Diversity of MHC DQB and DRB Genes in the Endangered Australian Sea Lion (Neophoca cinerea).

Major histocompatibility complex (MHC) class II molecules have an important role in vertebrate adaptive immunity, being responsible for recognizing, binding, and presenting specific antigenic peptides to T lymphocytes. Here, we study the MHC class II DQB and DRB exon 2 genes of the Australian sea lion (Neophoca cinerea), an endangered pinniped species that experiences high pup mortality. Following characterization of N. cinerea DQB and DRB by molecular cloning, and evaluation of diversity in pups across 2 colonies using variant screening (n = 47), 3 DQB alleles and 10 DRB variants (including 1 pseudogene allele) were identified. The higher diversity at DRB relative to DQB is consistent with other studies in marine mammals. Despite overall lower MHC class II allelic diversity relative to some other pinniped species, we observed similar levels of nucleotide diversity and selection in N. cinerea. In addition, we provide support for recent divergence of MHC class II alleles. The characterization of MHC class II diversity in the Australian sea lion establishes a baseline for further investigation of associations with disease, including endemic hookworm infection, and contributes to the conservation management of this species.

Health assessment of free-ranging endangered Australian sea lion (Neophoca cinerea) pups: effect of haematophagous parasites on haematological parameters.

Evaluation of the health status of free-ranging populations is important for understanding the impact of disease on individuals and on population demography and viability. In this study, haematological reference intervals were developed for free-ranging endangered Australian sea lion (Neophoca cinerea) pups within the context of endemic hookworm (Uncinaria sanguinis) infection and the effects of pathogen, host, and environment factors on the variability of haematological parameters were investigated. Uncinaria sanguinis was identified as an important agent of disease, with infection causing regenerative anaemia, hypoproteinaemia, and a predominantly lymphocytic-eosinophilic systemic inflammatory response. Conversely, the effects of sucking lice (Antarctophthirus microchir) were less apparent and infestation in pups appears unlikely to cause clinical impact. Overall, the effects of U. sanguinis, A. microchir, host factors (standard length, body condition, pup sex, moult status, and presence of lesions), and environment factors (capture-type and year of sampling) accounted for 26-65% of the total variance observed in haematological parameters. Importantly, this study demonstrated that anaemia in neonatal Australian sea lion pups is not solely a benign physiological response to host-environment changes, but largely reflects a significant pathological process. This impact of hookworm infection on pup health has potential implications for the development of foraging and diving behaviour, which would subsequently influence the independent survival of juveniles following weaning. The haematological reference intervals developed in this study can facilitate long-term health surveillance, which is critical for the early recognition of changes in disease impact and to inform conservation management.

Ivermectin treatment of free-ranging endangered Australian sea lion (Neophoca cinerea) pups: effect on hookworm and lice infection status, haematological parameters, growth, and survival.

A placebo-controlled study was used to investigate the effectiveness of ivermectin to treat hookworm (Uncinaria sanguinis) and lice (Antarctophthirus microchir) infections in free-ranging Australian sea lion (Neophoca cinerea) pups and to test the hypotheses that these parasitic infections cause anaemia, systemic inflammatory responses, and reduced growth, and contribute towards decreased pup survival. Ivermectin was identified as an effective and safe anthelmintic in this species. Pups administered ivermectin had significantly higher erythrocyte counts and significantly lower eosinophil counts compared to controls at 1-2 months post-treatment, confirming that U. sanguinis and/or A. microchir are causatively associated with disease and demonstrating the positive effect of ivermectin treatment on clinical health parameters. Higher growth rates were not seen in ivermectin-treated pups and, unexpectedly, relatively older pups treated with ivermectin demonstrated significantly reduced growth rates when compared to matched saline-control pups. Differences in survival were not identified between treatment groups; however, this was attributed to the unexpectedly low mortality rate of recruited pups, likely due to the unintended recruitment bias towards pups >1-2 months of age for which mortality due to hookworm infection is less likely. This finding highlights the logistical and practical challenges associated with treating pups of this species shortly after birth at a remote colony. This study informs the assessment of the use of anthelmintics as a tool for the conservation management of free-ranging wildlife and outlines essential steps to further the development of strategies to ensure the effective conservation of the Australian sea lion and its parasitic fauna.

Epidemiology of hookworm (Uncinaria sanguinis) infection in free-ranging Australian sea lion (Neophoca cinerea) pups.

