A WIDESPREAD NOVEL GAMMAHERPESVIRUS IN APPARENTLY HEALTHY WILD QUOKKAS (SETONIX BRACHYURUS): A THREATENED AND ENDEMIC WALLABY OF WESTERN AUSTRALIA.

Five genetically distinct macropodid marsupial herpesviruses have been reported [Macropodid alphaherpesviruses 1 and 2 (MaHV-1 and -2); Macropodid herpesviruses 3 to 5 (MaHV-3 to -5)]. MaHV-2 was originally isolated from an outbreak of fatal disease in captive quokkas (Setonix brachyurus) that were in contact with other macropodid species. This warranted a survey of the presence of herpesviruses in this threatened and endemic Western Australian (WA) wallaby. Blood samples from 142 apparently healthy quokkas were tested for exposure to MaHV-1 and -2 by serology. Of these 142, 121 [Rottnest Island (RI), n = 93; mainland WA, n = 28] were tested for herpesvirus infection by polymerase chain reaction (PCR). Antibodies to MaHV-1 and -2 were detected in one individual [prevalence, 0.7%; 95% confidence interval (CI), 0.1%-3.2%] from the mainland and none from RI. However, a novel gammaherpesvirus [designated Macropodid herpesvirus 6 (MaHV-6)] was detected by PCR in the blood of 13 of 121 individuals (11%; 95% CI, 6.2-17.2). Infection with MaHV-6 was significantly more prevalent on the mainland (7/28; i.e., 25%) compared with RI (6/93; i.e., 6.45%; difference in sample proportions, 95% CI, 6%-32%; P = 0.015). There was no association (P > 0.05) between infection with MaHV-6 and differences in hematology, blood chemistry, peripheral blood cell morphologies, or on clinical status. There was a significant association between infection with MaHV-6 and the presence of Theileria spp. in blood [odds ratio (OR) = 11.0; 95% CI, 2.31-52.3; P = 0.001] and yeast in the nasal lining (OR = 7.0; 95% CI, 1.54-31.8; P = 0.021), suggesting that quokkas may be more susceptible to infection with these microorganisms if also infected with MaHV-6. MaHV-6 infection may be a catalyst for vulnerability to disease with other infectious agents and may pose a significant threat to other macropods. These findings have implications for in situ and ex situ management programs of quokkas.

Spatial Distribution of Salmonella enterica in Poultry Shed Environments Observed by Intensive Longitudinal Environmental Sampling.

Detection of salmonellae within poultry environments is an important component of many food safety programs, but sampling approaches vary greatly and may not enable the detection of salmonellae when bacteria are present at a low prevalence or concentration. Intensive longitudinal sampling within caged sheds enabled us to undertake a longitudinal analysis of the spatial distribution of salmonellae in caged shed environments. Both the number of samples collected and location of sample collection within a poultry shed were important to ensure the best chance of detecting Salmonella spp. Differences in the within-shed spatial distribution of Salmonella enterica subspecies enterica serovar Typhimurium [χ2(27, 1,538) = 54.4; P < 0.001] and Salmonella enterica subspecies enterica serovar Infantis [χ2(27, 1,538) = 79.8; P < 0.0001] were identified. More than one Salmonella enterica serovar was detected in each shed on the same sampling occasion; 5% of all samples contained more than one serovar. Samples collected on the north side of the shed (odds ratio [OR], 1.77; 95% confidence interval [CI], 1.17-2.68), on the sheltered side of the shed (OR, 1.90; 95% CI, 1.26-2.89), and during winter (OR, 48.41; 95% CI, 23.56-104.19) were more likely to be positive for salmonellae. The within-shed differences observed in the both the sample prevalence and spatial location of the serovar detected indicate that there are important shed microenvironmental factors that influence the survival and/or distribution of salmonellae. These factors should be taken into consideration when environmental surveillance is undertaken for salmonellae in flocks housed in cage sheds.IMPORTANCE Routine epidemiological surveillance for salmonellae in poultry relies initially on environmental sampling. Intensive, spatially homogenous sampling, as conducted within this study, confirmed that the sampling methodology conducted within a poultry environment is a nontrivial part of sampling design. The frequency of sampling is especially important when the prevalence of Salmonella spp. is low. These factors must be taken into consideration in the design of studies for the detection of salmonellae in poultry sheds.

