Ocular Dirofilariasis in Migrant from Sri Lanka, Australia.

We describe a case of imported ocular dirofilariasis in Australia, linked to the Hong Kong genotype of Dirofilaria sp., in a migrant from Sri Lanka. Surgical extraction and mitochondrial sequences analyses confirmed this filarioid nematode as the causative agent and a Dirofilaria sp. not previously reported in Australia.

A molecular characterization of marsupial filarioid nematodes of the genus Breinlia.

Here we present the genetic relationships of 26 specimens of the genus Breinlia (Nematoda: Filarioidea) from a range of Australian marsupials using markers in the small subunit of nuclear ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes and compare them with morphological determinations. The molecular data support the validity of most of the morpho-species included in the study and provide provisional insights into the phylogeny of the genus in Australian mammals, with dasyuroid marsupials appearing to be the original hosts. The recent discovery of Breinlia annulipapillata in the eye of a human brings this genus of parasites into the group of emerging infectious parasitic diseases.

Citellinema (Nematoda: Heligmosomidae) from North America with descriptions of 2 new species from the red squirrel Tamiasciurus hudsonicus and 1 from the Canadian woodchuck, Marmota monax.

Citellinema Hall, 1918 includes 6 valid species of gastrointestinal nematodes of sciurids. Two species occur in the Palearctic and 4 in the Nearctic, 3 of which occur minimally across Colorado, Idaho and Oregon and 1, Citellinema bifurcatum, has a wide distribution across North America. Members of the genus are didelphic, possess a cephalic vesicle, a terminal spine-like process in females and feature robust spicules, consisting of a proximal end fused and semicylindrical shaft connected to a lamina supported by 2 terminal filiform processes. Typically, the size of the spicules is used to differentiate species. As part of the Beringian Coevolution Project, specimens provisionally identified as C. bifurcatum were collected through intensive field sampling of mammals and associated parasites from across localities spanning the Holarctic. These specimens revealed considerable genetic variability at both mitochondrial and nuclear loci, supporting the identification of deeply divergent clades. Examination of these new specimens, along with the holotypes of C. bifurcatum and Citellinema quadrivittati indicates that Citellinema monacis (previously synonymized with C. bifurcatum) should be resurrected and 3 additional species described. We suggest that the apparent bifurcated nature of the spicule should be considered a generic diagnostic trait, while the proportional length of the lamina relative to that of the spicule is used as a specific character. We demonstrate the critical need for continued inventory of often poorly known assemblages of hosts and parasites, contributing to a growing baseline of archival specimens, collections and information that make explorations of faunal structure and diversity possible.

Dipylidium caninum draft genome - a new resource for comparative genomic and genetic explorations of flatworms.

Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of ~13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular biology of D. caninum and a new resource for comparative genomic and genetic explorations of this and other flatworms.

Ocular Filariasis in Human Caused by Breinlia (Johnstonema) annulipapillata Nematode, Australia.

We report a human case of ocular filariasis, caused by a species of Breinlia nematode, from Queensland, Australia. Morphological and molecular evidence indicated that the nematode Breinlia (Johnstonema) annulipapillata, or a closely related taxon, likely transmitted from a macropodid marsupial host was involved, which might represent an accidental finding or an emerging zoonosis.

Age of first infection across a range of parasite taxa in a wild mammalian population.

Newborn mammals have an immature immune system that cannot sufficiently protect them against infectious diseases. However, variation in the effectiveness of maternal immunity against different parasites may couple with temporal trends in parasite exposure to influence disparities in the timing of infection risk. Determining the relationship between age and infection risk is critical in identifying the portion of a host population that contributes to parasite dynamics, as well as the parasites that regulate host recruitment. However, there are no data directly identifying timing of first infection among parasites in wildlife. Here, we took advantage of a longitudinal dataset, tracking infection status by viruses, bacteria, protists and gastro-intestinal worms in a herd of African buffalo (Syncerus caffer) to ask: how does age of first infection differ among parasite taxa? We found distinct differences in the age of first infection among parasites that aligned with the mode of transmission and parasite taxonomy. Specifically, we found that tick-borne and environmentally transmitted protists were acquired earlier than directly transmitted bacteria and viruses. These results emphasize the importance of understanding infection risk in juveniles, especially in host species where juveniles are purported to sustain parasite persistence and/or where mortality rates of juveniles influence population dynamics.

Enterocytozoon bieneusi genotypes in cats and dogs in Victoria, Australia.

BACKGROUND: Enterocytozoon bieneusi is one of the commonest microsporidians contributing to human microsporidiosis, and is frequently found in animals in various countries. However, there is limited epidemiological information on this microorganism in Australia. Here, we undertook the first molecular epidemiological study of E. bieneusi in cats and dogs in Victoria. RESULTS: Genomic DNAs were extracted from 514 individual faecal deposits from cats (n = 172) and dogs (n = 342) and then tested using PCR-based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. Four distinct genotypes (designated D, PtEb IX, VIC_cat1 and VIC_dog1) of E. bieneusi were identified in 20 of the 514 faecal samples (3.9%). Genotype D is known to have a broad host range (humans and other animals) and has a wide geographical distribution around the world. The identification of this genotype here suggests that companion animals might represent reservoir hosts that are able to transmit E. bieneusi infection to humans in Australia. A phylogenetic analysis of ITS sequence data revealed that the novel genotype VIC_cat1 is related to the known genotype type IV within Group 1, and the new genotype VIC_dog1 is linked to a contentious "Group 3", which includes genotypes reported previously in the published literature to represent Group 2 or 3. CONCLUSIONS: A future, large-scale phylogenetic study of all known E. bieneusi genotypes, including VIC_dog1, should aid in clarifying their relationships and assignment to Groups, and in the identification of new genotypes, thus assisting epidemiological investigations.

Enterocytozoon bieneusi Genotypes in Cattle on Farms Located within a Water Catchment Area.

Enterocytozoon bieneusi is a microsporidian found in humans and other animals around the world. Investigations in some countries, such as the U.S., have indicated the importance of E. bieneusi as a zoonotic water- and food-borne pathogen. However, there is scant epidemiological information on E. bieneusi in animals in many countries including Australia. Here, we conducted the first molecular epidemiological study of E. bieneusi in farmed cattle in Victoria, Australia, to assess whether these bovids are carriers of "zoonotic" genotypes of E. bieneusi. A total of 471 individual faecal samples were collected from calves of < 3 mo and of 3-9 mo of age. Genomic DNAs were extracted from individual faecal samples and then subjected to nested PCR-based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA to identify E. bieneusi and define genotypes. Enterocytozoon bieneusi was detected in 49 of the 471 samples (10.4%). An analysis of ITS sequence data revealed three known genotypes (BEB4, I, and J) and three novel genotypes (designated TAR_fc1 to TAR_fc3). Phylogenetic analysis showed that genotypes BEB4, I, J, TAR_fc1, and TAR_fc2 clustered with genotypes identified previously in humans, indicating that cattle are carriers of E. bieneusi with zoonotic potential.

Using PCR-Based Sequencing to Diagnose Haycocknema perplexum Infection in Human Myositis Case, Australia.

We report a case of myositis in a male patient in Australia who had progressive weakness and wasting in his left lower limb. Although clinical, pathologic, and laboratory assessments were inconclusive, a new, nested PCR-coupled sequencing method enabled the unequivocal diagnosis of myositis caused by the enigmatic nematode Haycocknema perplexum.

Molecular analysis of Cryptosporidium from cattle from five states of Peninsular Malaysia.

Despite the importance of the cattle industry in Malaysia, there are very few studies of the diversity and public health significance of bovine cryptosporidiosis in this country. In the present study, we used a PCR-based approach to detect and genetically characterize Cryptosporidium DNA in faecal samples from a cohort of 215 asymptomatic cattle (of different ages) from six farms from five states of Peninsular Malaysia. Cattle on four of the six farms were test-positive for Cryptosporidium, with an overall prevalence of 3.2%. Cryptosporidium bovis and Cryptosporidium ryanae were detected in two (0.9%) and five (2.3%) samples tested; this low prevalence likely relates to the age of the cattle tested, as most (73%) of the samples tested originated from cattle that were ≥2 years of age. Future studies should investigate the zoonotic potential of Cryptosporidium in pre-weaned and weaned calves in rural communities of Malaysia.

Phylogenetic relationships of species of the oesophageal parasitic nematode genera Cyclostrongylus and Spirostrongylus (Strongyloidea: Chabertiidae: Cloacininae) with their wallaby hosts (Marsupialia: Macropodidae).

