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Chlamydial disease continues to be one of the main factors threatening the long-term survival of the koala (Phascolarctos cinereus). Despite this, large epidemiological studies of chlamydial infection and disease in wild koala populations are lacking. A better understanding of the prevalence, transmission and pathogenesis is needed to improve control measures, such as the development of vaccines. We investigated the prevalence of Chlamydia pecorum infection and disease in 160 koalas in a peri-urban wild population in Queensland, Australia and found that 31% of koalas were Chlamydia PCR positive and 28% had clinically detectable chlamydial disease. Most infections were at the urogenital site (27%; both males and females) with only 14% at the ocular site. Interestingly, we found that 27% (4/15) of koalas considered to be sexually immature (9-13 months) were already infected with C. pecorum, suggesting that a significant percentage of animals are infected directly from their mother. Ocular infection levels were less prevalent with increasing age (8% in koalas older than 4 years), whereas the prevalence of urogenital tract infections remained high into older age (26% in koalas older than 4 years), suggesting that, after mother-to-young transmission, C. pecorum is predominantly a sexually transmitted infection. While 28% of koalas in this population had clinically detectable chlamydial disease (primarily urogenital tract disease), many PCR positive koalas had no detectable disease and importantly, not all diseased animals were PCR positive. We also observed higher chlamydial loads in koalas who were C. pecorum infected without clinical disease than in koalas who were C. pecorum infected with clinical disease. These results shed light on the potential mechanisms of transmission of C. pecorum in koalas and also guide future control measures, such as vaccination.

The virulence of chlamydial infection in wild koalas is highly variable between individuals. Some koalas can be infected (PCR positive) with Chlamydia for long periods but remain asymptomatic, whereas others develop clinical disease. Chlamydia in the koala has traditionally been studied without regard to coinfection with other pathogens, although koalas are usually subject to infection with koala retrovirus (KoRV). Retroviruses can be immunosuppressive, and there is evidence of an immunosuppressive effect of KoRV in vitro . Originally thought to be a single endogenous strain, a new, potentially more virulent exogenous variant (KoRV-B) was recently reported. We hypothesized that KoRV-B might significantly alter chlamydial disease outcomes in koalas, presumably via immunosuppression. By studying sub-groups of Chlamydia and KoRV infected koalas in the wild, we found that neither total KoRV load (either viraemia or proviral copies per genome), nor chlamydial infection level or strain type, was significantly associated with chlamydial disease risk. However, PCR positivity with KoRV-B was significantly associated with chlamydial disease in koalas ( p  = 0.02961). This represents an example of a recently evolved virus variant that may be predisposing its host (the koala) to overt clinical disease when co-infected with an otherwise asymptomatic bacterial pathogen ( Chlamydia ).

Southern hemisphere humpback whales are classified as high-fidelity Antarctic krill consumers and as such are vulnerable to variability and long-term changes in krill biomass. Evidence of heterogeneous feeding patterns of east coast of Australia migrating humpback whales has been observed, warranting a comprehensive assessment of interannual variability in their diet. We examined the lipid and fatty acid profiles of individuals of the east coast of Australia migrating stock sampled between 2008 and 2018. The use of live-sampled blubber biopsies showed that fatty acid profiles varied significantly among all years. The two trophic indicator fatty acids for Antarctic krill, 20:5ω3 and 22:6ω3 remained largely unchanged across the 10-year period, suggesting that Antarctic krill is the principal prey item. A distance-based linear model showed that 33% of the total variation in fatty acid profiles was explained by environmental variables and climate indices. Most of the variation was explained by the Southern Annular Mode (23.7%). The high degree of variability observed in this study was unexpected for a species that is thought to feed primarily on one prey item. We propose that the observed variability likely arises from changes in the diet of Antarctic krill rather than changes in the whale’s diet.

Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala’s gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-γ, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E’) that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas' gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.

MicroRNAs (miRNAs) are highly conserved small noncoding RNAs that regulate gene expression post‐transcriptionally. Circulating miRNAs – miRNAs that have been released from cells and circulate in the bloodstream – are relatively stable and interesting molecules for wildlife research, where they may form a proxy for gene expression as a function of the animal's state under a variety of environmental challenges. Aiming at providing initial baseline data on the circulating miRNAome in avian wildlife, we assessed the miRNA profiles of wild ruddy turnstones Arenaria interpres on their Australian non‐breeding grounds. The ruddy turnstone is a long‐distant migrant and a significant reservoir species for low pathogenic avian influenza virus (LPAIV). We therefore investigated both LPAIV‐infected and uninfected individuals for their specific miRNA profiles to potentially elucidate the species' molecular mechanisms underlying its response to LPAIV infection. De novo miRNA characterisation in the ruddy turnstone genome identified 161 conserved and two novel, bird‐specific miRNAs, with liver‐enriched miRNA‐122 being the most abundant. Z chromosome‐linked miR‐2954‐3p was significantly more abundant in serum from males (ZZ) than from females (ZW). Furthermore, we found a sex‐ and age‐associated effect of LPAIV infection on miRNA abundance in serum samples, including one novel miRNA. This circulating miRNA signature may reflect sex‐ and age‐specific differences in the host response, indicating that circulating miRNAs could serve as a valuable non‐destructive analytical tool for enhancing our understanding of avian infections in a wildlife context and should be explored further.

