Embedding research and enquiry in Australian DVM curriculum.

Research and enquiry (R&E) is an integral part of veterinary training. It is a foundation of evidence-based practice. In the University of Sydney Doctor of Veterinary Medicine degree R&E culminates in a cap-stone experience in Year 3: a 'professionally focused project', a student-driven and academic supported individual research project. The project provides an authentic experience within a veterinary discipline. Students work with an academic advisor who provides guidance for developing and achieving meaningful educational and professional goals. Successful advising depends upon a shared understanding of, and commitment to, the advising process by students, advisors and the university. The R&E mission can be broadly defined as - veterinarians recognise that evidence-based approach to practice, which is based on the scientific method, leads to the generation of new knowledge that underpins the veterinary medical profession.

Strongyloides stercoralis in two dogs from a household in temperate Australia.

Strongyloides stercoralis is parasite affecting both humans and dogs and is most prevalent in tropical and subtropical areas of Australia. This case report describes two dogs from a household in Sydney, New South Wales, one with chronic gastrointestinal signs and the other who was asymptomatic who were subsequently diagnosed with S. stercoralis. Diagnosis can be challenging in humans and dogs due to intermittent shedding and low worm burdens and in this case the symptomatic dog had Strongyloides spp. rhabitiform larvae detected on a direct faecal smear and PCR, the asymptomatic dog on PCR only. Obtained sequences from the symptomatic dog confirmed the presence of the S. stercoralis clade affecting both dogs and humans. Infection does not respond to commonly used deworming drugs for dogs. Treatment in both cases was undertaken using off-label doses of ivermectin and follow-up PCR testing was negative. This case report should increase practitioner awareness of this parasite as present and transmissible in temperate areas of Australia.

Unambiguous identification of Ancylostoma caninum and Uncinaria stenocephala in Australian and New Zealand dogs from faecal samples.

Hookworms (Ancylostomatidae) are well-known parasites in dogs due to their health impacts and zoonotic potential. While faecal analysis is the traditional method for detection, improvements in husbandry and deworming have decreased their prevalence in urban owned dogs. Drug resistance in Ancylostoma caninum is becoming a discussion point in small animal practices across the region. This study aimed to identify hookworm species present in Australian and New Zealand dogs using molecular techniques. The ITS-2 and isotype-1 ß-tubulin assays were used to identify and quantify hookworm species. Results showed absence of coinfection in Australian samples from Greater Sydney region belonging either to A. caninum or Uncinaria stenocephala, while New Zealand samples were a mixture of A. caninum and U. stenocephala. The amplified isotype-1 ß-tubulin sequences exhibited susceptibility to benzimidazole drugs. Rare mutations were identified in A. caninum and U. stenocephala sequences, representing a small percentage of reads. This study highlights the importance of molecular techniques in accurately identifying and quantifying hookworm species in dog populations.

Ecological drivers of helminth infection patterns in the Virunga Massif mountain gorilla population.

The Virunga Massif mountain gorilla population has been periodically monitored since the early 1970s, with gradually increasing effort. The population declined drastically in the 1970s, but the numbers stabilized in the 1980s. Since then, the population has been steadily increasing within their limited habitat fragment that is surrounded by a dense human population. We examined fecal samples collected during the Virunga 2015-2016 surveys in monitored and unmonitored gorilla groups and quantified strongylid and tapeworm infections using egg counts per gram to determine environmental and host factors that shape these helminth infections. We showed that higher strongylid infections were present in gorilla groups with smaller size of the 500-m buffered minimum-convex polygon (MCP) of detected nest sites per gorilla group, but in higher gorilla densities and inhabiting vegetation types occurring at higher elevations with higher precipitation and lower temperatures. On the contrary, the impact of monitoring (habituation) was minor, detected in tapeworms and only when in the interaction with environmental variables and MCP area. Our results suggest that the Virunga mountain gorilla population may be partially regulated by strongylid nematodes at higher gorilla densities. New health challenges are probably emerging among mountain gorillas because of the success of conservation efforts, as manifested by significant increases in gorilla numbers in recent decades, but few possibilities for the population expansion due to limited amounts of habitat.

Suspect 'baggage canine heartworm' case: canine heartworm disease in a dog from Sydney, New South Wales.

Locally acquired canine heartworm (Dirofilaria immitis) infection in the temperate southern climate zones of Australia is currently rare. We report a case of locally acquired canine heartworm from Sydney, New South Wales in a 12-year-old Fox Terrier × Jack Russell female that presented with coughing and breathing difficulties. Absence of heartworm prevention and no travel outside Sydney was noted. Blood sample was D. immitis positive using antigen test, but negative on Modified Knott's testing. PCR confirmed the presence of D. immitis DNA in circulating blood. Echocardiographic examination revealed multiple parallel echogenic lines separated by a hypoechoic region ('tram-tracks') in the right pulmonary artery. The patient was treated and clinical condition gradually improved over the following 12 months. Antigen test remained positive for D. immitis at ~7 months and became negative at ~15 months after the start of the treatment. The most plausible scenario is importation of infected mosquito(s) in the luggage arriving from Queensland, Australia, common holiday destination for many Sydney-siders. We consider this a case of 'baggage canine heartworm'. Canine heartworm in dogs who did not travel should be considered in the differential diagnosis and D. immitis antigen test coupled with Modified Knott's test or PCR must be considered.

Museum material of Rhipicephalus sanguineus sensu Roberts (1965) collected in 1902-1964 from Australia is identical to R. sanguineus sensu lato tropical lineage at the mitochondrial DNA 12S rRNA level.

Ticks identified as Rhipicephalus sanguineus (Latreille, 1806) have been recorded in Australia since 1896. It is now recognized that ticks identified as R. sanguineus belong to different lineages. Recently, the so-called temperate lineage has been redescribed as R. sanguineus sensu stricto with a designated neotype. In Australia, the evidence suggests that only R. sanguineus sensu lato tropical lineage exists. We present a genetic evaluation of R. sanguineus sensu Roberts (1965) from museum material that was evaluated or identified by Roberts. We evaluated 12 museum specimens collected between 1913 and 1964, from various locations around Australia at the molecular level. DNA isolation was successful for 4/12 specimens. All four museum specimens were 100% identical to each other and to the reference R. sanguineus s.l. tropical lineage. Measurements of morphologically significant structures for male and female museum and contemporary tick specimens were obtained. The morphologically significant structures revealed there was no significant difference (ANOVA, P > 0.05) between these groups of male and female ticks. This study updates and supplement the comprehensive descriptions provided by Roberts (1965), which genetically belong to R. sanguineus s.l. tropical lineage.

Fleas from domestic dogs and rodents in Rwanda carry Rickettsia asembonensis and Bartonella tribocorum.

Fleas (Siphonaptera) are ubiquitous blood-sucking parasites that transmit a range of vector-borne pathogens. The present study examined rodents (n = 29) and domestic dogs (n = 7) living in the vicinity of the Volcanoes National Park, Rwanda, for fleas, identified flea species from these hosts, and detected Bartonella (Rhizobiales: Bartonellaceae) and Rickettsia (Rickettsiales: Rickettsiaceae) DNA. The most frequently encountered flea on rodents was Xenopsylla brasiliensis (Siphonaptera: Pulicidae). In addition, Ctenophthalmus (Ethioctenophthalmus) calceatus cabirus (Siphonaptera: Hystrichopsyllidae) and Ctenocephalides felis strongylus (Siphonaptera: Pulicidae) were determined using morphology and sequencing of the cytochrome c oxidase subunit I and cytochrome c oxidase subunit II genes (cox1 and cox2, respectively). Bartonella tribocorum DNA was detected in X. brasiliensis and Rickettsia asembonensis DNA (a Rickettsia felis-like organism) was detected in C. felis strongylus. The present work complements studies that clarify the distributions of flea-borne pathogens and potential role of fleas in disease transmission in sub-Saharan Africa. In the context of high-density housing in central sub-Saharan Africa, the detection of B. tribocorum and R. asembonensis highlights the need for surveillance in both rural and urban areas to identify likely reservoirs.

Compensatory gastric stretching following subtotal gastric resection due to gastric adenocarcinoma in a diamond python (Morelia spilota spilota).

CASE REPORT: A 7-year-old male diamond python (Morelia spilota spilota) presented with a 2-month history of anorexia and a discrete intracoelomic mass, approximately 15 cm in length, located 90 cm from the head and approximately two-thirds of the snout to vent length. Physical examination determined the mass was likely to be stomach, testes or the right kidney. Radiographs showed a soft tissue opacity mass in the region of the stomach; fine needle aspirate demonstrated cellular debris admixed with bacteria and degenerate heterophils. Exploratory coeliotomy revealed a gastric mass involving 90% of the length of the stomach, partially occluding the gastric lumen. A subtotal gastrectomy was performed; the neoplastic tissue was removed with 2 cm margins, leaving 1 cm of stomach wall and the pyloric sphincter caudally that was anastomosed to the oesophagus. Four large nematodes were found within the necrotic lumen of the mass tightly adhered to the gastric mucosa. Ascarid nematodes were identified morphologically and further confirmed by molecular diagnostics as Ophidascaris spp. Histopathological evaluation of the excised mass revealed a gastric adenocarcinoma. Postoperatively the snake suffered from gastrointestinal dysfunction and maldigestion and was managed with slurry feeding for month. Three months postoperatively the snake was gaining weight, eating without assistance and digesting whole prey, which was incrementally increased in size. Gastroscopy 6 months postoperatively revealed the presence of a functional stomach with a functional pyloric sphincter and 8.5 cm of gastric mucosa caudal to the anastomosis between the oesophagus and stomach. CONCLUSION: This is the first report of almost complete subtotal gastric resection in an Australian python, with evidence of compensatory gastric stretching resulting in a functional stomach.

Shelter-housed cats show no evidence of faecal shedding of canine parvovirus DNA.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

Unusual presentation of neosporosis in a neonatal puppy from a litter of bulldogs.

CASE REPORT: A 17-day-old Bulldog puppy died soon after presentation for weakness and tachypnoea. Gross lesions included diffuse pulmonary oedema and a region of myocardial pallor that resembled an infarct. Inflammation was observed histopathologically in many organs, with numerous clusters of intracellular protozoa that stained positively using Neospora caninum immunohistochemistry. Myocarditis was severe and had associated necrosis of individual myocytes, but the tissue was not infarcted. The bitch had an antibody titre of 1 : 1600 for N. caninum. All six littermates were sold and reported to be healthy at 6 months of age. CONCLUSION: Unusual aspects of this case include the occurrence of clinical disease in only 1 of 7 neonatal puppies, widespread dissemination of the organism in multiple tissues, and regional pallor associated with myocarditis that gave a false gross appearance of infarction. This report also adds Bulldogs to the list of dog breeds shown to be susceptible to clinical neosporosis.

High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers.

The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché), is the most common flea species found on cats and dogs worldwide. We investigated the genetic identity of the cosmopolitan subspecies C. felis felis and evaluated diversity of cat fleas from Australia, Fiji, Thailand and Seychelles using mtDNA sequences from cytochrome c oxidase subunit I (cox1) and II (cox2) genes. Both cox1 and cox2 confirmed the high phylogenetic diversity and paraphyletic origin of C. felis felis. The African subspecies C. felis strongylus (Jordan) is nested within the paraphyletic C. felis felis. The south East Asian subspecies C. felis orientis (Jordan) is monophyletic and is supported by morphology. We confirm that Australian cat fleas belong to C. felis felis and show that in Australia they form two distinct phylogenetic clades, one common with fleas from Fiji. Using a barcoding approach, we recognize two putative species within C. felis (C. felis and C. orientis). Nucleotide diversity was higher in cox1 but COX2 outperformed COX1 in amino acid diversity. COX2 amino acid sequences resolve all phylogenetic clades and provide an additional phylogenetic signal. Both cox1 and cox2 resolved identical phylogeny and are suitable for population structure studies of Ctenocephalides species.

Histopathological survey of lesions and infections affecting sick ornamental fish in pet shops in New South Wales, Australia.

The objective of this study was to describe the frequency of histopathological lesions and categorize histopathologically evident infections in sick ornamental fish from pet shops in New South Wales, Australia. We examined 108 fish that had evidence of morbidity or mortality, including 67 cyprinids, 25 osphronemids, 11 poeciliids, 4 characids and 1 cichlid, sourced from 24 retail outlets. Conditions frequently observed in the study population included branchitis (62/86, 72.1%), visceral granulomas (41/108, 38.0%), dermatitis (17/55, 30.9%), wasting (31/108, 28.7%), and intestinal coccidiosis (18/104, 17.4 %). Branchitis and dermatitis were usually due to monogenean flukes, or flagellate or ciliate protozoa. Intralesional Microsporidia (16/41, 39.0%), mycobacteria (7/41, 17.%), or Myxosporidia (5/41, 12.2%) were identified in the majority of fish with visceral granulomas; however, special stains were critical in their identification. The proportion of histologically evident infections was remarkably high (77/108, 71.3%), and parasitic infections predominated. Many pathogens identified in the study have low host specificity and/or direct life cycles which would facilitate transmission to exposed naive fish populations, potentially posing a threat to native and commercial fish populations. Those caring for sick ornamental fish should take appropriate steps to investigate infectious disease and should take precautions that prevent the spread of pathogens.

Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum.

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.

Caryospora matatu n. sp., a new coccidian parasite (Apicomplexa: Eimeriidae) from the horned bush-viper, Atheris ceratophorus Werner, 1895 from Tanzania.

Caryospora matatu n. sp. is the first species of coccidia reported from the a horned bush viper, Atheris ceratophorus endemic to Tanzania. Oocysts are spherical or slightly subspherical, 19.8 (16-23) microns, a micropyle and an oocyst residuum are absent, a single polar granule is present. An oocyst wall is bilayered, approximately 1.5 microns thick, brownish and distinctly pitted. Sporocysts are ellipsoidal, 15.6 (12.5-17.0) x 10.1 (8-12) microns, tightly fitting an internal oocyst diameter, Stieda and substieda bodies are present. An experimental inoculation of SCID mice did not confirm a facultatively heteroxenous life cycle in the studied coccidium. Morphology and evolutionary history of Caryospora species from viperids is is reviewed and discussed.

Eimeria hajeki n. sp. (Apicomplexa: Eimeriidae), a new coccidian parasite of the pygmy chameleon, Rampholeon temporalis (Matschie, 1892) (Reptilia: Chamaeleonidae) from Usambara Mountains, Tanzania.

Fecal samples from 10 pygmy chameleons, Rampholeon temporalis (Matschie, 1892), an endemic species of the Usambara Mountains in northeastern Tanzania, were examined for coccidian parasites. Two (20%) chameleons were found to be passing oocysts of Eimerio Schneider. Comparison with other species of Eimeria indicates that the coccidian found represents a new species. Sporulated oocysts of Eimeria hajeki n. sp. are oval, 30.2 (29-31) by 23.5 (22-25) microm, with a shape index (length/width) of 1.3 (1.2-1.4) and a 2-microm-thick rough, bilayered wall. Micropyle and polar granule are absent. Sporocysts are oval to rhomboidal, 10.8 (9-11.5) by 8.8 (7.5-10) microm, with a shape index of 1.2 (1.15-1.3) and a wall composed of 2 valves joined by a suture.

Phylogenetic position of a renal coccidium of the European green frogs, 'Isospora' lieberkuehni Labbé, 1894 (Apicomplexa: Sarcocystidae) and its taxonomic implications.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.

Multiple origin of the dihomoxenous life cycle in sarcosporidia.

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.

Caryospora varaniornati sp. n. (Apicomplexa: Eimeriidae) in the Nile monitor, Varanus (Polydaedalus) niloticus species complex.

Parasitological examination of two ornate Nile monitors Varanus ornatus (Daudin, 1803) imported from Benin revealed the presence of a new species of Caryospora. Oocysts of Caryospora varaniornati sp. n. are spherical to slightly subspherical, 12.0 (11-12.5) x 11.5 (11-12) microm, without amicropyle and oocyst residuum, and occasionally possessing one small polar granule. Sporocysts are broadly ellipsoidal, 8.8 (8.5-9.5) x 6.7 (6.5-7) microm; a lentil-like Stieda body is present, ca. 0.5 x 1 microm; substieda body not visible. Experimental infection of a closely related host, Varanus niloticus (L.), did not lead to the oocyst excretion despite the fact that one of the experimentally inoculated monitors was immunosuppressed by dexamethasone. Histological examination did not reveal stages of coccidian development. Therefore, it is possible that C. varaniornati is strictly host specific.

Eimeria telekii n.sp. (Apicomplexa: Coccidia) from Lemniscomys striatus (Rodentia: Muridae): morphology, pathology and phylogeny.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20.4 x 15.7 (15.5-25.0 x 12.0-20.0) microm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11.2 x 7.8 (10.0-12.0 x 7.0-9.0) microm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.