Linda M. McInnes

Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites.

ABSTRACT Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala (Phascolarctos cinereus)

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

Novel trypanosome Trypanosoma gilletti sp. (Euglenozoa: Trypanosomatidae) and the extension of the host range of Trypanosoma copemani to include the koala (Phascolarctos cinereus)

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (± s.e.) at the 18S rDNA locus of 2.7 ± 0.5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (± s.e.) of T. gilletti was 8.7 ± 1.1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1.5%) of the koalas tested.

A survey of canine filarial diseases of veterinary and public health significance in India

BackgroundDirofilaria spp., Acanthocheilonema spp. and Brugia spp. have all been reported in Indian dogs. In previous studies, diagnosis was made by morphological identification only. This is the first geographically stratified cross-sectional study in India to determine the prevalence and geographical distribution of canine filarial species of veterinary and public health importance, using a combination of conventional and molecular diagnostic techniques.ResultsA total of 139 from 525 dogs (26.5%; 95% CI 22.7, 30.3) were positive for microfilariae. The most common species of canine filaria identified in this study was A. reconditum (9.3%) followed by D. repens (6.7%) and D. immitis (1.5%). Three out of 525 dogs were found to have mixed infections on PCR. The morphological and molecular evidence on the sequence of the 18S gene and phylogenetic analysis of the ITS-2 region provided strong evidence that the canine microfilariae discovered in the Himalayan city of Ladakh belong to a novel species of Acanthocheilonema. Two dogs in Ladakh were also found to have mixed infections of the novel species described above and a unique microfilaria which morphologically resembled Microfilaria auquieri Foley, 1921.ConclusionsAt least six species of filarial nematode are now known to infect dogs in India, two of which were reported for the first time in this study. The study also confirms and extends the geographical distribution of canine heartworm (D. immitis) which overlaps with D. repens, emphasising the importance for veterinary clinicians and diagnostic laboratories to utilise immunodiagnostic tests that will not cross-react between those two filarial species. From a public health viewpoint, the distribution and prevalences of these nematodes warrant an appropriate prophylaxis to be administered to dogs.

Identification of novel Babesia and Theileria genotypes in the endangered marsupials, the woylie (Bettongia penicillata ogilbyi) and boodie (Bettongia lesueur)

Piroplasms, which include the genera Theileria and Babesia, are blood-borne parasites transmitted mainly by tick vectors. Relatively little is known about their prevalence and clinical impact in Australian marsupials. In the present study the occurrence and molecular phylogeny of these parasites were studied in both wild and captive marsupials from Western Australia (WA) and Queensland (QLD). Blood samples were screened by microscopy and molecular methods, using PCR and DNA sequencing of the 18S ribosomal RNA gene (18S rDNA). Overall, 7.1% of the blood samples (8/113) were positive for piroplasm 18S rDNA. Theileria and Babesia rDNA was detected in 0.9% (1/113) and 6.2% (7/113) of the animals, respectively. The single Theileria positive was identified in one of three boodies (Bettongia lesueur) screened from a wildlife rehabilitation centre in WA, while all seven Babesia positives were detected in WA in wild captured woylies (Bettongia penicillata ogilbyi). Small intraerythrocytic inclusions were observed in blood films made from six of these individuals. This is the first report of a Babesia sp. in woylies, and Theileria sp. in boodies. Phylogenetic analysis indicated that the woylie-derived Babesia was genetically distinct and most closely related to Babesia occultans, the causative agent of a benign form of cattle babesiosis (genetic similarity 98.4%). The Theileria identified was most closely related to the marsupial-derived species Theileria penicillata from the woylie, Theileria brachyuri from the quokka (Setonix brachyurus), and Theileria sp. from the long-nosed potoroo (Potorous tridactylus).

Identification of novel trypanosome genotypes in native Australian marsupials.

In the present study, the occurrence and molecular phylogeny of trypanosome parasites were studied in both wild and captive marsupials from Western Australia and Queensland. Blood samples were screened by PCR at the 18S rDNA locus, and the glycosomal glyceraldehyde phosphate dehydrogenase gene. Overall, 5.3% of the blood samples were positive at the 18S rDNA locus. All positives belonged to wild-captured Western Australian individuals, where trypanosome-specific DNA was detected in 9.8% of the screened samples from wild marsupials, in common brushtail possums, and woylies. The detection rate of trypanosome DNA in these two host species was 12.5% and 20%, respectively. Phylogenetic analyses based on two loci, indicated that the possum-derived trypanosome isolates were genetically distinct, and most closely related to the Australian marsupial trypanosomes H25 from a kangaroo, and BRA2 from a bush rat. This is the first study to genetically characterise trypanosome isolates from possums. The analysis of the woylie-derived isolates demonstrated that this marsupial host can harbour multiple genotypes within the same geographical location and furthermore multiple genotypes within the same host, indicative of mixed infections. All the woylie-derived genotypes grouped with trypanosomes found in Australian marsupials, suggesting that they are more likely to belong to an endemic or Australasian trypanosome species. This is the first study to genetically characterise trypanosome isolates from possums (Trichosurus vulpecula). Although the clinical significance of these infections is currently unknown, the identification of these novel sequences may support future investigations on transmission, threats to endangered wildlife, and evolutionary history of the genus Trypanosoma.

