Treatment with potassium bromide mitigates ataxia and reduces tremor in lambs with perennial ryegrass toxicosis.

Aims: To assess the use of potassium bromide (KBr) as a therapeutic intervention for perennial ryegrass toxicosis (PRGT) in lambs fed ryegrass seed containing lolitrem B. Methods: Male lambs aged 10-12 months (n = 43) were assigned to receive ryegrass seed containing lolitrem B, at a dose of 0.16 mg/kg/day (Groups 2, 3 and 4), or lucerne chaff and molasses (Groups 1 and 5). Lambs in Groups 2 and 3 were observed for clinical signs and gait changes until defined signs of PGRT were observed, when they were transferred, with lambs in Group 1, to the Testing phase of the trial. Lambs in Group 3 were then treated with a single oral dose of 300 mg/kg bromide. Lambs in Groups 4 and 5 received KBr daily from the start of the trial (540 mg/kg bromide over 3 days then 20 mg/kg daily) and were transferred to the Testing phase after 18 days. Clinical examination and gait assessment, and surface electromyography of the triceps muscle, measuring root-mean-square (RMS) voltages, were carried out on Days 0, 1 and 2 of the Testing phase followed by necropsy, histopathology, measurement of concentrations of bromide in serum and CSF and faecal cortisol metabolites (FCM). Results: In Group 3 lambs, mean composite gait scores decreased between Testing phase Day 0 and Days 1 and 2 (p < 0.001), but increased in lambs in Group 2 between Day 0 and Day 2 (p = 0.015). Scores for lambs in Group 3 on Day 2 were lower than for lambs in Group 2 (p < 0.001). Mean RMS voltages on Day 2 were higher in lambs in Group 2 than Group 3 (p = 0.045). Mean concentrations of bromide in serum were >800 µg/mL in lambs in Groups 3 and 4 on Day 2. Concentrations of FCM were higher in lambs from Group 2 than in Groups 1 or 5, but were similar in Groups 2, 3 and 4. Histopathological findings in the cerebellum of lambs from Groups 2, 3 and 4 were similar, showing pyknosis of neurons within the granular layer of the cerebellum and Purkinje neuron proximal axonal spheroid formation. Conclusions and clinical relevance: A single oral dose of 300 mg/kg bromide in lambs with neurological signs of PRGT resulted in reduced composite gait scores and reduced RMS voltages, indicating a significant improvement in clinical signs of ataxia, movement disorder and muscle tremor associated with the neurotoxic effects of lolitrem B.

The first report of cerebral, pulmonary and gastrointestinal angiostrongyliasis in the water rat Hydromys chrysogaster from Queensland, Australia.

We describe the first case of angiostrongyliasis in a water rat, Hydromys chrysogaster, a large rodent adapted to aquatic life, which is endemic to Australia, New Guinea and adjacent islands.

Development of a model for investigation of perennial ryegrass toxicosis in sheep.

AIMS To develop a clinical model of perennial ryegrass toxicosis (PRGT) based on feeding a known dose of lolitrem B and ergotamine, and to produce a consistent clinical presentation for assessment of disease pathophysiology, neurological changes and neurohistopathology. METHODS Male lambs, aged between 10-12 months, were randomly assigned to either Treatment (n=9) or Control (n=9) groups. Lambs in the Treatment group received feed containing a novel endophyte-infested perennial ryegrass seed, commencing on Day 0 of the Feeding phase with a low induction dose, then increasing after 3 days to provide 0.16 mg/kg live bodywight (LBW)/day of lolitrem B and 0.054 mg/kg LBW/day ergotamine. Lambs were examined daily and when defined signs of PRGT were observed they were transferred to the Testing phase. Neurological examinations, assessment of gait, surface electromyography (EMG) and mechanosensory nociceptive threshold testing were carried out and blood samples collected during both phases of the trial, with a full necropsy, histopathological examination and measurement of faecal cortisol metabolites (FCM) performed on Day 2 of the Testing phase. RESULTS Typical clinical signs of PRGT, including ataxia of vestibulocerebellar origin leading to stumbling, were observed in all Treatment lambs. The median interval from the start of the Feeding phase to entry into the Testing phase was 21 (min 18, max 34) days. Histopathological characterisation of neurological lesions included the presence of Purkinje cell vacuolation, pyknotic granular layer neurons and proximal axonal Purkinje cell spheroids. Lesions were most apparent within the vestibulocerebellum. Mean root-mean-square voltages from triceps EMG increased in Treatment lambs between Feeding phase Day 0 and Testing phase Day 2 (p<0.001). Daily water intake during the Testing phase for the Treatment group was less than in Control group lambs (p=0.002), and concentrations of FCM at necropsy were higher in Treatment compared to Control lambs (p=0.02). CONCLUSIONS AND CLINICAL RELEVANCE Lolitrem B and ergotamine dosing in feed on a live weight basis combined with neurological/gait assessment provides an effective model for investigation of PRGT and potential therapeutics. Assessment of gait changes using defined criteria and RMS voltages from EMG appear to be useful tools for the assessment of the severity of neurological changes.

