Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

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.

Assessing risks for bovine and zoonotic tuberculosis through spatial analysis and a questionnaire survey in Fiji - A pilot study.

Mycobacterium bovis causes tuberculosis in cattle and when transmitted to humans typically causes extra-pulmonary tuberculosis (EPTB). Bovine tuberculosis (bTB) has a global distribution and is controlled in most countries to protect animal and public health. Recent studies revealed that bTB is established on dairy farms in Fiji where EPTB cases have been reported in people. The aims of this pilot investigation were to look for putative zoonotic TB (EPTB) cases in people and to evaluate practices that might contribute to the persistence and transmission of M. bovis between cattle and to humans. Existing data sets were shared between the Fiji Ministry of Agriculture and Ministry of Health and a questionnaire-based survey was implemented using One Health principles. Statistically significant co-location and close proximity of EPTB cases and bovine TB affected farms were identified. The bTB infection status of farms was significantly associated with unfenced water sources where cattle grazed. Of 247 households, 65 % shared drinking water sources with cattle and 36 % consumed raw milk without boiling, while 62 % of participants reported backyard slaughter of cattle. Several participants reported current symptoms potentially suggestive of TB (chronic cough) but the impact of smoking and history of previous TB treatment could not be evaluated. Farmers had limited understanding of the practices required to prevent bTB at farm level. Further study is recommended and should include an assessment of lifetime EPTB diagnoses, classification of farms based on more recent bTB test data and molecular typing of mycobacterial isolates from humans, cattle and the environment. A targeted awareness and education approach is required to reduce the future risk of zoonotic TB and to help ensure uptake of recommendations and practices aimed at controlling and preventing bTB.

Association between major histocompatibility complex haplotypes and susceptibility of unvaccinated and vaccinated cattle to paratuberculosis.

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.

Immune Priming of Pacific Oysters (Crassostrea gigas) to Induce Resistance to Ostreid herpesvirus 1: Comparison of Infectious and Inactivated OsHV-1 with Poly I:C.

Pacific oyster mortality syndrome (POMS), which is caused by Ostreid herpesvirus 1 (OsHV-1), causes economic losses in Pacific oyster (Crassostrea gigas) aquaculture in many countries. Reducing the mortality in disease outbreaks requires changing the host, pathogen and environment interactions to favor the host. Survivors of natural exposure to OsHV-1 are able to survive subsequent outbreaks. This has been replicated under laboratory conditions, suggesting the existence of an immune response. The aim of the present study is to compare the effects of prior exposure to infectious OsHV-1, heat-inactivated OsHV-1 and the chemical anti-viral immune stimulant poly I:C on mortality following exposure to virulent OsHV-1. All treatments were administered by intramuscular injection. Oysters were maintained at 18 °C for 14 days; then, the temperature was increased to 22 °C and the oysters were challenged with virulent OsHV-1. Heat-inactivated OsHV-1, infectious OsHV-1 and poly I:C all induced significant protection against mortality, with the hazard of death being 0.41, 0.18 and 0.02, respectively, compared to the controls, which had no immune priming. The replication of OsHV-1 on first exposure was not required to induce a protective response. While the underlying mechanisms for protection remain to be elucidated, conditioning for resistance to POMS by prior exposure to inactivated or infectious OsHV-1 may have practical applications in oyster farming but requires further development to optimize the dose and delivery mechanism and evaluate the duration of protection.

The Resistance to Lethal Challenge with Ostreid herpesvirus-1 of Pacific Oysters (Crassostrea gigas) Previously Exposed to This Virus.

Pacific oyster (Crassostrea gigas) aquaculture has been economically impacted in many countries by Pacific oyster mortality syndrome (POMS), a disease initiated by Ostreid herpesvirus 1. The objectives of this study were to determine whether naturally exposed, adult C. gigas could act as reservoirs for OsHV-1 and explain the recurrent seasonal outbreaks of POMS and to test whether or not they were resistant to OsHV-1. In a laboratory infection experiment using thermal shock, OsHV-1 replication was not reactivated within the tissues of such oysters and the virus was not transmitted to naïve cohabitating spat. The adult oysters were resistant to intramuscular injection with a lethal dose of OsHV-1 and had 118 times lower risk of mortality than naïve oysters. Considered together with the results of other studies in C. gigas, natural exposure or laboratory exposure to OsHV-1 may result in immunity during subsequent exposure events, either in the natural environment or the laboratory. While adult C. gigas can carry OsHV-1 infection for lengthy periods, reactivation of viral replication leading to mortality and transmission of the virus to naïve oysters may require specific conditions that were not present in the current experiment. Further investigation is required to evaluate the mechanisms responsible for resistance to disease in oysters previously exposed to OsHV-1, whether immunity can be exploited commercially to prevent POMS outbreaks and to determine the source of the virus for recurrent seasonal outbreaks.

