Toxoplasmosis epidemic in a population of urbanised allied rock-wallabies (Petrogale assimilis) on Magnetic Island (Yunbenun), North Queensland.

A mortality event involving 23 allied rock-wallabies (Petrogale assimilis) displaying neurological signs and sudden death occurred in late April to May 2021 in a suburban residential area directly adjacent to Magnetic Island National Park, on Magnetic Island (Yunbenun), North Queensland, Australia. Three allied rock-wallabies were submitted for necropsy, and in all three cases, the cause of death was disseminated toxoplasmosis. This mortality event was unusual because only a small, localised population of native wallabies inhabiting a periurban area on a tropical island in the Great Barrier Reef World Heritage Area were affected. A disease investigation determined the outbreak was likely linked to the presence of free-ranging feral and domesticated cats inhabiting the area. There were no significant deaths of other wallabies or wildlife in the same or other parts of Magnetic Island (Yunbenun) at the time of the outbreak. This is the first reported case of toxoplasmosis in allied rock-wallabies (Petrogale assimilis), and this investigation highlights the importance of protecting native wildlife species from an infectious and potentially fatal parasitic disease.

Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus).

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod#5 and #7). T. gondii DNA was obtained from the heart and lungs of Macropod#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod#5) and M. rufus (Macropod#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype#3, and TgRooCHn3 was ToxoDB genotyp#2. Both 104 TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod#5) and M. rufus (Macropod#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.

Verminous meningoencephalomyelitis in a red kangaroo associated with Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis is a zoonotic parasitic helminth that normally resides in the pulmonary arteries and the right ventricle of rats (Rattus sp.), the definitive host, where it causes little disease. Humans, dogs, opossums, and various zoo animals are "accidental" hosts. Here we report verminous meningoencephalomyelitis caused by A. cantonensis in a 9-mo-old male red kangaroo (Macropus rufus). The kangaroo was first presented lethargic, recumbent, and hypothermic, with severe muscle wasting. Within 3 wk, he progressed to non-ambulatory paraparesis and died. Gross examination revealed multifocal areas of dark-brown discoloration, malacia, and cavitation in the brain and the spinal cord. Histologically, there were several sections of nematodes surrounded by extensive areas of rarefaction, hemorrhage, spongiosis, neuronal necrosis, and gliosis. Based on size, morphology, and organ location, the nematodes were identified as subadult males and females. Interestingly, an eosinophilic response was largely absent, and the inflammatory response was minimal. A. cantonensis infection had not been reported previously in a red kangaroo in Louisiana or Mississippi, to our knowledge. Our case reaffirms the widespread presence of the helminth in the southeastern United States and indicates that A. cantonensis should be considered as a differential in macropods with neurologic clinical signs in regions where A. cantonensis is now endemic.

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.

Redescription of Rugopharynx australis (Mönnig, 1926) and the description of R. moennigi n. sp. (Nematoda: Strongyloidea) from kangaroos (Marsupialia: Macropodidae) in Australia.

Rugopharynx australis (Mönnig, 1926) (Nematoda: Strongyloidea) is redescribed based on specimens from the type host, Osphranter rufus (Desmarest), together with matching DNA sequence data. Additional hosts were Macropus giganteus Shaw and Osphranter robustus (Gould) with single occurrences in M. fuliginosus (Desmarest), Notamacropus dorsalis (Gray), Lagorchestes conspicillatus Gould and Petrogale xanthopus Gray. Rugopharynx moennigi n. sp., formerly included within R. australis, is distinguished by shorter but overlapping spicule lengths and in the morphology of the gubernaculum as well as by molecular data. Rugopharynx moennigi n. sp. appears to be primarily parasitic in M. fuliginosus throughout its geographical range, but also infects M. giganteus, O. robustus and O. rufus in areas of host sympatry.

Revision of Macroponema Mawson, 1978 (Nematoda: Strongylida) from macropodid marsupials with the description of two new species.