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778).

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.

Intracellular ciliated protozoal infection in silverlip pearl oysters, Pinctada maxima (Jameson, 1901).

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 microm (range of 2.73-7.47 microm, n=9) in width and 11.15 microm (range of 9.02-16.2 microm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p<0.001, C=0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38-50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection.

Sequence comparison of pigeon circoviruses.

The genome sequences of eight pigeon circoviruses (PiCV) were determined and compared with four previously published sequences. The viruses compared were from the USA, five European countries, China and Australia and included PiCVs from racing, feral, ornamental and meat pigeons and a Senegal dove (Streptopelia senegalensis). The 12 PiCV genomes, ranging from 2032 to 2040 nucleotides in length, displayed similar organizations. Pairwise comparisons showed that the genome nucleotide sequence identities ranged from 85.1% to 97.8% and that the amino acid identities of the putative replication associated (Rep) and putative capsid (Cap) proteins displayed ranges of 91.5-99.1% and 73.0-99.3%, respectively. Comparative analyses identified conserved nucleotide sequences within the Rep gene and 3' intergenic regions, which would be suitable for diagnostic PCR primers, and variable amino acid sequences within the capsid proteins, which should be considered when selecting virus isolates for vaccine development.

Spillover of avian haemosporidian parasites (Haemosporidia: Plasmodium) and death of captive psittacine species.

CASE REPORT: During February 2014, a yellow-tailed black cockatoo (Calyptorhynchus funereus) and glossy black cockatoo (C. lathami) housed in aviaries on a property in Wamuran, Queensland, were submitted for postmortem. Histopathology and molecular diagnostics demonstrated the presence of Plasmodium sp. infection. The Plasmodium isolate identified has previously only been reported as infecting a healthy wild rufous fantail (Rhipidura rufifrons) in Australia. CONCLUSION: To the authors' knowledge, these are the first reported cases of Plasmodium in Calyptorhynchus. We hypothesised that the maintenance of these two cockatoo species in ground level aviaries in a low-altitude geographic zone resulted in exposure of birds to mosquito vectors of endemic avian Plasmodium. Black cockatoos roost and forage in the mid to high canopy of forests in the wild, outside the likely spatiotemporal distribution of relevant haemosporidian vectors. It is therefore likely that these birds had immunological naivety and susceptibility to infection with Plasmodium circulating in wild passerines.

Genome sequence of an Australian strain of canid alphaherpesvirus 1.

OBJECTIVE: Characterisation of a complete genome sequence of an Australian strain of canid alphaherpesvirus 1 (CHV-1) and its phylogenetic relationship with other varicellovirus species. METHODS: Standard pathology and PCR methods were used to initially detect herpesvirus in hepatic tissue from an infected 4-week-old Labrador Retriever puppy. The complete CHV-1 genome was sequenced using next-generation sequencing technology followed by de novo and reference assembly, and genome annotation. RESULTS: The CHV-1 genome was 125 kbp in length and contained 74 predicted open reading frames encoding functional proteins, all of which have counterparts in other alphaherpesviruses. Phylogenetic analysis using the DNA polymerase gene revealed that the newly sequenced CHV-1 clustered with canid alphaherpesvirus isolated from the UK and shared a 99% overall nucleotide sequence similarity. CONCLUSION: This is the first complete genome of an Australian strain of CHV-1, which will contribute to our understanding of the genetics and evolution of herpesvirus.

Beak and feather disease virus carriage by Knemidocoptes pilae in a sulphur-crested cockatoo (Cacatua galerita).

BACKGROUND: This paper describes the pathology associated with psittacine beak and feather disease in a wild sulphur-crested cockatoo with concurrent knemidocoptic mange, cestodiasis and mycotic encephalitis. METHODS & RESULTS: Large numbers of Knemidocoptes pilae Lavoipierre and Griffiths, 1951 (Acari: Epidermoptidae, Knemidokoptinae) were identified in affected skin associated with enhanced expression of beak and feather disease virus (BFDV) determined by immunohistochemistry. Also, BFDV antigen was demonstrated in high concentration in the gut and faecal sacs of mites, raising the possibility of ectoparasites as fomites and vectors of BFDV transmission. Large numbers of Raillietina spp. cestodes were present in the intestines. Within the brain there was a focally extensive region of necrosis and inflammation associated with branching, septate, pigmented hyphae consistent with zygomycete fungal infection. CONCLUSION: This case highlights the potential immunosuppressive effects of BFDV infection and its potential as a keystone pathogen in the Australian environment.

Chronic shoulder osteoarthritis in a loggerhead turtle (Caretta caretta).

This report describes the lesions seen in an extremely weak and emaciated adult male loggerhead turtle (Caretta caretta) found stranded near Perth, Western Australia. Blood collected from a dorsal cervical sinus immediately prior to euthanasia demonstrated severe anaemia, markedly elevated plasma concentrations of creatinine kinase (2263 U/L), alkaline phosphatase (58 U/L), urea (18.1 mmol/L) and hypoglycaemia (glucose 0.7 mmol/L). Necropsy examination demonstrated a severe chronic osteoarthritis of the right shoulder joint with marked remodelling of the glenoid fossa, coracoid, scapula and humerus. There was marked synovial effusion and periarticular fibrosis. Cytological examination and culture of synovial fluid failed to demonstrate an infectious agent. Other findings were low numbers of trypanorhynch cysts present on the surface of the liver and numerous spirorchid eggs in the attached mesentery. Several large and small spirorchid flukes were present in the heart and there was a localised endarteritis in the left aortic arch associated with this infection. Numerous nematode eggs and at least three species of spirorchid eggs were seen microscopically in faeces collected from the terminal colon. The pathogenesis and significance of these lesions is discussed.

Airsac cystadenocarcinomas in cockatoos.

The clinical signs, radiographic and pathological findings of four histologically similar neoplasms that occurred as unilateral tumours projecting from the left axilla in three galahs (Eolophus roseicapillus) and one sulphur-crested cockatoo (Cacatua galerita) are described. In each case, the main reason for clinical presentation was respiratory distress. All cases were eventually fatal due to airway obstruction with evidence of extensive neoplastic invasion of the lungs, major airways and or humerus in all cases. A diagnosis of airsac cystadenocarcinoma was made in each bird on the basis of gross and histological appearance. The neoplasms were composed of fluid or air-filled sacs of proliferative cuboidal to squamous epithelial cells that stained positively with cytokeratin and negatively with vimentin. This was supported by a thin fibrovascular network although at least some areas in all four birds resembled airsac tissue. In some cases areas of haemorrhage, erythrophagocytosis, haemosiderosis and nodules of haemosiderophage infiltration with acicular cholesterol clefts were present in some parts of the sectioned tissue.

Infection with Photobacterium damselae subspecies damselae and Vibrio harveyi in snapper, Pagrus auratus with bloat.

OBJECTIVE: To diagnose the cause of chronic, low mortality associated with bloat in tanks of snapper at an aquaculture facility. DESIGN: A clinical, pathological and microbiological investigation into the cause of a low number of ongoing mortalities associated with bloat in snapper at an aquaculture facility is outlined. Necropsy, histology, microbiology and a comparison of haematology and water analysis from affected and unaffected fish and holding tanks, respectively were conducted. RESULTS: Affected moribund fish were found in lateral or dorsal recumbency floating on the water surface within 24 hours of death. Photobacterium damselae subspecies damselae was isolated from intestinal contents and Vibrio harveyi from the blood of affected fish and both were isolated from culture water. Both V harveyi and P damselae subspecies damselae isolates were sensitive to tetracycline, ciprofloxacin and sulphamethoxazole plus trimethoprim. Environmental parameters such as pH and dissolved oxygen were similar in tanks of affected and unaffected fish. Affected fish had gas distended swimbladders, anaemia, and the intestines were diffusely distended with a clear, pale yellowish fluid. Livers were mottled tan and green in a zonal pattern. Histologically the intestines of fish from tanks suffering mortality had a moderate granulocytic enteritis with oedema and infiltrations with eosinophilic granule cells that were also present as an infiltrate in the gills. There were elevated numbers of melanomacrophage centres and haemosiderin deposits in the spleen, kidney and liver of affected fish. CONCLUSION: Vibrio harveyi and Photobacterium damselae subspecies damselae infection should be recognised as potential pathogens of snapper held in water of less than optimal quality.

Surgical removal of an ovarian tumour in a koi carp (Cyprinus carpio).

Ultrasonography, radiography and exploratory coeliotomy were used to diagnose and treat a large intracoelomic neoplasm from a female koi carp (Cyprinus carpio) presented for abdominal enlargement of several months duration. Feed was withheld for 1 week immediately prior to surgery and the fish was sedated with isoeugenol (AQUI-S) at a dose rate of 10 mL/L to facilitate diagnostic imaging techniques. Surgical anaesthesia was induced by adding tricaine (MS-222) 50 mg/L to the water and an exploratory coeliotomy and tumour removal was performed. The fish was allowed to recover in fresh water at 18 degrees C and salt was added slowly to the water over a period of 1 hour to a concentration of 5 g/L This concentration was maintained in a recovery pond for 1 week postoperatively. Enrofloxacin was administered intramuscularly (10 mg/kg) immediately, 3 days and 1 week postoperatively. A diagnosis of undifferentiated ovarian carcinoma was made on the basis of the histological appearance of the neoplasm and immunohistochemical staining.

Review of psittacine beak and feather disease and its effect on Australian endangered species.

BACKGROUND: Since it was first described in the early 1980s, psittacine beak and feather disease (PBFD) has become recognised as the dominant viral pathogen of psittacine birds in Australia. Our aim was to evaluate and review the effect of PBFD and its position as a key threatening process to Australian psittacine bird species. We review the origin/evolutionary pathways and potential threat of PBFD to endangered psittacine bird populations and captive-breeding flocks. CONCLUSIONS: The most recent beak and feather disease virus (BFDV) phylogenetic analyses indicate that all endangered Australian psittacine bird species are susceptible to, and equally likely to be infected by, BFDV genotypes from a range of host psittacine species. Management of the disease in captive-breeding programs has relied on testing and culling, which has proven costly. The risk of PBFD should be considered very carefully by management teams contemplating the establishment of captive-breeding flocks for endangered species. Alternative disease prevention tools, including vaccination, which are increasingly being used in wildlife health, should be considered more seriously for managing and preventing PBFD in captive flocks of critically endangered species.

Herpesvirus in a captive Australian Krefft's river turtle (Emydura macquarii krefftii).

CASE REPORT: A mature, captive Krefft's river turtle (Emydura macquarii krefftii) was presented with severe proliferative and ulcerative lesions of the skin and shell. The areas were biopsied and histopathological examination demonstrated orthokeratotic hyperkeratosis with keratinocytes containing eosinophilic intranuclear inclusions. Molecular diagnostics confirmed the presence of a herpesvirus in the affected tissues. CONCLUSION: This is the first recorded case of herpesvirus infection in an Australian freshwater turtle species.

Beak and feather disease virus genotypes in Australian parrots reveal flexible host-switching.

OBJECTIVE: To discover beak and feather disease virus (BFDV) genotypes in Australian parrots that might threaten vulnerable and endangered psittacine bird species. METHODS: Phylogenetic analyses of new DNA sequence data from Australian birds including the Rep gene (n = 55) and nine whole genomes, were compared with all available published BFDV genomes to assess host- and geographically-based divergence as well as probable host-switch events. RESULTS: Strong support for flexible host-switching and recombination was detected, indicating active cross-species transmission in various subpopulations. CONCLUSION: The data suggested that all endangered Australian psittacine bird species are equally likely to be infected by BFDV genotypes from any other close or distantly related host reservoir species.

Assessment of liver function in chickens using galactose and indocyanine green clearances.

Values for galactose and indocyanine green (ICG) clearances, and plasma and serum biochemical markers of liver dysfunction were determined in normal chickens and following coeliotomy, and compared with birds after partial hepatectomy. Clearance tests, and serum and plasma biochemistry were performed 4h, and 4 and 7 days after surgery. Coeliotomy and manipulation of the liver did not delay clearance of either compound. Partial hepatectomy resulted in elevation of galactose single point concentrations but did not significantly alter galactose clearance (GEC) values. Clearance values of ICG were not significantly altered. Biochemical values were not significantly elevated in birds after a partial hepatectomy in comparison with birds after coeliotomy. Galactose single point concentrations have the potential to become a simple, relatively non-invasive method of screening for liver disease, with GEC tests having the potential to quantify the degree of loss of functional hepatic mass.

The pathology of normobaric oxygen toxicity in budgerigars (Melopsittacus undulatus).

The effects of normobaric oxygen exposure were investigated in budgerigars (Melopsittacus undulatus). Sixty birds were randomly divided into four equal groups of 15. These groups were randomly allocated as control, acute exposure, repeated acute exposure or chronic exposure. Control birds were exposed to 72 continuous hours of 21% oxygen in a sealed, enclosed chamber. Acute exposure, repeated acute exposure and chronic exposure groups were exposed oxygen at minimum concentration of 95% for a single 3-h period, a 3-h period daily for three sequential days, or a single 72-h period in a sealed, enclosed chamber, respectively. Oxygen exposure resulted in significant alteration in the histological morphology of respiratory exchange tissue, with severe oedema, and inflammatory cell infiltration. Electron micrographs revealed thickening of the blood-gas barrier with the tissue harmonic thickness increasing from 226 +/-90 nm in control birds to 639 +/-393nm following repeated acute exposure, with the total harmonic thickness increasing from a control value of 345 +/-146nm to 837 +/-423 nm at the same time. Chronic oxygen exposure resulted in significant changes in cell morphology including thickening of endothelial cells, ruffling of type I respiratory endothelial cells and interstitial vacuolation. These results indicate that budgerigars undergo significant morphological and ultrastructural changes in respiratory exchange tissue following exposure to 100% oxygen.

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.

Haematopoietic necrosis in a goldfish (Carassius auratus) associated with an agent morphologically similar to herpesvirus.

One of 14 goldfish (Carassius auratus) died 4 weeks after purchase and was investigated by necropsy and histological examination. Routine formalin fixation of the goldfish was followed by histopathology. Formalin fixed spleen and kidney from the fish was further processed by embedding in epoxy resin and examined by transmission electron microscopy (EM). Severe, diffuse necrosis of haematopoietic tissue in the spleen, thymus and kidney and severe, diffuse hyperplasia in the gill epithelial cells were seen. In the spleen there was severe, diffuse necrosis of lymphocytes and many nuclei with marginated chromatin and intranuclear inclusions were scattered throughout the necrotic tissue. EM of affected tissues demonstrated intranuclear particles morphologically similar to herpesvirus. The presence of an agent similar to a herpesvirus in a goldfish with severe haematopoietic necrosis suggests that the herpesvirus responsible for haematopoietic necrosis in cyprinid species throughout the world has entered the goldfish population in Australia.