ACATCA repeats. A formal subtype designation could not be made due to the short sequence length. More studies should be conducted to verify the common occurrence of this unusual C. parvum subtype and establish its genetic identity.

Cryptosporidium and Giardia in dam water on sheep farms - An important source of transmission?

Cryptosporidium and Giardia infections can negatively impact livestock health and reduce productivity, and some species and genotypes infecting livestock have zoonotic potential. Infection occurs via the faecal-oral route. Waterborne infections are a recognised source of infection for humans, but the role of livestock drinking water as a source of infection in livestock has not been described. This study aimed to determine whether contaminated drinking water supplies, such as farm dams, are a likely transmission source for Cryptosporidium and Giardia infections for extensively managed sheep. Dam water samples (n = 47) were collected during autumn, winter and spring from 12 farm dams located on six different farms in south west Western Australia, and faecal samples (n = 349) were collected from sheep with access to these dams. All samples were initially screened for Cryptosporidium spp. at the 18S locus and Giardia spp. at the gdh gene using qPCR, and oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR. Cryptosporidium-positive sheep faecal samples were typed and subtyped by sequence analysis of 18S and gp60 loci, respectively. Giardia-specific PCR and Sanger sequencing targeting tpi and gdh loci were performed on Giardia- positive sheep faecal samples to characterise Giardia duodenalis assemblages. To identify Cryptosporidium and Giardia spp. in dam water samples, next-generation sequencing analysis of 18S and gdh amplicons were performed, respectively. Two species of Cryptosporidium (Cryptosporidium xiaoi and Cryptospordium ubiquitum (subtype family XIIa)) were detected in 38/345 sheep faecal samples, and in water from 9/12 farm dams during the study period, with C. xiaoi the species most frequently detected in both faeces and dam water overall. Giardia duodenalis assemblages AI, AII and E were detected in 36/348 faecal samples and water from 10/12 farm dams. For dam water samples where oo/cysts were detected by qPCR, Cryptosporidium oocyst concentration ranged from 518-2429 oocysts/L (n = 14), and Giardia cyst concentration ranged from 102 to 1077 cysts/L (n = 17). Cryptosporidium and Giardia with zoonotic potential were detected in farm dam water, including C. ubiquitum, C. hominis, C. parvum, C. cuniculus, C. xiaoi, and G. duodenalis assemblages A, B and E. The findings suggest that dam water can be contaminated with Cryptosporidium species and G. duodenalis assemblages that may infect sheep and with zoonotic potential, and farm dam water may represent one source of transmission for infections.

Blood Parasites in Endangered Wildlife-Trypanosomes Discovered During a Survey of Haemoprotozoa from the Tasmanian Devil.

The impact of emerging infectious diseases is increasingly recognised as a major threat to wildlife. Wild populations of the endangered Tasmanian devil, Sarcophilus harrisii, are experiencing devastating losses from a novel transmissible cancer, devil facial tumour disease (DFTD); however, despite the rapid decline of this species, there is currently no information on the presence of haemoprotozoan parasites. In the present study, 95 Tasmanian devil blood samples were collected from four populations in Tasmania, Australia, which underwent molecular screening to detect four major groups of haemoprotozoa: (i) trypanosomes, (ii) piroplasms, (iii) Hepatozoon, and (iv) haemosporidia. Sequence results revealed Trypanosoma infections in 32/95 individuals. Trypanosoma copemani was identified in 10 Tasmanian devils from three sites and a second Trypanosoma sp. was identified in 22 individuals that were grouped within the poorly described T. cyclops clade. A single blood sample was positive for Babesia sp., which most closely matched Babesia lohae. No other blood protozoan parasite DNA was detected. This study provides the first insight into haemoprotozoa from the Tasmanian devil and the first identification of Trypanosoma and Babesia in this carnivorous marsupial.

Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in C. parvum biology remain un-elucidated. In this study, a member of the protein family, INS-15 of C. parvum encoded by cgd3_4260, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in Escherichia coli and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the C. parvum invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in E. coli, together with various degraded products. The cgd3_4260 gene had a peak expression at 2 h of in vitro C. parvum culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of C. parvum sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of in vitro growth indicate that INS-15 plays some roles in the invasion or early development of C. parvum.

Identification of eukaryotic microorganisms with 18S rRNA next-generation sequencing in wastewater treatment plants, with a more targeted NGS approach required for Cryptosporidium detection.

While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples (n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium-specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments.

Giardia: an under-reported foodborne parasite.

Foodborne zoonotic pathogens are a serious public health issue and result in significant global economic losses. Despite their importance to public health, epidemiological data on foodborne diseases including giardiasis caused by the enteric parasite, Giardia duodenalis, are lacking. This parasite is estimated to cause ∼28.2 million cases of diarrhoea each year due to contamination of food, but very few foodborne outbreaks have been documented due to the limitations of current detection as well as surveillance methods. The current method for the recovery of Giardia cysts from food matrices using immunomagnetic separation requires further standardisation and cost reduction before it can be widely used. It also should incorporate downstream molecular procedures for genotyping, and traceback and viability analyses. Foodborne giardiasis can be potentially controlled through improvements in national disease surveillance systems and the establishment of Hazard Analysis and Critical Control Point interventions across the food chain. Studies are needed to assess the true prevalence and public health impact of foodborne giardiasis.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the ß-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per µl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data.

