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.

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.

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.

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.

Dogs are reservoir hosts of the zoonotic Dirofilaria sp. 'hongkongensis' and potentially of Brugia sp. Sri Lanka genotype in Sri Lanka.

In 2016, the World Health Organization declared Sri Lanka as having successfully eliminated lymphatic filariasis as a public health concern. However, in recent decades, several infections with subperiodic filarial species suggestive of zoonotic infections have been recorded across the country. The arthropod-borne filarioids Dirofilaria repens, Brugia malayi, Brugia ceylonensis, and Acanthocheilonema reconditum are historically known to be endemic in dogs in Sri Lanka. Despite this, limited information on the prevalence, diversity, and predictors of filarial infections in dogs in the country has resulted in suboptimal control and prevention of these parasites, some of which are known to be zoonotic. To address this, whole blood and metadata were collected and analysed from 423 pet dogs across three geo-climatic zones within Sri Lanka. Blood samples were screened using the Modified Knott's Test (MKT) and PCR followed by Sanger sequencing. Multivariable logistic regression models were used to assess predictors for canine filarial infections. Dirofilaria sp. 'hongkongensis' (Dirofilaria sp. HK) and Brugia sp. Sri Lanka (SL) genotype were identified infecting dogs. The overall prevalence of filarial infection in pet dogs by PCR was 36.9% (95% CI 32.3-41.7%, n = 156), compared to 18.8% (95% CI 15.2-22.9%, n = 79) detected using the MKT. >80% of filarial-positive dogs were infected by Dirofilaria sp. HK, while the remaining dogs were infected by Brugia sp. SL genotype. Increasing age (p < 0.001) and residing in the low-country wet zone (p < 0.001), which includes regions that were endemic for human filariasis in Sri Lanka, were associated with filarial infections in dogs. No clear pathognomonic signs for filarial infection were identified, indicating that dogs act as reservoirs for these potentially zoonotic pathogens. Given the morphological similarity of Dirofilaria HK and Brugia sp. SL microfilariae with those of D. repens and B. malayi, respectively, it is likely that these species have been misidentified in the past. Prevention and control measures of these potentially zoonotic canine filarial infections are highly advocated to safeguard both canine and human health.

Advanced approaches for the diagnosis and chemoprevention of canine vector-borne pathogens and parasites-Implications for the Asia-Pacific region and beyond.

Vector-borne pathogens (VBPs) of canines are a diverse range of infectious agents, including viruses, bacteria, protozoa and multicellular parasites, that are pernicious and potentially lethal to their hosts. Dogs across the globe are afflicted by canine VBPs, but the range of different ectoparasites and the VBPs that they transmit predominate in tropical regions. Countries within the Asia-Pacific have had limited prior research dedicated to exploring the epidemiology of canine VBPs, whilst the few studies that have been conducted show VBP prevalence to be high, with significant impacts on dog health. Moreover, such impacts are not restricted to dogs, as some canine VBPs are zoonotic. We reviewed the status of canine VBPs in the Asia-Pacific, with particular focus on nations in the tropics, whilst also investigating the history of VBP diagnosis and examining recent progress in the field, including advanced molecular methods, such as next-generation sequencing (NGS). These tools are rapidly changing the way parasites are detected and discovered, demonstrating a sensitivity equal to, or exceeding that of, conventional molecular diagnostics. We also provide a background to the armoury of chemopreventive products available for protecting dogs from VBP. Here, field-based research within high VBP pressure environments has underscored the importance of ectoparasiticide mode of action on their overall efficacy. The future of canine VBP diagnosis and prevention at a global level is also explored, highlighting how evolving portable sequencing technologies may permit diagnosis at point-of-care, whilst further research into chemopreventives will be essential if VBP transmission is to be effectively controlled.

Genome-wide exploration reveals distinctive northern and southern variants of Clonorchis sinensis in the Far East.

Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis-a neglected tropical disease (NTD) affecting ~35 million people worldwide. No vaccine is available, and chemotherapy relies on one anthelmintic, praziquantel. This parasite has a complex life history and is known to infect a range of species of intermediate (freshwater snails and fish) and definitive (piscivorous) hosts. Despite this biological complexity and the impact of this biocarcinogenic pathogen, there has been no previous study of molecular variation in this parasite on a genome-wide scale. Here, we conducted the first extensive nuclear genomic exploration of C. sinensis individuals (n = 152) representing five distinct populations from mainland China, and one from Far East Russia, and revealed marked genetic variation within this species between "northern" and "southern" geographical regions. The discovery of this variation indicates the existence of biologically distinct variants within C. sinensis, which may have distinct epidemiology, pathogenicity and/or chemotherapic responsiveness. The detection of high heterozygosity within C. sinensis specimens suggests that this parasite has developed mechanisms to readily adapt to changing environments and/or host species during its life history/evolution. From an applied perspective, the identification of invariable genes could assist in finding new intervention targets in this parasite, given the major clinical relevance of clonorchiasis. From a technical perspective, the genomic-informatic workflow established herein will be readily applicable to a wide range of other parasites that cause NTDs.

Phylogenetic Relationships of the Strongyloid Nematodes of Australasian Marsupials Based on Mitochondrial Protein Sequences.

Australasian marsupials harbour a diverse group of gastrointestinal strongyloid nematodes. These nematodes are currently grouped into two subfamilies, namely the Cloacininae and Phascolostrongylinae. Based on morphological criteria, the Cloacininae and Phascolostrongylinae were defined as monophyletic and placed in the family Cloacinidae, but this has not been supported by molecular data and they are currently placed in the Chabertiidae. Although molecular data (internal transcribed spacers of the nuclear ribosomal RNA genes or mitochondrial protein-coding genes) have been used to verify morphological classifications within the Cloacininae and Phascolostrongylinae, the phylogenetic relationships between the subfamilies have not been rigorously tested. This study determined the phylogenetic relationships of the subfamilies Cloacininae and Phascolostrongylinae using amino acid sequences conceptually translated from the twelve concatenated mitochondrial protein-coding genes. The findings demonstrated that the Cloacininae and Phascolostrongylinae formed a well-supported monophyletic assemblage, consistent with their morphological classification as an independent family, Cloacinidae. Unexpectedly, however, the subfamily Phascolostrongylinae was split into two groups comprising the genera from macropodid hosts (kangaroos and wallabies) and those from vombatid hosts (wombats). Genera of the Cloacininae and Phascolostrongylinae occurring in macropodid hosts were more closely related compared to genera of the Phascolostrongylinae occurring in wombats that formed a sister relationship with the remaining genera from macropods. These findings provide molecular evidence supporting the monophyly of the family Cloacinidae and an alternative hypothesis for the origin of marsupial strongyloid nematodes in vombatid hosts that requires further exploration using molecular approaches and additional samples.

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.

Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

Thermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868.

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber's pole worm, and in other socioeconomically important parasitic nematodes. The "hit-to-target" workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

A Perspective on the Molecular Identification, Classification, and Epidemiology of Enterocytozoon bieneusi of Animals.

The microsporidian Enterocytozoon bieneusi is an obligate intracellular pathogen that causes enteric disease (microsporidiosis) in humans and has been recorded in a wide range of animal species worldwide. The transmission of E. bieneusi is direct and likely occurs from person to person and from animal to person via the ingestion of spores in water, food, or the environment. The identification of E. bieneusi is usually accomplished by molecular means, typically using the sequence of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Currently, ~820 distinct genotypes of E. bieneusi have been recorded in at least 210 species of vertebrates (mammals, birds, reptiles, and amphibians) or invertebrates (insects and mussels) in more than 50 countries. In this chapter, we provide a perspective on (1) clinical aspects of human microsporidiosis; (2) the genome and DNA markers for E. bieneusi as well as molecular methods for the specific and genotypic identification of E. bieneusi; (3) epidemiological aspects of E. bieneusi of animals and humans, with an emphasis on the genotypes proposed to be zoonotic, human-specific, and animal-specific; and (4) future research directions to underpin expanded molecular studies to better understand E. bieneusi and microsporidiosis.

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.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

A multipronged next-generation sequencing metabarcoding approach unearths hyperdiverse and abundant dog pathogen communities in Cambodia.

Recent surveys in Southeast Asia, including Cambodia, have identified canine vector-borne pathogens (VBPs), including those with zoonotic potential, as highly prevalent. The lack of veterinary care alongside the close association semidomesticated dogs have with humans in the region exacerbates these zoonotic risks. Nonetheless, the number of studies investigating such pathogens and the threats they pose to dog and human health is limited. Here, we utilize a next-generation sequencing (NGS)-based metabarcoding protocol to conduct an assumption-free characterization of the bacterial, apicomplexan, and kinetoplastid blood-borne pathogens of free-roaming dogs from across Cambodia. From 467 dogs at five field sites, 62% were infected with one of eight confirmed pathogens, comprising Anaplasma platys (32%), Ehrlichia canis (20%), Hepatozoon canis (18%), Babesia vogeli (14%), Mycoplasma haemocanis (13%), the zoonotic pathogen Bartonella clarridgeiae (3%), Candidatus Mycoplasma haematoparvum (0.2%), and Trypanosoma evansi (0.2%). Coinfections of between two and four VBPs were common with 28% of dogs found to have a mixed infection. Moreover, DNA from putatively infectious agents belonging to the bacterial family and genera Coxiella, Mycobacterium, Neisseria, Rickettsiaceae, Treponema, and two uncharacterized Mycoplasma species were identified, in addition to protozoan genera Colpodella, Parabodo, and Bodo. Using a multiple logistic regression model, the presence of ectoparasites, abnormal mucous membranes, anemia, and total protein were found as predictors of canine VBP exposure. This study represents the first time an NGS metabarcoding technique has been used to holistically detect the bacterial and protozoan hemoparasites communities of dogs through an in-depth survey, highlighting the power of such methods to unearth a wide spectrum of pathogenic organisms in an unbiased manner.

Phylogenetic relationships of the nematode subfamily Phascolostrongylinae from macropodid and vombatid marsupials inferred using mitochondrial protein sequence data.

BACKGROUND: The subfamily Phascolostrongylinae (Superfamily Strongyloidea) comprises nematodes that are parasitic in the gastrointestinal tracts of macropodid (Family Macropodidae) and vombatid (Family Vombatidae) marsupials. Currently, nine genera and 20 species have been attributed to the subfamily Phascolostrongylinae. Previous studies using sequence data sets for the internal transcribed spacers (ITS) of nuclear ribosomal DNA showed conflicting topologies between the Phascolostrongylinae and related subfamilies. Therefore, the aim of this study was to validate the phylogenetic relationships within the Phascolostrongylinae and its relationship with the families Chabertiidae and Strongylidae using mitochondrial amino acid sequences. METHODS: The sequences of all 12 mitochondrial protein-coding genes were obtained by next-generation sequencing of individual adult nematodes (n = 8) representing members of the Phascolostrongylinae. These sequences were conceptually translated and the phylogenetic relationships within the Phascolostrongylinae and its relationship with the families Chabertiidae and Strongylidae were inferred from aligned, concatenated amino acid sequence data sets. RESULTS: Within the Phascolostrongylinae, the wombat-specific genera grouped separately from the genera occurring in macropods. Two of the phascolostrongyline tribes were monophyletic, including Phascolostrongylinea and Hypodontinea, whereas the tribe Macropostrongyloidinea was paraphyletic. The tribe Phascolostrongylinea occurring in wombats was closely related to Oesophagostomum spp., also from the family Chabertiidae, which formed a sister relationship with the Phascolostrongylinae. CONCLUSION: The current phylogenetic relationship within the subfamily Phascolostrongylinae supports findings from a previous study based on ITS sequence data. This study contributes also to the understanding of the phylogenetic position of the subfamily Phascolostrongylinae within the Chabertiidae. Future studies investigating the relationships between the Phascolostrongylinae and Cloacininae from macropodid marsupials may advance our knowledge of the phylogeny of strongyloid nematodes in marsupials.

Molecular detection of Strongyloides sp. in Australian Thoroughbred foals.

BACKGROUND: Strongyloides westeri is found in the small intestine of young horses, mainly in foals up to about 16 weeks of age. The main source of infection for foals is through transmammary transmission, and foals can develop acute diarrhoea, weakness, dermatitis and respiratory signs. The epidemiology of S. westeri in Australia is largely unknown. Further, molecular techniques have never been employed for detection of S. westeri in horses. This pilot study aimed to assess the utility of a molecular phylogenetic method for the detection of S. westeri in the faeces of foals. METHODS: Faecal samples were collected from a foal of less than 2 months of age, and eggs of Strongyloides sp. were detected using the modified McMaster technique. DNA was extracted from purified eggs, and a partial fragment of the small subunit of the nuclear ribosomal DNA (18S) was characterised using polymerase chain reaction, DNA sequencing and phylogenetic methods. RESULTS: Microscopic examination of faeces revealed small ellipsoidal eggs typical of Strongyloides sp. The 18S sequence generated by PCR in this study revealed 98.4% identity with that of a reference sequence of S. westeri available from GenBank. Phylogenetic analyses revealed a polyphyletic clustering of S. westeri sequences. CONCLUSION: This is the first study reporting the detection of DNA of Strongyloides sp. in faeces of a foal using a molecular phylogenetic approach targeting the variable region of 18S rDNA. It is anticipated that this study will allow future molecular epidemiological studies on S. westeri in horses.

Detection of Breinlia sp. (Nematoda) in the Leadbeater's possum (Gymnobelideus leadbeateri).

The Leadbeater's possum (Gymnobelideus leadbeateri) is a critically endangered marsupial in south-eastern Australia. Among other conservation efforts, free-ranging animals in the two remaining geographically separate populations (highland and lowland) have been extensively studied; however, little is known about their health and mortality. Although some wild populations are frequently monitored, cadavers are rarely recovered for post mortem examination. In June 2019, a recently deceased, wild, adult male lowland Leadbeater's possum was collected from a nest box and a comprehensive post mortem examination was conducted. Microfilariae of a filarioid nematode were observed in testes, liver, lung and skin samples in tissue impression smears and upon histopathological examination. No gross or histological changes were seen associated with the parasites, except for a focal area of tissue damage in the skin, suggesting that the possum is a natural host. Using a PCR-coupled sequencing method the filarioid was identified as a species of Breinlia. Species of Breinlia occur in other Australian marsupials and rodents.

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.

Enterocytozoon bieneusi of animals-With an 'Australian twist'.

Enterocytozoon bieneusi is a microsporidian microorganism that causes intestinal disease in animals including humans. E. bieneusi is an obligate intracellular pathogen, typically causing severe or chronic diarrhoea, malabsorption and/or wasting. Currently, E. bieneusi is recognised as a fungus, although its exact classification remains contentious. The transmission of E. bieneusi can occur from person to person and/or animals to people. Transmission is usually via the faecal-oral route through E. bieneusi spore-contaminated water, environment or food, or direct contact with infected individuals. Enterocytozoon bieneusi genotypes are usually identified and classified by PCR-based sequencing of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA. To date, ~600 distinct genotypes of E. bieneusi have been recorded in ~170 species of animals, including various orders of mammals and reptiles as well as insects in >40 countries. Moreover, E. bieneusi has also been found in recreational water, irrigation water, and treated raw- and waste-waters. Although many studies have been conducted on the epidemiology of E. bieneusi, prevalence surveys of animals and humans are scant in some countries, such as Australia, and transmission routes of individual genotypes and related risk factors are poorly understood. This article/chapter reviews aspects of the taxonomy, biology and epidemiology of E. bieneusi; the diagnosis, treatment and prevention of microsporidiosis; critically appraises the naming system for E. bieneusi genotypes as well as the phylogenetic relationships of these genotypes; provides new insights into the prevalence and genetic composition of E. bieneusi populations in animals in parts of Australia using molecular epidemiological tools; and proposes some areas for future research in the E. bieneusi/microsporidiosis field.