Many persistent organic pollutants, such as polychlorinated biphenyls (PCBs), have high immunomodulating potentials. Exposure to them, in combination with virus infections, has been shown to aggravate outcomes of the infection, leading to increased viral titers and host mortality. Expression of immune-related microRNA (miR) signaling pathways (by host and/or virus) have been shown to be important in determining these outcomes; there is some evidence to suggest pollutants can cause dysregulation of miRNAs. It was thus hypothesized here that modulation of miRNAs (and associated cytokine genes) by pollutants exerts negative effects during viral infections. To test this, an in vitro study on chicken embryo fibroblasts (CEF) exposed to a PCB mixture (Aroclor 1260) and then stimulated with a synthetic RNA virus (poly(I:C)) or infected with a lymphoma-causing DNA virus (Gallid Herpes Virus 2 [GaHV-2]) was conducted. Using quantitative real-time PCR, expression patterns for mir-155, pro-inflammatory TNFα and IL-8, transcription factor NF-κB1, and anti-inflammatory IL-4 were investigated 8, 12, and 18 h after virus activation. The study showed that Aroclor1260 modulated mir-155 expression, such that a down-regulation of mir-155 in poly(I:C)-treated CEF was seen up to 12 h. Aroclor1260 exposure also increased the mRNA expression of pro-inflammatory genes after 8 h in poly(I:C)-treated cells, but levels in GaHV-2-infected cells were unaffected. In contrast to with Aroclor1260/poly(I:C), Aroclor1260/GaHV-2-infected cells displayed an increase in mir-155 levels after 12 h compared to levels seen with either individual treatment. While after 12 h expression of most evaluated genes was down-regulated (independent of treatment regimen), by 18 h, up-regulation was evident again. In conclusion, this study added evidence that mir-155 signaling represents a sensitive pathway to chemically-induced immunomodulation and indicated that PCBs can modulate highly-regulated innate immune system signaling pathways important in determining host immune response outcomes during viral infections.

Background Per- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2). Results The expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels. Conclusions Not only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.

Background Cetacean morbillivirus, herpesvirus, avian influenza virus (AIV) and Brucella spp. have been linked to numerous cetacean strandings in the Northeast (NE) Atlantic. Yet, their prevalence in free-living cetaceans remains insufficiently investigated, particularly in northern regions. Methods Between 2016 and 2025, humpback whales ( Megaptera novaeangliae ), sperm whales ( Physeter macrocephalus ) and, opportunistically, fin whales ( Balaenoptera physalus ) and a long-finned pilot whale ( Globicephala melas ), were sampled in two foraging grounds in northern Norway (Skjervøy and Andenes), in Iceland and in Cape Verde. Blow samples ( n  = 76), skin biopsies ( n  = 45), and organ samples from one stranded pilot whale were collected and screened for cetacean morbillivirus, herpesvirus, AIV and Brucella spp, via polymerase chain reaction (PCR). Results In northern Norway, cetacean morbillivirus, identified as the dolphin morbillivirus (DMV) strain, was detected in the blows of two asymptomatic groups of humpback whales, in the blow of one sperm whale in poor health and in the kidney of a stranded pilot whale. An alphaherpesvirus was detected in the blows of five humpback whale groups sampled in Norway, Iceland, and Cape Verde, while a gammaherpesvirus was detected in one humpback whale skin biopsy, sampled in Norway. No other samples tested positive to any of the pathogens, including AIV or Brucella spp. Conclusion Our results demonstrate that minimally invasive sampling, particularly blow sampling, can be used for pathogen surveillance in free-ranging cetaceans. They also provide new insights into the circulation of cetacean morbillivirus and herpesviruses in cetaceans from the NE Atlantic. Continuous monitoring of pathogen exposure, alongside other stressors, will be crucial to assess the cumulative health implications for these cetaceans.

Background Wild aquatic birds serve as the natural reservoir for avian influenza virus (AIV), a disease with significant implications for avian and mammalian health. Climate change is predicted to impact the dynamics of AIV, particularly in areas such as the Arctic, but the baseline data needed to detect these shifts is often unavailable. In this study, plasma from two species of gulls breeding on the high-Arctic Svalbard archipelago were screened for antibodies to AIV. Results AIV antibodies were found in black-legged kittiwake (Rissa tridactyla) samples from multiple years, as well as in glaucous gulls (Larus hyperboreous) samples. Conclusions Despite small sample sizes, evidence of exposure to AIV was found among Svalbard gulls. A wider survey of Svalbard avian species is warranted to establish knowledge on the extent of AIV exposure on Svalbard and to determine whether active infections are present.