A phylogeny for seven species of Cyclostrongylus and the monotypic genus Spirostrongylus (Nematoda: Chabertiidae), all highly host specific parasites of the oesophagi of wallabies (Marsupialia: Macropodidae), was constructed using sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. There was no evidence for co-speciation, or for the sympatric or synxenic speciation of Cyclostrongylus alatus and Cyclostrongylus perplexus, both of which are parasites of Macropus rufogriseus. Rather, host switching, correlating with geographical distributions, appeared to provide some explanation of the pattern of speciation observed.

Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) from the stomachs of macropodid marsupials defined by morphological and molecular criteria.

Pharyngostrongylus thylogale n. sp. (Nematoda: Strongylida) is described from the stomach of the red-legged pademelon, Thylogale stigmatica (Gould) (Marsupialia: Macropodidae) from north-eastern Queensland and Papua New Guinea, having formerly been confused with P. iota Johnston & Mawson, 1939. Pharyngostrongylus thylogale n. sp. differs from all congeners in having 12 labial crown elements rather than eight or 16. Pharyngostrongylus iota was found in T. stigmatica, but only in southern Queensland and northern New South Wales, in the subspecies T. s. wilcoxi, compared with P. thylogale n. sp. which was found in T. s. stigmatica in northern Queensland and T. s. oriomo in Papua New Guinea. Differences in the sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA of P. thylogale n. sp. and ten congeners support the erection of the new species, and the validity of the morphospecies examined. However, results of the phylogenetic analyses of the molecular data also provide evidence for the existence of cryptic species within P. kappa Mawson, 1965. No obvious co-evolutionary relationships were observed between parasite species and their macropodid marsupial hosts.

First human case of fatal Halicephalobus gingivalis meningoencephalitis in Australia.

Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool.

The phylogenetic relationships of endemic Australasian trichostrongylin families (Nematoda: Strongylida) parasitic in marsupials and monotremes.

The phylogenetic relationships of the endemic (or largely endemic) Australasian trichostrongylin nematode families Herpetostrongylidae, Mackerrastrongylidae and Nicollinidae as well as endemic trichostrongylin nematodes currently placed in the families Trichostrongylidae and Molineidae were examined using the complete large subunit (28S) ribosomal RNA gene. The Herpetostrongylinae proved to be monophyletic. However, representatives of the Nicollinidae nested with the Herpetostrongylinae. The Mackerrastrongylidae was also a monophyletic group and included Peramelistrongylus, currently classified within the Trichostrongylidae. The Globocephaloidinae, currently considered to be a subfamily of the Herpetostrongylidae, was excluded from the family in the current analysis. Ollulanus and Libyostrongylus, included for the first time in a molecular phylogenetic analysis, were placed within the Trichostrongylidae. This study provided strong support for the Herpetostrongylidae (including within it the Nicollinidae, but excluding the Globocephaloidinae) and the Mackerrastrongylidae as monophyletic assemblages. Additional studies are required to resolve the relationships of the remaining endemic Australasian trichostrongylin genera.

First genetic analysis of Cryptosporidium from humans from Tasmania, and identification of a new genotype from a traveller to Bali.

Little is known about the molecular composition of Cryptosporidium species from humans living in the insular state of Tasmania, Australia. In the present study, we genetically characterized 82 samples of Cryptosporidium from humans following conventional coproscopic testing in a routine, diagnostic laboratory. Using a PCR-coupled single-strand conformation polymorphism (SSCP) technique, targeting portions of the small subunit rRNA (SSU), and 60 kDa glycoprotein (gp60) loci, we identified two species of Cryptosporidium, including C. hominis (subgenotypes IbA10G2, IdA16, IeA12G3T3, and IfA19G1) and C. parvum (IIaA16G1R1 and IIaA18G3), and a new operational taxonomic unit (OTU) that genetically closely resembled C. wrairi. This OTU was further characterized using markers in the actin, Cryptosporidium oocyst wall protein (COWP), and 70 kDa heat shock protein (hsp70) genes. This study provides the first characterization of species and genotypes of Cryptosporidium from Tasmania, and presents clear genetic evidence, using five independent genetic loci, for a new genotype or species of Cryptosporidium in a Tasmanian person with a recent history of travelling to Bali, Indonesia. It would be interesting to undertake detailed molecular-based studies of Cryptosporidium in Indonesia and neighbouring countries, in conjunction with morphological and experimental investigations of new genotypes.