BACKGROUND: Species of Macroponema Mawson, 1978 are strongyloid nematodes which occur in the stomachs of macropodid marsupials in Australia. In this study, the genus Macroponema is revised, redescriptions of the two known species are provided, and two new species are added to the genus. METHODS: A molecular characterisation of the internal transcribed spacers of the nuclear ribosomal DNA of representative specimens of Macroponema from all known host species was undertaken to confirm the status of M. cf. comani. This resulted in the identification of a further new species within the genus. Consequently, a review of all available material in museum collections was undertaken. RESULTS: The two known species M. beveridgei Mawson, 1978 from Osphranter antilopinus (Gould) and O. robustus (Gould), and M. comani Mawson, 1978 from Macropus giganteus Shaw are re-described and their geographical distributions expanded. Two new species added to the genus are M. arundeli n. sp. from Ma. giganteus found in Queensland and the north east of New South Wales, and M. obendorfi n. sp. from O. antilopinus and O. robustus in the Northern Territory, the Kimberley Division of Western Australia and eastern Queensland. The latter species was formerly identified as M. cf. comani based on molecular studies. The specific identification of both of the new species is supported by ribosomal DNA sequence data. CONCLUSIONS: Based on the morphological and molecular characterisation of nematodes, this study has revealed the existence of four species within the genus Macroponema. The current phylogenetic data suggest that Macroponema spp. plausibly evolved by host switching; however, further studies are required to test this hypothesis.

New species of Macropostrongyloides Yamaguti, 1961 (Nematoda: Strongylida) and the redescription of Ma. baylisi (Wood, 1930) from Australian macropodid marsupials.

Specimens of four genetically distinct groups of Macropostrongyloides baylisi Wood, 1930 were analysed morphologically. Each genotype was found to represent a morphologically distinct species: Ma. baylisi from Osphranter robustus woodwardi (Thomas) and Osphranter robustus erubescens (Sclater); Ma. spearei n. sp. from Osphranter robustus robustus (Gould) and O. r. erubescens; Ma. mawsonae n. sp. from Macropus giganteus Shaw and Ma. woodi n. sp. from Osphranter rufus (Desmarest). The new species described here are differentiated primarily by several male-specific features that have been overlooked in previous taxonomic revisions. These features include striations on the terminal part of the spicule ala, the papillae surrounding the genital cone and the bursal striations. Furthermore, scanning electron photomicrographs have revealed greater details of previously undefined structures within the buccal cavity that warrant further investigations.

Genetic variation in Austrostrongylus thylogale Johnston & Mawson, 1940 (Nematoda: Trichostrongylida) from the tammar wallaby, Notamacropus eugenii (Gray), and the quokka, Setonix brachyurus (Quoy & Gaimard) (Marsupialia: Macropodidae) in Australia.

BACKGROUND: Australian marsupials harbour a diverse array of helminth parasites. Despite current attempts to assess the extent of this diversity in macropodid hosts, it has been suggested that unique parasite fauna of Australian wildlife is difficult to document comprehensively due to the common occurrence of cryptic species. This paper assessed genetic variation within Austrostrongylus thylogale Johnston & Mawson, 1940 from the tammar wallaby, Notamacropus eugenii (Gray), and the quokka, Setonix brachyurus (Quoy & Gaimard), from different localities using the molecular characterisation of the internal transcribed spacers (ITS) within the nuclear ribosomal DNA. METHODS: Thirty-seven specimens of A. thylogale collected from N. eugenii (from Parndana, Kangaroo Island, South Australia, and Perup, Western Australia) and S. brachyurus (from Wellington Dam, Western Australia) were characterised using a molecular-phylogenetic approach utilising the first (ITS1) and second (ITS2) internal transcribed spacers. RESULTS: Genetic variation was detected in both ITS1 and ITS2 between specimens of A. thylogale from N. eugenii and S. brachyurus; however, no variation was detected between specimens collected from N. eugenii from Parndana, South Australia, and Perup, Western Australia. Furthermore, the phylogenetic analyses of ITS sequences showed two clades of A. thylogale originating from two hosts, N. eugenii and S. brachyurus, suggesting the presence of cryptic species. CONCLUSIONS: This study provides evidence of genetic variation within A. thylogale based on collections from two different host species. Morphological studies are required to fully confirm the presence of a new species or cryptic species. Further molecular studies using a larger number of specimens are warranted to explore the genetic variation between A. thylogale from different geographical localities.

Molecular identification of Eimeria hestermani and Eimeria prionotemni from a red-necked wallaby (Macropodidae; Macropus rufogriseus) in Japan.

To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.

Gastrointestinal helminth parasites of the common wallaroo or euro, Osphranter robustus (Gould) (Marsupialia: Macropodidae) from Australia.