Understanding the fundamental factors influencing the epidemiology of wildlife disease is essential to determining the impact of disease on individual health and population dynamics. The host-pathogen-environment relationship of the endangered Australian sea lion (Neophoca cinerea) and the parasitic hookworm, Uncinaria sanguinis, was investigated in neonatal pups during summer and winter breeding seasons at two biogeographically disparate colonies in South Australia. The endemic occurrence of hookworm infection in Australian sea lion pups at these sites was 100% and post-parturient transmammary transmission is likely the predominant route of hookworm infection for pups. The prepatent period for U. sanguinis in Australian sea lion pups was determined to be 11-14 days and the duration of infection approximately 2-3 months. The mean hookworm infection intensity in pups found dead was 2138 ± 552 (n = 86), but a significant relationship between infection intensity and faecal egg count was not identified; infection intensity in live pups could not be estimated from faecal samples. Fluctuations in infection intensity corresponded to oscillations in the magnitude of colony pup mortality, that is, higher infection intensity was significantly associated with higher colony pup mortality and reduced pup body condition. The dynamic interaction between colony, season, and host behaviour is hypothesised to modulate hookworm infection intensity in this species. This study provides a new perspective to understanding the dynamics of otariid hookworm infection and provides evidence that U. sanguinis is a significant agent of disease in Australian sea lion pups and could play a role in population regulation in this species.

Uncinaria sanguinis sp. n. (Nematoda: Ancylostomatidae) from the endangered Australian sea lion, Neophoca cinerea (Carnivora: Otariidae).

This study investigates the identity of hookworms parasitising the Australian sea lion, Neophoca cinerea (Péron), from three colonies in South Australia, Australia. The Australian sea lion is at risk of extinction because its population is small and genetically fragmented. Using morphological and molecular techniques, we describe a single novel species, Uncinaria sanguinis sp. n. (Nematoda: Ancylostomatidae). The new species is most similar to hookworms also parasitic in otariid hosts, Uncinaria lucasi Stiles, 1901 and Uncinaria hamiltoni Baylis, 1933. Comparative morphometrics offered limited utility for distinguishing between species within this genus whilst morphological features and differences in nuclear ribosomal DNA sequences delineated U. sanguinis sp. n. from named congeners. Male specimens of U. sanguinis sp. n. differ from U. lucasi and U. hamiltoni by relatively shorter anterolateral and externodorsal rays, respectively, and from other congeners by the relative lengths and angulations of bursal rays, and in the shape of the spicules. Female specimens of U. sanguinis sp. n. are differentiated from Uncinaria spp. parasitic in terrestrial mammals by differences in vulval anatomy and the larger size of their eggs, although are morphologically indistinguishable from U. lucasi and U. hamiltoni. Molecular techniques clearly delimited U. sanguinis sp. n. as a distinct novel species. Obtaining baseline data on the parasites of wildlife hosts is important for the investigation of disease and the effective implementation and monitoring of conservation management.

Behavioural response of Australian fur seals (Arctocephalus pusillus doriferus) to vessel noise during peak and off-peak human visitation.

Vessel traffic is the greatest contributor to marine anthropogenic noise pollution, and particularly affects species utilising coastal areas. Seal Rocks (Victoria), the largest Australian fur seal breeding colony, has a relatively small vessel exclusion zone during the pupping and breeding season, when vessel traffic is at its peak. It is necessary to understand the impacts of visitation at sensitive marine sites to ensure they are adequately protected from human disturbance. This study assessed the behavioural response of hauled-out Australian fur seals at Seal Rocks to controlled vessel noise exposure during peak and off-peak vessel visitation periods. High level noise exposure (76-80 dB) resulted in increased vigilance and interruption to vital behaviours in both peak and off-peak periods. These behavioural responses were limited to the period of noise exposure but are nevertheless indicative of disturbance. It is important to ensure regulations intended to protect sensitive sites are up-to-date and evidence based.

Characterising Eastern Grey Kangaroos (Macropus giganteus) as Hosts of Coxiella burnetii.

Macropods are often implicated as the main native Australian reservoir hosts of Coxiella burnetii (Q fever); however, the maintenance and transmission capacity of these species are poorly understood. The objective of this cross-sectional study was to describe the epidemiology of C. burnetii in a high-density population of eastern grey kangaroos (Macropus giganteus) in a peri-urban coastal nature reserve in New South Wales, Australia. Blood, faeces and swabs were collected from forty kangaroos as part of a population health assessment. Frozen and formalin-fixed tissues were also collected from 12 kangaroos euthanised on welfare grounds. Specimens were tested for C. burnetii using PCR, serology, histopathology and immunohistochemistry. A total of 33/40 kangaroos were seropositive by immunofluorescence assay (estimated true seroprevalence 84%, 95% confidence interval [CI] 69% to 93%), with evidence of rising titres in two animals that had been tested four years earlier. The PCR prevalence was 65% (95% CI 48% to 79%), with positive detection in most sample types. There was no evidence of pathology consistent with C. burnetii, and immunohistochemistry of PCR-positive tissues was negative. These findings indicate that kangaroos are competent maintenance hosts of C. burnetii, likely forming a significant part of its animal reservoir at the study site.

Trends in Rescue and Rehabilitation of Marsupials Surviving the Australian 2019-2020 Bushfires.

The 2019-2020 Australian bushfire season had a devastating impact on native wildlife. It was estimated that 3 billion native animals were impacted by the fires, yet there are few estimates of the number of animals that were rescued and rehabilitated post-fire. Focusing on the state of New South Wales (NSW) and Kangaroo Island, South Australia, we used a case study approach to determine the number of marsupials that were reported rescued due to the 2019-2020 bushfires in these areas and analysed species-specific trends in rescue and release success. In NSW, we found 889 reports of fire-affected marsupials in 2019-2020, mostly comprising kangaroos and wallabies (macropods; n = 458), koalas (n = 204), and possums (n = 162), with a smaller number of wombats (n = 43) and other marsupial species. Most reports of fire-affected marsupials occurred 6-8 weeks after fire ignition, and there was no difference in temporal frequency of rescues between marsupial groups. For the three main groups, the probability of survival and subsequent release differed, with macropods having the lowest probability of release after rescue (0.15 ± 0.04) compared to koalas (0.47 ± 0.04) and possums (0.55 ± 0.10). The type of injury was the main predictor of survival during rehabilitation for all three marsupial groups, with those malnourished/moribund or with traumatic injuries less likely to survive rehabilitation. Death or euthanasia occurred on the day of rescue for 77% of macropods, 48% of possums and 15% of koalas. Koalas most often died during rehabilitation rather than on the day of rescue, with 73% either dying or being euthanised between day 1 and 30 post-rescue, representing a potential welfare concern. On Kangaroo Island, koalas were the most frequently rescued marsupial species; most euthanasia cases and deaths occurred in a hospital, whereas other marsupials were mostly euthanised at triage. In both jurisdictions, koalas were over-represented while possums were under-represented relative to baseline population densities and wildlife rescue trends in the years before the 2019-2020 bushfires. These species differences in presentation post-fire warrant further investigation, as do the differences in triage, survival and release outcomes. It is hypothesised that the high intensity and large scale of the 2019-2020 fires impeded marsupial fire evasion tactics, as evidenced by the small number of animals found for rescue, and the differing rates of presentation relative to underlying population densities for the main marsupial groups. Based on our findings, there is a need for detailed record keeping and data sharing, development of consistent and evidence-based triage, treatment and euthanasia guidelines and deployment of trained wildlife emergency rescue teams with advanced search techniques to minimise animal suffering where safe to do so.

Mitigating disease risk in an endangered pinniped: early hookworm elimination optimizes the growth and health of Australian sea lion pups.

The Australian sea lion (Neophoca cinerea) experiences high pup mortality of seasonally alternating severity, partly attributed to endemic hookworm (Uncinaria sanguinis) infection. To further explore health outcomes of early hookworm elimination, a treatment trial was conducted at Seal Bay Conservation Park, South Australia, over consecutive lower and higher mortality breeding seasons (2019, 19.2%; 2020-1; 28.9%). Pups (n = 322) were stratified into two age cohorts (median 14 d and 24 d recruitment ages) and randomly assigned to treated (topical ivermectin 500 µg/kg) or control (untreated) groups. A younger prepatent cohort <14 d old (median 10 d) was identified a posteriori. A seasonally independent growth benefit resulted from hookworm elimination across all age cohorts. The greatest relative improvements (bodyweight + 34.2%, standard length + 42.1%; p ≤ 0.001) occurred in the month post-treatment, in the youngest prepatent cohort. A significant benefit of lesser magnitude (bodyweight + 8.6-11.6%, standard length + 9.5-18.4%; p ≤ 0.033) persisted up to 3 months across all age cohorts - greatest in the youngest pups. Treatment resulted in immediate improvement in hematological measures of health - decreased anemia and inflammation severity (p ≤ 0.012). These results enhance our understanding of host-parasite-environment interactions within the context of hematological ontogenesis, confirm the seasonally independent benefits of hookworm disease intervention, and further inform conservation recommendations for this endangered species.

Implications of Escherichia coli community diversity in free-ranging Australian pinniped pups.

Escherichia coli is a widely studied bacterium, commonly used as an indicator of faecal contamination. Investigations into the structure and diversity of E. coli in free-ranging wildlife species has been limited. The objective of this study was to characterise intra-individual and inter-species E. coli phylotype and B2 sub-type diversity in free-ranging Australian pinniped pups, to determine whether a single E. coli colony is representative of the phylotype and B2 sub-type diversity in these hosts. Faecal samples were collected from free-ranging Australian fur seal (Arctocephalus pusillus doriferus), Australian sea lion (Neophoca cinerea) and long-nosed fur seal (Arctocephalus forsteri) pups from three breeding colonies between 2018 and 2021. Faecal swabs from thirty randomly selected pups (n = 10 from each species) were cultured and ten E. coli colonies were selected from each culture based on morphology and separation between colonies on agar plates. Molecular screening techniques were utilised to assign isolates to phylotypes and B2 sub-types. There was no significant difference (p > 0.05) in either intra-individual or inter-species E. coli phylotype and B2 sub-type diversity. The B2 phylotype was the most dominant, with 78% of isolates (n = 234) assigned to this phylotype. Host factors (species, weight [kg] and standard length [cm]) did not significantly affect phylotype diversity. The absence of intra-individual and inter-species differences in E. coli diversity at a phylotype level suggests that a single E. coli colony could be used as an indicator of overall diversity of E. coli at a phylotype level in A. p. doriferus, N. cinerea and A. forsteri pups. These findings can be used to simplify and improve the efficiency of sampling protocols for ongoing monitoring of human-associated E. coli phylotypes in free-ranging pinniped populations.

Gut microbiota of endangered Australian sea lion pups is unchanged by topical ivermectin treatment for endemic hookworm infection.

The gut microbiota is essential for the development and maintenance of the hosts' immune system. Disturbances to the gut microbiota in early life stages can result in long-lasting impacts on host health. This study aimed to determine if topical ivermectin treatment for endemic hookworm (Uncinaria sanguinis) infection in endangered Australian sea lion (Neophoca cinerea) pups resulted in gut microbial changes. The gut microbiota was characterised for untreated (control) (n = 23) and treated (n = 23) Australian sea lion pups sampled during the 2019 and 2020/21 breeding seasons at Seal Bay, Kangaroo Island. Samples were collected pre- and post-treatment on up to four occasions over a four-to-five-month period. The gut microbiota of untreated (control) and treated pups in both seasons was dominated by five bacterial phyla, Fusobacteria, Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. A significant difference in alpha diversity between treatment groups was seen in pups sampled during the 2020/21 breeding season (p = 0.008), with higher richness and diversity in treated pups. Modelling the impact of individual pup identification (ID), capture, pup weight (kg), standard length (cm), age and sex on beta diversity revealed that pup ID accounted for most of the variation (35% in 2019 and 42% in 2020/21), with pup ID, capture, and age being the only significant contributors to microbial variation (p < 0.05). There were no statistically significant differences in the composition of the microbiota between treatment groups in both the 2019 and 2020/21 breeding seasons, indicating that topical ivermectin treatment did not alter the composition of the gut microbiota. To our knowledge, this is the first study to characterise the gut microbiota of free-ranging Australian pinniped pups, compare the composition across multiple time points, and to consider the impact of parasitic treatment on overall diversity and microbial composition of the gut microbiota. Importantly, the lack of compositional changes in the gut microbiota with treatment support the utility of topical ivermectin as a safe and minimally invasive management strategy to enhance pup survival in this endangered species.

Carriage of antibiotic resistant bacteria in endangered and declining Australian pinniped pups.

