Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Testing the intrinsic mechanisms driving the dynamics of Ross River Virus across Australia.

The mechanisms driving dynamics of many epidemiologically important mosquito-borne pathogens are complex, involving combinations of vector and host factors (e.g., species composition and life-history traits), and factors associated with transmission and reporting. Understanding which intrinsic mechanisms contribute most to observed disease dynamics is important, yet often poorly understood. Ross River virus (RRV) is Australia's most important mosquito-borne disease, with variable transmission dynamics across geographic regions. We used deterministic ordinary differential equation models to test mechanisms driving RRV dynamics across major epidemic centers in Brisbane, Darwin, Mandurah, Mildura, Gippsland, Renmark, Murray Bridge, and Coorong. We considered models with up to two vector species (Aedes vigilax, Culex annulirostris, Aedes camptorhynchus, Culex globocoxitus), two reservoir hosts (macropods, possums), seasonal transmission effects, and transmission parameters. We fit models against long-term RRV surveillance data (1991-2017) and used Akaike Information Criterion to select important mechanisms. The combination of two vector species, two reservoir hosts, and seasonal transmission effects explained RRV dynamics best across sites. Estimated vector-human transmission rate (average ß = 8.04x10-4per vector per day) was similar despite different dynamics. Models estimate 43% underreporting of RRV infections. Findings enhance understanding of RRV transmission mechanisms, provide disease parameter estimates which can be used to guide future research into public health improvements and offer a basis to evaluate mitigation practices.

Exposure to Persistent Organic Pollutants in Australian Waterbirds.

There is growing worldwide recognition of the threat posed by persistent organic pollutants (POPs) to wildlife populations. We aimed to measure exposure levels to POPs in a Southern Hemisphere aquatic waterbird species, the nomadic gray teal (Anas gracilis), which is found across Australia. We collected wings from 39 ducks harvested by recreational hunters at two sites (one coastal, one inland) in Victoria, southeastern Australia, in 2021. We examined three groups of POPs: nine congeners of polychlorinated biphenyls (PCBs), 13 organochlorine pesticides (OCPs), and 12 polycyclic aromatic hydrocarbons (PAHs). The PCBs, OCPs, and PAHs were detected at quantifiable levels in 13%, 72%, and 100% of birds, respectively. Of the congeners we tested for in PCBs, OCPs, and PAHs, 33%, 38%, and 100% were detected at quantifiable levels, respectively. The highest levels of exposure to POPs that we found were to the PAH benzo[b]fluoranthene, occurring at a concentration range of 1.78 to 161.05 ng/g wet weight. There were some trends detected relating to differences between geographical sites, with higher levels of several PAHs at the coastal versus inland site. There were several strong, positive associations among PAHs found. We discuss potential sources for the POPs detected, including industrial and agricultural sources, and the likely role of large-scale forest fires in PAH levels. Our results confirm that while Australian waterbirds are exposed to a variety of POPs, exposure levels are currently relatively low. Additional future investigations are required to further characterize POPs within Australian waterbird species. Environ Toxicol Chem 2024;43:736-747. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Field epidemiology capacity of the national veterinary services of Lao PDR: An online survey.

Introduction: Capacity in veterinary epidemiology is critical to detect, respond and control infectious diseases. Laos veterinary service is limited by having only a small number of veterinarians who graduated from overseas. Animal science graduates support the majority of the Laos veterinary services. The veterinary program was established in 2009 at the National University of Laos. We aimed to understand the national veterinary epidemiology capacity and identify gaps and training needs. Method: A cross-sectional online study was conducted in 2021 targeting central (DLF), provincial (PAFO) and district (DAFO) government animal health officers and veterinary/animal science academics (n = 332). The questionnaire addressed skills, experiences and perceived training needs in outbreak investigation, disease surveillance, data management and analysis, epidemiological surveys, One Health, leadership and communication and biosecurity. A descriptive analysis was performed and associations between demographic factors and epidemiological skills were examined. Results and discussion: In total, 205 respondents completed the questionnaire (61.8% response rate). Respondents reported low or no skills and experience in data management and analysis, epidemiological surveys and One Health. In contrast, higher but limited skills and experiences were reported in outbreak investigation, disease surveillance and biosecurity. Previous epidemiology training was primarily associated with stronger experiences in veterinary epidemiology competencies, followed by respondents that had completed a veterinary degree, highlighting the value of the currently available epidemiology training and veterinary-trained personnel in Lao PDR. This study could help inform the Laos government in its policy planning for field veterinary epidemiology capacity and future training.

A repeatable CT protocol for quantifying caudal vena cava growth in medium and large breed dogs.

Developmental malformations can cause stunted or abnormal growth and clinical disease in dogs. In humans, measurements of the inferior vena cava are used as methods for detecting abnormal growth trajectories. The objectives of this retrospective, multicenter, analytical, cross-sectional study were to develop a repeatable protocol to measure the caudal vena cava (CVC) and generate growth curves in medium and large-breed dogs during development. Contrast-enhanced CT DICOM images from 438 normal dogs, aged from 1 to 18 months, from five specific breeds were included. A "best guess" measurement protocol was created. Dogs were stratified into medium or large breed groups based on growth rate trajectories. Linear regression models and logarithmic trend lines were used to evaluate the CVC growth over time. The CVC measurements were analyzed from four anatomical regions: thorax, diaphragm, intra-hepatic, and renal. The thoracic segment produced the most repeatable measurements with the highest explanatory power. The CVC thoracic circumference ranged from 2.5 to 4.9 cm from 1 to 18 months of age. Medium and large breeds had similar CVC growth trajectories, with comparable estimated marginal means, however medium dogs reached 80% of predicted final CVC size approximately 4 weeks earlier than large breed dogs. This new protocol provides a standardized technique for evaluation of the CVC circumference over time using contrast-enhanced CT and is most repeatable when taken at the thoracic level. This approach could be adapted for other vessels to predict their growth trajectories, generating healthy reference population data for comparison against patients with vascular anomalies.

Estimation of a Within-Herd Transmission Rate for African Swine Fever in Vietnam.

We describe results from a panel study in which pigs from a 17-sow African swine fever (ASF) positive herd in Thái Bình province, Vietnam, were followed over time to record the date of onset of ASF signs and the date of death from ASF. Our objectives were to (1) fit a susceptible-exposed-infectious-removed disease model to the data with transmission coefficients estimated using approximate Bayesian computation; (2) provide commentary on how a model of this type might be used to provide decision support for disease control authorities. For the outbreak in this herd, the median of the average latent period was 10 days (95% HPD (highest posterior density interval): 2 to 19 days), and the median of the average duration of infectiousness was 3 days (95% HPD: 2 to 4 days). The estimated median for the transmission coefficient was 3.3 (95% HPD: 0.4 to 8.9) infectious contacts per ASF-infectious pig per day. The estimated median for the basic reproductive number, R0, was 10 (95% HPD: 1.1 to 30). Our estimates of the basic reproductive number R0 were greater than estimates of R0 for ASF reported previously. The results presented in this study may be used to estimate the number of pigs expected to be showing clinical signs at a given number of days following an estimated incursion date. This will allow sample size calculations, with or without adjustment to account for less than perfect sensitivity of clinical examination, to be used to determine the appropriate number of pigs to examine to detect at least one with the disease. A second use of the results of this study would be to inform the equation-based within-herd spread components of stochastic agent-based and hybrid simulation models of ASF.

Lead contamination in Australian game meat.

Lead-based ammunition (gunshot and bullets) frequently leaves small lead fragments embedded in the meat of wild-shot game animals. Australia produces several commercial game meat products from wild animals harvested with lead-based ammunition and has a growing population of recreational hunters. However, no studies have previously investigated the frequency of lead fragments or lead concentrations in Australian game meat. We examined 133 Australian minced game meat items of four types for evidence of lead contamination. Samples were meat from kangaroos (Macropus and Osphranter spp.; n=36) and Bennett's wallabies (Notamacropus rufogriseus; n=28) sold for human consumption, and deer ('venison'; multiple spp.; n=32) and stubble quail (Coturnix pectoralis; n=37) harvested for private consumption by recreational hunters. All packages were studied by digital radiography to detect the presence of radio-dense fragments, assumed to be lead fragments from ammunition. Visible fragments were absent in commercially available kangaroo products, but were present in 4%, 28% and 35% of wallaby, venison and quail, respectively. Mean meat lead concentrations (mg/kg wet weight) were 0.01 ± 0.01 for kangaroo, 0.02 ± 0.01 for wallaby, 0.12 ± 0.07 for venison, and 1.76 ± 3.76 for quail. The Australian food standards threshold for livestock meat (0.1 mg/kg w.w.) was not exceeded by any kangaroo or wallaby products but was exceeded by 53% and 86% of venison and quail, respectively. Radiography only detected 35% of samples that were above the food safety threshold. While average lead concentrations in commercially available macropod (kangaroo and wallaby) meat were low, those in recreationally harvested game meat may pose health risks for hunters and associated consumers.

Performance Evaluation and Validation of Air Samplers To Detect Aerosolized Coxiella burnetii.

Coxiella burnetii, the etiological agent of Q fever, is an intracellular zoonotic pathogen transmitted via the respiratory route. Once released from infected animals, C. burnetii can travel long distances through air before infecting another host. As such, the ability to detect the presence of C. burnetii in air is important. In this study, three air samplers, AirPort MD8, BioSampler, and the Coriolis Micro, were assessed against a set of predetermined criteria in the presence of three different aerosolized C. burnetii concentrations. Two liquid collection media, phosphate-buffered saline (PBS) and alkaline polyethylene glycol (Alk PEG), were tested with devices requiring a collection liquid. Samples were tested by quantitative polymerase chain reaction assay (qPCR) targeting the single-copy com1 gene or multicopy insertion element IS1111. All air samplers performed well at detecting airborne C. burnetii across the range of concentrations tested. At high nebulized concentrations, AirPort MD8 showed higher, but variable, recovery probabilities. While the BioSampler and Coriolis Micro recovered C. burnetii at lower concentrations, the replicates were far more repeatable. At low and intermediate nebulized concentrations, results were comparable in the trials between air samplers, although the AirPort MD8 had consistently higher recovery probabilities. In this first study validating air samplers for their ability to detect aerosolized C. burnetii, we found that while all samplers performed well, not all samplers were equal. It is important that these results are further validated under field conditions. These findings will further inform efforts to detect airborne C. burnetii around known point sources of infection. IMPORTANCE Coxiella burnetii causes Q fever in humans and coxiellosis in animals. It is important to know if C. burnetii is present in the air around putative sources as it is transmitted via inhalation. This study assessed air samplers (AirPort MD8, BioSampler, and Coriolis Micro) for their efficacy in detecting C. burnetii. Our results show that all three devices could detect aerosolized bacteria effectively; however, at high concentrations the AirPort performed better than the other two devices, showing higher percent recovery. At intermediate and low concentrations AirPort detected at a level higher than or similar to that of other samplers. Quantification of samples was hindered by the limit of quantitation of the qPCR assay. Compared with the other two devices, the AirPort was easier to handle and clean in the field. Testing air around likely sources (e.g., farms, abattoirs, and livestock saleyards) using validated sampling devices will help better estimate the risk of Q fever to nearby communities.

Validation of an Indirect Immunofluorescence Assay and Commercial Q Fever Enzyme-Linked Immunosorbent Assay for Use in Macropods.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

Risk factors for campylobacteriosis in Australia: outcomes of a 2018-2019 case-control study.

