Genomics and pathogenesis of the avian coronavirus infectious bronchitis virus.

Infectious bronchitis virus (IBV) is a member of the family Coronaviridae, together with viruses such as SARS-CoV, MERS-CoV and SARS-CoV-2 (the causative agent of the COVID-19 global pandemic). In this family of viruses, interspecies transmission has been reported, so understanding their pathobiology could lead to a better understanding of the emergence of new serotypes. IBV possesses a single-stranded, non-segmented RNA genome about 27.6 kb in length that encodes several non-structural and structural proteins. Most functions of these proteins have been confirmed in IBV, but some other proposed functions have been based on research conducted on other members of the family Coronaviridae. IBV has variable tissue tropism depending on the strain, and can affect the respiratory, reproductive, or urinary tracts; however, IBV can also replicate in other organs. Additionally, the pathogenicity of IBV is also variable, with some strains causing only mild clinical signs, while infection with others results in high mortality rates in chickens. This paper extensively and comprehensibly reviews general aspects of coronaviruses and, more specifically, IBV, with emphasis on protein functions and pathogenesis. The pathogenicity of the Australian strains of IBV is also reviewed, describing the variability between the different groups of strains, from the classical to the novel and recombinant strains. Reverse genetic systems, cloning and cell culture growth techniques applicable to IBV are also reviewed.

Water consumption and wastage behaviour in pigs: implications for antimicrobial administration and stewardship.

Daily water use and wastage patterns of pigs have major effects on the efficacy of in-water antimicrobial dosing events when conducted for metaphylaxis or to treat clinical disease. However, daily water use and wastage patterns of pigs are not routinely quantified on farms and are not well understood. We conducted a prospective, observational 27-day study of the daily water use and wastage patterns of a pen group of 15 finisher pigs reared in a farm building. We found that the group of pigs wasted a median of 36.5% of the water used per day. We developed models of the patterns of water used and wasted by pigs over each 24-h period using a Bayesian statistical method with the brm() function in the brms package. Both patterns were uni-modal, peaking at 1400-1700, and closely aligned. Wastage was slightly greater during hours of higher water use. We have shown that it is feasible to quantify the water use and wastage patterns of pigs in farm buildings using a system that records and aggregates data, and analyses them using hierarchical generalised additive models. This system could support more efficacious in-water antimicrobial dosing on farms, and better antimicrobial stewardship, by helping to reduce the quantities of antimicrobials used and disseminated into the environment.

Antimicrobial stewardship in companion animal practice: an implementation trial in 135 general practice veterinary clinics.

BACKGROUND: Antimicrobial stewardship programmes (ASPs) have been widely implemented in medical practice to improve antimicrobial prescribing and reduce selection for multidrug-resistant pathogens. OBJECTIVES: To implement different antimicrobial stewardship intervention packages in 135 veterinary practices and assess their impact on antimicrobial prescribing. METHODS: In October 2018, general veterinary clinics were assigned to one of three levels of ASP, education only (CON), intermediate (AMS1) or intensive (AMS2). De-identified prescribing data (1 October 2016 to 31 October 2020), sourced from VetCompass Australia, were analysed and a Poisson regression model fitted to identify the effect of the interventions on the incidence rates of antimicrobial prescribing. RESULTS: The overall incidence rate (IR) of antimicrobial prescribing for dogs and cats prior to the intervention was 3.7/100 consultations, which declined by 36% (2.4/100) in the implementation period, and by 50% (1.9/100) during the post-implementation period. Compared with CON, in AMS2 there was a 4% and 6% reduction in the overall IR of antimicrobial prescribing, and a 24% and 24% reduction in IR of high importance antimicrobial prescribing, attributable to the intervention in the implementation and post-implementation periods, respectively. A greater mean difference in the IR of antimicrobial prescribing was seen in high-prescribing clinics. CONCLUSIONS: These AMS interventions had a positive impact in a large group of general veterinary practices, resulting in a decline in overall antimicrobial use and a shift towards use of antimicrobials rated as low importance, with the greatest impact in high-prescribing clinics.

Review: Water medication of growing pigs: sources of between-animal variability in systemic exposure to antimicrobials.

On many Australian commercial pig farms, groups of growing pigs are mass-medicated through their drinking water with selected antimicrobials for short periods to manage herd health. However, delivery of medication in drinking water cannot be assumed to deliver an equal dose to all animals in a group. There is substantial between-animal variability in systemic exposure to an antimicrobial (i.e. the antimicrobial concentration in plasma), resulting in under-dosing or over-dosing of many pigs. Three sources of this between-animal variability during a water medication dosing event are differences in: (1) concentration of the active constituent of the antimicrobial product in water available to pigs at drinking appliances in each pen over time, (2) medicated water consumption patterns of pigs in each pen over time, and (3) pharmacokinetics (i.e. oral bioavailability, volume of distribution and clearance between pigs and within pigs over time). It is essential that factors operating on each farm that influence the range of systemic exposures of pigs to an antimicrobial are factored into antimicrobial administration regimens to reduce under-dosing and over-dosing.