IP10 is a predictor of successful vaccine protection against paratuberculosis infection in sheep.

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 relationships 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 exposure (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 experimental 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 vaccinated sheep and could be a biomarker for a DIVA test in sheep.

Diversity and molecular epidemiology of Ostreid herpesvirus 1 in farmed Crassostrea gigas in Australia: Geographic clusters and implications for "microvariants" in global mortality events.

Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or "microvariant", i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10-3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

Correlates of vaccine protection against Mycobacterium avium sub-species paratuberculosis infection revealed in a transcriptomic study of responses in Gudair® vaccinated sheep.

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.

A survey to understand farmers' perceptions and risk factors for hoof diseases including footrot in sheep in New South Wales, Australia.

The aims of this study were to develop an understanding of farmers' perceptions and risk factors for footrot, including its less severe forms, and other hoof diseases in sheep in New South Wales (NSW). A questionnaire was developed and administered to sheep farmers in Local Land Services (LLS) regions across NSW. LLS staff selected sheep farmers who met the inclusion criteria which included farmers with a minimum of 100 sheep, a history of having had foot problems in their flock or having expressed an interest in improving sheep health and production. Farmers completed the questionnaire either by telephone or via the REDCap online survey platform. Descriptive analyses and multivariable logistic regression models were created. The survey was completed by 43 sheep farmers with a median farm size of 1,500 Ha and flock size of 2,300; footrot was present on 39% of farms while 75.6% had other hoof diseases. A flock of >3,000 sheep were more likely to have footrot than a smaller flock (OR = 11.99, 90% CI = 3.02-63.92, P-value = 0.005) and footrot was less likely to be present on farms when an Animal Health Statement was requested while purchasing sheep (OR = 0.10, 90% CI = 0.01-0.56, P-value = 0.04). Hoof conditions other than footrot were likely to be present in flocks when foot inspections were conducted at a time other than weekly inspections (OR = 0.13, 90% CI = 0.01-0.68, P-value = 0.04) and flocks kept on undulating ground were more likely to have diseases other than footrot compared to those kept on flat ground (OR = 3.72, 90% CI = 1.02-15.80, P-value = 0.09). Most farmers agreed that footrot including its less severe forms can cause production losses and negatively affect animal health and welfare. Limitations of the study were the sample size and dry environmental conditions prior to and during study period in many regions of NSW which limited the expression of footrot.

Bovine tuberculosis control in Fiji: Retrospective study findings for 2015 to 2020.

Control of bovine tuberculosis (bTB) is a priority for animal health, biosecurity, and human health authorities in Fiji as evident from the long-term funding of the Bovine Brucellosis and Tuberculosis Eradication and Control program (BTEC) and notable improvements to the program described in this paper. To evaluate the performance of the Fiji BTEC program from 2015 to 2020, all available bTB data for cattle were analyzed. Data sources included BTEC bTB testing records, abattoir records and laboratory records. We integrated all information to quantify the bTB tests applied, bTB positive farms and animals, meat inspection and laboratory findings. Test coverage was highest among dairy cattle in Central Division (~73%), where bTB was highly prevalent with 7.8% of dairy cattle and 61.7% of dairy farms found to be positive between 2015 and 2020. There was no visible downward trend in the apparent prevalence of bTB over the 6-year period. During 2019 and 2020, only 21.3% (51/239) of the tested dairy farms maintained their clear status, another 8.4% (20/239) reverted to infected status after 1 year or more of being bTB clear, and most farms remained infected during these 2 years. Factors observed to be contributing to this situation were persistent infections, related in part to the significant number of untested animals, uncontrolled animal movements, and larger farm size. Similar to other developing countries, bTB remains a serious concern and further strengthening of the program targeting the main contributors to bTB persistence, along with maintenance of a comprehensive reporting and traceability system, industry awareness and government support are needed. Control of bTB in Fiji is a long-term objective that must have multiple stakeholder engagement and regular review to measure success.