Traditional Salmonella Typhimurium typing tools (phage typing and MLVA) are sufficient to resolve well-defined outbreak events only.

Between 1991 and 2014 the per capita notification rate of salmonellosis in Australia increased from 31.9 to 69.7 cases per 100,000 people. Salmonella Typhimurium accounted for nearly half the human cases until the end of 2014. In this study, we used cluster analysis tools to compare S. Typhimurium isolates from a chicken-meat study with those reported to the National Enteric Pathogen Surveillance System (NEPSS) from the coincident human and non-human populations. There was limited phage type diversity within all populations and a lack of specificity of MLVA profiling within phage types. The chicken-meat study isolates were not significantly clustered with the human cases and at least 7 non-human sources, based on typing profiles (PT/MLVA combination), could be implicated as a source of human cases during the same period. In the absence of a strong surveillance system representative of all putative sources, MLVA and phage typing alone or in combination are insufficient to identify the source of human cases.

A longitudinal study of serological responses to Coxiella burnetii and shedding at kidding among intensively-managed goats supports early use of vaccines.

Vaccination against Coxiella burnetii, the cause of Q fever, is reportedly the only feasible strategy of eradicating infection in ruminant herds. Preventive vaccination of seronegative goats is more effective in reducing shedding of C. burnetii than vaccinating seropositive goats. The age at which goats born on heavily-contaminated farms first seroconvert to C. burnetii has not yet been documented. In a 16-month birth cohort study, the age at which goats seroconverted against C. burnetii was investigated; 95 goats were bled every 2 weeks and tested for antibodies against C. burnetii. Risk factors for seroconversion were explored and goats shedding C. burnetii were identified by testing vaginal swabs taken at the goats' first kidding using a com1 polymerase chain reaction assay. The first surge in the number of goats with IgM to C. burnetii was observed at week 9. Thus, a first vaccination not later than 8 weeks of age to control C. burnetii in highly contaminated environments is indicated. The odds of seroconversion were 2.0 times higher [95% confidence interval (CI) 1.2, 3.5] in kids born by does with serological evidence of recent infection (IgM seropositive) compared to kids born by IgM seronegative does, suggesting either in utero transmission or peri-parturient infection. The rate of seroconversion was 4.5 times higher (95% CI 2.1, 9.8) during than outside the kidding season, highlighting the risk posed by C. burnetii shed during kidding, even to goats outside the kidding herd. Shedding of C. burnetii at kidding was detected in 15 out of 41 goats infected before breeding.

Antimicrobial use in laboratory rodent facilities in Australia and New Zealand- a cross-sectional survey of veterinarians and facility managers.

This cross-sectional study surveyed veterinarians and facility managers to characterise the use of antimicrobials in laboratory rodent facilities within Australia and New Zealand. Most facilities (71%) reported routine administration of antimicrobials. The indications for antibiotic use reflected those described in publications and differed significantly to reasons for use in non-laboratory animals. Antimicrobials used include those of critical importance to human health, and access to these drugs is unregulated, as prescription-only classes are ordered through research catalogues, without human or veterinary physician prescriptions. The ways in which antimicrobials are used in Australian and New Zealand rodent facilities are likely contributing to antimicrobial resistance within rodent populations, particularly as they are largely administered in drinking water, risking subtherapeutic dosing. Much antimicrobial use reported is unnecessary and could be replaced with changes to husbandry and handling. The generation of resistance in both pathogenic and commensal microbes may also represent a work health and safety issue for humans working with these animals. Reported disposal of antimicrobials included discharge into wastewater, without inactivation, and some respondents reported disposal of substrate, or soiled bedding, nesting material, and disposable enrichment items, from treated animals and medicated feed into landfill, without prior inactivation. Environmental contamination with resistant microbes and antimicrobials is a significant driver of antimicrobial resistance. As such, significant opportunities exist to implement judicious and responsible use of antimicrobials within research rodent facilities in Australia and New Zealand, with a particular focus on instituting aseptic surgery, optimising dosing regimens, and inactivation of medicated water and substrate before disposal.

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.

Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen.

Classical swine fever (CSF) is a highly contagious and severe viral disease of swine resulting in substantial production losses in different farming systems in many regions of the world. The accurate and rapid detection of CSF outbreaks is reliant on sensitive and specific laboratory testing and is a key component of disease control. Specific detection of CSF virus can be achieved by virus isolation in tissue culture, antigen capture or the detection of viral RNA using molecular techniques. In order to reduce the time taken to achieve a diagnostic result and simplify testing methods, an antigen capture ELISA using immunomagnetic beads (IMB) as the solid phase was developed and compared to a microplate-based antigen capture (AC)-ELISA. The IMB-ELISA has up to 64-fold greater analytical sensitivity than the AC-ELISA and initial estimates of diagnostic sensitivity and specificity are 100%. The IMB-ELISA has a highly robust, rapid and stable test format and is simpler to perform than the AC-ELISA. The IMB-ELISA has the added advantage that a result can be sensitively and specifically determined by eye, lending it to the possibility of adaptation to a near-to-field test with minimal equipment or expertise needed.

Salmonella spp. transmission in a vertically integrated poultry operation: Clustering and diversity analysis using phenotyping (serotyping, phage typing) and genotyping (MLVA).

The transmission of Salmonella enterica within a vertically integrated poultry operation was investigated longitudinally over an 18-month period (2013-2014). Thirty six percent of all samples collected (1503 of 4219) were positive for salmonellae with seven Salmonella enterica subsp. enterica serovars, and one Salmonella enterica subsp. salamae serovar detected. Both Salmonella enterica subsp. enterica serovars Infantis and Typhimurium were detected in all locations sampled. Salmonella Typhimurium was the most frequently detected serovar (63% of serotyped samples) with 8 phage types (PT) and 41 multiple-locus variable-number tandem-repeats analysis (MLVA) profiles identified. The most frequently identified phage types were PT135a and DT135. A total of 62 PT/MLVA combinations were observed. MLVA profiles 03-14-10-09-525 and 03-15-11-11-525 were the most frequently identified and 83% of the isolates shared at least one MLVA profile with an isolate from another phage type. The use of phage typing and MLVA profiling, on their own or in combination, were insufficient to understand the complexity of the epidemiological relationships between locations within this production system. Despite the high level of apparent diversity, cluster analysis was unable to differentiate the transmission pathways of all S. Typhimurium variants detected within the integrated enterprise. Using additional epidemiological information, the parent breeder rearing site was identified as the most likely point of introduction of two S. Typhimurium isolates into the production system with subsequent dissemination to the broiler flocks via the hatchery. This complexity is unable to be resolved in the absence of intensive sampling programs at all generations of the production system.

Peripartum dynamics of Coxiella burnetii infections in intensively managed dairy goats associated with a Q fever outbreak in Australia.

Coxiella burnetii may cause reproduction disorders in pregnant animals but subclinical infection in other animals. Unrecognised disease may delay implementation of control interventions, resulting in transmission of infection to other livestock and to humans. Seroreactivity to C. burnetii phase-specific antigens, is routinely used to interpret the course of human Q fever. This approach could be similarly useful in identifying new and existing infections in livestock herds to help describe risk factors or production losses associated with the infections and the implementation of disease-control interventions. This study aimed to elucidate the dynamics of C. burnetii infections using seroreactivity to phase-specific antigens and to examine the impact of infection on milk yield in goats in an endemically-infected farm that was associated with a Q fever outbreak in Australia. Seroreactivity pre- and post-partum and milk yield were studied in 164 goats (86 nulliparous and 78 parous). Post-partum, the seroprevalence of antibodies to C. burnetti increased from 4.7% to 31.4% throughout goats' first kiddings and from 47.4% to 55.1% in goats kidding for the second or greater time. Of 123 goats that were seronegative pre-partum, 26.8% seroconverted over the three-month peri-partum period, highlighting the importance of controlling infection throughout this time. The risk of seroconversion was comparable in first or later kidders, suggesting constant risk irrespective of parity. No loss in milk production associated with seroconversion to phase 2 was observed within the first nine weeks of lactation. However, seroconversion to only phase 1 was associated with extra 0.276L of milk per day (95% Confidence Interval: 0.010, 0.543; P=0.042), which warrants further investigation to ascertain whether or not the association is causal. Further studies on seroreactivity and milk production over longer periods are required, as milk production loss caused by C. burnetti may be an additional reason to control the disease in goat herds.

