Aquatic pathogens and biofouling: pilot study of ostreid herpesvirus 1 translocation by bivalves.

Ostreid herpesvirus 1 (OsHV-1) has caused mass mortalities in Pacific oysters (Crassostrea gigas) in Europe, Australia, and New Zealand. While aquaculture-associated movements of infected Pacific oysters are a well-known cause of OsHV-1 spread once established in a region, translocation via biofouling of aquaculture equipment or vessels needs further investigation to explain the more distant spread of OsHV-1. Laboratory experiments were designed to test for transmission of OsHV-1 between infected and naïve Pacific oysters via a simulated biofouling translocation scenario. Three common biofouling species [Sydney rock oysters (Saccostrea glomerata), Mediterranean mussels (Mytilus galloprovincialis) and Pacific oysters] were tested as intermediaries using a cohabitation challenge with Pacific oysters infected by injection. Transmission occurred, albeit for one of eight replicates when Pacific oysters were the intermediary species. This demonstrated a possible pathway for pathogen spread via biofouling containing Pacific oysters while highlighting the complexity of OsHV-1 transmission. Such complexities require further investigation to inform future risk assessments and management of fouled aquaculture equipment and vessels.

The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster (Crassostrea gigas) microbiome.

AIMS: This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. METHODS AND RESULTS: Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. CONCLUSIONS: Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

Design standards for experimental and field studies to evaluate diagnostic accuracy of tests for infectious diseases in aquatic animals.

Design and reporting quality of diagnostic accuracy studies (DAS) are important metrics for assessing utility of tests used in animal and human health. Following standards for designing DAS will assist in appropriate test selection for specific testing purposes and minimize the risk of reporting biased sensitivity and specificity estimates. To examine the benefits of recommending standards, design information from published DAS literature was assessed for 10 finfish, seven mollusc, nine crustacean and two amphibian diseases listed in the 2017 OIE Manual of Diagnostic Tests for Aquatic Animals. Of the 56 DAS identified, 41 were based on field testing, eight on experimental challenge studies and seven on both. Also, we adapted human and terrestrial-animal standards and guidelines for DAS structure for use in aquatic animal diagnostic research. Through this process, we identified and addressed important metrics for consideration at the design phase: study purpose, targeted disease state, selection of appropriate samples and specimens, laboratory analytical methods, statistical methods and data interpretation. These recommended design standards for DAS are presented as a checklist including risk-of-failure points and actions to mitigate bias at each critical step. Adherence to standards when designing DAS will also facilitate future systematic review and meta-analyses of DAS research literature.

Age dependency of nervous necrosis virus infection in barramundi Lates calcarifer (Bloch).

Age-dependent susceptibility to nervous necrosis virus (NNV) was demonstrated for barramundi (Lates calcarifer). The experiment used juvenile barramundi produced from a single spawning that were challenged consecutively by immersion with a redspotted grouper nervous necrosis virus (RGNNV) isolate. The dose and environmental conditions (35 ppt salinity and 30 °C) were constant. Fish and water were sampled longitudinally for histopathology and RT-qPCR analysis to examine the evolution of the disease, virus replication, immune response and release of virus into water. Viral nervous necrosis (VNN) disease occurred in barramundi challenged at 3 and 4 weeks of age while fish challenged at 5, 7 and 9 weeks of age developed subclinical infection. Replication of NNV occurred faster and the concentration of virus reached higher concentrations in the younger fish with clinical disease. Virus isolation and qPCR tests indicated that infectious NNV was released from carcasses into water when fish were affected with clinical disease but not when NNV infection was subclinical. Based on these observations, we consider that carcasses from clinically infected fish have a potentially important role in the horizontal transmission of NNV, and barramundi juveniles should be protected from exposure to NNV until they are 5 weeks of age and reach the disease resistance threshold.

Milk as a diagnostic fluid to monitor viral diseases in dairy cattle.

BACKGROUND: Infectious viral diseases in dairy cattle have substantial implications for milk production, quality and overall animal health. Diagnostic tools providing reliable results are crucial for effective disease control at the farm and industry level. Pooled or bulk tank milk (BTM) can be used as a cost-effective aggregate sample to assess herd disease status in dairy farms. FINDINGS: Detection of pathogens or specific antibodies in milk can be used for monitoring endemic diseases within-farm, region or country-level disease surveillance and to make informed decisions on farm management. The suitability of assays applied to pooled milk samples relies on validation data of fit-for-purpose tests to design an optimal testing strategy. Diverse approaches and variable scope of studies determining test accuracy need to be critically appraised before sourcing the parameters to design sampling strategies and interpreting surveys. Determining if BTM or pooled milk is the best approach for a disease management programme should carefully consider several aspects that will impact the accuracy and interpretation, for example, the size of the lactating herd, the risk of infection in the lactating and non-lactating groups, the expected within-herd prevalence, the duration of infection, the duration and concentration of antibodies in milk and use of vaccination. CONCLUSIONS: There are examples of tests on BTM samples providing efficient assessments of the herd disease status and supporting disease control programmes for viral diseases. However, challenges arise in pooled milk testing due to the need for accurate estimates of the imperfect sensitivity and specificity of the assays. Integration of new biotechnologies could enhance multiplexing and data interpretation for comprehensive surveillance. The development of highly sensitive assays is necessary to meet the demands of larger dairy herds and improve disease detection and assessment.

Ostreid herpesvirus-1 microvariant surveillance in Pacific oysters (Magallana gigas, Thunberg, 1793) in Australia in 2011.

OBJECTIVE: To demonstrate that OsHV-1 microvariant was limited to the known infected areas in New South Wales at the time of the survey in 2011. DESIGN: A 2-stage survey to demonstrate probability of infection at 2% design prevalence within oyster growing regions and to detect at least one infected region (4% design prevalence) with 95% confidence. SAMPLE POPULATION: Magallana gigas in nominated oyster growing regions in New South Wales, South Australia and Tasmania as approved by the Aquatic Consultative Committee on Emergency Animal Diseases and documented in a national surveillance plan. PROCEDURE: Field sampling for active surveillance and laboratory selection of appropriate tissues using methods to minimize potential for cross contamination. Published methods for qPCR and conventional PCR for OsHV-1 microvariant. Stochastic analysis of survey results to demonstrate probability of detection in the areas tested. RESULTS AND CONCLUSIONS: OsHV-1 microvariant was not detected in a total 4121 samples according to the case definition developed for the survey. However, in NSW a screening qPCR for OsHV-1 detected 13 samples that reacted. These samples were negative at 2 laboratories in the qPCR and conventional PCR assays used in the case definition for the survey. We concluded that oyster production areas of Australia outside the infected area in NSW met the criteria for self-declaration of freedom at the time of the survey in 2011. CLINICAL RELEVANCE: This activity illustrated achievements in surveillance for an emerging emergency animal pathogen where epidemiological and test validation data were limited, but where data was required to inform the emergency disease response. It also illustrated the challenges faced by investigators in interpreting surveillance results using tests with limited validation. It was guided by and has informed improvements in surveillance and emergency disease preparedness.

Clinical and epidemiological features of West Nile virus equine encephalitis in New South Wales, Australia, 2011.

BACKGROUND: Between February and June 2011, more than 300 horses with unexplained neurological disease were observed in New South Wales, Australia. A virulent strain of West Nile virus (WNVNSW2011 ), of Australian origin, was shown to be the cause of many of these cases. METHODS: We reviewed the clinical descriptions provided by veterinary practitioners and the associated laboratory results. Although there was a range of clinical signs described, ataxia was the only sign that was consistently described in laboratory-confirmed cases. RESULTS: WNV was detected in brain samples by real-time reverse transcription PCR assay and virus isolation. For serological confirmation of clinical cases, an equine IgM ELISA specific for WNV was shown to be the most effective tool. CONCLUSION: A state-wide serological survey undertaken after the outbreak indicated that, contrary to expectation, although infection had been widespread, the seroprevalence of antibodies to WNV was very low, suggesting that there could be a significant risk of future disease outbreaks.

Transcutaneous detection of relative changes in artery diameter.

The extent of the excursions of the arterial walls during the cardiac cycle depends on both the compliance of the vessel wall and the local pressure fluctuations. Simultaneous assessment of the relative change in artery diameter in combination with the velocity distribution along the vessel cross section can reveal the cause of loss of distensibility. As will be demonstrated, a multigate pulsed Doppler system with a high spatial resolution can perform simultaneously both functions. The relative change in diameter during the cardiac cycle is obtained by taking the ratio of the distension and the diameter of the artery as observed along the ultrasound beam. It can be shown that this ratio will be angle independent. Statistical and experimental evaluations demonstrate that the system allows the assessment of the relative change in diameter of major peripheral arteries as a continuous function of time with an accuracy of about 0.5%.