Sheep vaccinated against paratuberculosis have increased levels of B cells infiltrating the intestinal tissue.

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.

Reduction in Virulence over Time in Ostreid herpesvirus 1 (OsHV-1) Microvariants between 2011 and 2015 in Australia.

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014-2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

Engineering Synthetic Lipopeptide Antigen for Specific Detection of Mycobacterium avium subsp. paratuberculosis Infection.

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

A comparison of multivalent and bivalent vaccination strategies for the control of virulent ovine footrot.

Virulent footrot is a significant economic and animal welfare concern. The disease can be treated, controlled, and eliminated with vaccine, but selecting the appropriate vaccination strategy can be challenging. There are two main strategies: outbreak (serogroup)-specific univalent or bivalent vaccination, or use of a multivalent vaccine containing up to nine of the most common serogroups. The objective of this study was to compare these approaches in sheep flocks infected with multiple Dichelobacter nodosus serogroups. In the first phase, we undertook an immunogenicity trial in which we compared four pre-commercial multivalent recombinant fimbrial vaccines containing six (A, B, C, G, H, I) or nine (A, B, C, D, E, F, G, H, I) serogroups, and compared them to commercial bivalent vaccines. Two multivalent vaccines stimulated significantly higher antibody responses than two other multivalent vaccines but the number of serogroups included in the multivalent vaccine formulations did not have a significant effect. In the first phase, we also compared inter-vaccination intervals of two- and three-months between sequential bivalent vaccines, and found that a two-month interval was sufficient to avoid antigenic competition. In the second phase, the most immunogenic multivalent vaccine (nine serogroups) was compared to sequential bivalent vaccines and monthly foot-bathing in a field trial in four commercial Merino flocks. The duration of protection afforded by the multivalent vaccine was likely to be less than that of the bivalent vaccines, as the antibody titres stimulated were lower and less persistent.

Prevalence of Infectious Spleen and Kidney Necrosis Virus (ISKNV), Nervous Necrosis Virus (NNV) and Ectoparasites in Juvenile Epinephelus spp. Farmed in Aceh, Indonesia.

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0-12) to 73% (95% CI: 54-88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4-31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.

Detection of ostreid herpesvirus-1 in plankton and seawater samples at an estuary scale.

Ostreid herpesvirus-1 (OsHV-1) is known to associate with particles in seawater, leading to infection and disease in the Pacific oyster Crassostrea gigas. The estuarine environment is highly complex and changeable, and this needs to be considered when collecting environmental samples for pathogen detection. The aims of this study were to (1) compare different aspects of collecting natural seawater and plankton samples for detection of OsHV-1 DNA and (2) determine whether detection of OsHV-1 DNA in such environmental samples has merit for disease risk prediction. The results of one experiment suggest that sampling on the outgoing tide may improve the detection of OsHV-1 DNA in seawater and plankton tow samples (odds ratio 2.71). This statistical comparison was not possible in 2 other experiments. The method (plankton tow or beta bottle) and depth of collection (range: 250-1250 mm) had no effect on the likelihood of detection of OsHV-1. OsHV-1 DNA was found at low concentrations in plankton tow and seawater samples, and only when outbreaks of mortality associated with OsHV-1 were observed in nearby experimental or farmed populations of C. gigas. This suggests that single point in time environmental samples of seawater or plankton are not sufficient to rule out the presence of OsHV-1 in an estuary. The association of OsHV-1 with particles in seawater needs to be better understood in order to determine whether more selective and sensitive methods can be devised to detect it, before environmental samples can be reliably used in disease risk prediction.

Australian Veterinarians' Perceptions Regarding the Zoonotic Potential of Mycobacterium avium Subspecies Paratuberculosis.