BACKGROUND: We aimed to identify risk factors for sporadic campylobacteriosis in Australia, and to compare these for Campylobacter jejuni and Campylobacter coli infections. METHODS: In a multi-jurisdictional case-control study, we recruited culture-confirmed cases of campylobacteriosis reported to state and territory health departments from February 2018 through October 2019. We recruited controls from notified influenza cases in the previous 12 months that were frequency matched to cases by age group, sex, and location. Campylobacter isolates were confirmed to species level by public health laboratories using molecular methods. We conducted backward stepwise multivariable logistic regression to identify significant risk factors. RESULTS: We recruited 571 cases of campylobacteriosis (422 C. jejuni and 84 C. coli) and 586 controls. Important risk factors for campylobacteriosis included eating undercooked chicken (adjusted odds ratio [aOR] 70, 95% CI 13-1296) or cooked chicken (aOR 1.7, 95% CI 1.1-2.8), owning a pet dog aged < 6 months (aOR 6.4, 95% CI 3.4-12), and the regular use of proton-pump inhibitors in the 4 weeks prior to illness (aOR 2.8, 95% CI 1.9-4.3). Risk factors remained similar when analysed specifically for C. jejuni infection. Unique risks for C. coli infection included eating chicken pâté (aOR 6.1, 95% CI 1.5-25) and delicatessen meats (aOR 1.8, 95% CI 1.0-3.3). Eating any chicken carried a high population attributable fraction for campylobacteriosis of 42% (95% CI 13-68), while the attributable fraction for proton-pump inhibitors was 13% (95% CI 8.3-18) and owning a pet dog aged < 6 months was 9.6% (95% CI 6.5-13). The population attributable fractions for these variables were similar when analysed by campylobacter species. Eating delicatessen meats was attributed to 31% (95% CI 0.0-54) of cases for C. coli and eating chicken pâté was attributed to 6.0% (95% CI 0.0-11). CONCLUSIONS: The main risk factor for campylobacteriosis in Australia is consumption of chicken meat. However, contact with young pet dogs may also be an important source of infection. Proton-pump inhibitors are likely to increase vulnerability to infection.

Bayesian latent class analysis to estimate the optimal cut-off for the MilA ELISA for the detection of Mycoplasma bovis antibodies in sera, accounting for repeated measures.

The MilA ELISA has been identified as a highly effective diagnostic tool for the detection of Mycoplasma bovis specific antibodies and has been validated for serological use in previous studies. This study aimed to estimate the optimal cut-off and corresponding estimates of diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the MilA ELISA for testing bovine serum. Serum samples from 298 feedlot cattle from 14 feedlots across four Australian states were tested on entry into the feedlot and approximately 42 days later. The paired serum samples were tested with the MilA ELISA, BIO K302 (Bio-X Diagnostics, Belgium) and BIO K260 (Bio-X Diagnostics, Belgium). A cut-off of 135 AU was estimated to be optimal using Bayesian latent class analysis with three tests in multiple populations, accounting for conditional dependence between tests. At this cut-off, the DSe and DSp of the MilA ELISA were estimated to be 92.1 % (95 % highest probability density [HPD] interval: 87.4, 95.8) and 95.5 % (95 % HPD: 92.4, 97.8), respectively. The DSes of the BIO K260 and BIO K302 ELISAs were estimated to be 60.5 % (95 % HPD: 54.0, 66.9) and 44.6 % (95 % HPD: 38.7, 50.7), respectively. DSps were 95.6 % (95 % HPD: 92.9, 97.7) and 97.8 % (95 % HPD: 95.9, 99.0), respectively. Mycoplasma bovis seroprevalence was remarkably high at follow-up after 42 days on the feedlots. Overall, this study estimated a cut-off, DSe and DSp for the MilA ELISA with less dependence on prior information than previous analyses and demonstrated that the MilA ELISA has higher DSe than the BIO K260 and BIO K302 assays.

Evaluation of the MilA ELISA for the diagnosis of herd infection with Mycoplasma bovis using bulk tank milk and estimation of the prevalence of M. bovis in Australia.