Methods to evaluate the sample volume of pulsed Doppler systems.

The spatial resolution of a pulsed Doppler system heavily depends on both the characteristics of the ultrasound transducer and the processing circuitry. The local characteristics of the ultrasound beam give the lateral sample function while the way the signals are processed affects the axial shape of the sample volume. A simple way to explore the size of the sample volume is the employment of a running string target. However, the detected Doppler signals will be the sum of all the Doppler signals returned by the string along its course through the sample volume. Therefore, it will be complicated to separate the axial and lateral contribution to the actual size of the sample volume. A scanning system capable of visualizing the cross-sectional distribution of the ultrasound intensity allows the assessment of the sample sensitivity in lateral direction. A single vibrating point target enables the exploration of both the axial and lateral sampling function. The latter approach is especially suited to evaluate routinely the spatial performance of pulsed Doppler systems in clinical practice.

Limitations of Doppler spectral broadening in the early detection of carotid artery disease due to the size of the sample volume.

In the present study the criteria of spectral broadening used in pulsed Doppler systems to diagnose minor disease in the cervical carotid arteries were evaluated in relation to the size of the sample volume. For this purpose the Doppler audio spectra as recorded in asymptomatic volunteers with an ATL Duplex scanner (relatively large sample volume) and a multi-gate pulsed Doppler system (relatively small sample volume) were compared with each other. The volunteers, randomly selected from the population registers, varied in age from 20-69 yr. Only in the common carotid artery at younger age, were normal spectra recorded, especially with the multi-gate system. In the internal carotid artery the number of spectra, supposed to be associated with low grade stenosis, increased significantly, especially when recorded with the Duplex scanner. More pronounced spectral broadening was found when the spectra were recorded distally in this artery. In the cervical carotid arteries normal spectra were found only when sampled in the middle of the bloodstream. These findings indicate that for the detection of vascular lesions in the cervical carotid arteries at an early stage of the disease on the basis of spectral broadening, small sample volumes and sampling in the centre of the vessel are required.

The diagnosis of minor to moderate atherosclerotic lesions in the carotid artery bifurcation by means of spectral broadening combined with the direct detection of flow disturbances using a multi-gate pulsed Doppler system.

The diagnostic accuracy of noninvasive detection of carotid artery lesions with a diameter of less than 50% (minor to moderate) and greater than 50% reduction (severe) with the use of a multi-gate pulsed Doppler system (spectral analysis combined with the detection of flow disturbances) was studied in patients with focal neurological symptoms. Biplane arteriography was used as a reference. For all lesions the diagnostic accuracy (observed agreement), sensitivity and specificity was 88, 92, and 84%, respectively, and 84, 83, and 84%, respectively, when only minor to moderate lesions were considered. Kappa, a chance-corrected measure of agreement, which takes into account the marginal distribution of the data and, hence, may be considered to be a better parameter to describe diagnostic accuracy, was found to be 82.2%. The results in this study compare favorably with those obtained in studies employing duplex techniques (B-mode image combined with single-gate pulsed Doppler) and indicate that minor to moderate carotid artery lesions are detected rather accurately with the use of a multi-gate pulsed Doppler system.

Carotid artery wall properties in normotensive and borderline hypertensive subjects of various ages.

The vessel wall properties of the common carotid artery were studied noninvasively in normotensive and borderline hypertensive male volunteers of various ages with the use of a multi-gate pulsed Doppler system. In the younger age group (20-35 y) both distensibility and cross-sectional compliance were significantly less in the borderline hypertensive group. In the older borderline hypertensive subjects (50-69 y) distensibility (p less than 0.05) and cross-sectional compliance (p = 0.06) were also less than in the control subjects. The reduced distensibility and cross-sectional compliance likely result from a decrease in arterial wall elasticity because the relative increase in common carotid artery diameter during systole is diminished in borderline hypertensives, despite the fact that their pulse pressure is similar to or higher than that in control subjects. The less pronounced differences in vessel wall properties between borderline hypertensive and normotensive volunteers in the older age group may be a consequence of the changes in these properties with age, partly masking the alterations due to borderline hypertension. Since the stiffer behavior of the common carotid artery in borderline hypertensives is associated with relatively slight changes in blood pressure, the question can be raised whether the alterations in arterial wall properties are really a result of the elevated arterial blood pressure; these alterations might develop independent of the blood pressure elevation.

Differences in carotid artery wall properties between presumed-healthy men and women.

The vessel wall properties of the common carotid artery were noninvasively studied in 109 normotensive, presumed normal male and female volunteers of various ages (20-69 y) with the use of a multi-gate pulsed Doppler system. This system allows the on-line recording of velocity profiles and the relative changes in carotid artery diameter during the cardiac cycle (delta d/d). From the width of these profiles the systolic internal diameter (d) of the carotid artery can be determined. With the use of d, the maximal increase in delta d/d during systole and the pulse pressure, as measured in the brachial artery (delta p), the distensibility coefficient and the cross-sectional compliance were calculated. Both distensibility and cross-sectional compliance were significantly lower in females than in males, irrespective of age. This likely results from a stiffer arterial wall behavior in females, because the relative increase in vessel diameter during the cardiac cycle is lower in females than in males, despite the fact that their pulse pressure values are not significantly different. The carotid artery diameters were similar in men and women. The findings in the present study indicate that the common carotid artery is stiffer in females than in males of comparable age. Therefore, sex-dependent differences have to be considered in the interpretation of arterial distensibility and cross-sectional compliance.

Age-related changes in carotid artery wall properties in men.

Changes in distensibility and cross-sectional compliance of the common carotid artery with age were studied in 80 presumed healthy volunteers, varying in age between 20 and 69 y. The distensibility was assessed from the relative increase in arterial diameter during systole normalized with respect to the arterial pulse pressure. The cross-sectional compliance was obtained by multiplying the distensibility by the arterial diameter. The relative diameter changes of the common carotid artery during the cardiac cycle were recorded on-line with a high resolution multigate pulsed Doppler system. The arterial diameter was assessed from the width of the velocity profiles which can also be recorded on-line with this system. Arterial pulse pressure was determined from brachial artery cuff blood pressure measurements. Both distensibility and cross-sectional compliance of the common carotid artery decreased linearly with age, starting in the third age decade. The reduction in the latter parameter was less pronounced, probably as a consequence of an increase in arterial diameter with age.

A simple centrifugation method for improving the detection of Ostreid herpesvirus-1 (OsHV-1) in natural seawater samples with an assessment of the potential for particulate attachment.

Ostreid herpesvirus-1 (OsHV-1) is responsible for massive mortality events in commercially farmed Pacific oysters (Crassostrea gigas) in Australia, New Zealand, Europe and the USA. Economic losses have been severe in many countries since 2008, associated with a strain known as OsHV-1µ-var. Despite intensive studies of the virus itself, there is almost no information on its detection in natural seawater, how it is spread over wide geographic distance in water or on how it is transmitted from oyster to oyster via seawater. The aim of the current work was to (1) assess and compare several centrifugation methods in order to detect OsHV-1 in natural seawater samples using real-time quantitative PCR, in such a way that large numbers of samples could be processed efficiently and (2) assess the potential for particulate attachment of OsHV-1 using filtration. Compared to testing unprocessed seawater samples, centrifugation of seawater at 1000×g for 20 min with testing of the pellet improved OsHV-1 detection rates by two fold. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles, free virus or free viral DNA. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.

Coronavirus infection in intensively managed cattle with respiratory disease.

BACKGROUND: A detailed laboratory investigation identified bovine coronavirus (BCoV) as the aetiological agent in an outbreak of respiratory disease at a semi-intensive beef cattle feedlot in south-east Australia. The outbreak caused 30% morbidity in the resident population and also affected two cohorts of cattle that were newly introduced to the property. METHODS: At slaughter, pulmonary consolidation and inflammatory lesions in the trachea were identified in 15 of 49 animals. Pasteurella multocida or Histophilus somni was cultured from 3 of 7 animals with lesions. Histopathological examination revealed multifocal non-suppurative bronchointerstitial pneumonia with formation of epithelial syncytial cells, sometimes associated with suppurative bronchopneumonia. RESULTS: BCoV was detected in nasal swabs and pulmonary lesions using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay and virus isolation. There was serological evidence of previous exposure to bovine viral diarrhoea virus, bovine respiratory syncytial virus and bovine parainfluenza virus type 3, but not to bovine herpesvirus type 1. None of these viral pathogens or Mycoplasma bovis was identified by qRT-PCR. CONCLUSION: This is believed to be the first report of BCoV in association with bovine respiratory disease complex in Australia.