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.

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.

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.

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.

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.

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.

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.

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.

Draft Whole-Genome Sequences of Five Klebsiella pneumoniae Isolates from the Subantarctic Islands of New Zealand.

Klebsiella pneumoniae is a Gram-negative bacterium that can be found in the environment, as well as on mucosal surfaces of humans and animals. Here, we report the genome sequence of five K. pneumoniae isolates from substrate samples and bird feces collected in the Subantarctic Islands of New Zealand.

Draft Whole-Genome Sequences of Seven Isolates of Klebsiella pneumoniae from New Zealand Sea Lions.

Klebsiella pneumoniae is a Gram-negative bacterium that may cause infection in a broad range of hosts. We report here the genome sequences of seven K. pneumoniae isolates from New Zealand sea lions.

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.

Interspecies differences in plasma concentrations of 25-hydroxyvitamin D3 and dermal Vitamin D synthesis of kiwi (Apteryx mantelli), tuatara (Sphenodon punctatus), and New Zealand sea lions (Phocarctos hookeri).

Vitamin D plays a central role in calcium homeostasis of most vertebrates, and is obtained in different species through diet, dermal synthesis, or a combination of both. The aim of this study was to determine the predominant routes of Vitamin D synthesis in three disparate species, brown kiwi (Apteryx mantelli), tuatara (Sphenodon punctatus), and New Zealand sea lions (Phocarctos hookeri). We surveyed plasma concentrations of 25-hydroxyvitamin D2 and D3, analysed environmental conditions and life history factors, and determined the ability of skin samples to synthesise Vitamin D3 on exposure to ultraviolet-B radiation. There was variation in the plasma/serum 25-hydroxyvitamin D3 concentrations between and within the species studied, with wild kiwi having the lowest concentrations and NZ sea lions the highest. Kiwi skin produced small but measurable amounts of Vitamin D3, while tuatara skin produced Vitamin D3 concentrations higher than that of kiwi. New Zealand sea lion skin produced the highest amount of Vitamin D3 and differed from the other two species in this study in that Vitamin D3 was present in skin before UV-B exposure. The results from this study show that all three species studied retained the ability to use both dietary and dermal sources of Vitamin D, although there was interspecies variation in the magnitude of dermal synthesis. Comparisons between these species show that there are differences in their Vitamin D pathways, but suggest that there are more factors contributing to these pathways than might be expected solely from life history characteristics.

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.

Impact of race training on volumetric bone mineral density and its spatial distribution in the distal epiphysis of the third metatarsal bone of 2-year-old horses.

The aim of the study was to use spatial and multiple correspondence analysis (MCA) to describe and compare the regional proportion and spatial pattern of volumetric bone mineral density (BMDv) values within loaded regions of the plantar metatarsal epiphysis of young horses in race training. A single 2 mm transverse peripheral quantitative computed tomography 'slice', 10 mm proximal from the distal limit of the sagittal ridge of the distal metatarsal epiphysis was obtained from 14 2-year-old Thoroughbred fillies (7 exercised and 7 controls). Six regions of interest were generated and examined for relative BMDv using MCA. The spatial distribution of BMDv was statistically examined at two sites loaded by the proximal sesamoid bones using geographical information software. The BMDv response was focal with distinct regional differences in relation to load. Deposition of new bone within existing high density bone contributed to a greater bone fraction and the distinct profile of clusters of uniformly distributed high density bone as well as a lower proportion of lower density bone in exercised horses. The MCA and spatial analysis provided statistical techniques to quantify and describe non-invasively the exercise induced changes in bone that had previously been described using microradiography of thin slices and by block-face imaging. These statistical techniques may prove useful in quantifying spatial patterns of response to load.

Postmortem stability of S100B in the aqueous humor of northern fur seals (Callorhinus ursinus).