The gastrointestinal helminth parasites of 170 common wallaroos or euros, Osphranter robustus (Gould), collected from all mainland states in which the species occurs as well as the Northern Territory, are presented, including previously published data. A total of 65 species of helminths were encountered, including four species of anoplocephalid cestodes found in the bile ducts and small intestine, and 61 species of strongylid nematodes, all but two of which occurring in the stomach, and with the remainder occurring in the terminal ileum, caecum and colon. Among the mainland subspecies of O. robustus, 52 species of helminths were encountered in O. r. robustus, compared with 30 species in O. r. woodwardi and 35 species in O. r. erubescens. Of the parasite species encountered, only 17 were specific to O. robustus, the remaining being shared with sympatric host species. Host-specific species or species occurring in O. robustus at a high prevalence can be classified as follows: widely distributed; restricted to northern Australia; restricted to the northern wallaroo, O. r. woodwardi; found only in the euro, O. r. erubescens; found essentially along the eastern coast of Australia, primarily in O. r. robustus; and species with highly limited regional distributions. The data currently available suggest that the acquisition of a significant number of parasites is due to co-grazing with other macropodids, while subspeciation in wallaroos as well as climatic variables may have influenced the diversification of the parasite fauna.

Four new species of Rugopharynx Mönnig, 1927 (Nematoda: Strongyloidea) parasitic in the stomachs of macropodid marsupials from Australia.

Four new species of Rugopharynx Mönnig, 1927 are described from macropodid marsupials in Australia, some identified initially using molecular methods. Rugopharynx thetidis n. sp. from Thylogale thetis (Lesson) was initially included within R. sigma Chilton, Beveridge & Andrews, 1993 but was identified as being distinctive using molecular methods and is differentiated morphologically from R. sigma by the more anterior position of the deirid and by the distinctive curvature of the spicule tips. Rugopharynx thylogale n. sp., also from Thylogale thetis, is distinguished by the extreme elongation of the dorsal lobe of the bursa and spicule length (1.07-1.23 mm). Rugopharynx solitarius n. sp., again from Thylogale thetis, was initially included within Rugopharynx zeta (Johnston & Mawson, 1939) but differs morphologically in the features of the bursa. Rugopharynx disiunctus n. sp. from Macropus fuliginosus (Desmarest) was initially included within Rugopharynx rho Beveridge & Chilton, 1999 but was identified as distinctive based on DNA sequence differences. The new species differs from R. rho in the pattern of striations on the buccal capsule and in the lengths of the spicules. Additional records of species of Rugopharyx in M. fuliginosus in Western Australia are provided.

New species of Wallabinema Beveridge, 1983 (Nematoda: Strongyloidea) and a redescription of W. gallardi (Johnston & Mawson, 1939) from Australian macropodid marsupials.

Two new species of Wallabinema Beveridge, 1983 are described from the sacculated fore-stomachs of macropodid marsupials in Australia. Wallabinema petrogale n. sp. from the rock wallabies Petrogale penicillata (J. Gray) and P. inornata Gould, from Queensland, differs from all congeners in having its four sub-median lips subdivided at the base. Wallabinema macropodis n. sp. from the black stripe wallaby Macropus dorsalis (J. Gray) and the red-necked pademelon Thylogale thetis (Lesson), also from Queensland, is most similar to congeners with the nerve-ring encircling the oesophageal isthmus (W. parvispiculare Beveridge, 1983, W. tasmaniense Beveridge, 1983 and W. thylogale Beveridge, 1983), but differs in the length of the spicules. Wallabinema gallardi (Johnston & Mawson, 1939) is redescribed from T. thetis in Queensland and New South Wales, with T. thetis considered to be the type-host.

Gastric and Caecal Amoebiasis in a Red Kangaroo (Macropus rufus) with Disseminated Toxoplasmosis.

A 1-year-old male red kangaroo (Macropus rufus) with an acute clinical history of lethargy, depression and increased respiratory rate was presented for necropsy examination. Gross lesions in the digestive tract were a distended stomach with watery content and multifocal raised ulcers covered by fibrinous pseudomembranes in the forestomach and caecum. On histopathology, there was necrotizing and ulcerative gastritis and typhlitis with intralesional amoebic trophozoites and Toxoplasma gondii zoites. Lesions due to T. gondii infection were observed in multiple organs and diagnosis was confirmed by fluorescent antibody test and immunohistochemistry. Both toxoplasmosis and gastric amoebiasis are diseases described in macropods. In this case report, we describe concurrent disease caused by both protozoa in a red kangaroo. Lesions of amoebiasis were also observed in the caecum. Both toxoplasmosis and amoebiasis should be considered as differential diagnoses of ulcerative lesions in the gastrointestinal tract in macropods during necropsy examination. Amoebiasis should be suspected especially when ulcerative lesions are observed in the forestomach.

Genetic variation within the genus Macropostrongyloides (Nematoda: Strongyloidea) from Australian macropodid and vombatid marsupials.