Appraisal of the Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery in dogs and cats.

The Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery on dogs and cats are evidence-based guidelines for veterinary practitioners. Validation of these guidelines is necessary to ensure quality and implementability. Two validated tools, used for medical guideline appraisal, were chosen to assess the guidelines. The terminology from the GuideLine Implementability Appraisal (GLIA) and the Appraisal of Guidelines for Research and Evaluation version 2 (AGREE II) were adapted for use by veterinarians. A two-phase evaluation approach was conducted. In the first phase of the evaluation, the GLIA tool was used by two specialist veterinary surgeons in clinical practice. The results of this phase were then used to modify the guidelines. In the second phase, the AGREE II tool was used by 6 general practitioners and 6 specialists to appraise the guidelines. In phase 1, the specialist surgeons either agreed or strongly agreed that the guidelines were executable, decidable, valid and novel, and that the guidelines would fit within the process of care. The surgeons were neutral on flexibility and measurability. Additional clarity around one common surgical procedure was added to the guidelines, after which the surgeons agreed that the guidelines were sufficiently flexible. In phase 2, 12 veterinarians completed the assessment using the AGREE II tool. In all sections the scaled domain score was greater than 70%. The overall quality of the guidelines was given a global scaled score of 76%. This assessment has demonstrated that the guidelines for antimicrobial prophylaxis for companion animal surgery are valid and appear implementable.

Survey of veterinary prescribing for poultry disease.

BACKGROUND: To enable better antimicrobial stewardship, it is important to know what the patterns of prescribing behaviour are and what diseases or reasons antibiotics are being prescribed. A prescribing guideline (i.e. recommended best practice for writing prescriptions) developed by the Australian poultry veterinarians exists. However, it is not a prescribing guideline detailing treatments for the commonly observed bacterial diseases in commercial poultry. METHODS: An online survey was deployed to all registered veterinarians and members of the Australian Veterinary Poultry Association to identify prescribing behaviours for the most frequently observed bacterial diseases of poultry. RESULTS: A total of 39 survey responses were received. Most surveys were started but not completed; 13 (33%) were completed with 18 (46%) containing some information on prescribing. The most frequent treatment responses were for Escherichia coli in both layers and broilers, chronic respiratory disease (CRD), fowl cholera and spotty liver in layers and necrotic enteritis in broilers. Treatments described were for products registered for poultry use, within the recommended label dose and duration of treatment (Tables 1, 2). Unsurprisingly, tetracyclines and amoxycillin, followed by lincomycin and trimethoprim sulfonamide products were the most frequently reported treatment options. Inappropriate treatments were reported for salmonellosis and one veterinarian recommended the use of enrofloxacin for the treatment of fowl cholera. CONCLUSION: Information provided by respondents will enable the initial development of prescribing guidelines for both commercial and small poultry flocks. Importantly, it identified less than optimal prescribing behaviour for some diseases, a reliance on one class of antibiotic more than others and a failure to utilise all antimicrobial classes potentially available for treatment. Critically, the survey identified a lack of treatment options for bacterial disease in poultry. The most important bacterial diseases of poultry remain the same; effective alternatives for antibiotic treatment are required and old diseases, thought once gone, are reinventing themselves as problems for the future. Surveys of prescribing behaviours are essential for identifying diseases of high priority, changes in treatments and response to treatment and to identify areas for targeted antimicrobial stewardship, and research needs.

Characterisation of the course of Mycoplasma bovis infection in naturally infected dairy herds.

Mycoplasma bovis causes bovine respiratory disease, mastitis, arthritis and otitis. The importance of M. bovis has escalated because of recent outbreaks and introductions into countries previously free of M. bovis. We characterized the course of M. bovis infection on 19 recently infected dairy farms over 24 months. Our objective was to identify diagnostic tools to assess the efficacy of control measures to assess low risk infection status on M. bovis infected farms. PCR assays and culture were used to detect M. bovis, and in-house and BioX ELISAs were used to follow antibody responses. Cows and young stock were sampled on four separate occasions, and clinical cases were sampled when they arose. On 17 farms, a few cases of clinical mastitis were detected, mostly within the first eight weeks after the index case. Antibodies detected by in-house ELISA persisted in the serum of cows at least for 1.5 years on all farms, regardless of the M. bovis infection status or signs of clinical disease or subclinical mastitis on the farm. Six out of 19 farms became low risk as the infection was resolved. Our results suggest that, for biosecurity purposes, regular monitoring should be conducted on herds by screening for M. bovis in samples from cows with clinical mastitis and calves with pneumonia, in conjunction with testing young stock by screening longitudinally collected nasal swabs for M. bovis and sequential serum samples for antibody against recombinant antigen.

Antimicrobial dosing for common equine drugs: a content review and practical advice for veterinarians in Australia.

BACKGROUND: Appropriate dosing with antimicrobial agents is critical for effective treatment and to prevent the development of antimicrobial resistance. METHODS: A review was undertaken of equine journal articles (Equine Veterinary Journal, Equine Veterinary Education, Australian Veterinary Journal, Australian Equine Veterinarian, Journal of Veterinary Internal Medicine and Journal of Equine Veterinary Science) between January 2015 and August 2018. Those with dosing regimens for procaine penicillin G, gentamicin or trimethoprim-sulfonamide in adult horses were examined and evaluated. Pharmacokinetics and -dynamics of these drugs were also reviewed. RESULTS & CONCLUSION: The most frequently reported doses for penicillin, gentamicin and trimethoprim-sulfonamide were 20-25,000 IU/kg, 6.6 mg/kg and 30 mg/kg, respectively. Veterinarians treating equine patients in Australia should be aware of the current recommended doses and inter-dosing intervals to ensure efficacy in therapy and to preserve the usefulness of these antimicrobials for the future.

First detection of bovine noroviruses and detection of bovine coronavirus in Australian dairy cattle.

BACKGROUND AND OBJECTIVE: Noroviruses have been recognised as a significant cause of neonatal enteritis in calves in many countries, but there has been no investigation of their occurrence in Australian cattle. This study aimed to establish whether bovine noroviruses could be detected in faecal samples from Australian dairy cattle. It also sought to determine whether bovine coronaviruses, also associated with neonatal enteritis in calves, could be detected in the same faecal samples. METHODS: A selection of faecal samples that were negative for rotaviruses from dairy farms located in three geographically distinct regions of Victoria were pooled and tested by reverse transcription-PCR for the presence of noroviruses (genogroup III), neboviruses and bovine coronaviruses. RESULTS AND CONCLUSION: Genetically distinct genogroup III noroviruses were detected in two sample pools from different geographic regions and bovine coronavirus was detected in a third pool of samples. This is the first report of bovine norovirus infection in Australian cattle and suggests that future work is required to determine the significance of these agents as a cause of bovine enteric disease in Australia.

Antimicrobial labelling in Australia: a threat to antimicrobial stewardship?

Antimicrobial resistance is a public health emergency, placing veterinary antimicrobial use under growing scrutiny. Antimicrobial stewardship, through appropriate use of antimicrobials, is a response to this threat. The need for antimicrobial stewardship in Australian veterinary practices has had limited investigation. A 2016 survey undertaken to investigate antimicrobial usage patterns by Australian veterinarians found that antimicrobial dose rates were varied and often inappropriate. Doses of procaine penicillin in horses and cattle were often low, with 68% and 90% of respondents, respectively, reporting doses that were unlikely to result in plasma concentrations above minimum inhibitory concentrations for common equine and bovine pathogens. Frequency of penicillin administration was also often inappropriate. Gentamicin doses in horses were largely appropriate (89% of dose rates appropriate), but 9% of respondents reported twice daily dosing. Amoxycillin and amoxycillin-clavulanate were administered at the appropriate doses, or above, to dogs and cats by 54% and 70% of respondents, respectively. Here, we explore the potential reasons for inappropriate antimicrobial dose regimens and report that antimicrobial labels often recommend incorrect dose rates and thus may be contributing to poor prescribing practices. Changes to legislation are needed to ensure that antimicrobial drug labels are regularly updated to reflect the dose needed to effectively and safely treat common veterinary pathogens. This will be especially true if changes in legislation restrict antimicrobial use by veterinarians to the uses and doses specified on the label, thus hampering the current momentum towards improved antimicrobial stewardship.

Antimicrobial susceptibility testing by Australian veterinary diagnostic laboratories.