Bycatch (accidental drowning in fishing nets) is a significant problem for some marine mammal species, but can be difficult to diagnose as there are no pathognomonic gross or histological lesions. In human medicine, biomarkers such as S100B are increasingly being used to investigate hypoxic-ischemic syndromes, but, to the authors' knowledge, studies using this marker have not been reported for marine mammal species. The aims of the current study were to determine baseline postmortem S100B levels in a pinniped species, and to determine whether S100B levels were stable over a postmortem interval of 48 hr. Aqueous humor, which is simple to collect and avoids many of the problems associated with postmortem collection of blood, was used as a surrogate for serum. S100B was detected in the aqueous humor of acute deaths (<15 min) and was stable for up to 48 hr, with a wider variation in values at the 48-hr time interval.

Body regional distribution and stratification of fatty acids in the blubber of New Zealand sea lions: implications for diet predictions.

Fatty acids (FAs) from blubber are often analysed to assess the diet of marine mammals. However, distribution of blubber FAs is not necessarily uniform along the body. It is therefore important to understand the deposition of dietary fat to be able to estimate the diet. We analysed the FA compositions of the thoracic ventral (T region) blubber of 28 New Zealand (NZ) sea lions Phocarctos hookeri by-caught by the southern arrow squid Nototodarus sloani fishery. Each blubber sample was divided into an inner and an outer layer. For 16 of these 28 animals, the pelvic dorsal (P) region was also sampled. The influence of body region and layer was statistically tested on the distribution of blubber FAs. We found minimal differences between the P and T regions (3 out of 29 FAs). The outer blubber layer was more concentrated in short-chain monounsaturated FAs, and less concentrated in saturated FAs, but the degree of stratification was small. Diet predictions from quantitative FA signature analysis (QFASA) applied on different body regions were similar. When applied to different blubber layers, QFASA gave some variation in the contribution of rattails (~25 % in outer blubber vs. ~12 % in inner blubber). Nonetheless, diet predicted from both layers was dominated by similar prey species: octopus, hoki and rattails. Hoki and rattails shared a similar ecological niche. Therefore, feeding ecology of NZ sea lions inferred from the inner or the outer blubber would lead to the same conclusions. In the case of NZ sea lions, the outer layer of blubber, if the only sample accessible, could be a useful tissue for diet inference from FAs.

The role of lead in a syndrome of clenched claw paralysis and leg paresis in Swamp Harriers (Circus approximans).

We investigated the hypothesis that lead poisoning was the cause of the clinical syndrome of clenched feet paralysis and leg paresis in wild raptors. Swamp Harriers (Circus approximans) are one of three extant native raptor species in New Zealand. Harriers with the syndrome were found to have statistically significantly higher blood lead concentrations than those without clenched feet (t-test; t=-4.06, df=5, P=0.01). However, elevated blood lead concentrations were also present in 60% of wild harriers without the clinical syndrome of clenched feet paralysis and leg paresis. There were features of the response to chelation treatment, electroneurodiagnostics, and pathology that were inconsistent with lead poisoning as reported in other birds of prey. We conclude that lead may be a factor in the expression of this clinical syndrome of clenched claw paralysis but that other factors not identified in our study play a role in the expression of the disease.

Serologic evaluation of New Zealand sea lions for exposure to Brucella and Leptospira spp.

A serologic survey of anti-Brucella and antileptospiral antibodies was conducted on 147 adult, female New Zealand sea lions (Phocarctos hookeri). Most sea lions (n=138) were sampled at Sandy Bay, Enderby Island, Auckland Islands (50°30'S, 166°17'E), January 2000-March 2005. Nine were sampled at Otago, New Zealand (46°0'S, 170°40'E); four in April 2008 and five in March 2009. Serum from one of the Enderby Island females was weakly positive for antibodies to Brucella abortus using the competitive enzyme-linked immunosorbent assay, and one female had a low titer for Leptospira interrogans serovar pomona using the microscope agglutination test. All serum samples from Otago animals were negative. Brucellosis and leptospirosis are therefore considered unlikely to play a major role in population dynamics of these populations, and the low antibody prevalence of these agents suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand.