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The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

SUMMARYThis study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenicpotential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats,is unknown. In other species, including humans, cellular transformation by gammaherpesvirusesis typically mediated by viral genes expressed during latency. We analysed tumour RNA, fromdiffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and felineimmunodeficiency virus (FIV) (n = 10). Analysis was done by high throughput transcriptomesequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identifiedin five tumors, including homologs of oncogenic latency-associated transcripts, latency-associatednuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORFunique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplantlymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single casein which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcriptscan be detected in some FIV-associated lymphomas, but with a low copy number, precludingassessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1transcriptome in clinical samples might employ viral enrichment and greater sequencing depth toenhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-celltumors and large granular lymphocyte lymphoma, for study.

High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation.

Despite an apparent increase in cases of angiostrongyliasis in humans and animals in Australia, the epidemiology of infection with the two species of Angiostrongylus that co-exist in this country, namely A. cantonensis and A. mackerrasae, is poorly understood. This knowledge gap is particularly important with respect to A. mackerrasae, a species evidently native to Australia, as its ability to cause disease in humans is unknown. Likewise, there is little information on the roles of native and introduced species of rodents and molluscs as hosts of Angiostrongylus species in Australia. This review focuses on the gaps in the knowledge about the two species, highlighting the need for epidemiological and pathogenesis studies on the native lungworm A. mackerrasae.

Despite an apparent increase in cases of angiostrongyliasis in humans and animals in Australia, the epidemiology of infection with the two species of Angiostrongylus that co-exist in this country, namely A. cantonensis and A. mackerrasae, is poorly understood. This knowledge gap is particularly important with respect to Angiostrongylus mackerrasae, a species evidently native to Australia, as its ability to cause disease in humans is unknown. Likewise, there is little information on the roles of native and introduced species of rodents and molluscs as hosts of Angiostrongylus species in Australia. This thesis focuses on the gaps in the knowledge about the epidemiology of two species in southeast Queensland, highlighting the hotspots for transmission of the parasite in urban Brisbane. A comprehensive survey of rats belonging to the species Rattus rattus, Rattus norvegicus and Rattus fuscipes was conducted. The prevalence of infection with Angiostrongylus spp. was 16.5% in Rattus spp. trapped in urban Brisbane and surrounds which is much higher than estimates of earlier studies. This highlights the possible risk of zoonotic infection in children, dogs and wildlife in this region and indicates the necessity for public awareness as well as more detailed epidemiological studies on this parasite in eastern Australia. Of the two Australian species, Angiostrongylus mackerrasae appears to be adapted to life in the native bush rat, Rattus fuscipes. This species evidently has an identical life cycle to that of Angiostrongylus cantonensis. A. mackerrasae may have greater potential to infect non-rodent hosts as it has been found as adult form in the lungs, as has been recorded for an individual flying fox. This raises concerns as to the potential of this native angiostrongylid to infect domestic animals and humans. To date, data on the taxonomy, epidemiology and population genetics of A. mackerrasae are poor or unknown. Here, we describe the mitochondrial (mt) genome of A. mackerrasae with the aim of addressing some of these knowledge gaps. The complete mitochondrial (mt) genome of A. mackerrasae was amplified from a single morphologically identified adult worm, by long-PCR in two overlapping amplicons (8 kb and 10 kb). The amplicons were sequenced using the MiSeq Illumina platform and annotated using an in-house pipeline. Amino acid sequences inferred from individual protein coding genes of the mt genomes were concatenated and then subjected to phylogenetic analysis using Bayesian inference. The mt genome of A. mackerrasae is 13,640 bp in size and contains 12 protein coding genes (cox1-3, nad1-6, nad4L, atp6 and cob), and two ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) genes. The mt genome of A. mackerrasae has similar characteristics to those of other Angiostrongylus species which is explained in detail in chapter 3. Phylogenic comparisons reveal that A. mackerrasae is closely related to A. cantonensis and the two sibling species may have recently diverged compared with all other species in the genus with a highly specific host selection. This mt genome will provide a source of genetic markers for explorations of the epidemiology, biology and population genetics of A. mackerrasae. The potential pathogenicity of A. mackerrasae for human and animals was also investigated in this PhD and the results reveal that A. mackerrasae causes severe eosinophilic meningitis in mice and guinea pigs and can potentially cause similar neurological disease in human and animals. It was also revealed that mice immune response is less responsive to A. cantonensis comparing to the similar species A. mackerrasae, as A. cantonensis live for a longer time in meninges of the accidental host and this can help the parasite causing a more patent infection in human and animals. The data of this project raises the possibility that cerebral meningitis remains a potential problem in South Eastern Queensland, particularly as people encroach onto forested areas. Angiostrongylus mackerassae appears to be a species that diverged from A. cantonensis after invasion of the Australian mainland. This species may have greater pathogenesis both in definitive and accidental hosts.

© 2015 Chan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Angiostrongylus cantonensis and Angiostrongylus mackerrasae are metastrongyloid nematodes that infect various rat species. Terrestrial and aquatic molluscs are intermediate hosts of these worms while humans and dogs are accidental hosts. Angiostrongylus cantonensis is the major cause of angiostrongyliasis, a disease characterised by eosinophilic meningitis. Although both A. cantonensis and A. mackerrasae are found in Australia, A. cantonensis appears to account for most infections in humans and animals. Due to the occurrence of several severe clinical cases in Sydney and Brisbane, the need for epidemiological studies on angiostrongyliasis in this region has become apparent. In the present study, a conventional PCR and a TaqMan assay were compared for their ability to amplify Angiostrongylus DNA from DNA extracted from molluscs. The TaqMan assay was more sensitive, capable of detecting the DNA equivalent to one hundredth of a nematode larva. Therefore, the TaqMan assay was used to screen molluscs (n=500) of 14 species collected from the Sydney region. Angiostrongylus DNA was detected in 2 of the 14 mollusc species; Cornu aspersum [14/ 312 (4.5%)], and Bradybaenia similaris [1/10 (10%)], which are non-native terrestrial snails commonly found in urban habitats. The prevalence of Angiostrongylus spp. was 3.0% ±0.8% (CI 95%). Additionally, experimentally infected Austropeplea lessoni snails shed A. cantonensis larvae in their mucus, implicating mucus as a source of infection. This is the first Australian study to survey molluscs using real-time PCR and confirms that the garden snail, C. aspersum, is a common intermediate host for Angiostrongylus spp. in Sydney.

Ninety-four European brown bear (Ursus arctos) faecal samples, collected in three counties of Croatia, were examined for the presence of gastrointestinal parasites. Five genera were identified, including the nematodes Baylisascaris transfuga and Syngamus sp., and the protozoan enteropathogens Cryptosporidium sp., Eimeria sp., and Giardia sp. Ancylostomatid eggs were also recovered. Cestodes, trematodes and acanthocephalan eggs were absent from all samples. This is the first parasite survey of brown bears in Croatia in thirty years and the first report of Giardia in this species from the region., Devedeset i četiri uzorka izmeta europskoga smeđeg medvjeda (Ursus arctos), sakupljena u tri županije u Hrvatskoj, pregledana su na prisutnost želučano-crijevnih nametnika. Utvrđeno je ukupno 5 rodova, uključujući nematode Baylisascaris transfuga i Syngamus sp., te enteropatogene protozoe Cryptosporidium sp., Eimeria sp. i Giardia sp. Utvrđena su i jaja ankilostomatida dok jaja cestoda, trematoda i akantocefala nisu pronađena. Ovo je prvo parazitološko istraživanje smeđeg medvjeda u Hrvatskoj u posljednjih 30 godina i prvi nalaz Giardia spp. u ove vrste s ovog područja.

Angiostrongylus vasorum is a parasitic nematode that can cause serious and potentially fatal disease in dogs and other canids. The aim of this study was to determine the intermediate slug species infected in nature by sampling sites in Greater London and Hertfordshire located within a known hyperendemic region. Overall, A. vasorum larvae were recovered from 6/381 slugs (1.6 %) by tissue digestion, and their identity was confirmed by PCR. Infected slugs originated from three different sites in the Greater London area: one in Waltham Forest and two in Bromley. Slugs parasitised by A. vasorum were identified by a combination of external morphological characteristics and molecular techniques and belonged to three different families: the Arionidae, the Milacidae and the Limacidae. This includes two new host records for the parasite: Arion distinctus and Tandonia sowerbyi. This is the first record of A. vasorum in the family Milacidae, indicating that the parasite has a broader intermediate host range than previously recognised.

The European brown bear (Ursus arctos) is a species present in limited areas of Europe and several small populations are considered endangered. This species can be affected by a range of parasites. In particular, the genus Baylisascaris is an emerging parasite of wild animals which can cause severe larva migrans syndrome in aberrant hosts, which include 100 species of birds, mammals and also humans. Baylisascaris transfuga is the species reported from bears, and with the exception of a few laboratory trials, little is known about the capacity of this species to infect other animals. Furthermore, the identification of this species has traditionally been based on light microscopy, using either morphology of the adults at necropsy or detection of the eggs in faeces, which are methods limited by the experience and the efforts of the observer. The current work was aimed at developing a specific PCR to detect the parasite directly from faecal samples of naturally infected brown bears in the field, without the need for previous flotation. Using eggs and adults of B. transfuga collected in Croatia, we first developed a PCR to detect a portion of the second internal transcribed spacer region (ITS-2) of ribosomal DNA and then applied it to bear faecal samples spiked with a known number of B. transfuga eggs. We show here for the first time that this method allows the detection of a minimum of two Baylisascaris eggs in 25 mg of faecal material, thus it demonstrates a high diagnostic capacity that could be applied to evaluate the prevalence of the parasite in faecal samples from wild populations of brown bears.