BACKGROUND: The national strategy for tackling antimicrobial resistance highlights the need for antimicrobial stewardship in veterinary practice and for surveillance of antimicrobial susceptibility in veterinary pathogens. Diagnostic laboratories have an important role in facilitating both of these processes, but it is unclear whether data from veterinary diagnostic laboratories are similar enough to allow for compilation and if there is consistent promotion of appropriate antimicrobial use embedded in the approaches of different laboratories to susceptibility testing. METHODS: A cross-sectional study of antimicrobial susceptibility testing and reporting procedures by Australian veterinary diagnostic laboratories was conducted in 2017 using an online questionnaire. All 18 veterinary diagnostic laboratories in Australia completed the questionnaire. RESULTS: Kirby-Bauer disc diffusion was the method predominantly used for antimicrobial susceptibility testing and was used to evaluate 86% of all isolates, although two different protocols were used across the 18 laboratories (CLSI 15/18, CDS 3/18). Minimum inhibitory concentrations were never reported by 61% of laboratories. Common isolates were consistently reported on across all species, except for gram-negative isolates in pigs, for which there was some variation in the approach to reporting. There was considerable diversity in the panels of antimicrobials used for susceptibility testing on common isolates and no consistency was apparent between laboratories for any bacterial species. CONCLUSION: We recommend that nationally agreed and consistent antimicrobial panels for routine susceptibility testing should be developed and a uniform set of guidelines should be adopted by veterinary diagnostic laboratories in Australia.

Barriers to and enablers of implementing antimicrobial stewardship programs in veterinary practices.

BACKGROUND: Antimicrobial stewardship (AMS) programs are yet to be widely implemented in veterinary practice and medical programs are unlikely to be directly applicable to veterinary settings. OBJECTIVE: To gain an in-depth understanding of the factors that influence effective AMS in veterinary practices in Australia. METHODS: A concurrent explanatory mixed methods design was used. The quantitative phase of the study consisted of an online questionnaire to assess veterinarians' attitudes to antimicrobial resistance (AMR) and antimicrobial use in animals, and the extent to which AMS currently is implemented (knowingly or unknowingly). The qualitative phase used semi-structured interviews to gain an understanding of the barriers to and enablers of AMS in veterinary practices. Data were collected and entered into NVivo v.11, openly coded and analyzed according to mixed methods data analysis principles. RESULTS: Companion animal, equine, and bovine veterinarians participated in the study. Veterinary practices rarely had antimicrobial prescribing policies. The key barriers were a lack of AMS governance structures, client expectations and competition between practices, cost of microbiological testing, and lack of access to education, training and AMS resources. The enablers were concern for the role of veterinary antimicrobial use in development of AMR in humans, a sense of pride in the service provided, and preparedness to change prescribing practices. CONCLUSION AND CLINICAL IMPORTANCE: Our study can guide development and establishment of AMS programs in veterinary practices by defining the major issues that influence the prescribing behavior of veterinarians.

Antimicrobials used for surgical prophylaxis by equine veterinary practitioners in Australia.

BACKGROUND: Antimicrobials are widely used in Australian veterinary practices, but no investigation into the classes of antimicrobials used, or the appropriateness of use in horses, has been conducted. OBJECTIVES: The aim of the study was to describe antimicrobial use for surgical prophylaxis in equine practice in Australia. STUDY DESIGN: Cross-sectional questionnaire survey. METHODS: An online questionnaire was used to document antimicrobial usage patterns. Information solicited in the questionnaire included demographic details of the respondents, the frequency with which antimicrobials were used for specific surgical conditions (including the dose, timing and duration of therapy) and practice antimicrobial use policies and sources of information about antimicrobials and their uses. RESULTS: A total of 337 members of the Australian veterinary profession completed the survey. Generally, the choice of antimicrobial was appropriate for the specified equine surgical condition, but the dose and duration of therapy varied greatly. While there was poor optimal compliance with British Equine Veterinary Association guidelines in all scenarios (range 1-15%), except removal of a nonulcerated dermal mass (42%), suboptimal compliance (compliant antimicrobial drug selection but inappropriate timing, dose or duration of therapy) was moderate for all scenarios (range 48-68%), except for an uninfected contaminated wound over the thorax, where both optimal and suboptimal compliance was very poor (1%). Veterinarians practicing at a university hospital had higher odds of compliance than general practice veterinarians (Odds ratio 3.2, 95% CI, 1.1-8.9, P = 0.03). MAIN LIMITATIONS: Many survey responses were collected at conferences which may introduce selection bias, as veterinarians attending conferences may be more likely to have been exposed to contemporary antimicrobial prescribing recommendations. CONCLUSIONS: Antimicrobial use guidelines need to be developed and promoted to improve the responsible use of antimicrobials in equine practice in Australia. An emphasis should be placed on antimicrobial therapy for wounds and appropriate dosing for procaine penicillin.

Comparative Metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum Reveals Fundamental Differences in Active Metabolic Pathways and Suggests Novel Gene Annotations.

Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here, we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of mycoplasma metabolite extracts revealed significant differences in the steady-state levels of many metabolites in central carbon metabolism, while 13C stable isotope labeling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilization, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterizing significant differences in the metabolism of different bacterial species and in improving genome annotation. IMPORTANCE Mycoplasmas are pathogenic bacteria that cause serious chronic infections in production animals, resulting in considerable losses worldwide, as well as causing disease in humans. These bacteria have extremely reduced genomes and are thought to have limited metabolic flexibility, even though they are highly successful persistent parasites in a diverse number of species. The extent to which different Mycoplasma species are capable of catabolizing host carbon sources and nutrients, or synthesizing essential metabolites, remains poorly defined. We have used advanced metabolomic techniques to identify metabolic pathways that are active in two species of Mycoplasma that infect distinct hosts (poultry and cattle). We show that these species exhibit marked differences in metabolite steady-state levels and carbon source utilization. This information has been used to functionally characterize previously unknown genes in the genomes of these pathogens. These species-specific differences are likely to reflect important differences in host nutrient levels and pathogenic mechanisms.

Survey of Victorian small ruminant herds for mycoplasmas associated with contagious agalactia and molecular characterisation of Mycoplasma mycoides subspecies capri isolates from one herd.

OBJECTIVE: Regarded as one of the most expensive production diseases of dairy sheep and goats, contagious agalactia (CA) is caused by any of four agents: Mycoplasma agalactiae, M. mycoides subspecies capri (Mmc), M. capricolum subspecies capricolum (Mcc) and M. putrefaciens. Although CA is worldwide in distribution, it has not been reported in Australia, even though studies between the 1950s and 1980s isolated each agent from sheep or goats without any clinical signs associated with it. The aim of this study was to examine sheep and goats in Victoria, Australia, for the presence of CA-associated mycoplasmas and to investigate the evolutionary relationships of these isolates by comparing their genetic differences with their counterparts from other parts of the world. METHODS: A 3-year epidemiological survey of small ruminant populations in Victoria, Australia, was conducted for the presence of CA-associated mycoplasmas and the isolates obtained were genotyped by multilocus sequence typing (MLST). RESULTS: Mmc was the only CA-associated agent isolated from the 1358 samples analysed in the study, but was not associated with CA on the property where it was found. MLST analyses of Mmc strains revealed a distinct clustering of Australian isolates into a novel clade, with the closest relatives being strains from Europe. The distinct clustering is consistent with the absence of clinical disease in Australia. CONCLUSION: The isolation of Mmc indicates that this subspecies persists in Australian small ruminant populations. However, full genome sequencing and in vitro animal experimentation are needed to unequivocally demonstrate the avirulence of Australian strains.

Antimicrobial Prescribing in Dogs and Cats in Australia: Results of the Australasian Infectious Disease Advisory Panel Survey.

BACKGROUND: Investigations of antimicrobial use in companion animals are limited. With the growing recognition of the need for improved antimicrobial stewardship, there is urgent need for more detailed understanding of the patterns of antimicrobial use in this sector. OBJECTIVES: To investigate antimicrobial use for medical and surgical conditions in dogs and cats by Australian veterinarians. METHODS: A cross-sectional study was performed over 4 months in 2011. Respondents were asked about their choices of antimicrobials for empirical therapy of diseases in dogs and cats, duration of therapy, and selection based on culture and susceptibility testing, for common conditions framed as case scenarios: 11 medical, 2 surgical, and 8 dermatological. RESULTS: A total of 892 of the 1,029 members of the Australian veterinary profession that completed the survey satisfied the selection criteria. Empirical antimicrobial therapy was more common for acute conditions (76%) than chronic conditions (24%). Overall, the most common antimicrobial classes were potentiated aminopenicillins (36%), fluoroquinolones (15%), first- and second-generation cephalosporins (14%), and tetracyclines (11%). Third-generation cephalosporins were more frequently used in cats (16%) compared to dogs (2%). Agreement with Australasian Infectious Disease Advisory Panel (AIDAP) guidelines (generated subsequently) was variable ranging from 0 to 69% between conditions. CONCLUSIONS AND CLINICAL IMPORTANCE: Choice of antimicrobials by Australian veterinary practitioners was generally appropriate, with relatively low use of drugs of high importance, except for the empirical use of fluoroquinolones in dogs, particularly for otitis externa and 3rd-generation cephalosporins in cats. Future surveys will determine whether introduction of the 2013 AIDAP therapeutic guidelines has influenced prescribing habits.

Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.