MYCOBACTERIAL DISEASE AND SUBSEQUENT DIAGNOSTIC INVESTIGATIONS IN A GROUP OF CAPTIVE PINNIPEDS IN NEW ZEALAND.

This case series includes a single case of disseminated tuberculous disease due to Mycobacterium pinnipedii in a New Zealand fur seal (Arctocephalus forsteri), which was being cared for by a zoo in New Zealand. The remaining five pinnipeds in the colony underwent extensive mycobacterial disease surveillance over the following 4 yr, involving a total of 26 anesthetic procedures and numerous diagnostic tests that included comparative intradermal tuberculin skin tests, mycobacterial antibody serology, respiratory and gastric lavages, and computed tomography (CT) scans. An additional case of chronic sinusitis due to Mycobacterium marinum and Pseudomonas aeruginosa was identified in a California sea lion (Zalophus californianus). Results from CT and the respiratory lavages were the most helpful antemortem diagnostic tests for active mycobacterial disease in this case series. Of the remaining four animals, two were euthanatized and two remain alive, and none of them had evidence of active mycobacterial disease. Further mycobacterial disease surveillance in staff and animals was performed, and no other case was identified. There are no validated mycobacterial surveillance tests available for pinnipeds and so it remains unknown whether the two surviving pinnipeds are truly negative or whether they have latent mycobacterial infection that could develop into active mycobacterial disease in the future. For this reason, increased levels of biosecurity and quarantine remain permanently in place for the pinniped colony.

Comparison of PCR assays to detect Toxoplasma gondii oocysts in green-lipped mussels (Perna canaliculus).

Toxoplasma gondii is recognised as an important pathogen in the marine environment, with oocysts carried to coastal waters in overland runoff. Currently, there are no standardised methods to detect T. gondii directly in seawater to assess the extent of marine ecosystem contamination, but filter-feeding shellfish may serve as biosentinels. A variety of PCR-based methods have been used to confirm presence of T. gondii DNA in marine shellfish; however, systematic investigations comparing molecular methods are scarce. The primary objective of this study was to evaluate analytical sensitivity and specificity of two nested-PCR (nPCR) assays targeting dhps and B1 genes and two real-time (qPCR) assays targeting the B1 gene and a 529-bp repetitive element (rep529), for detection of T. gondii. These assays were subsequently validated for T. gondii detection in green-lipped mussel (Perna canaliculus) haemolymph using oocyst spiking experiments. All assays could reliably detect 50 oocysts spiked into mussel haemolymph. The lowest limit of detection was 5 oocysts using qPCR assays, with the rep529 primers performing best, with good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Assay specificity was evaluated by testing DNA from closely related protozoans, Hammondia hammondi, Neospora caninum, and Sarcocystis spp. Both nPCR assays were specific to T. gondii. Both qPCR assays cross-reacted with Sarcocystis spp. DNA, and the rep529 primers also cross-reacted with N. caninum DNA. These studies suggest that the rep529 qPCR assay may be preferable for future mussel studies, but direct sequencing is required for definitive confirmation of T. gondii DNA detection.

First report of Toxoplasma gondii sporulated oocysts and Giardia duodenalis in commercial green-lipped mussels (Perna canaliculus) in New Zealand.

Pollution of marine ecosystems with the protozoan parasites Toxoplasma gondii, Cryptosporidium spp. and Giardia duodenalis can be studied using bivalve shellfish as biosentinels. Although evidence suggests that these parasites are present in New Zealand coastal waters, the extent of protozoal pollution has not been investigated. This study used optimised molecular methods to detect the presence of Cryptosporidium spp., G. duodenalis and T. gondii in commercially sourced green-lipped mussel (Perna canaliculus), an endemic species found throughout coastal New Zealand. A nested polymerase chain reaction was validated for detection of T. gondii DNA and applied to 104 commercially sourced mussels. Thirteen mussels were positive for T. gondii DNA with an estimated true prevalence of 16.4% using Bayesian statistics, and the presence of T. gondii in mussels was significantly associated with collection during the summer compared with that in the winter (P = 0.003). Consumption of contaminated shellfish may also pose a health risk for humans and marine wildlife. As only sporulated T. gondii oocysts can be infectious, a reverse transcriptase-polymerase chain reaction was used to confirm presence of a sporozoite-specific marker (SporoSAG), detected in four mussels. G. duodenalis assemblage B, known to be pathogenic in humans, was also discovered in 1% mussels, tested by polymerase chain reaction (n = 90). Cryptosporidium spp. was not detected in the sampled mussel haemolymph. Results suggest that New Zealand may have high levels of coastal contamination with T. gondii, particularly in summer months, and that naturally exposed mussels can ingest and retain sporulated oocysts, further establishing shellfish consumption as a health concern.