Genetic identification of an oxyurid from a captive, black-handed spider monkey--implications for treatment and control.

Parasites are of major clinical significance in captive primates in zoos, particularly those with direct life cycles. Oxyurid nematodes can be a persistent problem, as infection intensity and environmental contamination with infective eggs are usually high. Observations at the Basel Zoo in Switzerland have revealed that particularly black-handed spider monkeys (Ateles geoffroyi) exhibit continuous oxyurid nematode infection(s), despite regular deworming with anthelmintics. In the present study, using a molecular approach, we were able to identify the nematode (Trypanoxyuris atelis) causing this ongoing problem, and we are now evaluating a practical treatment and control regimen to tackle this parasite problem.

New records of Hepatozoon and Oswaldofilaria from saltwater crocodiles (Crocodylus porosus) in Australia.

Diseases affecting wild Australian saltwater crocodiles (Crocodylus porosus) are rarely reported due to the difficulty in capturing animals and obtaining samples. In this investigation, we identified two haemoparasites (Hepatozoon and a filarial nematode) in saltwater crocodiles in Darwin, Australia. Light microscopic examination identified Hepatozoon in 7/7 (100%) wild crocodiles and in 2/20 (10%) of captive ones. When genomic DNAs from these same samples were further investigated using polymerase chain reaction (PCR)-based sequencing, we detected Hepatozoon in all 27 blood samples. Using both microscopy and PCR-based sequencing, we detected a filarial worm (proposed to be Oswaldofilaria) in one of 20 captive crocodiles. The sequence data were compared with sequence data available in public databases, and phylogenetic analyses indicated that the operational taxonomic units of Hepatozoon and Oswaldofilaria discovered here in these crocodiles are likely new species. This study is the first to use molecular tools to explore haemoparasites in Australian saltwater crocodiles and highlights the importance of health investigations in poorly studied vertebrate hosts.

Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing-phylogenetic approach.

Blastocystis is a genus of intestinal stramenopiles that infect vertebrates, and may cause disease of the alimentary tract. Currently, at least 40 genotypes ("subtypes") of Blastocystis are recognised worldwide based on sequence data for the small subunit of the nuclear ribosomal RNA (SSU-rRNA) gene. Despite the numerous studies of Blastocystis worldwide, very few studies have explored Blastocystis in wild animals, particularly in Australia. Here, we used a PCR-based next generation sequencing (NGS)-phylogenetic approach to genetically characterise and classify Blastocystis variants from selected wildlife in the Australian state of Victoria. In total, 1658 faecal samples were collected from nine host species, including eastern grey kangaroo, swamp wallaby, common wombat, deer, European rabbit, canines and emu. Genomic DNA was extracted from these samples, a 500 bp region of the SSU-rRNA gene amplified by polymerase chain reaction (PCR) and, then, a subset of samples sequenced using Illumina technology. Primary PCR detected Blastocystis in 482 of the 1658 samples (29%), with the highest percentage in fallow deer (63%). Subsequent, Illumina-based sequencing of a subset of 356 samples revealed 55 distinct amplicon sequence variants (ASVs) representing seven currently-recognised subtypes (STs) [ST13 (prominent in marsupials), ST10, ST14, ST21, ST23, ST24 and ST25 (prominent in deer)] and two novel STs (ST45 and ST46) in marsupials. Mixed infections of different STs were observed in macropods, deer, emu and canids (fox, feral dog or dingo), but no infection was detected in rabbits or wombats. This study reveals marked genetic diversity within Blastocystis in a small number of species of wild animals in Australia, suggesting complexity in the genetic composition and transmission patterns of members of the genus Blastocystis in this country.

Mitochondrial genome of the fluke pond snail, Austropeplea cf. brazieri (Gastropoda: Lymnaeidae).

BACKGROUND: Lymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria. METHODS: The mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes. RESULTS: The assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low. CONCLUSIONS: The representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.

Understanding temporal and spatial distribution of intestinal nematodes of horses using faecal egg counts and DNA metabarcoding.

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses.