Detection of ostreid herpesvirus-1 in plankton and seawater samples at an estuary scale.

Ostreid herpesvirus-1 (OsHV-1) is known to associate with particles in seawater, leading to infection and disease in the Pacific oyster Crassostrea gigas. The estuarine environment is highly complex and changeable, and this needs to be considered when collecting environmental samples for pathogen detection. The aims of this study were to (1) compare different aspects of collecting natural seawater and plankton samples for detection of OsHV-1 DNA and (2) determine whether detection of OsHV-1 DNA in such environmental samples has merit for disease risk prediction. The results of one experiment suggest that sampling on the outgoing tide may improve the detection of OsHV-1 DNA in seawater and plankton tow samples (odds ratio 2.71). This statistical comparison was not possible in 2 other experiments. The method (plankton tow or beta bottle) and depth of collection (range: 250-1250 mm) had no effect on the likelihood of detection of OsHV-1. OsHV-1 DNA was found at low concentrations in plankton tow and seawater samples, and only when outbreaks of mortality associated with OsHV-1 were observed in nearby experimental or farmed populations of C. gigas. This suggests that single point in time environmental samples of seawater or plankton are not sufficient to rule out the presence of OsHV-1 in an estuary. The association of OsHV-1 with particles in seawater needs to be better understood in order to determine whether more selective and sensitive methods can be devised to detect it, before environmental samples can be reliably used in disease risk prediction.

Mycobacterium avium subspecies paratuberculosis is able to manipulate host lipid metabolism and accumulate cholesterol within macrophages.

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Closely related pathogenic mycobacteria such as M. tuberculosis are capable of altering host lipid metabolism, highlighting the need to explore the role of lipid metabolism contributing to intracellular survival. This study aimed to identify whether MAP is able to manipulate host lipid metabolic pathways and accumulate host cholesterol during early infection. Macrophages were exposed to four different MAP strains and non-pathogenic M. phlei for up to 72 h, with changes to lipid metabolism examined using fluorescent microscopy and gene expression. MAP-infected macrophages displayed strain-dependent differences to intracellular cholesterol levels during early infection, however showed similarly increased intracellular cholesterol at later timepoints. Gene expression revealed that MAP strains similarly activate the host immune response in a conserved manner compared to M. phlei. MAP significantly upregulated host genes associated with lipid efflux and endocytosis. Moreover, lipid biosynthesis genes were differentially regulated in a strain-dependent manner following MAP infection. Collectively, these results demonstrate that MAP manipulates host lipid metabolism during early infection, however the extent of these modulations are strain-dependent. These findings reflect a conserved pathway contributing to intracellular MAP survival.

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.

The humoral immune response is essential for successful vaccine protection against paratuberculosis in sheep.

BACKGROUND: The role played by the humoral immune response in animals vaccinated against a mycobacterial disease such as paratuberculosis, is not well understood. Sheep vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP) can still become infected and in some cases succumb to clinical disease. The strength and location of the humoral immune response following vaccination could contribute to the ability of sheep to clear MAP infection. We examined the peripheral antibody response along with the localised humoral response at the site of paratuberculosis infection, the ileum, to better understand how this contributes to MAP infection of sheep following vaccination and exposure. RESULTS: Through assessing MAP specific serum IgG1 and IgG levels we show that the timing and strength of the humoral immune response directly relates to prevention of infection following vaccination. Vaccinated sheep that subsequently became infected had significantly reduced levels of MAP specific serum IgG1 early after vaccination. In contrast, vaccinated sheep that did not subsequently become infected had significantly elevated MAP specific serum IgG1 following vaccination. Furthermore, at 12 months post MAP exposure, vaccinated and subsequently uninfected sheep had downregulated expression of genes related to the humoral response in contrast to vaccinated infected sheep where expression levels were upregulated. CONCLUSIONS: The timing and strength of the humoral immune response following vaccination against paratuberculosis in sheep directly relates to subsequent infection status. An initial strong IgG1 response following vaccination was crucial to prevent infection. Additionally, vaccinated uninfected sheep were able to modulate that response following apparent MAP clearance, unlike vaccinated infected animals where there was apparent dysregulation of the humoral response, which is associated with progression to clinical disease.

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.

Detection and Serogrouping of Dichelobacter nodosus Infection by Use of Direct PCR from Lesion Swabs To Support Outbreak-Specific Vaccination for Virulent Footrot in Sheep.