The rapid emergence of antimicrobial resistance (AMR) is a major concern for wildlife and ecosystem health globally. Genetic determinants of AMR have become indicators of anthropogenic pollution due to their greater association with humans and rarer presence in environments less affected by humans. The objective of this study was to determine the distribution and frequency of the class 1 integron, a genetic determinant of AMR, in both the faecal microbiome and in Escherichia coli isolated from neonates of three pinniped species. Australian sea lion (Neophoca cinerea), Australian fur seal (Arctocephalus pusillus doriferus) and long-nosed fur seal (Arctocephalus forsteri) pups from eight breeding colonies along the Southern Australian coast were sampled between 2016-2019. DNA from faecal samples (n = 309) and from E. coli (n = 795) isolated from 884 faecal samples were analysed for class 1 integrons using PCRs targeting the conserved integrase gene (intI) and the gene cassette array. Class 1 integrons were detected in A. p. doriferus and N. cinerea pups sampled at seven of the eight breeding colonies investigated in 4.85% of faecal samples (n = 15) and 4.52% of E. coli isolates (n = 36). Integrons were not detected in any A. forsteri samples. DNA sequencing of the class 1 integron gene cassette array identified diverse genes conferring resistance to four antibiotic classes. The relationship between class 1 integron carriage and the concentration of five trace elements and heavy metals was also investigated, finding no significant association. The results of this study add to the growing evidence of the extent to which antimicrobial resistant bacteria are polluting the marine environment. As AMR determinants are frequently associated with bacterial pathogens, their occurrence suggests that these pinniped species are vulnerable to potential health risks. The implications for individual and population health as a consequence of AMR carriage is a critical component of ongoing health investigations.

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.

Non-essential heavy metals and protective effects of selenium against mercury toxicity in endangered Australian sea lion (Neophoca cinerea) pups with hookworm disease.

The endangered Australian sea lion, Neophoca cinerea, faces ongoing population decline. Identification of key threats to N. cinerea population recovery, including disease and pollutants, is an objective of the species' recovery plan. Previous studies have identified Uncinaria sanguinis, an intestinal nematode, as a significant cause of disease and mortality in N. cinerea pups. Given the impact of heavy metals on the immune response, investigation of these pollutants is critical. To this end, the concentrations of arsenic (As), total mercury (Hg), cadmium (Cd), chromium (Cr), lead (Pb) and selenium (Se) were determined in blood collected from N. cinerea pups sampled during the 2017/18, 2019 and 2020/21 breeding seasons at Seal Bay Conservation Park, South Australia. Significant differences (p < 0.05) in Hg, As, Cr, and Se concentrations and molar ratio of Se:Hg were seen between breeding seasons. Pup age, maternal parity and inter-individual foraging behaviour were considered factors driving these differences. The concentrations of Hg (357, 198 and 241 µg/L) and As (225, 834 and 608 µg/L) were high in 2017/18, 2019 and 2020/21 respectively with Hg concentrations in the blood of N. cinerea pups above toxicological thresholds reported for marine mammals. The concentration of Se (1332, 647, 763 µg/L) and molar ratio of Se:Hg (9.47, 7.98 and 6.82) were low compared to other pinniped pups, indicating potential vulnerability of pups to the toxic effects of Hg. Significant (p < 0.05) negative associations for Pb and Cd with several red blood cell parameters suggest they could be exacerbating the anaemia caused by hookworm disease. Temporal (age-related) changes in element concentrations were also seen, such that pup age needs to be considered when interpreting bioaccumulation patterns. Further investigation of the role of elevated heavy metal concentrations on N. cinerea pup health, disease and development is recommended, particularly with respect to immunological impacts.

A systematic review of factors affecting wildlife survival during rehabilitation and release.

