The intracellular bacterium, Coxiella burnetii , causes Q fever in humans and coxiellosis in animals. Cell-mediated immunity (CMI) has been shown to be important in Q fever; however, few studies have investigated the role of CMI in coxiellosis in cattle. Therefore, we aimed to optimize a whole-blood cytokine recall assay (CRA) and to evaluate C. burnetii –specific interferon-gamma (IFNγ) and interleukin 10 (IL10) responses in naturally infected cattle. Duplicate blood samples were collected from cows in an endemically infected dairy herd (as determined by serologic and molecular testing as well as through microbiologic culture) and subsequently transported at ambient temperature or on ice before stimulation with phase 1 and 2 C. burnetii antigen at various concentrations for 24 or 48 h; IFNγ and IL10 were measured in supernatant by ELISA. Using the optimized assay, the cytokine response was measured at calving and ~8 wk post-calving. The effect was assessed using generalized linear models and Spearman correlation coefficient (ρ). The greatest mean cytokine responses were elicited when blood samples were transported at ambient temperature and stimulated for 48 h with the highest C. burnetii antigen concentration. There was a significantly greater mean IFNγ (2.04 times) and IL10 (1.86 times) response at 8 wk post-calving compared to calving. At 8 wk post-calving, the correlation between cytokine response to phase 1 and 2 C. burnetii was ρ = 0.84 for IFNγ and ρ = 0.91 for IL10. Ultimately, our optimization study will guide informed use of the CRA in cattle research, allowing effective investigation of CMI in coxiellosis.

There is an unmet need for a stable and nonreactogenic vaccine against the bioterrorism agent, Coxiella burnetii , causing Q fever. Here a safe, effective, and non‐reactogenic Q fever vaccine is developed by employing self‐assembled particles (CPs) composed of cross‐reacting material 197, a nontoxic variant of diphtheria toxin. CPs are designed that incorporate selected C. burnetii antigens and assemble them inside engineered Escherichia coli at high yields. A cost‐effective manufacturing process enables the production of CP‐based Q fever vaccine candidates. Four vaccine candidates are developed, including a T‐cell epitope‐based vaccine (CP‐COX), and one that comprises two immunodominant antigens, Com1 and YbgF. The latter is tested separately (CP‐Com1, CP‐YbgF) or as a mixture (CP‐Com1/CP‐YbgF). Initial immunogenicity studies in mice reveal that the mixed CP‐Com1/YbgF elicits the highest antibody titers with a half maximal effective concentration (EC50) value of ≈100 000 and induction of T H 1 and T H 2 cytokines. CP‐Com1/YbgF is further evaluated in guinea pigs, demonstrating its safety and efficacy, as shown by the absence of adverse reactions and a significant reduction in febrile responses compared to alum upon challenge with C. burnetii . Together, the study shows the potential of CPs for the development of a safe and immunogenic subunit Q fever vaccine.

Coxiella burnetti is an intracellular bacterium that causes Q fever, a disease of worldwide importance. Q-VAX® , the approved human Q fever vaccine, is a whole cell vaccine associated with safety concerns. Here a safe particulate subunit vaccine candidate is developed that is ambient-temperature stable and can be cost-effectively manufactured. Endotoxin-free Escherichia coli is bioengineered to efficiently self-assemble biopolymer particles (BPs) that are densely coated with either strings of 18 T-cell epitopes (COX-BP) or two full-length immunodominant antigens (YbgF-BP-Com1) all derived from C. burnetii. BP vaccine candidates are ambient-temperature stable. Safety and immunogenicity are confirmed in mice and guinea pig (GP) models. YbgF-BP-Com1 elicits specific and strong humoral immune responses in GPs with IgG titers that are at least 1 000 times higher than those induced by Q-VAX® . BP vaccine candidates are not reactogenic. After challenge with C. burnetii, YbgF-BP-Com1 vaccine leads to reduced fever responses and pathogen burden in the liver and the induction of proinflammatory cytokines IL-12 and IFN-γ inducible protein (IP-10) when compared to negative control groups. These data suggest that YbgF-BP-Com1 induces functional immune responses reducing infection by C. burnetii. Collectively, these findings illustrate the potential of BPs as effective antigen carrier for Q fever vaccine development.

Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Bovine Johne’s disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease (JD), results in a chronic enteritis affecting ruminants worldwide and presents to severe economic and welfare issues. Pathogenic mycobacteria such as MAP are able to manipulate host transcriptomic pathways to establish infection and cause disease. Following exposure to pathogenic mycobacteria, host microRNA (miRNA) are modulated, effecting downstream gene expression. miRNA have been utilised as circulating biomarkers for several human diseases and cancers, however their applicability to MAP infection in ruminants remains relatively unexplored. Here, we use a combination of small RNA sequencing and microarray data to explore circulating miRNA and mRNA responses of sheep infected with and resilient to MAP. A total of 58 differentially expressed miRNA were identified in only MAP infected sheep and 25 in only resilient sheep. Early and late timepoint specific signatures were also observed between infected and resilient sheep. Together, our data presents a novel miRNA signature of MAP infection, and resilience to infection. Further, we identify key signalling and immune pathways regulated by these miRNA which may contribute to the resilience phenotype and the ability of these sheep to prevent infection.

The cell mediated immune response and ability of immune cells to migrate to the site of infection are both key aspects of protection against many pathogens. Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen and the causative agent of paratuberculosis, a chronic wasting disease of ruminants. Current commercial vaccines for paratuberculosis reduce the occurrence of clinical disease but not all animals are protected from infection. Therefore, there is a need to understand the immune responses triggered by these vaccines at the site of infection, in circulating immune cells and their rela-tionships to vaccine-mediated protection. The magnitude and location of gene expression related to the cell mediated immune response and cellular migration were studied in the ileum of sheep. In addition, longitudinal IP10 (also known as IP10) secretion by circulating immune cells was examined in the same sheep. Animals were grouped based on vaccination status (vaccinated vs non-vaccinated) and MAP expo-sure (experimentally exposed vs unexposed). Vaccination of unexposed sheep increased the expression of IP10, CCL5 and COR1c. Sheep that were successfully protected by vaccination (uninfected following exper-imental exposure) had significantly reduced expression of IP10 in the ileum at 12 months post exposure compared to vaccine non-responders (those that became infected) and non-vaccinated infected sheep. Successfully protected sheep also had significantly increased secretion of IP10 in in vitro stimulated immune cells from whole blood compared to vaccine non responders at 4 months post exposure. Therefore, the IP10 recall response has the potential to be used as marker for infection status in vacci-nated sheep and could be a biomarker for a DIVA test in sheep.(c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The 2019/2020 Australian bushfires were unprecedented in terms of total area burned and impact on livestock and wildlife populations. However, there is currently limited literature available relating the consequences of bushfire or smoke exposure to livestock health, welfare, and carcase quality. A retrospective cross-sectional study was conducted using historical monitoring data from a Meat Standards Australia (MSA) accredited abattoir located on the mid-north coast of New South Wales, Australia. The spatiotemporal association between exposure to bushfire smoke (specifically the duration of the bushfires, distance from closest bushfire, annual bushfire season, proportion of the property of origin burned during the bushfires and time frame since exposure to bushfires) and effects on carcase meat quality metrics and pathology were measured, by building linear, generalised linear and cumulative link mixed models. Our findings indicate that hot carcase weight increased as the distance between the property of origin and the closest bushfire became greater and decreased with exposure to bushfires of longer duration or when greater proportions of the property of origin were burnt during bushfires. Subcutaneous rib fat of carcases also increased with an increasing distance of properties from the closest recorded bushfire and decreased with exposure to bushfires during the 2019/2020 season. Higher meat colour scores (darker meat colour) were associated with exposure to bushfires during the 2019/2020 season and exposure to bushfires of longer durations. There was only a weak association between increasing distance to the closest bushfire and higher marbling scores. Evidence of pneumonia in carcases was associated with exposure to bushfires of longer duration, specifically increasing risk of pneumonia was associated with fires of longer durations. Greater periods of time since exposure (i.e., > 6 months) to bushfires were also associated with a higher risk of evidence of pneumonia at the time of processing. With increasing incidence of bushfires in Australia forecasted as a result of climate change, there is an urgent need to understand the impact of bushfires on livestock, to limit the effects on livestock health and mitigate the risk of significant socioeconomic impacts to the livestock industry. By providing a greater understanding of the impact of bushfires, the findings of this study can support producers to make informed decisions to mitigate the effects of bushfires on livestock health and carcase meat quality.

A critical hindrance in the development of effective vaccine strategies to combat infectious disease is lack of knowledge about correlates of protection and of the host responses necessary for successful adaptive immunity. Often vaccine formulations are developed by stepwise experimentation, with incomplete investigation of the fundamental mechanisms of protection. Gudair® is a commercially available vaccine registered for use in sheep and goats for controlling spread of Mycobacterium avium sub-species paratuberculosis (MAP) infections and reduces mortality by up to 90%. Here, using an experimental infection model in sheep, we have utilized a transcriptomics approach to identify white blood cell gene expression changes in vaccinated, MAP-exposed Merino sheep with a protective response in comparison to those vaccinated animals that failed to develop immunity to MAP infection. This methodology facilitated an overview of gene-associated functional pathway adaptations using an in-silico analysis approach. We identified a group of genes that were activated in the vaccine-protected animals and confirmed stability of expression in samples obtained from naturally exposed commercially maintained sheep. We propose these genes as correlates of vaccine induced protection.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Background The 2019/2020 Australian bushfires were the largest bushfire event in modern Australian history. While actions to mitigate risk to homes from bushfires are well reported, there is very little research reported on the impacts of bushfires on livestock. With an increasing incidence of bushfires predicted, there is an urgent need to identify how farmers can best protect their livestock. Objectives Compare bushfire affected farms with and without injured livestock to identify associations between risk factors and bushfire injury. Infer management approaches that can be used to reduce bushfire injury in livestock. Method A case‐control study using a structured interview questionnaire, delivered in late 2020 to cattle and sheep farmers in south‐eastern Australia (New South Wales and Victoria) whose farmland was burnt in the 2019/2020 Australian bushfires. Case farms were farms with bushfires injured or killed livestock. Control farms were farms that had no bushfire injured livestock but that still had fire present on the farm. Interview responses were summarised and information theoretical approaches were used to identify potential risk factors for livestock bushfire injury and protective actions that could inform future fire‐preparation recommendations. Results and discussion Of 46 farms in the case‐control study, 21 (46%) reported bushfire injured or killed livestock. Apparent protective factors identified included: preparation (having a bushfire plan and more than two farm bushfire fighting units), backburning and receiving assistance from fire authorities. Combined beef and sheep grazing enterprises appeared to have an increased risk of bushfire injury to livestock.

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence.

Systemic immunisation delivered subcutaneously is currently used to control paratuberculosis, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). These vaccines do not provide complete protection and a small cohort of animals still succumb to clinical disease. The aim of this study was to assess mycobacterial infection site-specific variations in immune cells in vaccinated sheep that did or did not develop the disease following controlled exposure to MAP. Immunohistochemical staining of terminal ileum demonstrated that vaccination increased infiltration of CD4 + T cells and B cells. Infiltration of large numbers of CD4 + T and B cells was also seen in sheep that successfully cleared infection. Vaccination promoted the polarisation of macrophages to an M1 activation state. The presence of certain cells at the site of infection, especially CD4 + T cells, is likely to contribute to vaccine success by increasing the speed and potency of the local immune response. Systemic immunisation against MAP can alter the composition of innate and adaptive immune cell populations at the predilection site for MAP infection in the ileum one year after vaccination. This informs understanding of the impact of vaccination at the site of infection and also the duration of vaccine-elicited changes. This information may assist vaccine development and allow targeting of protective immune responses in the gut of ruminants.

Q fever is caused by the bacterium Coxiella burnetii and is spread to humans from infected animals especially goats, sheep and cattle, predominantly when giving birth. There is an effective human vaccine (Q-VAX) against Q fever, and although Q fever is a worldwide problem, the vaccine is only used in Australia due to difficulties associated with its use and the risk of adverse reactions. The desire to protect humans, particularly farmers and abattoir workers, from Q fever prompted the development of a new safe and effective human vaccine without all the difficulties associated with the current vaccine. Candidate vaccines were prepared using purified O-specific polysaccharide (OSP) extracted from the lipopolysaccharide of virulent (phase 1) C. burnetii, strain Nine Mile, which was then conjugated to a tetanus toxoid (TT) carrier protein. Two vaccines were prepared using OSP from C. burnetii grown in embryonated eggs (vaccine A) and axenic media (vaccine B). Vaccines with or without alum adjuvant were used to vaccinate guinea pigs, which were later challenged by intranasal inoculation with virulent C. burnetii. Both vaccines protected guinea pigs from fever and loss of weight post challenge. Post-mortem samples of the spleen, liver and kidney of vaccinated guinea pigs contained substantially less C. burnetii DNA as measured by PCR than those of the unvaccinated control animals. This study demonstrated that a C. burnetii OSP-TT conjugate vaccine is capable of inducing protection against virulent C. burnetii in guinea pigs. Additionally, OSP derived from C. burnetii grown in axenic media compared to OSP from embryonated eggs is equivalent in terms of providing a protective immune response.

In 2019/2020, Australia experienced a severe bushfire event, with many tens of thousands of livestock killed or euthanized. Little systematic research has occurred to understand livestock bushfire injuries, risk factors for injury, or how to make decisions about management of bushfire-injured livestock. Addressing this research gap is important as there is an increasing bushfire incidence globally. This paper presents qualitative research findings about bushfire-injured and killed livestock in the south-east of Australia after the 2019/2020 Australian bushfires. We describe observed pathology, treatments used, and risk factors for injury, then use thematic analysis to understand decision making about managing fire-injured livestock. Livestock injured by the fires showed pathology predominantly associated with the common integument (feet, hooves and skin) and signs of acute respiratory damage. It could take several days for the full extent of burns to become apparent, leaving prognostic doubt. Treatment strategies included immediate euthanasia, salvage slaughter, retention for later culling, treatment and recovery on farm, hospitalization and intensive treatment, or no intervention. Risk factors reported for livestock injury included lack of warnings about an impending fire, the type and amount of vegetation around livestock and the weather conditions on the day the fire reached livestock. Moving stock to an area with little vegetation before fire arrived was seen as protective. Decision making regarding injured livestock appeared influenced by three main themes: (1) observations on the severity of pathology, clinical signs and level of prognostic doubt, (2) pre-existing beliefs about animal welfare (responsibility to minimize unnecessary suffering) and (3) assumptions about the future. The management of livestock was largely appropriate due to the rapid provision of veterinary expertise. However, it is likely that some injured livestock were euthanized due to conservative veterinary advice driven by a lack of opportunity to re-assess stock, with impacts on farmers. In future, resourcing regular revisits of injured livestock to manage risks of gradual progression of burn pathology may facilitate more accurate prognostic assessment, provided injured animals can receive appropriate pain relief. In addition, a more comprehensive burns classification system linked to prognosis that can be rapidly applied in the field may assist assessments.

Mycobacterium avium is separated into four subspecies: M. avium subspecies avium (MAA), M. avium subspecies silvaticum (MAS), M. avium subspecies hominissuis (MAH), and M. avium subspecies paratuberculosis (MAP). Understanding the mechanisms of host and tissue adaptation leading to their clinical significance is vital to reduce the economic, welfare, and public health concerns associated with diseases they may cause in humans and animals. Despite substantial phenotypic diversity, the subspecies nomenclature is controversial due to high genetic similarity. Consequently, a set of 1,230 M. avium genomes was used to generate a phylogeny, investigate SNP hotspots, and identify subspecies-specific genes. Phylogeny reiterated the findings from previous work and established that Mycobacterium avium is a species made up of one highly diverse subspecies, known as MAH, and at least two clonal pathogens, named MAA and MAP. Pan-genomes identified coding sequences unique to each subspecies, and in conjunction with a mapping approach, mutation hotspot regions were revealed compared to the reference genomes for MAA, MAH, and MAP. These subspecies-specific genes may serve as valuable biomarkers, providing a deeper understanding of genetic differences between M. avium subspecies and the virulence mechanisms of mycobacteria. Furthermore, SNP analysis demonstrated common regions between subspecies that have undergone extensive mutations during niche adaptation. The findings provide insights into host and tissue specificity of this genetically conserved but phenotypically diverse species, with the potential to provide new diagnostic targets and epidemiological and therapeutic advances.

Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations ( n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.