Virulent footrot is an economically significant disease in most sheep-rearing countries. The disease can be controlled with vaccine targeting the fimbriae of virulent strains of the essential causative agent, Dichelobacter nodosus However, the bacterium is immunologically heterogeneous, and 10 distinct fimbrial serogroups have been identified. Ideally, in each outbreak the infecting strains would be cultured and serogrouped so that the appropriate serogroup-specific mono- or bivalent vaccine could be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at the flock level. The aims of this study were to develop a PCR-based procedure for detecting and serogrouping D. nodosus directly from foot swabs and to determine whether this could be done accurately from the same cultured swab. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in southeastern Australia were evaluated. DNA extracts taken from putative pure cultures of D. nodosus and directly from the swabs were evaluated in PCR assays for the 16S rRNA and fimA genes of D. nodosus Pure cultures were tested also by the slide agglutination test. Direct PCR using extracts from swabs was more sensitive than culture for detecting and serogrouping D. nodosus strains. Using the most sensitive sample collection method of the use of swabs in lysis buffer, D. nodosus was more likely to be detected by PCR in active than in inactive lesions, and in lesions with low levels of fecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in modified Stuart's transport medium that had already been used to inoculate culture plates had lower sensitivity. Therefore, if culture is required to enable virulence tests to be conducted, it is recommended that duplicate swabs be collected from each foot lesion, one in transport medium for culture and the other in lysis buffer for PCR.

Counting the dead to determine the source and transmission of the marine herpesvirus OsHV-1 in Crassostrea gigas.

Marine herpesviruses are responsible for epizootics in economically, ecologically and culturally significant taxa. The recent emergence of microvariants of Ostreid herpesvirus 1 (OsHV-1) in Pacific oysters Crassostrea gigas has resulted in socioeconomic losses in Europe, New Zealand and Australia however, there is no information on their origin or mode of transmission. These factors need to be understood because they influence the way the disease may be prevented and controlled. Mortality data obtained from experimental populations of C. gigas during natural epizootics of OsHV-1 disease in Australia were analysed qualitatively. In addition we compared actual mortality data with those from a Reed-Frost model of direct transmission and analysed incubation periods using Sartwell's method to test for the type of epizootic, point source or propagating. We concluded that outbreaks were initiated from an unknown environmental source which is unlikely to be farmed oysters in the same estuary. While direct oyster-to-oyster transmission may occur in larger oysters if they are in close proximity (< 40 cm), it did not explain the observed epizootics, point source exposure and indirect transmission being more common and important. A conceptual model is proposed for OsHV-1 index case source and transmission, leading to endemicity with recurrent seasonal outbreaks. The findings suggest that prevention and control of OsHV-1 in C. gigas will require multiple interventions. OsHV-1 in C. gigas, which is a sedentary animal once beyond the larval stage, is an informative model when considering marine host-herpesvirus relationships.

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.

Evaluation of Genotypic and Phenotypic Protease Virulence Tests for Dichelobacter nodosus Infection in Sheep.

Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%).

Detection of Ostreid herpesvirus-1 microvariants in healthy Crassostrea gigas following disease events and their possible role as reservoirs of infection.

Ostreid herpesvirus-1 microvariants (OsHV-1) cause severe mortalities in farmed Crassostrea gigas in Europe, New Zealand and Australia. Outbreaks are seasonal, recurring in the warmer months of the year in endemic estuaries. The reference genotype and microvariant genotypes of OsHV-1 have been previously detected in the tissues of apparently healthy adult oysters naturally exposed to OsHV-1 in the field. However, the role of such oysters as reservoirs of infection for subsequent mortality outbreaks remains unclear. The aims of this study were: (1) to identify the optimal sample type to use for the detection of OsHV-1 DNA in apparently healthy C. gigas; and (2) to assess whether live C. gigas maintained on-farm after an OsHV-1 related mortality event remain infected and could act as a reservoir host for subsequent outbreaks. OsHV-1 DNA was detected in the hemolymph, gill, mantle, adductor muscle, gonad and digestive gland of apparently healthy adult oysters. The likelihood of detecting OsHV-1 DNA in hemolymph was equivalent to that in gill and mantle, but the odds of detecting OsHV-1 DNA in hemolymph and gill were more than 8 times that of adductor muscle. Gill had the highest viral loads. Compared to testing whole gill homogenates, testing snippets of the gill improved the detection of OsHV-1 DNA by about four fold. The prevalence of OsHV-1 in gill and mantle was highest after the first season of OsHV-1 exposure; it then declined to low or negligible levels in the same cohorts in subsequent seasons, despite repeated seasonal exposure in monitoring lasting up to 4years. The hemolymph of individually identified oysters was repeatedly sampled over 15months, and OsHV-1 prevalence declined over that time frame in the youngest cohort, which had been exposed to OsHV-1 for the first time at the start of that season. In contrast, the prevalence in two cohorts of older oysters, which had been exposed to OsHV-1 in prior seasons, was consistently low (<10%). Viral loads were <104 DNA copies per mg tissue or µL hemolymph, suggesting that OsHV-1 was not being maintained at or amplified to high quantities. Therefore, while OsHV-1 may persist within apparently healthy oysters that have survived an outbreak of disease, they may not be a major reservoir host for the virus for subsequent outbreaks. However, further investigation is required to ascertain whether OsHV-1 replication occurs in surviving oysters, and whether transmission from them to naive oysters and induction of clinical disease is possible.

Surveillance for nervous necrosis virus-specific antibodies in barramundi Lates calcarifer in Australian hatcheries.

We conducted single point-in-time and repeated cross-sectional studies of the prevalence of antibodies against nervous necrosis virus (NNV) in populations of adult barramundi Lates calcarifer in Australia. Serum samples collected between 2002 and 2012 were analyzed with indirect ELISA (n = 468). Most of the samples were sourced from broodstock with unknown exposure history, and these were compared with reference populations with confirmed history of exposure to NNV. Non-lethally collected gonad fluid samples from economically valuable barramundi broodstock (n = 164) were tested for the presence of NNV using RT-quantitative PCR at the time of blood sampling to compare infectivity with serostatus, but no virus was detected. NNV-specific immunoreactivity in broodstock was significantly lower than that for immunized and persistently infected populations. Seroprevalence increased over time in broodstock sampled longitudinally, probably reflecting repeated exposure to NNV in a region where the virus was endemic. The seroprevalence for the broodstock was 23.8% over the entire sample period while a cross-sectional survey conducted in 2012 found a seroprevalence of 34.5% with no significant difference between populations based on the geographic region or the history of occurrence of viral nervous necrosis (VNN) disease in the progeny in the respective hatcheries. Although serological surveillance was useful for studying the history of exposure of barramundi to NNV, the lack of association between serostatus in broodstock and the subsequent occurrence of VNN disease in their progeny indicates that ELISA tests for anti-NNV antibodies are not suitable for the purpose of preventing vertical transmission of NNV in barramundi.

Detection of ostreid herpesvirus 1 microvariant DNA in aquatic invertebrate species, sediment and other samples collected from the Georges River estuary, New South Wales, Australia.

Ostreid herpesvirus 1 microvariants (OsHV-1) present a serious threat to the Australian Crassostrea gigas industry. Of great concern is the propensity for mortality due to the virus recurring each season in farmed oysters. However, the source of the virus in recurrent outbreaks remains unclear. Reference strain ostreid herpesvirus 1 (OsHV-1 ref) and other related variants have been detected in several aquatic invertebrate species other than C. gigas in Europe, Asia and the USA. The aim of this study was to confirm the presence or absence of OsHV-1 in a range of opportunistically sampled aquatic invertebrate species inhabiting specific locations within the Georges River estuary in New South Wales, Australia. OsHV-1 DNA was detected in samples of wild C. gigas, Saccostrea glomerata, Anadara trapezia, mussels (Mytilus spp., Trichomya hirsuta), whelks (Batillaria australis or Pyrazus ebeninus) and barnacles Balanus spp. collected from several sites between October 2012 and April 2013. Viral loads in non-ostreid species were consistently low, as was the prevalence of OsHV-1 DNA detection. Viral concentrations were highest in wild C. gigas and S. glomerata; the prevalence of detectable OsHV-1 DNA in these oysters reached approximately 68 and 43%, respectively, at least once during the study. These species may be important to the transmission and/or persistence of OsHV-1 in endemically infected Australian estuaries.

Serology in Finfish for Diagnosis, Surveillance, and Research: A Systematic Review.

Historically, serological tests for finfish diseases have been underused when compared with their use in terrestrial animal health. For years the nonspecific immune response in fish was judged to make serology unreliable and inferior to the direct measurement of agent analytes. We conducted a systematic review of peer-reviewed publications that reported on the development, validation, or application of serological tests for finfish diseases. A total of 168 articles met the screening criteria; most of them were focused on salmonid pathogens (e.g., Aeromonas spp. and viral hemorrhagic septicemia virus). Before the 1980s, most publications reported the use of agglutination tests, but our review indicates that enzyme-linked immunosorbent assay (ELISA) has more recently become the dominant serological test. The main application of serological tests has been in the assessment of vaccine efficacy, with few applications for surveillance or demonstration of freedom from disease, despite the advantages of serological tests over direct detection at the population level. Nonlethal sampling, low cost, and postinfection persistence of antibodies make serological assays the test of choice in surveillance, especially of valuable broodstock. However, their adoption has been constrained by poor characterization and validation. The number of publications in our review reporting diagnostic sensitivity and specificity of serological tests in finfish was small (n = 7). Foreseeing a wider use of serological tests in the future for diagnostic end purposes, we offer recommendations for mitigating deficiencies in the development and evaluation of serological tests, including optimization, control of nonspecific reactions, informed cutoff points, diagnostic accuracy, and serological baseline studies. Achieving these goals will facilitate greater international recognition of serological testing in programs supporting aquatic animal health. Received March 21, 2016; accepted September 24, 2016.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays.

UNLABELLED: Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE: Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

Susceptibility of Australian Redfin Perch Perca fluviatilis Experimentally Challenged with Epizootic Hematopoietic Necrosis Virus (EHNV).

The ranavirus epizootic hematopoietic necrosis virus (EHNV) is endemic to Australia and is listed by the Office International des Epizooties. Clinical outbreaks have only been observed in wild populations of Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and farmed populations of Rainbow Trout Oncorhynchus mykiss. The initial outbreaks of EHNV describe all age-classes of Redfin Perch as being susceptible and can lead to epidemic fish kills. Subsequently, experimental challenge studies using either cohabitation with the virus or injection exposures resulted in mixed susceptibilities across various age-groupings of Redfin Perch. We used an experimental bath challenge model to investigate the susceptibility of Redfin Perch collected from areas with and without a history of EHNV outbreaks. The median survival time for fish from Blowering Dam in New South Wales, a zone with a history of EHNV outbreaks, was 35 d, compared with fish from other areas, which had a median survival between 12 and 28 d postexposure. Redfin Perch from Blowering Dam demonstrated an increased mortality associated with epizootic hematopoietic necrosis up to approximately day 14 after exposure, and then there was a significantly reduced risk of mortality until the end of the trial compared with all other fish. Redfin Perch from Blowering Dam had markedly decreased susceptibility to EHNV, and less than 40% became infected following a bath challenge. In contrast, Redfin Perch from neighboring (e.g., Bethungra Dam and Tarcutta Creek) and distant water bodies (e.g., in Western Australia) with no previous history of EHNVdisplayed moderate to high susceptibility when given a bath challenge. Potential factors for the observed changes in the host-pathogen relationship include intense positive selection pressure for resistant fish following epizootic hematopoietic necrosis outbreaks and subsequent attenuation of the virulence of the virus in resistant fish. Received August 22, 2015; accepted February 13, 2016.

Efficient, validated method for detection of mycobacterial growth in liquid culture media by use of bead beating, magnetic-particle-based nucleic acid isolation, and quantitative PCR.

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis.

Experimental infections of Pacific oyster Crassostrea gigas using the Australian ostreid herpesvirus-1 (OsHV-1) µVar strain.

In Australia, the spread of the ostreid herpesvirus-1 microvariant (OsHV-1 µVar) threatens the Pacific oyster industry. There is an urgent need to develop an experimental infection model in order to study the pathogenesis of the virus under controlled laboratory conditions. The present study constitutes the first attempt to use archived frozen oysters as a source of inoculum, based on the Australian OsHV-1 µVar strain. Experiments were conducted to test (1) virus infectivity, (2) the dose-response relationship for OsHV-1, and (3) the best conditions in which to store infective viral inoculum. Intramuscular injection of a viral inoculum consistently led to an onset of mortality 48 h post-injection and a final cumulative mortality exceeding 90%, in association with high viral loads (1 × 105 to 3 × 107 copies of virus mg-1) in dead individuals. For the first time, an infective inoculum was produced from frozen oysters (tissues stored at -80°C for 6 mo). Storage of purified viral inoculum at +4°C for 3 mo provided similar results to use of fresh inoculum, whereas storage at -20°C, -80°C and room temperature was detrimental to infectivity. A dose-response relationship for OsHV-1 was identified but further research is recommended to determine the most appropriate viral concentration for development of infection models that would be used for different purposes. Overall, this work highlights the best practices and potential issues that may occur in the development of a reproducible and transferable infection model for studying the pathogenicity of the Australian OsHV-1 strain in Crassostrea gigas under experimental conditions.

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.