Genetic characterization of flea-derived Bartonella species from native animals in Australia suggests host-parasite co-evolution.

Fleas are important arthropod vectors for a variety of diseases in veterinary and human medicine, and bacteria belonging to the genus Bartonella are among the organisms most commonly transmitted by these ectoparasites. Recently, a number of novel Bartonella species and novel species candidates have been reported in marsupial fleas in Australia. In the present study the genetic diversity of marsupial fleas was investigated; 10 species of fleas were collected from seven different marsupial and placental mammal hosts in Western Australia including woylies (Bettongia penicillata), western barred bandicoots (Perameles bougainville), mardos (Antechinus flavipes), bush rats (Rattus fuscipes), red foxes (Vulpes vulpes), feral cats (Felis catus) and rabbits (Oryctolagus cuniculus). PCR and sequence analysis of the cytochrome oxidase subunit I (COI) and the 18S rRNA genes from these fleas was performed. Concatenated phylogenetic analysis of the COI and 18S rRNA genes revealed a close genetic relationship between marsupial fleas, with Pygiopsylla hilli from woylies, Pygiopsylla tunneyi from western barred bandicoots and Acanthopsylla jordani from mardos, forming a separate cluster from fleas collected from the placental mammals in the same geographical area. The clustering of Bartonella species with their marsupial flea hosts suggests co-evolution of marsupial hosts, marsupial fleas and Bartonella species in Australia.

Theileria gilberti n. sp. (Apicomplexa: Theileriidae) in the Gilbert's Potoroo (Potorous gilbertii)

ABSTRACT. The morphology and genetic characterisation of a new species of piroplasm identified in the blood of the Gilberts potoroo (Potorous gilbertii) from the Two Peoples Bay Nature Reserve near Albany, Western Australia, is described from blood and tissue samples from 16 Gilberts potoroos. Microscopy of blood showed these parasites are highly pleomorphic with a mean length of 1.8 μm and mean width of 0.85 μm. Phylogenetic analysis of 18S rRNA sequence data identified the piroplasm as a new species of Theileria that is closely related to other Australian marsupial piroplasm species. Based on biological and molecular data, it is proposed that the parasite from Gilberts potoroo be given the name Theileria gilberti n. sp.

Piroplasms of New Zealand seabirds

Blood and ectoparasitic ticks were collected from migratory seabirds in New Zealand, including Australasian gannets (n = 13) from two sites and red-billed gulls (n = 9) and white-fronted terns (n = 2) from a third location. Blood smears were screened for parasite presence by microscopy, while DNA from blood samples was subjected to PCR for the presence of tick-transmitted protozoan haemoparasites belonging to the order Piroplasmida. Parasites were identified by comparing small subunit ribosomal RNA (18S rDNA) gene sequences to related sequences on GenBank. Analyses indicated that nine birds were infected with unknown variants of a Babesia poelea-like parasite (recorded as genotypes I and II), while four harboured a piroplasm that was genetically similar to Babesia kiwiensis. There was no parasite stratification by bird species; both the gannets and gulls were positive for all three parasites, while the terns were positive for the B. kiwiensis-like and the B. poelea-like (genotype I) parasites. The B. kiwiensis-like parasite found in the birds was also found in two species of ticks: Carios capensis and Ixodes eudyptidis. This represents the first report of Babesia-positive ticks parasitising seabirds in New Zealand. The lack of host specificity and evidence of wide ranging distributions of the three piroplasm genotypes suggests there is a high degree of haemoparasite transmission occurring naturally between New Zealand seabird populations and species.

Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao, Philippines.

Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is the causative agent of the livestock disease known as surra. A total of 140 out of 179 T. evansi isolates collected between 2006 and 2007 from 44 villages (comprising of 16 reported surra outbreaks) in 3 provinces (Agusan del Sur (ADS), Surigao del Sur (SDS) and Agusan del Norte (ADN)) in Mindanao, Philippines were each successfully genotyped using a suite of 7 polymorphic microsatellites. The study identified 16 multi locus genotypes (MLG) within the T. evansi isolates and evidence of the spread of surra outbreaks from one village to another, most likely due to the movement of infected animals. Genotyping provided evidence of population sub-structuring with 3 populations (I, II and III (only 1 isolate)) identified. The most abundant population was II, which was the predominant population in ADS and SDS (p=0.022). In addition, buffalo mortality was statistically higher in outbreak areas associated with isolates from population I (13.6%) than with isolates from population II (6.9%) (p=0.047). The present study has highlighted the utility of microsatellite loci to improve understanding of the epidemiology of T. evansi and in tracking surra outbreaks.