The genetic variation and taxonomic status of the four morphologically-defined species of Macropostrongyloides in Australian macropodid and vombatid marsupials were examined using sequence data of the ITS+ region (=first and second internal transcribed spacers, and the 5.8S rRNA gene) of the nuclear ribosomal DNA. The results of the phylogenetic analyses revealed that Ma. baylisi was a species complex consisting of four genetically distinct groups, some of which are host-specific. In addition, Ma. lasiorhini in the common wombat (Vombatus ursinus) did not form a monophyletic clade with Ma. lasiorhini from the southern hairy-nosed wombat (Lasiorhinus latifrons), suggesting the possibility of cryptic (genetically distinct but morphologically similar) species. There was also some genetic divergence between Ma. dissimilis in swamp wallabies (Wallabia bicolor) from different geographical regions. In contrast, there was no genetic divergence among specimens of Ma. yamagutii across its broad geographical range or between host species (i.e. Macropus fuliginosus and M. giganteus). Macropostrongyloides dissimilis represented the sister taxon to Ma. baylisi, Ma. yamagutii and Ma. lasiorhini. Further morphological and molecular studies are required to assess the species complex of Ma. baylisi.

New species and new records of species of Cloacina von Linstow, 1898 (Nematoda: Strongylida) parasitic in the western scrub wallaby, Notamacropus irma (Jourdan) (Marsupialia: Macropodidae) from Western Australia.

The helminth parasites of the western scrub wallaby or black-glove wallaby, Notamacropus irma (Jourdan) which occurs in Western Australia are relatively poorly documented. Six new species of the strongyloid genus Cloacina von Linstow, 1898 (Strongylida: Chabertiidae) are described namely C. asymmetrica n. sp., C. brazellei n. sp., C. harriganae n. sp., C. hobbsi n. sp., C. middletoni n. sp. and C. woodi n. sp. A redescription of C. laius Beveridge, 1999 from the same host species is included. Molecular sequence data (ITS1 and ITS2 ribosomal DNA) were obtained for C. asymmetrica, C. brazellei, C. hobbsi, C. middletoni and from the previously described species C. themis Beveridge, 1998 occurring in the same host species. Phylogenetically, C. asymmetrica, C. hobbsi and C. middletoni formed a distinct clade, suggesting the possibility of within-host speciation. Cloacina themis clustered with a group of morphologically distinctive species in a separate clade and C. brazellei clustered in a third clade but with poor support. This pattern of congeners in a single host species occurring in multiple clades mirrors the situation in other kangaroos and wallabies. Species of Cloacina from N. irma reported thus far therefore consist of a series of species found only in this host, with two species (C. brazellei and C. laius) shared with the sympatric macropodid Setonix brachyurus (Quoy & Gaimard).

Morphological and molecular diagnosis of Eimeria sp. that caused fatality in a red-necked wallaby (Macropus rufogriseus) in Korea.

A red-necked wallaby (Macropus rufogriseus) in the Republic of Korea, introduced from Australia, died in 12 d after exhibiting anorexia and diarrhea. Postmortem examination revealed that the wallaby died due to coccidiosis by Eimeria sp. Morphologically, Eimeria sp. identified closely resembled E. mykytowyczi. The 18S rRNA sequence analysis showed that Eimeria sp. identified in this study has a 98.5% identity with that in Australian red kangaroo (M. rufus). However, owing to insufficient molecular information on marsupial-specific Eimeria, exact species could not be determined. Phylogenetically, Eimeria sp. identified in this study belonged to clade five of the marsupial group.

Phylogenetic relationships of three tribes of cloacinine nematodes (Strongylida: Chabertiidae) from macropodid marsupials.

The phylogenetic relationships of 42 species of cloacinine nematodes belonging to three tribes (Coronostrongylinea, Macropostrongylinea and Zoniolaiminea) were examined based on sequence data of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. All nematodes examined are parasites of Australian macropodid marsupials. None of the three nematode tribes was monophyletic. Paraphyly was also encountered in three genera: Papillostrongylus, Monilonema and Wallabinema. Species within the genus Thallostonema were limited to a single host genus (i.e. Thylogale), whereas species within the five principal genera (Coronostrongylus, Macropostrongylus, Popovastrongylus, Wallabinema and Zoniolaimus) were found to occur in multiple host genera. Potential modes of evolution among these nematodes are discussed.

New species of Cloacina von Linstow, 1898 (Nematoda: Strongyloidea) parasitic in the stomachs of wallaroos, Osphranter spp. (Marsupialia: Macropodidae) from northern Australia.