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

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.

Brucellosis in Endangered Hector's Dolphins (Cephalorhynchus hectori).

Brucella spp infections of marine mammals are often asymptomatic but have been associated with reproductive losses and deaths. Zoonotic infections originating from marine isolates have also been described. Hector's dolphins (Cephalorhynchus hectori) are an endangered species with a declining population, and the role of infectious disease in population dynamics is not fully understood. In this study, 27 Hector's dolphins found dead around the New Zealand coastline between November 2006 and October 2010 were evaluated for lesions previously associated with cetacean brucellosis. Tissues were examined using histological, immunohistochemical, and molecular (polymerase chain reaction [PCR]) techniques. Seven of 27 dolphins (26%) had at least 1 tissue that was positive on PCR for Brucella spp. Lesions consistent with brucellosis were present in 10 of 27 (37%) dolphins, but in 8 of these dolphins Brucella infection could not be demonstrated in lesional tissues. Two dolphins (7%) were diagnosed with active brucellosis: 1 female with placentitis and metritis, and 1 stillborn male fetus. Brucella identified in these 2 dolphins had genetic similarity (99%) to Brucella pinnipedialis. The omp2a gene amplicon from the uterus of the female had 100% homology with ST27 genotype isolates from a human in New Zealand and a bottlenose dolphin of Pacific origin. The remaining 5 PCR-positive dolphins were assessed as having asymptomatic or latent infection. While most Brucella infections identified in this study appeared to be subclinical, the finding of 2 dolphins with reproductive disease due to Brucella infection suggests that this disease has the potential to affect reproductive success in this species.

Four cases of fatal toxoplasmosis in three species of endemic New Zealand birds.

Four cases of fatal toxoplasmosis in three endemic New Zealand avian species are reported. Between 2009 and 2012, two kereru (Hemiphaga novaeseelandiae), one North Island brown kiwi (Apteryx mantelli), and one North Island kaka (Nestor meridionalis) were submitted for necropsy examination. On gross postmortem, the kiwi had marked hepatosplenomegaly while the kaka and two kereru had swollen, slightly firm, deep-red lungs. Histologically there was extensive hepatocellular necrosis in the liver of the kiwi while the kaka and kereru showed severe fibrinous bronchointerstitial pneumonia. In the kiwi, protozoal organisms were present within both hepatocytes and Kupffer cells of the liver and within the epithelial cells and macrophages of the interstitium of the lungs in the kaka and two kereru. The diagnosis of toxoplasmosis was confirmed with immunohistochemistry and PCR of paraffin-embedded formalin-fixed tissue of the liver, lungs, or both. Genotyping of up to seven markers revealed that an atypical Type II isolate of Toxoplasma gondii was present in at least three of the cases. This study provides evidence that T. gondii can cause mortality in these endemic species and suggests further research is needed to determine the full extent of morbidity and mortality caused by this parasite in New Zealand's unique avifauna.

Blood lead concentration in an urban parrot: Nestling Kaka (Nestor meridionalis) demonstrate evidence of exposure to lead via eggs and parental feeding.

Lead is a persistent, highly toxic heavy metal known to affect physiological function and survival in birds. Nestlings are particularly at risk as exposure during critical stages of development can result in life-long deficits. Urban environments are increasingly associated with high levels of contamination and lead exposure at the urban-wildlife interface can have significant population health effects on wildlife. Wellington has an established population of Kaka (Nestor meridionalis) and provides the ideal opportunity to study the risks of lead exposure in an urban context. We sampled 139 nestlings over two breeding seasons (2015/16 and 2016/17) and examined concentrations of lead in blood samples. Nestlings were subjected to a clinical and neurological examination. Lead concentrations of egg shells were measured to evaluate maternal transfer of lead to nestlings. Overall, 36.7 % of nestlings showed evidence of lead exposure based on blood lead concentrations, ranging from <3.3µg/dL to 42.9µg/dL, with no detectable clinical signs of toxicity. The pattern of exposure in the majority of nestlings is indicative of exposure from hatch via eggshells and also direct parental feeding of lead following hatch. Lead concentrations in this cohort of Kaka have the potential to contribute to morbidity and mortality in this species. The lack of measurable neurological or physiological deficits associated with lead exposure is suggestive of an innate tolerance to these concentrations of lead in this population. However, the well-described subclinical and persistent effects of lead suggests a need for continued monitoring of this toxicant and its effects on Kaka behaviour and neurodevelopment.

