Revisiting the Importance of Orthobunyaviruses for Animal Health: A Scoping Review of Livestock Disease, Diagnostic Tests, and Surveillance Strategies for the Simbu Serogroup.

Orthobunyaviruses (order Bunyavirales, family Peribunyaviridae) in the Simbu serogroup have been responsible for widespread epidemics of congenital disease in ruminants. Australia has a national program to monitor arboviruses of veterinary importance. While monitoring for Akabane virus, a novel orthobunyavirus was detected. To inform the priority that should be given to this detection, a scoping review was undertaken to (1) characterise the associated disease presentations and establish which of the Simbu group viruses are of veterinary importance; (2) examine the diagnostic assays that have undergone development and validation for this group of viruses; and (3) describe the methods used to monitor the distribution of these viruses. Two search strategies identified 224 peer-reviewed publications for 33 viruses in the serogroup. Viruses in this group may cause severe animal health impacts, but only those phylogenetically arranged in clade B are associated with animal disease. Six viruses (Akabane, Schmallenberg, Aino, Shuni, Peaton, and Shamonda) were associated with congenital malformations, neurological signs, and reproductive disease. Diagnostic test interpretation is complicated by cross-reactivity, the timing of foetal immunocompetence, and sample type. Serological testing in surveys remains a mainstay of the methods used to monitor the distribution of SGVs. Given significant differences in survey designs, only broad mean seroprevalence estimates could be provided. Further research is required to determine the disease risk posed by novel orthobunyaviruses and how they could challenge current diagnostic and surveillance capabilities.

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

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

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

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

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus.

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

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

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

Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Alien Smooth Newts (Lissotriton vulgaris) in Australia Are Infected with Batrachochytrium dendrobatidis but Test Negative for Ranaviruses.

Smooth newts (Lissotriton vulgaris) established recently in Melbourne, Australia. Previously, the population's disease status was unknown. Samples from 34 adults and 78 larvae, collected 2011-16, were tested for two pathogens driving the global amphibian extinction crisis. The fungus Batrachochytrium dendrobatidis was identified (6.3% quantitative PCR positive); ranaviruses were not detected.

Factors associated with bovine respiratory disease case fatality in feedlot cattle.

Bovine respiratory disease (BRD) is the primary cause of morbidity and mortality in cattle feedlots. There is a need to understand what animal health and production factors are associated with increased mortality risk due to BRD. The aim of the present study was to explore factors associated with BRD case fatality in feedlot cattle. Four pens totaling 898 steers were monitored daily for visual signs of BRD such as difficult breathing and coughing, and animals exhibiting signs of BRD were taken to the hospital shed for further examination and clinical measures. Blood samples were obtained at feedlot entry and at time of first BRD pull from animals diagnosed with BRD (n = 121) and those that died due to BRD confirmed by postmortem examination (n = 16; 13.2% case fatality rate). Mixed-effects linear regression models were used to estimate differences in animal health and production factors and the relative concentrations of 34 identified blood metabolites between animals that survived versus those that died. Generalized linear mixed-effects models were used to obtain the odds of being seronegative (at both feedlot entry and first BRD pull) to 5 BRD viruses and having a positive nasal swab result at the time of first pull in died and survived animals. Animals that died from BRD had lower average daily gain (ADG), reduced weight at first BRD pull, higher visual BRD scores and received more treatments for BRD compared with animals that survived BRD (P < 0.05). The odds of being seronegative for bovine viral diarrhea virus 1 (BVDV-1) were 5.66 times higher for animals that died compared with those that survived (P = 0.013). The odds of having a positive bovine coronavirus nasal swab result were 13.73 times higher in animals that died versus those that survived (P = 0.007). Animals that died from BRD had higher blood concentrations of α glucose chain, ß-hydroxybutyrate, leucine, phenylalanine, and pyruvate compared with those that survived (P < 0.05). Animals that died from BRD had lower concentrations of acetate, citrate, and glycine compared with animals that survived (P < 0.05). The results of the current study suggest that ADG to first BRD pull, weight at first BRD pull, visual BRD score, the number of BRD treatments, seronegativity to BVDV-1, virus positive to BCoV nasal swab, and that certain blood metabolites are associated with BRD case fatality risk. The ability of these measures to predict the risk of death due to BRD needs further research.

Community engagement strengthens pig disease knowledge and passive surveillance in Timor-Leste.