Three new species of the parasitic nematode genus Cloacina von Linstow, 1898 (Strongyloidea: Cloacininae) are described from the stomachs of wallaroos, Osphranter spp. (Marsupialia: Macropodidae), from northern Australia. Cloacina spearei n. sp. is described from O. robustus woodwardi (Thomas) and O. antilopinus (Gould) and is distinguished from congeners by the shape of the cephalic papillae, the shallow buccal capsule, the presence of an oesophageal denticle and the convoluted but non-recurrent vagina in the female. Cloacina longibursata n. sp. also from O. robustus woodwardi and O. antilopinus is distinguished from congeners by the elongate dorsal lobe of the bursa, with the origin of the lateral branchlets posterior to the principal bifurcation, in the features of the spicule tip, the lack of bosses lining the oesophagus and the absence of an oesophageal denticle. Cloacina crassicaudata n. sp., from the same two host species was formerly identified as C. cornuta (Davey & Wood, 1938). Differences in the cephalic cuticle (inflation lacking in the new species), the shape of the cephalic papillae, the dorsal oesophageal tooth and the spicule tips, as well as differences in the sequences of the internal transcribed spacers of the nuclear ribosomal DNA, indicate that this is an independent species. The geographical distribution of this species is disjunct with populations in both the Northern Territory and Queensland. Possible reasons for the disjunct distribution are discussed.

Molecular and morphological characterisation of Pharyngostrongylus kappa Mawson, 1965 (Nematoda: Strongylida) from Australian macropodid marsupials with the description of a new species, P. patriciae n. sp.

BACKGROUND: Pharyngostrongylus kappa Mawson, 1965 is a nematode (Strongyloidea: Cloacininae), endemic to the sacculated forestomachs of Australian macropodid marsupials (kangaroos and wallaroos). A recent study revealed genetic variation within the internal transcribed spacer region of the nuclear ribosomal DNA among P. kappa specimens collected from Macropus giganteus Shaw and Osphranter robustus (Gould). This study aimed to characterise the genetic and morphological diversity within P. kappa from four macropodid host species, including M. giganteus, O. robustus, O. antilopinus (Gould) and O. bernardus (Rothschild). METHODS: Specimens of P. kappa from M. giganteus and Osphranter spp. from various localities across Australia were examined. The first and second internal transcribed spacers (ITS1 and ITS2, respectively) were amplified using polymerase chain reaction and sequenced. Phylogenetic methods were used to determine the interspecific diversification within P. kappa and its evolutionary relationship with other congeners. RESULTS: Morphological examination revealed that P. kappa from M. giganteus, the type-host, can be distinguished from those in Osphranter spp. by the greater length and number of striations on the buccal capsules. DNA sequences showed that P. kappa from M. giganteus was genetically distinct from that in Osphranter spp., thereby supporting the morphological findings. Based on these finding, a new species from Osphranter spp., Pharyngostrongylus patriciae n. sp., is described. CONCLUSION: Pharyngostrongylus patriciae n. sp. from Osphranter spp. is distinguished from P. kappa based on molecular and morphological evidence. The study highlights the importance of combining molecular and morphological techniques for advancing the nematode taxonomy. Although ITS genetic markers have proven to be effective for molecular prospecting as claimed in previous studies, future utilisation of mitochondrial DNA to validate ITS data could further elucidate the extent of speciation among macropodid nematodes.

Identification of Theileria fuliginosa-like species in Ixodes australiensis ticks from western grey kangaroos (Macropus fuliginosus) in Western Australia.

Piroplasms, including the genera Babesia and Theileria, are intra-erythrocytic protozoa that are generally transmitted by ticks and are the aetiological agents for piroplasmosis in animals, as well as humans, worldwide. In Australia, numerous studies have been conducted on piroplasms in domestic animals; however, less is known about these protozoa in ticks from native wildlife. The present study characterised piroplasms in Ixodes australiensis (n = 119) and Amblyomma triguttatum (n = 35) ticks collected from kangaroos in Western Australia (WA). Approximately 7.6% (9/119) (95% CI 2.8-12.2) of the I. australiensis ticks were positive for piroplasms using nested-PCR at the 18S rRNA locus, whereas no piroplasm 18S rDNA was detected in the A. triguttatum ticks. All sequences from I. australiensis ticks were identical. Using a 852 bp multiple nucleotide alignment at the 18S rRNA variable region, sequences shared 97.6%, 94.3%, 93.5% and 93.4% pairwise identity with Theileria fuliginosa, Theileria brachyuri, Theileria penicillata, and a Theileria sp. (K1), derived from a burrowing bettong or boodie (Bettongia lesueur), respectively. Phylogenetic analysis revealed that the Theileria sp. from I. australiensis clustered together in the marsupial-associated Theileria group, with T. fuliginosa as closest sister species. Hence, we conclude that this is the first observation of T. fuliginosa-like species in I. australiensis ticks parasitising kangaroos in WA.