Estimating the intra-cluster correlation coefficient for evaluating an educational intervention program to improve rabies awareness and dog bite prevention among children in Sikkim, India: A pilot study.

BACKGROUND: Educational initiatives targeting at-risk populations have long been recognized as a mainstay of ongoing rabies control efforts. Cluster-based studies are often utilized to assess levels of knowledge, attitudes and practices of a population in response to education campaigns. The design of cluster-based studies requires estimates of intra-cluster correlation coefficients obtained from previous studies. This study estimates the school-level intra-cluster correlation coefficient (ICC) for rabies knowledge change following an educational intervention program. METHODS: A cross-sectional survey was conducted with 226 students from 7 schools in Sikkim, India, using cluster sampling. In order to assess knowledge uptake, rabies education sessions with pre- and post-session questionnaires were administered. Paired differences of proportions were estimated for questions answered correctly. A mixed effects logistic regression model was developed to estimate school-level and student-level ICCs and to test for associations between gender, age, school location and educational level. RESULTS: The school- and student-level ICCs for rabies knowledge and awareness were 0.04 (95% CI: 0.01, 0.19) and 0.05 (95% CI: 0.2, 0.09), respectively. These ICCs suggest design effect multipliers of 5.45 schools and 1.05 students per school, will be required when estimating sample sizes and designing future cluster randomized trials. There was a good baseline level of rabies knowledge (mean pre-session score 71%), however, key knowledge gaps were identified in understanding appropriate behavior around scared dogs, potential sources of rabies and how to correctly order post rabies exposure precaution steps. After adjusting for the effect of gender, age, school location and education level, school and individual post-session test scores improved by 19%, with similar performance amongst boys and girls attending schools in urban and rural regions. The proportion of participants that were able to correctly order post-exposure precautionary steps following educational intervention increased by 87%. CONCLUSION: The ICC estimates presented in this study will aid in designing cluster-based studies evaluating educational interventions as part of disease control programs. This study demonstrates the likely benefits of educational intervention incorporating bite prevention and rabies education.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants.

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses.

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.

Detection and identification of a gammaherpesvirus in Antechinus spp. in Australia.

We detected herpesvirus infection in a male yellow-footed antechinus (Antechinus flavipes) and male agile antechinus (Antechinus agilis) during the period of postmating male antechinus immunosuppression and mortality. Histopathologic examination of tissues revealed lesions consistent with herpesvirus infection in the prostate of both animals. Herpesvirus virions were observed by transmission electron microscopy in the prostate tissue collected from the male yellow-footed antechinus. Herpesvirus DNA was detected in prostate, liver, lung, kidney, spleen, and ocular/nasal tissues using a pan-herpesvirus PCR targeting the viral DNA polymerase. Nucleotide sequencing identified a novel herpesvirus from the Gammaherpesvirinae subfamily that we have tentatively designated dasyurid herpesvirus 1 (DaHV-1).

Isolation and characterization of a novel herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteus).

We isolated a macropodid herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteous) displaying clinical signs of respiratory disease and possibly neurologic disease. Sequence analysis of the herpesvirus glycoprotein G (gG) and glycoprotein B (gB) genes revealed that the virus was an alphaherpesvirus most closely related to macropodid herpesvirus 2 (MaHV-2) with 82.7% gG and 94.6% gB amino acid sequence identity. Serologic analyses showed similar cross-neutralization patterns to those of MaHV-2. The two viruses had different growth characteristics in cell culture. Most notably, this virus formed significantly larger plaques and extensive syncytia when compared with MaHV-2. No syncytia were observed for MaHV-2. Restriction endonuclease analysis of whole viral genomes demonstrated distinct restriction endonuclease cleavage patterns for all three macropodid herpesviruses. These studies suggest that a distinct macropodid alphaherpesvirus may be capable of infecting and causing disease in eastern grey kangaroos.

Detection of a second novel gammaherpesvirus in a free-ranging koala (Phascolarctos cinereus).

A second novel gammaherpesvirus was detected in a free-ranging koala (Phascolarctos cinereus) shown previously to be infected with phascolarctid herpesvirus 1. Analysis of the DNA polymerase gene showed that the virus was genetically distinct from all known gammaherpesviruses. This is the first reported dual gammaherpesvirus infection in an Australian marsupial.