Infection with Mycoplasma bovis has been identified as a growing threat in dairy industries worldwide and there is an urgent need for an inexpensive and accurate herd-level screening tool to identify herds that have been exposed to M. bovis. This study aimed to evaluate the use of the MilA ELISA for testing bulk tank milk (BTM) samples for antibodies against M. bovis and estimate a suitable cut-off and diagnostic sensitivity (DSe) and specificity (DSp) for this assay. An optimal cut-off was then applied for investigating the geographical and seasonal distribution of infection with M. bovis in Australia. A total of 5554 BTM samples from 2683 dairy herds were collected during March, August and December 2017. BTM samples were tested in the MilA ELISA and a cut-off of 29 antibody units (AU) was estimated to be optimal using Bayesian latent class analysis which makes no assumption about the true disease status of herds under investigation. At this cut-off, the DSe and DSp were estimated to be 96.6% (95% highest probability density [HPD] interval: 87.0, 99.8) and 94.2% (95% HPD: 89.9, 97.4), respectively. The diagnostic specifications were found to vary markedly with stage of the production cycle, suggesting that targeted sampling was needed to maximize accuracy. We also found distinct differences in the apparent prevalence of M. bovis in different dairying regions, as well as seasonal variation. The highest apparent prevalence of M. bovis was observed in samples collected in March and an overall drop in the proportion of positive herds was seen from March to December. Overall, this study provides insights into the dynamics of BTM antibodies against M. bovis in Australian dairy herds and how the MilA ELISA can be applied for bulk tank milk testing.

A randomised controlled trial of the immunogenicity and safety of a formaldehyde-inactivated Coxiella burnetii vaccine in 8-week-old goats.

Coxiella burnetii causes Q fever in individuals exposed to infected ruminants. Vaccination in 3-4-month-old goats, has been reported to result in significantly greater reduction in C. burnetii shedding compared to goats vaccinated one month before breeding, the most commonly used strategy of controlling Q fever on infected intensively-managed herds. It is possible that an even greater reduction in the number of animals shedding C. burnetii could be achieved if vaccination were administered shortly after protection from colostrum antibodies wanes and animals become susceptible to infection with C. burnetii. This study aimed to evaluate the immunogenicity and safety of a formaldehyde-inactivated phase 1 C. burnetii vaccine in 8-week-old goats. Two injections, four weeks apart, elicited specific IgM and IgG responses in all vaccinated goats (n = 6), while no antibodies were detected in two control groups (n = 12). Swelling at the site of inoculation was observed in all the vaccinated and in 10/11 of the placebo-treated goats but receded after 3 weeks. Weight change and rectal temperatures were also comparable between vaccinated and control goats. The data indicated that this vaccine could be suitable for immunising 8-week-old goats, although further trials to determine level of protection against challenge are required.

Optimising predictive modelling of Ross River virus using meteorological variables.

BACKGROUND: Statistical models are regularly used in the forecasting and surveillance of infectious diseases to guide public health. Variable selection assists in determining factors associated with disease transmission, however, often overlooked in this process is the evaluation and suitability of the statistical model used in forecasting disease transmission and outbreaks. Here we aim to evaluate several modelling methods to optimise predictive modelling of Ross River virus (RRV) disease notifications and outbreaks in epidemiological important regions of Victoria and Western Australia. METHODOLOGY/PRINCIPAL FINDINGS: We developed several statistical methods using meteorological and RRV surveillance data from July 2000 until June 2018 in Victoria and from July 1991 until June 2018 in Western Australia. Models were developed for 11 Local Government Areas (LGAs) in Victoria and seven LGAs in Western Australia. We found generalised additive models and generalised boosted regression models, and generalised additive models and negative binomial models to be the best fit models when predicting RRV outbreaks and notifications, respectively. No association was found with a model's ability to predict RRV notifications in LGAs with greater RRV activity, or for outbreak predictions to have a higher accuracy in LGAs with greater RRV notifications. Moreover, we assessed the use of factor analysis to generate independent variables used in predictive modelling. In the majority of LGAs, this method did not result in better model predictive performance. CONCLUSIONS/SIGNIFICANCE: We demonstrate that models which are developed and used for predicting disease notifications may not be suitable for predicting disease outbreaks, or vice versa. Furthermore, poor predictive performance in modelling disease transmissions may be the result of inappropriate model selection methods. Our findings provide approaches and methods to facilitate the selection of the best fit statistical model for predicting mosquito-borne disease notifications and outbreaks used for disease surveillance.