Smallholder pig production in Timor-Leste is culturally and economically important for most households. However, regular and ongoing disease surveillance and pig husbandry training for farmers are limited. This article describes collaborative social and diagnostic research followed by a pilot community engagement program to improve farmer and technician knowledge, skills, and working relationships. There were three phases: (1) A qualitative study in 2020 to explore the experiences and knowledge of 133 pig farmers, 6 village leaders, and 16 district veterinary technicians on pig diseases and reporting, treatment methods, and access to information or assistance. (2) A pilot community engagement program in 3 villages in 2021 with the diagnostic investigation with samples analyzed from 27 dead pigs, and (3) Evaluation of community engagement and training outcomes. Results of the qualitative study revealed limited reporting of sick or dead pigs by farmers to veterinary technicians due to a lack of trust in the veterinary diagnostic system. Most technicians lacked experience with sampling or post-mortems so diagnostic training was undertaken for the pilot disease investigation. Evaluation results showed improved knowledge, motivation, and confidence of government staff and farmers. The credibility of veterinary technicians improved and gave them more confidence to work with communities. Farmers felt supported because all aspects of pig husbandry were addressed, and they were more willing to report dead or sick pigs. The project indicates that improved passive disease surveillance can be achieved by engaging communities in smallholder pig farming in Timor-Leste. Further research and testing of the approach in other districts and countries is recommended.

Optimizing surveillance for early disease detection: Expert guidance for Ostreid herpesvirus surveillance design and system sensitivity calculation.

To keep pace with rising opportunities for disease emergence and spread, surveillance in aquaculture must enable the early detection of both known and new pathogens. Conventional surveillance systems (designed to provide proof of disease freedom) may not support detection outside of periodic sampling windows, leaving substantial blind spots to pathogens that emerge in other times and places. To address this problem, we organized an expert panel to envision optimal systems for early disease detection, focusing on Ostreid herpesvirus 1 (OsHV-1), a pathogen of panzootic consequence to oyster industries. The panel followed an integrative group process to identify and weight surveillance system traits perceived as critical to the early detection of OsHV-1. Results offer a road map with fourteen factors to consider when building surveillance systems geared to early detection; factor weights can be used by planners and analysts to compare the relative value of different designs or enhancements. The results were also used to build a simple, but replicable, model estimating the system sensitivity (SSe) of observational surveillance and, in turn, the confidence in disease freedom that negative reporting can provide. Findings suggest that optimally designed observational systems can contribute substantially to both early detection and disease freedom confidence. In contrast, active surveillance as a singular system is likely insufficient for early detection. The strongest systems combined active with observational surveillance and engaged joint industry and government involvement: results suggest that effective partnerships can generate highly sensitive systems, whereas ineffective partnerships may seriously erode early detection capability. Given the costs of routine testing, and the value (via averted losses) of early detection, we conclude that observational surveillance is an important and potentially very effective tool for health management and disease prevention on oyster farms, but one that demands careful planning and participation. This evaluation centered on OsHV-1 detection in farmed oyster populations. However, many of the features likely generalize to other pathogens and settings, with the important caveat that the pathogens need to manifest via morbidity or mortality events in the species, life stages and environments under observation.

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

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

Predicting bovine respiratory disease outcome in feedlot cattle using latent class analysis.

Bovine respiratory disease (BRD) is the most significant disease affecting feedlot cattle. Indicators of BRD often used in feedlots such as visual signs, rectal temperature, computer-assisted lung auscultation (CALA) score, the number of BRD treatments, presence of viral pathogens, viral seroconversion, and lung damage at slaughter vary in their ability to predict an animal's BRD outcome, and no studies have been published determining how a combination of these BRD indicators may define the number of BRD disease outcome groups. The objectives of the current study were (1) to identify BRD outcome groups using BRD indicators collected during the feeding phase and at slaughter through latent class analysis (LCA) and (2) to determine the importance of these BRD indicators to predict disease outcome. Animals with BRD (n = 127) were identified by visual signs and removed from production pens for further examination. Control animals displaying no visual signs of BRD (n = 143) were also removed and examined. Blood, nasal swab samples, and clinical measurements were collected. Lung and pleural lesions indicative of BRD were scored at slaughter. LCA was applied to identify possible outcome groups. Three latent classes were identified in the best model fit, categorized as non-BRD, mild BRD, and severe BRD. Animals in the mild BRD group had a higher probability of having visual signs of BRD compared with non-BRD and severe BRD animals. Animals in the severe BRD group were more likely to require more than 1 treatment for BRD and have ≥40 °C rectal temperature, ≥10% total lung consolidation, and severe pleural lesions at slaughter. Animals in the severe BRD group were also more likely to be naïve at feedlot entry and the first BRD pull for Bovine Viral Diarrhoea Virus, Bovine Parainfluenza 3 Virus, and Bovine Adenovirus and have a positive nasal swab result for Bovine Herpesvirus Type 1 and Bovine Coronavirus. Animals with severe BRD had 0.9 and 0.6 kg/d lower overall ADG (average daily gain) compared with non-BRD animals and mild BRD animals (P < 0.001). These results demonstrate that there are important indicators of BRD severity. Using this information to predict an animal's BRD outcome would greatly enhance treatment efficacy and aid in better management of animals at risk of suffering from severe BRD.