Clinical parameters of hypervirulent Klebsiella pneumoniae disease and ivermectin treatment in New Zealand sea lion (Phocarctos hookeri) pups.

Hypervirulent Klebsiella pneumoniae infection causes significant mortality of endangered New Zealand sea lion pups at Enderby Island, Auckland Islands. Gross necropsy and histopathology findings are well reported, but little is known about the clinical course of disease in affected pups. To determine factors feasible as clinical screening tools for hypervirulent K. pneumoniae in live pups, 150 pups over two field seasons (2016-18) were recruited shortly after birth for a prospective cohort study. A randomised controlled clinical treatment trial with the anthelmintic ivermectin was conducted concurrently and risk factor data and biological samples were collected approximately fortnightly. Treatment with ivermectin has been demonstrated to reduce the risk of hypervirulent K. pneumoniae mortality in pups, so effects on clinical parameters between the treated and control cohorts were also investigated. A broader sample of pups were monitored for clinical signs to investigate the course of disease in affected pups. Clinical signs, haematology and oral and rectal swabs to detect gastrointestinal carriage of hypervirulent K. pneumoniae were not useful for detection of disease prior to death. Of those pups that died due to hypervirulent K. pneumoniae, only 26.1% (18/69) had any clinical signs prior, likely a reflection of the peracute course of disease. On comparison of haematological parameters between ivermectin-treated and control pups, significantly lower total plasma protein and higher eosinophil counts were seen in control versus treated pups, however standard length as a surrogate for age was a more important influence on parameters overall than ivermectin treatment. This study also highlighted a cohort of pups with severe clinical signs suggestive of hypervirulent K. pneumoniae infection were lost to follow up at the end of the monitored season, which could be contributing to cryptic juvenile mortality.

PREVALENCE AND PATHOGEN LOAD OF EIMERIA IN WILD YELLOW-EYED PENGUINS (MEGADYPTES ANTIPODES) AND THE MORPHOLOGIC CHARACTERIZATION OF A NOVEL EIMERIA SPECIES.

Coccidia infections in wild birds rarely cause clinical signs; however, disease and mortality can occur with predisposing environmental and host conditions. The Yellow-eyed Penguin (Megadyptes antipodes) is an endangered species endemic to New Zealand that has seen significant ongoing population decline. The aim of this study was to examine the host-pathogen dynamics of coccidian parasites in two wild populations of Yellow-eyed Penguin: the mainland (South Island) population and the sub-Antarctic (Enderby Island) population. There was weak evidence for a difference in the prevalence of the Eimeria sp. in birds from Enderby Island (76.6%; 36/47; 95% confidence interval [CI] 62.78-86.4%) and the South Island of New Zealand (58.54%; 24/41; 95% CI 43.37-72.24%). The mean pathogen load in penguins on Enderby Island was 9,723 oocysts/g of feces (SE=5831 oocysts/g) and from the South Island of New Zealand was 1,050 oocysts/g (SE=398 oocysts/g). No evidence of an association was found between pathogen load and body weight in either study population. The morphology of the sporulated coccidial oocysts was consistent with a novel species of Eimeria. There was statistically significant variation between the oocysts collected from the two sites in all measurements apart from the oocyst wall thickness. However, the standard technique of assessing linear regressions of the length and width of oocysts from both sampling sites was 0.80, and therefore above the standard R2>0.5 used to indicate variation within a single population of oocysts, suggesting that only a single species of Eimeria was present at both sampling locations. The prevalence and pathogen load of Eimeria sp. was substantially higher than previous reports of coccidial oocysts in Yellow-eyed Penguins and free-living Sphenisciformes globally. This host-parasite relationship deserves further investigation, as the impact of this novel organism on the population remains unclear.

ENVIRONMENTAL FACTOR INVESTIGATION OF EXUDATIVE CLOACITIS IN KAKAPO (STRIGOPS HABROPTILUS) ON WHENUA HOU (CODFISH ISLAND), NEW ZEALAND.