Serological evidence for the presence of wobbly possum disease virus in Australia.

Wobbly possum disease virus (WPDV) is an arterivirus that was originally identified in common brushtail possums (Trichosurus vulpecula) in New Zealand, where it causes severe neurological disease. In this study, serum samples (n = 188) from Australian common brushtail, mountain brushtail (Trichosurus cunninghami) and common ringtail (Pseudocheirus peregrinus) possums were tested for antibodies to WPDV using ELISA. Antibodies to WPDV were detected in possums from all three species that were sampled in the states of Victoria and South Australia. Overall, 16% (30/188; 95% CI 11.0-22.0) of possums were seropositive for WPDV and 11.7% (22/188; 95% CI 7.5-17.2) were equivocal. The frequency of WPDV antibody detection was the highest in possums from the two brushtail species. This is the first reported serological evidence of infection with WPDV, or an antigenically similar virus, in Australian possums, and the first study to find antibodies in species other than common brushtail possums. Attempts to detect viral RNA in spleens by PCR were unsuccessful. Further research is needed to characterise the virus in Australian possums and to determine its impact on the ecology of Australian marsupials.

Transmission network reconstruction for foot-and-mouth disease outbreaks incorporating farm-level covariates.

Transmission network modelling to infer 'who infected whom' in infectious disease outbreaks is a highly active area of research. Outbreaks of foot-and-mouth disease have been a key focus of transmission network models that integrate genomic and epidemiological data. The aim of this study was to extend Lau's systematic Bayesian inference framework to incorporate additional parameters representing predominant species and numbers of animals held on a farm. Lau's Bayesian Markov chain Monte Carlo algorithm was reformulated, verified and pseudo-validated on 100 simulated outbreaks populated with demographic data Japan and Australia. The modified model was then implemented on genomic and epidemiological data from the 2010 outbreak of foot-and-mouth disease in Japan, and outputs compared to those from the SCOTTI model implemented in BEAST2. The modified model achieved improvements in overall accuracy when tested on the simulated outbreaks. When implemented on the actual outbreak data from Japan, infected farms that held predominantly pigs were estimated to have five times the transmissibility of infected cattle farms and be 49% less susceptible. The farm-level incubation period was 1 day shorter than the latent period, the timing of the seeding of the outbreak in Japan was inferred, as were key linkages between clusters and features of farms involved in widespread dissemination of this outbreak. To improve accessibility the modified model has been implemented as the R package 'BORIS' for use in future outbreaks.

Comparison of the modified agglutination test and real-time PCR for detection of Toxoplasma gondii exposure in feral cats from Phillip Island, Australia, and risk factors associated with infection.

Toxoplasma gondii is considered a disease risk for many native Australian species. Feral cats are the key definitive host of T. gondii in Australia and therefore, investigating the epidemiology of T. gondii in cat populations is essential to understanding the risk posed to wildlife. Test sensitivity and specificity are poorly defined for diagnostic tests targeting T. gondii in cats and there is a need for validated techniques. This study focused on the feral cat population on Phillip Island, Victoria, Australia. We compared a novel real-time PCR (qPCR) protocol to the modified agglutination test (MAT) and used a Bayesian latent class modelling approach to assess the diagnostic parameters of each assay and estimate the true prevalence of T. gondii in feral cats. In addition, we performed multivariable logistic regression to determine risk factors associated with T. gondii infection in cats. Overall T. gondii prevalence by qPCR and MAT was 79.5% (95% confidence interval 72.6-85.0) and 91.8% (84.6-95.8), respectively. Bayesian modelling estimated the sensitivity and specificity of the MAT as 96.2% (95% credible interval 91.8-98.8) and 82.1% (64.9-93.6), and qPCR as 90.1% (83.6-95.5) and 96.0% (82.1-99.8), respectively. True prevalence of T. gondii infection in feral cats on Phillip Island was estimated as 90.3% (83.2-95.1). Multivariable logistic regression analysis indicated that T. gondii infection was positively associated with weight and this effect was modified by season. Cats trapped in winter had a high probability of infection, regardless of weight. The present study suggests qPCR applied to tissue is a highly sensitive, specific and logistically feasible tool for T. gondii testing in feral cat populations. Additionally, T. gondii infection is highly prevalent in feral cats on Phillip Island, which may have significant impacts on endemic and introduced marsupial populations.

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).

Unravelling animal exposure profiles of human Q fever cases in Queensland, Australia, using natural language processing.

Q fever, caused by the zoonotic bacterium Coxiella burnetii, is a globally distributed emerging infectious disease. Livestock are the most important zoonotic transmission sources, yet infection in people without livestock exposure is common. Identifying potential exposure pathways is necessary to design effective interventions and aid outbreak prevention. We used natural language processing and graphical network methods to provide insights into how Q fever notifications are associated with variation in patient occupations or lifestyles. Using an 18-year time-series of Q fever notifications in Queensland, Australia, we used topic models to test whether compositions of patient answers to follow-up exposure questionnaires varied between demographic groups or across geographical areas. To determine heterogeneity in possible zoonotic exposures, we explored patterns of livestock and game animal co-exposures using Markov Random Fields models. Finally, to identify possible correlates of Q fever case severity, we modelled patient probabilities of being hospitalized as a function of particular exposures. Different demographic groups consistently reported distinct sets of exposure terms and were concentrated in different areas of the state, suggesting the presence of multiple transmission pathways. Macropod exposure was commonly reported among Q fever cases, even when exposure to cattle, sheep or goats was absent. Males, older patients and those that reported macropod exposure were more likely to be hospitalized due to Q fever infection. Our study indicates that follow-up surveillance combined with text modelling is useful for unravelling exposure pathways in the battle to reduce Q fever incidence and associated morbidity.

Spatiotemporal and risk analysis of H5 highly pathogenic avian influenza in Vietnam, 2014-2017.

The aim of this study was to describe the spatiotemporal distribution of H5 HPAI outbreak reports for the period 2014-2017 and to identify factors associated with H5 HPAI outbreak reports. Throughout the study period, a total of 139 outbreaks of H5 HPAI in poultry were reported, due to either H5N1 (96 outbreaks) or H5N6 (43 outbreaks) subtype viruses. H5N1 HPAI outbreaks occurred in all areas of Vietnam while H5N6 HPAI outbreaks were only reported in the northern and central provinces. We counted the number of H5N1 and H5N6 outbreak report-positive districts per province over the four-year study period and calculated the provincial-level standardized morbidity ratio for H5N1 and H5N6 outbreak reports as the observed number of positive districts divided by the expected number. A mixed-effects, zero-inflated Poisson regression model was developed to identify risk factors for outbreak reports of each H5N1 and H5N6 subtype virus. Spatially correlated and uncorrelated random effects terms were included in this model to identify areas of the country where outbreak reports occurred after known risk factors had been accounted-for. The presence of an outbreak report in a province in the previous 6-12 months increased the provincial level H5N1 outbreak report risk by a factor of 2.42 (95% Bayesian credible interval [CrI] 1.27-4.60) while 1000 bird increases in the density of chickens decreased provincial level H5N6 outbreak report risk by a factor of 0.65 (95% CrI 0.38 to 0.97). We document distinctly different patterns in the spatial and temporal distribution of H5N1 and H5N6 outbreak reports. Most of the variation in H5N1 report risk was accounted-for by the fixed effects included in the zero-inflated Poisson model. In contrast, the amount of unaccounted-for risk in the H5N6 model was substantially greater than the H5N1 model. For H5N6 we recommend that targeted investigations should be carried out in provinces with relatively large spatially correlated random effect terms to identify likely determinants of disease. Similarly, investigations should be carried out in provinces with relatively low spatially correlated random effect terms to identify protective factors for disease and/or reasons for failure to report.