Millions of native animals around the world are rescued and rehabilitated each year by wildlife rehabilitators. Triage and rehabilitation protocols need to be robust and evidence-based, with outcomes consistently recorded, to promote animal welfare and better understand predictors of wildlife survival. We conducted a global systematic review and meta-analysis of 112 articles that reported survival rates of native mammals and birds during rehabilitation and after release to determine intrinsic and extrinsic factors associated with their survival. We assessed survival during rehabilitation and in the short- and long-term post-release, with the hypothesis that survival will vary as a function of species body size, diel activity pattern, trophic level and study location (region of the world). We aimed to determine the direction of effect of these factors on survival to assist in decision-making during triage and rehabilitation. Results showed that mammals and birds were equally likely to survive all stages of rehabilitation, and survival rates varied between locations. Birds in North America had the poorest survival rates post-release, particularly long-term, as did diurnal and carnivorous birds in the short-term post-release. Anthropogenic factors such as motor vehicle collisions and domestic or feral animal attack contributed to morbidity and post-release mortality in 45% (168 of 369) of instances. The reasons for rescue and associated severity of diagnosis were commonly reported to affect the likelihood of survival to release, but factors affecting survival were often species-specific, including bodyweight, age, and characteristics of the release location. Therefore, evidence-based, species-specific, and context-specific protocols need to be developed to ensure wildlife survival is maximised during rehabilitation and post-release. Such protocols are critical for enabling rapid, efficient rescue programs for wildlife following natural disasters and extreme weather events which are escalating globally, in part due to climate change.

Developing Immune Profiles of Endangered Australian Sea Lion (Neophoca cinerea) Pups Within the Context of Endemic Hookworm (Uncinaria sanguinis) Infection.

As a top predator, the endangered Australian sea lion (Neophoca cinerea) is a sentinel of ecosystem change, where population trends can reflect broader shifts in the marine environment. The population of this endemic pinniped was historically diminished by commercial sealing, and recovery has been slowed by fishery interactions, disease and, potentially, pollutants. Hookworm infects 100% of neonatal pups and has been identified as a contributor to population decline. Here, a multivariable approach using traditional serological and novel molecular tools such as qPCR and ddPCR was used to examine immune phenotypes of developing Australian sea lion pups infected with the endemic hookworm (Uncinaria sanguinis) from two South Australian colonies. Results show changing immunophenotypes throughout the patent period of infection represented by pro-inflammatory cytokines (IL-6), IgG and acute-phase proteins. Although cytokines may prove useful as markers of resistance, in this study, IL-6 is determined to be an early biomarker of inflammation in Australian sea lion pups, excluding the alternative hypothesis. Additionally, immunological differences between animals from high- and low-intensity hookworm seasons, as well as ivermectin-treated animals, indicate hookworm infection modulation of the host immune response, as evidenced by a lower IL-6 mRNA expression in the non-treated groups. This study of the Australian sea lion is an example of an ecoimmunological approach to disease investigation, which can be applied to evaluate the impact of environmental and anthropogenic factors on susceptibility to infectious diseases in free-ranging species.

Sustained reduction in numbers of Australian fur seal pups: Implications for future population monitoring.

Fur seal populations in the Southern Hemisphere were plundered in the late 1700s and early 1800s to provide fur for a clothing industry. Millions of seals were killed resulting in potentially major ecosystem changes across the Southern Hemisphere, the consequences of which are unknown today. Following more than a century of population suppression, partly through on-going harvesting, many of the fur seal populations started to recover in the late 1900s. Australian fur seals (Arctocephalus pusillus doriferus), one of the most geographically constrained fur seal species, followed this trend. From the 1940s to 1986, pup production remained at approximately 10,000 per year, then significant growth commenced. By 2007, live pup abundance had recovered to approximately 21,400 per year and recovery was expected to continue However, a species-wide survey in 2013 recorded a 20% decline, to approximately 16,500 live pups. It was not known if this decline was due to 2013 being a poor breeding year or a true population reduction. Here we report the results of a population-wide survey conducted in 2017 and annual monitoring at the most productive colony, Seal Rocks, Victoria that recorded a large decline in live pup abundance (-28%). Sustained lower pup numbers at Seal Rocks from annual counts between 2012-2017 (mean = 2908 ± 372 SD), as well as the population-wide estimate of 16,903 live pups in 2017, suggest that the pup numbers for the total population have remained at the lower level observed in 2013 and that the 5-yearly census results are not anomalies or representative of poor breeding seasons. Potential reasons for the decline, which did not occur range-wide but predominantly in the most populated and long-standing breeding sites, are discussed. To enhance adaptive management of this species, methods for future monitoring of the population are also presented. Australian fur seals occupy several distinct regions influenced by different currents and upwellings: range-wide pup abundance monitoring enables comparisons of ecosystem status across these regions. Forces driving change in Australian fur seal pup numbers are likely to play across other marine ecosystems, particularly in the Southern Hemisphere where most fur seals live.

A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea).

We detail a novel presentation of tuberculosis associated with intestinal perforation in an endangered Australian sea lion (Neophoca cinerea) from South Australian waters and confirm the presence of this disease in the region of highest pup production. In February 2017, a 3-yr-old juvenile male died shortly after hauling out at the Kingscote beach on Kangaroo Island. On postmortem examination, we found a mid-jejunal intestinal perforation and partial obstruction (from a strangulating fibrous and granulomatous mesenteric mass), a marked multicentric abdominal fibrosing granulomatous lymphadenitis, and a large volume serosanguinous peritoneal effusion. Acid-fast bacteria were detected postmortem in cytologic preparations of the mesenteric lymph node and in histologic sections of jejunum and the encircling mass. Mycobacterial infection was confirmed by positive culture after 3 wk. Molecular typing using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing with 12-locus analysis identified Mycobacterium pinnipedii. This case highlights the need for vigilance of zoonotic disease risk when handling pinnipeds, including in the absence of specific respiratory signs or grossly apparent pulmonary pathology. Increased serologic population surveillance is recommended to assess the species' risk from this and other endemic diseases, especially given its endangered status.

Biological variation of total thyroxine (T4), free T4 and thyroid-stimulating hormone in 11 clinically healthy cats.

OBJECTIVES: The aim of this study was to investigate the biological variation of total thyroxine (T4), free T4 (fT4) and thyroid-stimulating hormone (TSH) in 11 clinically healthy cats aged between 3 and 15 years old, in Sydney, Australia. METHODS: Blood was collected weekly for up to 6 weeks and serum T4, fT4 and TSH concentrations were analysed using canine-specific reagents. Restricted maximum likelihood was used to estimate within-subject, between-subject and analytical variance components, which were recorded in terms of the related coefficients of variation. The index of individuality and reference change values were then calculated for each analyte. RESULTS: T4 and TSH had intermediate individuality, indicating both subject-based and population-based reference intervals (RIs) could be used, with the knowledge that population-based RIs are suboptimally sensitive. fT4 had high individuality, indicating subject-based RIs are more appropriate than population-based RIs. CONCLUSIONS AND RELEVANCE: This study has demonstrated that subject-based RIs could be more sensitive than population-based RIs for the diagnosis of thyroid dysfunction in cats.

Topical ivermectin is a highly effective seal 'spot-on': A randomised trial of hookworm and lice treatment in the endangered Australian sea lion (Neophoca cinerea).

The Australian sea lion (Neophoca cinerea) is an endangered and declining otariid species, with a high rate of pup mortality associated with endemic hookworm (Uncinaria sanguinis) infection a suspected contributor to this decline. Injected ivermectin is an effective treatment for Uncinaria sp. in otariids, with optimal outcomes achieved by the early treatment of pups prior to disease development. This randomised controlled trial evaluated the effectiveness of the novel use of a topical ivermectin formulation against hookworm infection and lice (Antarctophthirus microchir) infestation, in comparison with injected ivermectin. During the 2017 breeding season at Dangerous Reef, South Australia, pups ≤ 70 cm in standard length (≤ 2 weeks of age; n = 85) were randomised to single dose topical (500 µg/kg spot-on; n = 27) or injected (200 µg/kg subcutaneous; n = 29) ivermectin treatment groups, or to an untreated control group (n = 29). Topical ivermectin was highly effective for U. sanguinis elimination, and not significantly different to the injected formulation (estimated effectiveness 96.4% and 96.8%, respectively; P > 0.05). Its application resulted in an 81.6% reduction and 62.7% additional clearance for A. microchir infestation by 15-24 days post-treatment, compared with untreated control pups (also not significantly different to injected ivermectin; 83.1% and 59.4%, respectively; P > 0.05). Treatment with either ivermectin formulation significantly ameliorated increases in inflammatory markers detected in the blood of untreated control pups - peripheral blood eosinophil counts (persisting to 36-41 days post-recruitment P < 0.05) and increased plasma protein concentrations (15-24 days post-recruitment; P < 0.05). Further, an initial short-term decrease in body condition in the control group was not observed in either of the treatment groups. This study demonstrates that topical ivermectin is an effective antiparasitic treatment in N. cinerea. It offers an alternative administration method for ivermectin delivery to a young pup cohort in this species, and an alternative, minimally invasive management tool for species conservation.