Kakapo (Strigops habroptilus) are critically endangered nocturnal parrots endemic to New Zealand. Exudative cloacitis is a disease only affecting the breeding population of Kakapo on Whenua Hou (Codfish Island), for which a consistent primary pathogenic organism involved has not been identified. This epidemiological study was conducted to identify the environmental factors contributing to the initiation of this disease in Kakapo by 1) producing and describing a case series; 2) mapping the geographic distribution of exudative cloacitis cases; 3) investigating the chemical characteristics of Kakapo roosting sites; and 4) assessing the effects of climatic factors on the incidence of exudative cloacitis each year. Soil samples from the Kakapo roost sites and other areas of the Whenua Hou were examined for pH, ammonium, and moisture contents. From 2002 to 2017, 22 sporadic cases of exudative cloacitis have been diagnosed and the disease distribution on Whenua Hou overlaps the Kakapo distribution. A mixed group of adults and juveniles was affected and there was no evidence of spatial or temporal clustering of the disease. Current findings on the chemical characteristics of Kakapo roosting sites do not show any evidence that these factors are involved in the initiation of the exudative cloacitis. Nevertheless, the results suggest that the ammonium and moisture levels of the roosts are worthy of more detailed study in future cases. We were not able to demonstrate any significant influence of broad measures of climate on the incidence of exudative cloacitis on Whenua Hou. Prospective data collection would help for a complete epidemiological investigation of this disease in future cases.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV-67, bla SHV-11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

SEABIRDS AS POSSIBLE RESERVOIRS OF ERYSIPELOTHRIX RHUSIOPATHIAE ON ISLANDS USED FOR CONSERVATION TRANSLOCATIONS IN NEW ZEALAND.

Erysipelothrix rhusiopathiae, the causative agent of the disease erysipelas, is a gram-positive bacillus, and an opportunistic pathogen in diverse species of animals. In New Zealand, E. rhusiopathiae has killed endangered birds on offshore islands, including Kakapo (Strigops habroptilus), Takahe (Porphyrio hochstetteri), and Kiwi (Apteryx spp.). The source of infection is uncertain, and the prevalence of E. rhusiopathiae among wild birds is currently unknown. During October 2018 to December 2018, we surveyed dead and live seabirds that visit two of New Zealand's offshore islands used for Kakapo conservation with the goal of determining the prevalence of E. rhusiopathiae. Bone marrow from dead birds was cultivated on selective agar, and organisms cultured were identified using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. The prevalence of E. rhusiopathiae was calculated in different species for each island. We surveyed live birds using PCR with Erysipelothrix spp.-specific primers on blood samples. The prevalence of E. rhusiopathiae in dead seabirds on Whenua Hou and Te Hauturu-o-Toi was 3.4% (3/86) and 11.4% (5/44), respectively. On Whenua Hou, E. rhusiopathiae was detected in Sooty Shearwaters (Puffinus griseus; 5.9%, 2/34) and Mottled Petrels (Pterodroma inexpectata; 2.7%, 1/36) while it was detected only in Cook's Petrels (Pterodroma cookie; 13.5%, 5/37) on Te Hauturu-o-Toi. Blood samples were collected from two seabird species; only one of 50 Mottled Petrels (2.0%) was positive for the presence of Erysipelothrix spp. Our findings confirm that burrowing seabirds are possible reservoirs of E. rhusiopathiae on both islands studied and may be the source of spillover to other species on the island. The differences in observed prevalence suggest the species composition of the reservoir of E. rhusiopathiae may vary geographically.

Detection of Foot-and-Mouth Disease Virus in the Absence of Clinical Disease in Cattle and Buffalo in South East Asia.

