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Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus).

Abstract CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summer of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds. CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birdsbelonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater). DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined. CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.

The prevalence of beak and feather disease virus infection in wild populations of parrots and cockatoos in New Zealand

Abstract The prevalence of beak and feather disease virus infection in wild populations of parrots and cockatoos in New Zealand METHODS: Eastern rosellas (Platycercus eximius, n=162) were caught from Te Puke, Wellington and Dunedin, using mistnets, between April 2004 and February 2006, and sulphurcrested cockatoos (Cacatua galerita, n=255) were captured for pet-trading from November 2001 to September 2004. Feathers from both species were tested for BFDV, using an established polymerase chain reaction (PCR) test. Post-mortem examinations were conducted on some of the eastern rosellas, and selected tissues from 24 birds positive for BFDV were examined using routine histological methods for the presence of characteristic inclusion bodies. RESULTS: Of the eastern rosellas, 24/162 (14.8%) were positive for BFDV, and the 95% confidence interval (CI) for true prevalence was estimated as 8.6–20.4%, which varied between regions. Eastern rosellas that were positive for BFDV showed no clinical or histological signs of disease or inclusion bodies. Of the sulphur-crested cockatoos, 70/255 (28%) were positive for BFDV, and the 95% CI for true prevalence was calculated as 22–33%. CONCLUSIONS: The surprisingly high prevalence of BFDV in wild eastern rosellas and sulphur-crested cockatoos has serious implications for the conservation of native parrots and the export of wild-trapped parrots and cockatoos from New Zealand. Serological studies for BFDV in wild exotic parrots, and molecular studies of virus genotype, are recommended to further characterise the origin and epidemiology of the disease in populations of wild exotic parrots and cockatoos in New Zealand.

Evaluation of the pathogenicity of avipoxvirus strains isolated from wild birds in New Zealand and the efficacy of a fowlpox vaccine in passerines

Avipoxvirus (APV) infection is a highly contagious disease of birds and has been reported in more than 200 bird species, affecting both domesticated and free-ranging birds around the world. In New Zealand, at least three different strains of Avipoxvirus (APV) have been identified in a range of bird species.The pathogenicityof two APV strains isolated from wild birds in New Zealand, representing subclade A1 and subclade B1 were compared in zebra finches (Taeniopygia guttata). The efficacy of fowlpox vaccine at preventing clinical disease in passerines was also evaluated. Twenty-five zebra finches were divided into five groups (I-IV and a control group). Birds from Groups II and IV were vaccinated using fowl poxvirus vaccine prior to challenge. Subsequently two groups (I and II) were inoculated with a silvereye isolate (A1) and the other two groups (Group III and IV) were inoculated with a blackbird isolate (B1). Both inocula were previously propagated in chicken fibroblast cell culture. Birds in the control group were inoculated with sterile PBS. Skin thickness at the inoculation sites was measured and the development of additional skin lesions was monitored. Antibody development was measured by ELISA pre- and post virus inoculation. Both APV strains caused either swelling or hyperplasia at the inoculation site of non-vaccinated birds (4/5 in Group I and 5/5 in Group III). The swelling was milder and no foot lesions were observed in vaccinated birds before or after challenge with the silvereye or blackbird APV strains. These findings indicated that the fowlpox vaccine provided safe and appropriate protection for zebra finches exposed to the two wild APV strains and suggest that the vaccine has the potential to be used where APV threatens the captive management or translocation of endangered passerines.

The prevalence of psittacine beak and feather disease virus infection in native parrots in New Zealand

Abstract AIM: To determine the prevalence of psittacine beak and feather disease virus (BFDV) infection in native parrots in New Zealand. METHODS: One hundred and sixty-nine wild native parrots and 143 captive native parrots throughout New Zealand were examined for the presence of BFDV, from June 2003 to January 2005. Feathers of each bird, and blood samples from 15 birds, were collected and submitted for PCR assay to detect BFDV. RESULTS: All of the samples from wild native parrots were negative for BFDV by PCR assays. Similarly, of the 143 PCR tests from captive native parrots 139 (97%) were negative for BFDV. However, a pair of red-crowned parakeets and two Antipodes Island parakeets from different captive facilities were found to be infected with BFDV. The infected birds showed no clinical signs suggestive of psittacine beak and feather disease (PBFD), although the second Antipodes Island parakeet was found dead, and had pathological changes consistent with acute septicaemia. CONCLUSIONS: The results indicate a very low prevalence of BFDV among free-living native parrots although captive birds, in particular native parakeets, are susceptible to BFDV infection, and the Antipodes Island parakeets may be susceptible to PBFD.

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle.

Abstract CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types. LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR. A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%). DIAGNOSIS: Bovine haemoplasmosis. CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.

Avipoxvirus infections in brown kiwi (Apteryx mantelli)

Abstract CASE HISTORY: Nodular lesions were found on the skin of two immature brown kiwi (Apteryx mantelli) less than 6 months of age living freely on Ponui Island off the North Island of New Zealand. The lesions were observed during routine external examination undertaken as a part of the management of other research projects, one in 2006 and the other in 2011. Apart from the skin lesions, both birds showed no signs of illness and the lesions resolved spontaneously over a 2-month period. PATHOLOGICAL FINDINGS: The first case showed several 3-mm diameter firm, brown nodules located on the skin below the hock of both legs. The second case had a single multinodular mass that measured 7×20 mm, on the base of the bill. A portion of the mass and scab samples were collected for diagnosis. Histological examination of the nodules revealed severe ballooning degeneration of keratinocytes and epithelial hyperplasia. Round eosinophilic structures resembling avipoxvirus (APV) intracytoplasmic inclusion bodies (Bollinger bodies) were observed in the layers of keratinocytes. In deeper layers of the epidermis, there was evidence of secondary bacterial growth and inflammation. DIAGNOSIS: DNA was extracted from tissue samples and subjected to PCR analysis. Avipoxvirus 4b core protein gene was detected in both samples by PCR. Bootstrap analysis of APV 4b core protein gene revealed that APV isolates from two kiwi comprised two different subclades. One isolate displayed 100% sequence homology to subclade B1, and the other presented 100% sequence homology to subclade A3. CLINICAL RELEVANCE: This study confirmed that kiwi are susceptible to APV infection and that at least two different strains of APV are present in the population examined. Since there is no information on the origin, virulence, or prevalence of APV in kiwi, a seroprevalence study would be useful to elucidate the degree of exposure and immune response to the disease. This would allow a more informed approach to risk management of the disease in wild and captive populations.

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations. CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara. LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA. DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates. CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.

Nocardiosis in freshwater reared Chinook salmon (Oncorhynchus tshawytscha)

Abstract CASE HISTORY: An investigation was conducted to identify the cause of mortalities in freshwater reared Chinook salmon (Oncorhynchus tshawytscha). Mortalities occurred in juvenile salmon, at a salmon rearing facility in the South Island of New Zealand. The affected fish were from a pen inside the facility with no surrounding pens or other year classes affected. CLINICAL FINDINGS: Clinically affected fish presented with skin lesions. The majority of skin lesions were unruptured, boil-like, raised circular masses up to 4 cm in diameter, particularly on the dorsolateral aspects and the flank. A number of fish presented with large ulcers resulting from rupturing of the raised lesions described above. This clinical presentation showed similarities to that of furunculosis caused by typical Aeromonas salmonicida, a bacterium exotic to New Zealand. LABORATORY FINDINGS: Samples were taken from two representative fish in the field for histopathology, bacterial culture and molecular testing. Histopathological findings included granulomatous lesions in the kidney, liver, spleen and muscle. When stained with Fite-Faraco modified acid fast stain filamentous branching rods were identified within these granulomas. Following bacterial culture of kidney swabs pure growth of small white matt adherent colonies was observed. This isolate was identified as a Nocardia species by biochemical testing and nucleotide sequencing of the partial 16S rRNA gene. All samples were negative for A. salmonicida based on bacterial culture and PCR testing. DIAGNOSIS: Nocardiosis caused by a Nocardia species. CLINICAL RELEVANCE: Nocardiosis in these fish was caused by a previously undescribed Nocardia species that differs from the species known to be pathogenic to fish: N. asteroides, N. salmonicida and N. seriole. This bacterium is likely to be a new or unnamed environmental species of Nocardia that has the potential to cause disease in Chinook salmon under certain conditions. The clinical presentation of this Nocardia species manifested as raised, boil-like skin lesions which has similarities to the presentation of furunculosis caused by the bacterium typical A. salmonicida, a species exotic to New Zealand.

Investigation of a mortality cluster in wild adult yellow-eyed penguins (Megadyptes antipodes) at Otago Peninsula, New Zealand.

ABSTRACT We investigated an epidemic mortality cluster of yellow-eyed penguins (Megadyptes antipodes) that involved 67 moribund or dead birds found on various beaches of the Otago Peninsula, New Zealand, between 21 January and 20 March 2013. Twenty-four carcases were examined post-mortem. Histological lesions of pulmonary, hepatic and splenic erythrophagocytosis and haemosiderosis were found in 23 of 24 birds. Fifteen birds also had haemoglobin-like protein droplets within renal tubular epithelial cells. Despite consistent histological lesions, a cause of death could not be established. Virology, bacteriology and molecular tests for avian influenza, avian paramyxovirus-1, avipoxvirus, Chlamydia psittaci, Plasmodium spp., Babesia spp., Leucocytozoon spp. and Toxoplasma gondii were negative. Tissue concentrations of a range of heavy metals (n = 4 birds) were consistent with low level exposure, while examination of proventricular contents and mucus failed to detect any marine biotoxins or Clostridium botulinum toxin. Hepatic concentrations of total polycyclic aromatic hydrocarbons (PAHs) (n = 5 birds) were similar to background concentrations of polycyclic aromatic hydrocarbons previously found in yellow-eyed penguins from the South Island of New Zealand, but there were significantly higher concentrations of 1-methylnapthelene and 2-methylnapthelene in the birds found dead in this mortality cluster. The biological significance of this finding is unclear. A temporal investigation of the epidemic did not indicate either a common source or propagative epidemic pattern. Although our investigation did not definitively implicate a toxic or infectious agent, we could not rule out causes such as toxic marine organisms or mycoplasmosis. Further investigations should therefore by carried out in the event of future mortality clusters.

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis. METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results. RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.