Public concerns over exposure to Mycobacterium avium subspecies paratuberculosis (MAP) or MAP components via foods of animal origin could have negative trade consequences, despite the absence of conclusive scientific evidence of a causal association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease (CD). This study was conducted among Australian veterinarians to understand (a) their perceptions regarding the role of MAP in the causation of CD (an ordinal outcome), and (b) their consideration of the adoption of the precautionary principle against Johne's disease (JD; a binary outcome). Ordinal and binary logistic regression analyses were performed to evaluate the association of explanatory variables with the above outcomes, respectively. Almost one-third of the respondents (32.2%) considered that MAP was likely to be involved in the causation of CD whereas more than two-thirds (69.8%) agreed with the adoption of the precautionary principle against JD. Veterinarians who were concerned about exposure to and/or getting infected with MAP were more likely to consider MAP as a causative agent of CD (odds ratio: 7.63; 95% CI: 1.55, 37.63) and favor the adoption of the precautionary principle against JD (odds ratio: 6.20; 95% CI: 1.90, 20.25). Those perceiving MAP as a causative agent of CD were also more likely to favor the adoption of the precautionary principle against JD (odds ratio: 13.2; 95% CI: 1.26, 138.90). The results suggest that Australian veterinarians, particularly those who consider MAP as a causative agent of CD are concerned about exposure to MAP and favor the adoption of the precautionary principle against JD. These findings can be useful for animal health authorities for designing JD control programs and policies.

Mongoose (Herpestes auropunctatus) May Not Be Reservoir Hosts for Mycobacterium bovis in Fiji Despite High Population Density and Direct Contact with Cattle.

The presence of a wildlife reservoir for Mycobacterium bovis complicates the eradication of bovine tuberculosis (BTB) from domestic cattle populations. For the BTB eradication program in Fiji, there is concern about the small Indian mongoose (Herpestes auropunctatus), which is overabundant and in direct contact with cattle. Consequently, a survey of mongooses trapped on three BTB affected dairy farms led to necropsy of 85 mongooses during January-February 2017. Thirty (35%) mongooses had gross pathological changes including possible granulomas detected at necropsy, and tissues from these animals were taken for histopathological examination. Granulomatous lesions were present in 53% of animals examined histopathologically but acid-fast bacilli were not observed and the majority of lesions in lung and kidney were associated with the nematodes Pulmostrongylus herpestis and Capillaria sp., respectively. Nevertheless, assuming test sensitivity of 35% for the current study, from this sample of 85 mongooses it can be concluded with 95% confidence that if present in the mongoose population susceptible to trapping, M. bovis prevalence was ≤10%. The prevalence of intercurrent lesions raised concerns about gross pathology as a screening test for M. bovis infection in mongooses in Fiji, and therefore pathogen detection methods such as bacterial culture and direct tissue PCR are recommended for future surveys. These are needed to completely rule out the mongoose as a reservoir host for M. bovis in Fiji.

Different in vivo growth of ostreid herpesvirus 1 at 18 °C and 22 °C alters mortality of Pacific oysters (Crassostrea gigas).

Seasonally recurrent outbreaks of mass mortality in Pacific oysters (Crassostrea gigas) caused by microvariant genotypes of ostreid herpesvirus 1 (OsHV-1) occur in Europe, New Zealand and Australia. The incubation period for OsHV-1 under experimental conditions is 48-72 hours and depends on water temperature, as does the mortality. An in vivo growth curve for OsHV-1 was determined by quantifying OsHV-1 DNA at 10 time points between 2 and 72 hours after exposure to OsHV-1. The peak replication rate was the same at 18 °C and 22 °C; however, there was a longer period of amplification leading to a higher peak concentration at 22 °C (2.34 × 107 copies/mg at 18 hours) compared to 18 °C (1.38 × 105 copies/mg at 12 hours). The peak viral concentration preceded mortality by 72 hours and 20 hours at 18 °C and 22 °C, respectively. Cumulative mortality to day 14 was 45.9% at 22 °C compared to 0.3% at 18 °C. The prevalence of OsHV-1 infection after 14 days at 18 °C was 33.3%. No mortality from OsHV-1 occurred when the water temperature in tanks of oysters challenged at 18 °C was increased to 22 °C for 14 days. The influence of water temperature prior to exposure to OsHV-1 and during the initial virus replication is an important determinant of the outcome of infection in C. gigas.