The effects of annual cycle, source population, and body condition on leukocyte profile and immune challenge in a basal reptile, the tuatara (Sphenodon punctatus).

Leukocyte profiles are broadly used to assess the health status of many species. Reference intervals, and an understanding of the factors that may influence these intervals, are necessary for adequate interpretation of leukograms. Using a data set that spans over three decades, we investigated variation in leukocyte profile in several populations of the evolutionarily unique reptile, the tuatara (Sphenodon punctatus). To do this, we first established reference intervals for each leukocyte type according to best practices. Next, we determined that source population and sampling date were the two most important predictors of leukocyte makeup. We found significant differences in the ratio of heterophils: lymphocytes (H:L) between populations, with tuatara on the more resource-stressed sampling island having a significantly higher ratio of H:L. Finally, we found that sampling location, sex, and life stage did not explain variation in the responses of tuatara to stimulation with Concanavalin A and lipopolysaccharide in both 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide and Griess assay experiments. Our results offer important insight into the function of leukocytes in reptiles.

Molecular Evidence of Leptospira spp. in Isolated Fijian Bats.

Bats host several zoonotic pathogens. Island biogeography and epidemiologic theory predict small remote islands have lower infection diversity. Molecular studies of urine and feces from three species at 10 sites from three islands suggest multiple pathogenic Leptospira, but not coronavirus, paramyxovirus, or Histoplasma, circulate in isolated Pacific Fijian bat populations.

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.

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.

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.

Leveraging an existing whole-genome resequencing population data set to characterize toll-like receptor gene diversity in a threatened bird.

Species recovery programs are increasingly using genomic data to measure neutral genetic diversity and calculate metrics like relatedness. While these measures can inform conservation management, determining the mechanisms underlying inbreeding depression requires information about functional genes associated with adaptive or maladaptive traits. Toll-like receptors (TLRs) are one family of functional genes, which play a crucial role in recognition of pathogens and activation of the immune system. Previously, these genes have been analysed using species-specific primers and PCR. Here, we leverage an existing short-read reference genome, whole-genome resequencing population data set, and bioinformatic tools to characterize TLR gene diversity in captive and wild tchuriwat'/tuturuatu/shore plover (Thinornis novaeseelandiae), a threatened bird endemic to Aotearoa New Zealand. Our results show that TLR gene diversity in tchuriwat'/tuturuatu is low, and forms two distinct captive and wild genetic clusters. The bioinformatic approach presented here has broad applicability to other threatened species with existing genomic resources in Aotearoa New Zealand and beyond.

Gut microbiota of the threatened takahe: biogeographic patterns and conservation implications.

BACKGROUND: The Aotearoa New Zealand takahe (Porphyrio hochstetteri), once thought to be extinct, is a nationally threatened flightless rail under intensive conservation management. While there has been previous research into disease-related microbes in takahe, little is known about the microbes present in the gastrointestinal tract. Given the importance of gut-associated microbes to herbivore nutrition and immunity, knowledge of these communities is likely to be of considerable conservation value. Here we examined the gut microbiotas of 57 takahe at eight separate locations across Aotearoa New Zealand. RESULTS: Faecal samples, taken as a proxy for the hindgut bacterial community, were subjected to 16S rRNA gene amplicon sequencing using Illumina MiSeq. Phylogenetic analysis of > 2200 amplicon sequence variants (ASVs) revealed nine main bacterial phyla (Acidobacteriota, Actinobacteriota, Bacteroidota, Campilobacterota, Firmicutes, Fusobacteriota, Planctomycetota, Proteobacteria, and Verrucomicrobiota) that accounted for the majority of sequence reads. Location was a significant effect (p value < 0.001, 9999 permutations) that accounted for 32% of the observed microbiota variation. One ASV, classified as Lactobacillus aviarius, was present in all samples at an average relative abundance of 17% (SD = 23.20). There was strong evidence (p = 0.002) for a difference in the abundance of the genus Lactobacillus between locations. A common commensal bacterium previously described in takahe, Campylobacter spp., was also detected in most faecal samples. CONCLUSIONS: Location plays a pivotal role in the observed variation among takahe gut bacterial communities and is potentially due to factors such as supplemental feeding and medical treatment experienced by birds housed in captivity at one of the eight sampled sites. These data present a first glimpse of the previously unexplored takahe gut microbiota and provide a baseline for future microbiological studies and conservation efforts.

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.

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.

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.

Serum prolactin and testosterone levels in captive and wild brown kiwi (Apteryx mantelli) during the prebreeding, breeding, and incubation periods.

In brown kiwi (Apteryx mantelli), the male is the primary incubator, a trait that is relatively rare among birds. The maintenance of avian incubation behavior is controlled by the protein hormone prolactin (PRL). Although steroid hormone concentrations in both wild and captive kiwi have previously been reported, this study is the first to report levels of PRL in captive and wild male and female kiwi through the prebreeding and breeding seasons, and to directly compare testosterone (T) concentrations between captive and wild males during the breeding and incubation periods. Female PRL concentrations increased at the time of oviposition, whereas male PRL concentrations rose gradually between the prebreeding and incubation periods. Although males are considered the main incubator, an increase in PRL levels could help females maintain behaviors such as nest guarding, or to take over incubation the event of mate loss. A gradual increase in PRL allows the male to be ready for incubation during the long breeding season. Interestingly, T concentrations in captive males did not decrease during incubation and was significantly higher than in wild males. Continual elevated T could have an impact on sperm production through negative feedback, thereby contributing to the low egg fertility seen in captive kiwi. Therefore, determining the underlying reason for the differences in hormone levels could be significant, if not vital, for improving the success of captive kiwi breeding programs.