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

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

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals.

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population-level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease-specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer-reviewed research. A systematic review identified only 12 peer-reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence-based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer-reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.

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

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

Influence of environment on the pathogenesis of Ostreid herpesvirus-1 (OsHV-1) infections in Pacific oysters (Crassostrea gigas) through differential microbiome responses.

The oyster microbiome is thought to contribute to the pathogenesis of mass mortality disease in Pacific oysters, associated with OsHV-1. As filter-feeders, oysters host a microbiota that can be influenced by the estuarine environment. This may alter susceptibility to OsHV-1 infections, causing variable mortality. This study aimed at: (1) differences in the microbiome of Pacific oysters with a common origin but grown in geographically distinct estuaries; (2) evaluating changes occurring in the microbiota, especially in Vibrio, and (3) differential responses of the oyster microbiome, in response to an OsHV-1 infection. Pacific oysters sourced from a single hatchery but raised separately in Patonga Creek, Shoalhaven River and Clyde River of NSW, Australia, were used and challenged with OsHV-1. The initial microbiome composition was different in the three batches and changed further, post-injection (p < 0.05). The Patonga oysters with the highest mortality also had higher OsHV-1 and Vibrio quantities compared to the other two batches (p < 0.05). The higher initial bacterial diversity in Patonga oysters decreased in moribund oysters which was not observed in the other two batches (p < 0.05). The microbiome of survivors of OsHV-1 infection and negative control oysters of two batches, did not show any changes with the relevant pre-challenged microbiome. A strong correlation was observed between the OsHV-1 and Vibrio quantities in OsHV-1 infected oysters (r = 0.6; p < 0.001). In conclusion, the Pacific oyster microbiome differed in different batches despite a common hatchery origin. Different microbiomes responded differently with a differential outcome of OsHV-1 challenge. The higher Vibrio load in oysters with higher OsHV-1 content and higher mortality, suggests a role in Vibrio in the pathogenesis of this mortality disease. This study provided insights of the potential of different estuarine environments to shape the Pacific oyster microbiome and how different microbiomes are associated with different outcomes of OsHV-1 infection.

The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus.

Movements of large volumes and species varieties make the ornamental fish industry a high-risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

Host, agent and environment interactions affecting Nervous necrosis virus infection in Australian bass Macquaria novemaculeata.

Australian bass Macquaria novemaculeata were challenged by immersion with nervous necrosis virus (NNV) at different ages and under controlled conditions to investigate factors affecting disease expression. Fish challenged at 3 weeks of age with 103 TCID50 /ml and higher doses developed clinical disease; a lower dose of 102 TCID50 /ml resulted in incidence below 100% and 101 TCID50 /ml was insufficient to cause infection. Additionally, fish were challenged at 5, 6 and 13 weeks of age at 17 and 21°C to assess the role of the age of the host and water temperature on disease expression. Although Australian bass challenged at all ages had evidence of replication of NNV, only those challenged at 3 weeks of age (20 and 24 days post-hatch [dph]) developed clinical disease. Higher water temperature had an additive effect on disease expression in larvae challenged at 24 dph, but it did not affect the disease outcome in older fish. Finally, isolates of NNV derived from fish with clinical or subclinical disease presentations caused similar cumulative mortality and clinical signs when larvae at 24 dph were challenged, suggesting that agent variation was not responsible for variation in clinical presentation in these field outbreaks of NNV infection.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses.

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.