Prevalence of Cryptosporidium species and subtypes in paediatric oncology and non-oncology patients with diarrhoea in Jordan.

Cryptosporidiosis is a protozoan parasitic disease which affects human and animals worldwide. In adult immunocompetent individuals, cryptosporidiosis usually results in acute and self-limited diarrhoea; however, it can cause life threatening diarrhoea in children and immunocompromised individuals. In the present study, we compared the prevalence of Cryptosporidium species and gp60 subtypes amongst paediatric oncology patients with diarrhoea (n=160) from King Hussein Medical Centre for Cancer in Jordan, and non-oncology paediatric patients with diarrhoea (n=137) from Al-Mafraq paediatric hospital. Microscopy results using modified acid fast staining identified a significantly (p≤0.05) higher prevalence of Cryptosporidium in paediatric oncology patients with diarrhoea (14.4% - 23/160), compared to non-oncology paediatric patients with diarrhoea only (5.1% - 7/137). With the exception of one sample, all microscopy-positive samples (n=29) and an additional 3/30 microscopy-negative controls were typed to species and subtype level at the 18S and gp60 loci, respectively. All Cryptosporidium positives were typed as C. parvum. Of the 22 typed Cryptosporidium positives from the paediatric oncology patients, 21 were subtyped as IIaA17G2R1 and one as IIaA16G2R1 C. parvum subtypes. The 7 typed positives from the paediatric patients from Al-Mafraq hospital were subtyped as IIaA17G2R1 (n=5) and IIaA16G2R1 (n=2). The 3 additional positives from the 30 microscopy negative control samples were subtyped as IIaA17G2R1. The high prevalence of the IIaA17G2R1 subtype, particularly amongst oncology patients, suggests that an outbreak of cryptosporidiosis may have been occurring in oncology patients during the collection period (April to December, 2016). New therapies for cryptosporidiosis in immunocompromised patients are urgently required.

Molecular typing of Giardia duodenalis in humans in Queensland - first report of Assemblage E.

Little is known about the genetic diversity of the protozoan parasite, Giardia duodenalis, infecting humans in Queensland, Australia. The present study typed 88 microscopically Giardia-positive isolates using assemblage-specific primers at the triose phosphate isomerase (tpi) gene and sequenced a subset of isolates at the glutamate dehydrogenase (gdh) gene (n = 30) and tpi locus (n = 27). Using the tpi-assemblage specific primers, G. duodenalis assemblage A and assemblage B were detected in 50% (44/88) and 38·6% (34/88) of samples, respectively. Mixed infections with assemblages A and B were identified in 4·5% (4/88) and assemblage E was identified in 6·8% (6/88) of samples. Sequence analysis at the gdh and tpi loci also confirmed the presence of assemblage E in these isolates. Cyst numbers per gram of feces (g-1) were determined using quantitative polymerase chain reaction and of the isolates that were typed as assemblage E, cyst numbers ranged 13·8-68·3 × 106 cysts g-1. This is the first report of assemblage E in humans in Australia, indicating that in certain settings, this assemblage may be zoonotic.

Characterization of two complete Isospora mitochondrial genomes from passerine birds: Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner.

The genus term Isospora is now applied specifically to parasites of birds, with the term Cystoisospora preferred for parasites which infect mammals. Isospora is a common parasitic coccidian in birds worldwide, especially in passerine birds, in which it can cause systemic coccidiosis. The complete mitochondrial genome sequences from two recently identified Isospora species; Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner, were sequenced and compared with those of other closely related coccidian species. The complete mitochondrial genome sequence for Isospora serinuse is 6260bp in size and 6223bp for Isospora manorinae. The mitochondrial genomes of Isospora serinuse and Isospora manorinae include three protein-coding genes (COI, COIII and CytB), 19 LSU and 14 SSU rDNA fragments, including one newly identified putative LSU fragment in Isospora sp. The arrangement of coding regions in these two Isospora species were identical to that of available Isospora sp. and Eimeria spp. mitochondrial genomes and the start codon usage for protein coding genes was conservative. Phylogenetic analysis of the mt genome of the two Isospora species based on the three coding regions further support that the monophyletic nature of avian Isospora.

Morphological and molecular characterization of an uninucleated cyst-producing Entamoeba spp. in captured Rangeland goats in Western Australia.

Uninucleated Entamoeba cysts measuring 7.3×7.7µm were detected in faecal samples collected from wild Rangeland goats (Capra hircus) after arrival at a commercial goat depot near Geraldton, Western Australia at a prevalence of 6.4% (8/125). Sequences were obtained at the 18S rRNA (n=8) and actin (n=5) loci following PCR amplification. At the 18S locus, phylogenetic analysis grouped the isolates closest with an E. bovis isolate (FN666250) from a sheep from Sweden with 99% similarity. At the actin locus, no E. bovis sequences were available, and the isolates shared 94.0% genetic similarity with E. suis from a pig in Western Japan. This is the first report to describe the morphology and molecular characterisation of Entamoeba from Rangeland goats in Western Australia and the first study to produce actin sequences from E. bovis-like Entamoeba sp.

Molecular characterisation of a disseminated Cryptosporidium infection in a Koi carp (Cyprinus carpio).

Cryptosporidium is a protozoan parasite that infects a wide range of hosts, yet relatively little is known about the epidemiology of cryptosporidiosis in fish. Here we report a disseminated Cryptosporidium infection in a male Koi carp (Cyprinus carpio), with parasite stages identified deep within the epithelium of the intestine, kidneys, spleen, liver and gills causing severe granulomatous inflammatory lesions. Molecular characterization at two loci; 18S ribosomal RNA (rRNA) and actin, revealed this to be a novel Cryptosporidium genotype, most closely related to Cryptosporidium molnari.

Chronic Cystoisospora belli infection in an immunocompetent Myanmar refugee - microscopy is not sensitive enough.

BACKGROUND: Cystoisosporiasis is an opportunistic infection seen more commonly in patients with acquired immunodeficiency syndrome. Although uncommon, Cystoisospora infection can occur in immunocompetent individuals but tend to be benign and self-limiting. Chronic infection however, has been described but diagnosis can often be challenging and requires a high clinical index of suspicion. CASE PRESENTATION: We present a case of delayed diagnosis of Cystoisospora belli (C. belli) in an immunocompetent 28-year-old refugee from Myanmar. She had a history of chronic diarrhea where exhaustive investigations over many years failed to reveal a diagnosis. Cystoisospora belli cysts were finally detected in stool 4 years after investigation commenced, and PCR testing on stored colon biopsies amplified a molecular product with 99 % sequence homology to C. belli. The patient improved promptly with trimethoprim-sulfamethoxazole treatment. CONCLUSION: In the appropriate clinical context we suggest molecular testing for C. belli or an empirical therapeutic trial.

First Molecular Characterization of Theileria ornithorhynchi Mackerras, 1959: yet Another Challenge to the Systematics of the Piroplasms.

Piroplasms, tick-transmitted Apicomplexa of the genera Theileria, Babesia and Cytauxzoon, are blood-borne parasites of clinical and veterinary importance. The order Piroplasmida shows a puzzling systematics characterized by multiple clades, soft polytomies and paraphyletic/polyphyletic genera. In the present study, screening of platypuses (Ornithorhynchus anatinus), was performed to infer the parasite molecular phylogeny. DNA was extracted from blood, ectoparasites and tick eggs and the 18S rRNA- hsp70-genes were used for the phylogenetic reconstructions. Microscopic analyses detected pleomorphic intra-erythrocytic organisms and tetrads consistent with previous descriptions of Theileria ornithorhynchi Mackerras, 1959, but observation of possible schizonts could not be confirmed. DNA sequences obtained from blood and ticks allowed resolving the systematics of the first piroplasm infecting a monotreme host. Molecularly, T. ornithorhynchi formed a novel monophyletic group, basal to most known piroplasms' clades. The ancestral position of this clade, isolated from an ancient lineage of mammalian host appears particularly fascinating. The present paper discusses the inadequacies of the current molecular systematics for the Piroplasmida and the consequences of incomplete sampling, morphology-based classification and ambiguous microscopic identifications. Likely when the current sampling bias is rectified and more sequence data is made available, the phylogenetic position of T. ornithorhynchi will be further contextualized without ambiguity.

Molecular characterisation of Cryptosporidium and Giardia in cats (Felis catus) in Western Australia.

Little is known of the prevalence of Cryptosporidium and Giardia in domestic cats in Western Australia and their potential role as zoonotic reservoirs for human infection. In the present study, a total of 345 faecal samples from four different sources were screened for the presence of Cryptosporidium and Giardia by PCR and genotyped by sequence analysis. Oocyst numbers and cyst numbers for Cryptosporidium and Giardia respectively were also determined using quantitative PCR assays. Cryptosporidium and Giardia were detected in 9.9% (95% CI 6.7-13.0) and 10.1% (95% CI 7.0-13.3) of cats in Western Australia respectively. Sequence analysis at the 18S rRNA locus identified five Cryptosporidium species/genotypes; C. felis (n = 8), C. muris (n = 1), C. ryanae (n = 1), Cryptosporidium rat genotype III (n = 5) and a novel genotype most closely related to Cryptosporidium rat genotype III in one isolate. This is the first report of C. ryanae and Cryptosporidium rat genotype III in cats. For Giardia, assemblage F the most commonly identified species, while only 1 assemblage sequence was detected. Since most human cases of cryptosporidiosis are caused by C. parvum and C. hominis and human cases of giardiasis are caused by G. duodenalis assemblage A and B, the domestic cats in the present study are likely to be of low zoonotic risk to pet owners in Perth. Risk analyses identified that elderly cats (more than 6 years) were more prone to Cryptosporidium and Giardia infections than kittens (less than 6 months) (P = 0.009). Clinical symptoms were not associated with the prevalence of Cryptosporidium and Giardia infections in cats.