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.

Lead exposure in an urban, free-ranging parrot: Investigating prevalence, effect and source attribution using stable isotope analysis.

Anthropogenic lead use has resulted in widespread environmental lead contamination known to affect wildlife populations worldwide. Ecotoxicological investigations in wild birds have thus far prioritised waterfowl and raptor species and primarily addressed contamination in natural ecosystems. Urban areas are increasingly associated with high levels of heavy metal contamination, however the risk of lead exposure in urban wildlife is less well known. This study investigates lead exposure in an urban population of North Island Kaka (Nestor meridionalis septentrionalis), an endemic New Zealand parrot. The inquisitive nature of these birds, an expanding urban population and increased availability of food in the urban environment are the primary factors implicated in their dispersal into urban areas where there is increased exposure to anthropogenic sources of lead. Blood lead concentrations were assessed in free-ranging birds to quantify the prevalence and magnitude of lead exposure. The impact of lead on physiological and neurological function was assessed using behavioural and clinical examinations. Finally, lead stable isotope analysis was employed to investigate lead in roofing material as a potential source of exposure in the urban environment. Results indicate a significant prevalence of lead exposure in this population (43.2%) with a maximum recorded plasma concentration of 50.7µg/dL. Although no mortality was observed during this study, lead exposure was associated with reduced body condition in kaka. Behavioural changes were present in one individual with the highest recorded blood lead concentration. Lead isotope values of roof-collected rainwater overlapped with kaka blood lead isotope values, suggesting this to be an important source of exposure in this population. The prevalence of lead exposure observed in this study suggests that lead is a toxin of importance to kaka in this urban area. Wildlife intoxications largely result from anthropogenic lead sources and this study identifies a previously undescribed urban source of lead for wildlife.

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.

Genomic Epidemiology and Management of Salmonella in Island Ecosystems Used for Takahe Conservation.

Translocation and isolation of threatened wildlife in new environments may have unforeseen consequences on pathogen transmission and evolution in host populations. Disease threats associated with intensive conservation management of wildlife remain speculative without gaining an understanding of pathogen dynamics in meta-populations and how location attributes may determine pathogen prevalence. We determined the prevalence and population structure of an opportunistic pathogen, Salmonella, in geographically isolated translocated sub-populations of an endangered New Zealand flightless bird, the takahe (Porphyrio hochstetteri). Out of the nine sub-populations tested, Salmonella was only isolated from takahe living on one private island. The apparent prevalence of Salmonella in takahe on the private island was 32% (95% CI 13-57%), with two serotypes, Salmonella Mississippi and Salmonella houtenae 40:gt-, identified. Epidemiological investigation of reservoirs on the private island and another island occupied by takahe identified environmental and reptile sources of S. Mississippi and S. houtenae 40:gt- on the private island. Single nucleotide polymorphism analysis of core genomes revealed low-level diversity among isolates belonging to the same serotype and little differentiation according to host and environmental source. The pattern observed may be representative of transmission between sympatric hosts and environmental sources, the presence of a common unsampled source, and/or evidence of a recent introduction into the ecosystem. This study highlights how genomic epidemiology can be used to ascertain and understand disease dynamics to inform the management of disease threats in endangered wildlife populations.

Analgesic effects of morphine and butorphanol in broiler chickens.

OBJECTIVE: To evaluate analgesic efficacies of morphine and butorphanol in lame broiler chickens. STUDY DESIGN: Double blind, randomized, controlled experimental study. ANIMALS: In study 1, 36 lame and 36 sound chickens. In study 2, 48 lame and 48 sound chickens. METHODS: Sound and lame chickens were gait scored and randomly assigned into four groups: sound-drug, sound-placebo, lame-drug, and lame-placebo in study 1. In study 2, an additional lame and sound handling control group was included. Chickens in drug groups were injected with either morphine or butorphanol 2 mg kg-1 intravenously. Chickens in placebo groups were injected with an equal volume of normal saline. All birds underwent an obstacle course (OC) and latency-to-lie (LTL) test before injection and at 30 minutes and 2 hours after injection, to assess their walking ability and their standing ability. The time taken to finish the OC and the standing time in the LTL test were recorded. Friedman tests with Dunn's correction were used to identify significant differences. RESULTS: Lame chickens finished the OC faster (mean ± standard deviation 36 ± 8 c.f. 69 ± 18 seconds) after the injection of butorphanol. Morphine caused sedation with an increase in time taken to finish the OC, even in sound chickens. In the lame handling control and placebo groups the OC times increased and the LTL times decreased with each observation. CONCLUSION: Intravenous butorphanol (2 mg kg-1) may be analgesic in chickens for up to 2 hours. Morphine caused sedation.

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.