Foot-and-mouth disease virus (FMDV) is widespread throughout much of the world, including parts of South East Asia. Surveillance is often limited in endemic areas, relying predominantly on passive outbreak reporting. As part of the World Organisation for Animal Health (OIE)'s South East Asia and China Foot-and-Mouth Disease Project (SEACFMD), field sampling was performed to help understand evidence of widespread virus exposure observed in previous studies. Serum and dry mucosal swabs were collected to evaluate the presence of FMDV RNA on the nasal, oral, and dorsal nasopharyngeal mucosal surfaces of 262 healthy cattle (n = 84 in Laos; n = 125 in Myanmar) and buffalo (n = 48 in Laos; n = 5 in Myanmar) immediately following slaughter in three slaughterhouses. Swabs and serum were tested by the OIE/FAO World Reference Laboratory for foot-and-mouth disease (WRLFMD) using pan-serotypic real-time reverse transcription-PCR (rRT-PCR) and serum was evaluated using the FMD PrioCHECK non-structural protein (NSP) ELISA. In total, 7.3% of animals had detectable FMDV RNA in one or more of the three sites including 5.3% of nasopharyngeal swabs, 2.3% of oral swabs, and 1.5% of nasal swabs. No FMDV RNA was detected in serum. Overall, 37.8% of animals were positive for NSP antibodies, indicating likely past natural exposure to FMDV. Results were comparable for Laos and Myanmar, and for both cattle and buffalo, and were not significantly different between age groups. Detectable FMDV RNA present on the oral and nasal mucosa of clinically-healthy large ruminants in Laos and Myanmar demonstrates the importance of sampling asymptomatic animals as part of surveillance, and may indicate that subclinical infection plays a role in the epidemiology of FMD in these countries.

Nematode larva migrans caused by Toxocara cati in the North Island brown kiwi (Apteryx mantelli).

Sporadic cases of visceral and neural nematode larva migrans have been diagnosed at necropsy in the endangered New Zealand kiwi (Apteryx spp.), but the causative organisms have not yet been definitively identified. From an initial group of five affected kiwi, PCR was performed on DNA extracted from archival formalin-fixed paraffin-embedded tissue sections in which larval nematodes had been histologically identified. Sequencing of positive results from four out of the five kiwi aligned with sequences from Toxocara cati, a nematode parasite whose definitive host is the domestic cat. PCR was then performed on a second group of 12 kiwi that had histologic inflammatory lesions consistent with larva migrans, but variable larval presence. Repeatable positive PCR results were only achieved in one tissue, in which larval organisms were histologically confirmed. This study supports the use of PCR as an alternative or adjunct to the morphological identification of nematode larvae in formalin-fixed histopathological samples, as well as showing that in investigation of larva migrans, PCR has greatest chance of success from sections where nematode larvae are evident histologically. The identification of Toxocara cati from lesions of larva migrans in kiwi reflects an indirect, parasite-mediated effect of an invasive mammalian species on a native species.

Ventral dermatitis in rowi (Apteryx rowi) caused by cutaneous capillariasis.

In 2013 there was an outbreak of crusting ventral dermatitis among a group of juvenile rowi (Apteryx rowi), a species of the endangered New Zealand kiwi, that were being raised on an off-shore island sanctuary. Biopsies taken at the time found nematodes migrating within the epidermis of affected skin but the specific identity and origin of the organisms was not established, and sporadic cases of similar skin disease continue to occur on the island. On examination of additional sections from the original skin biopsies, adult nematodes and eggs were identified, the histomorphology of which was consistent with Capillaria sensu lato. PCR was performed on DNA extracted from archived formalin-fixed, paraffin-embedded tissue blocks of skin from eight affected rowi, using primers targeting the 18S region of nuclear ribosomal DNA and the COI gene of mitochondrial DNA of capillarid nematodes. The 18S sequences from all rowi samples were identical and matched sequences from members of the genus Eucoleus. In contrast, two distinct capillarid COI sequences were obtained, in one case both from the same rowi skin biopsy. While there were no close matches, both COI sequences also aligned nearest to sequences identified as Eucoleus spp. It is considered unlikely that two different nematode species are involved in the rowi skin lesions and the possible amplification of a COI pseudogene or "numt" is discussed. A species-level identification of the capillarid nematodes causing skin disease in rowi was not obtained, however based on histological evaluation the infections include reproductively-active adult nematodes. This finding indicates the possibility of perpetuation of the skin disease in the absence of the original source, as well as raising potential for the transfer of infection from the island when the juvenile rowi are translocated to their new habitats.

Comparison of the Pathogenic Potential of Campylobacter jejuni, C. upsaliensis and C. helveticus and Limitations of Using Larvae of Galleria mellonella as an Infection Model.

