Evaluation of the accuracy of the IDvet serological test for Mycoplasma bovis infection in cattle using latent class analysis of paired serum ELISA and quantitative real-time PCR on tonsillar swabs sampled at slaughter.

Mycoplasma bovis (Mbovis) was first detected in cattle in New Zealand (NZ) in July 2017. To prevent further spread, NZ launched a world-first National Eradication Programme in May 2018. Existing diagnostic tests for Mbovis have been applied in countries where Mbovis is endemic, for detecting infection following outbreaks of clinical disease. Diagnostic test evaluation (DTE) under NZ conditions was thus required to inform the Programme. We used Bayesian Latent Class Analysis on paired serum ELISA (ID Screen Mycoplasma bovis Indirect from IDvet) and tonsillar swabs (qPCR) for DTE in the absence of a gold standard. Tested samples were collected at slaughter between June 2018 and November 2019, from infected herds depopulated by the Programme. A first set of models evaluated the detection of active infection, i.e. the presence of Mbovis in the host. At a modified serology positivity threshold of SP%> = 90, estimates of animal-level ELISA sensitivity was 72.8% (95% credible interval 68.5%-77.4%), respectively 97.7% (95% credible interval 97.3%-98.1%) for specificity, while the qPCR sensitivity was 45.2% (95% credible interval 41.0%-49.8%), respectively 99.6% (95% credible interval 99.4%-99.8%) for specificity. In a second set of models, prior information about ELISA specificity was obtained from the National Beef Cattle Surveillance Programme, a population theoretically free-or very low prevalence-of Mbovis. These analyses aimed to evaluate the accuracy of the ELISA test targeting prior exposure to Mbovis, rather than active infection. The specificity of the ELISA for detecting exposure to Mbovis was 99.9% (95% credible interval 99.7%-100.0%), hence near perfect at the threshold SP%=90. This specificity estimate, considerably higher than in the first set of models, was equivalent to the manufacturer's estimate. The corresponding ELISA sensitivity estimate was 66.0% (95% credible interval 62.7%-70.7%). These results confirm that the IDvet ELISA test is an appropriate tool for determining exposure and infection status of herds, both to delimit and confirm the absence of Mbovis.

Use of scenario tree modelling to plan freedom from infection surveillance: Mycoplasma bovis in New Zealand.

Since mid-2018, the New Zealand (NZ) Ministry for Primary Industries (MPI) has been operating an eradication program for an incursion of Mycoplasma bovis. Although NZ is still delimiting the outbreak, consideration is being given to how freedom from M. bovis will be demonstrated. Rapid demonstration of freedom will minimise the length of the program, significantly reducing its financial burden. This collaborative research was undertaken to help inform planning of surveillance to demonstrate freedom after M. bovis is believed eradicated. Scenario tree modelling (STM) involves assimilating multiple surveillance system components to determine whether disease is absent. STM has infrequently been used to plan appropriate surveillance but this was the approach used here. A stochastic simulation model was implemented in R. The model represented the NZ commercial dairy and non-dairy cattle industries and the current surveillance components that are also planned to be used to gather evidence of absence of M. bovis once it is eradicated. Different surveillance intensities and risk based versus random surveillance were simulated and compared for probability of freedom, financial cost of sampling and testing and the time to demonstrate freedom. The results indicate that the current surveillance components will enable demonstration of freedom. Surveillance components included bulk tank milk testing, herd testing and testing at meat processing plants, predominantly using an imperfect ELISA. Several combinations of surveillance components appeared most efficient achieving >95 % confidence of freedom over 2-4 years, whilst sampling 4-7 % of the non-dairy herds and less than 25 % of dairy herds annually. The results indicate that surveillance intensity can be lower than is currently occurring to support the delimiting phase, thereby saving significant resources in the post eradication phase (proof of freedom phases). Further consideration is required to enable the assumption of 100 % herd specificity made in the model to be achieved. The ELISA used is very specific, but will yield some false positives that must be resolved to their true status. This may occur for example through modified diagnostic test interpretation (e.g. cut point optimisation at individual and herd level) or resolution of putative false positive herds with epidemiological investigation. In conclusion this research demonstrates the utility of STM for planning surveillance programs, and in this instance has highlighted efficient and effective surveillance components for demonstrating freedom from M. bovis in NZ. It also highlights the need to achieve 100 % specificity for M. bovis in herds tested during the proof of freedom phases.

Synchronous shedding of multiple bat paramyxoviruses coincides with peak periods of Hendra virus spillover.

Within host-parasite communities, viral co-circulation and co-infections of hosts are the norm, yet studies of significant emerging zoonoses tend to focus on a single parasite species within the host. Using a multiplexed paramyxovirus bead-based PCR on urine samples from Australian flying foxes, we show that multi-viral shedding from flying fox populations is common. We detected up to nine bat paramyxoviruses shed synchronously. Multi-viral shedding infrequently coalesced into an extreme, brief and spatially restricted shedding pulse, coinciding with peak spillover of Hendra virus, an emerging fatal zoonotic pathogen of high interest. Such extreme pulses of multi-viral shedding could easily be missed during routine surveillance yet have potentially serious consequences for spillover of novel pathogens to humans and domestic animal hosts. We also detected co-occurrence patterns suggestive of the presence of interactions among viruses, such as facilitation and cross-immunity. We propose that multiple viruses may be interacting, influencing the shedding and spillover of zoonotic pathogens. Understanding these interactions in the context of broader scale drivers, such as habitat loss, may help predict shedding pulses of Hendra virus and other fatal zoonoses.

Development of multiplexed bead arrays for the simultaneous detection of nucleic acid from multiple viruses in bat samples.

Virus surveillance of wildlife populations is important for identifying, monitoring, and predicting the emergence of pathogens that pose a potential threat to animal and human health. Bats are identified as important wildlife hosts of many viruses capable of causing fatal human disease, including members of the henipaviruses, coronaviruses, rhabdoviruses and filoviruses. As global warming and habitat change are thought to impact upon pathogen transmission dynamics and increase the risk of spillover, virus surveillance in bat populations remains a significant component of efforts to improve the prediction and control of potential future disease outbreaks caused by bat-borne viruses. In this study we have developed two fluid bead array assays containing customized panels that target multiple bat-borne viruses. These assays detect up to 11 viral RNA's simultaneously in urine samples collected from wild bat populations in Australia and Bangladesh. The assays developed show high specificity for the target viruses and the analytical sensitivity compares favorably to qRT-PCR. These assays enhance the ability to monitor multi-pathogen dynamics and identify patterns of virus shedding from bat populations, thus informing key approaches to outbreak response and control.

Complete genome sequence of teviot paramyxovirus, a novel rubulavirus isolated from fruit bats in australia.

The causative agents of a number of emerging zoonotic diseases have been identified as paramyxoviruses originating in bats. We report here the complete genome sequence of two Teviot paramyxoviruses, novel rubulaviruses isolated from urine samples collected from pteropid bats in Australia. The zoonotic potential of Teviot paramyxovirus is undetermined.