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. Other species cause campylobacteriosis relatively infrequently; while this could be attributed to bias in diagnostic methods, the pathogenicity of non-jejuni/coli Campylobacter spp. such as C. upsaliensis and C. helveticus (isolated from dogs and cats) is uncertain. Galleria mellonella larvae are suitable models of the mammalian innate immune system and have been applied to C. jejuni studies. This study compared the pathogenicity of C. jejuni, C. upsaliensis, and C. helveticus isolates. Larvae inoculated with either C. upsaliensis or C. helveticus showed significantly higher survival than those inoculated with C. jejuni. All three Campylobacter species induced indistinguishable histopathological changes in the larvae. C. jejuni could be isolated from inoculated larvae up to eight days post-inoculation whereas C. upsaliensis and C. helveticus could only be isolated in the first two days. There was a significant variation in the hazard rate between batches of larvae, in Campylobacter strains, and in biological replicates as random effects, and in species and bacterial dose as fixed effects. The Galleria model is applicable to other Campylobacter spp. as well as C. jejuni, but may be subject to significant variation with all Campylobacter species. While C. upsaliensis and C. helveticus cannot be considered non-pathogenic, they are significantly less pathogenic than C. jejuni.

Fatal fulminant hepatitis in a chimpanzee (Pan troglodytes) receiving cyproterone acetate.

Cyproterone acetate is a steroidal anti-androgen that has been used in human medicine for contraceptive purposes as well as treatment of medical conditions responsive to suppression of testosterone production. While serious side effects are considered to be rare, sporadic cases of severe hepatitis have been reported, including several fatal cases. This report describes a case of fatal fulminant hepatitis in one of four male chimpanzees (Pan troglodytes) that were undergoing trial treatment with cyproterone acetate to decrease inter-male aggression.

Pup mortality in New Zealand sea lions (Phocarctos hookeri) at Enderby Island, Auckland Islands, 2013-18.

New Zealand sea lions (Phocarctos hookeri) are an endemic and endangered species. Pup mortality at Enderby Island (50.5°S, 166.28°E) in the New Zealand sub-Antarctic has been well studied, with subsequent investigations yielding more intricate detail of the causes of mortality, as new diagnostic methods become available. Klebsiella pneumoniae was first reported in 2001-02 at this site, causing a pup mortality epizootic and is now known to be present at several colonies. This bacterium is a common mucosal commensal of humans and animals, however the agent found in pups at necropsy is a hypervirulent strain, readily recognised in microbial culture as being hypermucoviscous. Infection causes septicaemia with a common syndrome of subsequent meningitis and polyarthritis. This investigation uses histopathology and microbiology, with new modalities such as matrix assisted laser desorption/ionisation-time of flight mass spectrometry to show that Klebsiella septicaemia could have historically been, and continues to be, the most important cause of pup mortality, but has been previously underrepresented due to the often cryptic presentation and sometimes peracute course of disease. Hypermucoviscous K. pneumoniae should be considered a serious threat to pup survival in the species, causing on average 60.2% of pup deaths annually at Enderby Island between 2013 and 2018, with likely more continuing mortality following pup dispersal and the cessation of the summer monitoring season. Less common causes of death included starvation (14.8%), trauma/asphyxiation (9.9%) and other infections (7%). This study forms the basis for further evaluation of risk factors for pup mortality in the species, with a view to developing active mitigation.

Pathology and molecular epidemiology of Mycobacterium pinnipedii tuberculosis in native New Zealand marine mammals.

Mycobacterium pinnipedii causes tuberculosis in a number of pinniped species, and transmission to cattle and humans has been reported. The aims of this study were to: characterize the pathology and prevalence of tuberculosis in New Zealand marine mammals; use molecular diagnostic methods to confirm and type the causal agent; and to explore relationships between type and host characteristics. Tuberculosis was diagnosed in 30 pinnipeds and one cetacean. Most affected pinnipeds had involvement of the pulmonary system, supporting inhalation as the most common route of infection, although ingestion was a possible route in the cetacean. PCR for the RD2 gene confirmed M. pinnipedii as the causal agent in 23/31 (74%) cases (22 using DNA from cultured organisms, and one using DNA from formalin-fixed paraffin-embedded (FFPE) tissue), including the first published report in a cetacean. RD2 PCR results were compared for 22 cases where both cultured organisms and FFPE tissues were available, with successful identification of M. pinnipedii in 7/22 (31.8%). In cases with moderate to large numbers of acid-fast bacilli, RD2 PCR on FFPE tissue provided a rapid, inexpensive method for confirming M. pinnipedii infection without the need for culture. VNTR typing distinguished New Zealand M. pinnipedii isolates from M. pinnipedii isolated from Australian pinnipeds and from common types of M. bovis in New Zealand. Most (16/18) M. pinnipedii isolates from New Zealand sea lions were one of two common VNTR types whereas the cetacean isolate was a type detected previously in New Zealand cattle.