Efficacy of bivalent fimbrial vaccines to control and eliminate intermediate forms of footrot in sheep.

OBJECTIVE: The aim of this study was to determine the efficacy of serogroup-specific bivalent fimbrial vaccines in the control and elimination of relatively mild (intermediate) forms of footrot in sheep flocks in NSW, there being some evidence that such forms are difficult to control. METHODS: Four flocks of sheep with history of footrot of intermediate virulence were selected based on clinical and bacteriological diagnoses. Dichelobacter nodosus serogroups included in bivalent vaccines at each farm were based on on-farm serogroup-prevalence data. Two doses of bivalent vaccine were administered with a 4-week interval between doses. Repeated post-vaccination inspections of all feet of between 100 and 119 animals per mob were conducted and foot swabs were collected for bacteriological testing. Blood samples were collected from 10 to 24 individually identified animals per flock at each inspection to check for agglutinating antibody responses. RESULTS: In the majority of animals, antibody levels for serogroups included in each vaccine were above the level believed to be required for protective immunity. Footrot disappeared on farm 1 prior to vaccination, but did not reappear postvaccination. Footrot was controlled but not eliminated on farms 2, 3, and 4, where the prevalence and severity of the disease and number of serogroups present were reduced. CONCLUSION: Serogroup-specific bivalent vaccines can be effective at controlling footrot caused by intermediate strains of D. nodosus.

The severity of footrot lesions induced by aprV2-positive strains of Dichelobacter nodosus varies between strains.

OBJECTIVES: The primary objective of this study was to evaluate the clinical virulence of aprV2-positive lesser virulent field isolates of footrot bacteria Dichelobacter nodosus in comparison with an aprV2-positive clinically virulent reference strain. Correlations between the clinical expression of the disease and the presence of aprV2 (detected using PCR tests) have been inconsistent. A second objective was to evaluate the elimination of D. nodosus following treatment of sheep as some strains of D. nodosus have been reported to be difficult to eliminate. METHODS: The virulence of three aprV2-positive field isolates of D. nodosus which had lesser virulent phenotypes, and an aprV2-positive virulent reference strain was evaluated in a sheep trial using a pasture-based experimental infection model. In the second phase of the study, treatments including footbathing and a long-acting antibiotic were administered and their efficacy in elimination of these strains was evaluated. RESULTS: Severe underrun (score 4) lesions developed in sheep infected with the aprV2-positive virulent reference strain but not in sheep infected with the field isolates; they had mild lesions (score 2 or 3). The three field isolates and the virulent reference strain of D. nodosus were eliminated by intensive foot bathing and antibiotic therapy in combination with housing the animals in dry conditions post-treatment. CONCLUSION: The results suggest that the presence of aprV2 gene in isolates of D. nodosus may not be a reliable indicator of virulence and that further investigation of the factors that determine clinical virulence is required. While the treatment regime was successful, based on a range of considerations, the use of such an intensive treatment involving antibiotics should be limited to small groups of high-value animals, such as rams.

L'impact des microvariants du virus herpétique ostreid herpesvirus 1 sur la culture d'huîtres creuses dans les hémisphères Nord et Sud depuis 2008.

Mollusc farming is the third most productive aquaculture activity in the world, and the Pacific oyster (Crassostrea gigas) is one of the most important farmed species. Since 2008, mass mortalities in C. gigas due to ostreid herpesvirus 1 microvariants have challenged the viability of this industry in Europe, New Zealand and Australia. Ten years after the emergence of this disease, there is evidence that the industry has become consolidated into fewer, larger companies, with the displacement of small farming enterprises and loss of employment in coastal communities. Rather than seeking technical solutions, the industry has turned to compensatory production strategies, such as increasing the number of spat placed on farms, higher market prices for table oysters and direct marketing, which appear to have allowed profitability. Biosecurity policies and responses to outbreaks, including those from within the industry, have had unintended consequences for hatcheries and farmers in areas free of disease, mainly caused by restrictions on animal movements, and have not prevented global spread. There may be opportunities for better coordination of industry and government responses to epizootic disease emergence in aquaculture. There is certainly a need for increased adoption of technical advances from research, once these solutions have been adequately verified.

L'élevage de mollusques occupe le troisième rang mondial parmi les activités de l'aquaculture en termes de production ; l'une des principales espèces élevées est l'huître creuse (Crassostrea gigas). Depuis 2008, la rentabilité des élevages de C. gigas en Europe, en Nouvelle-Zélande et en Australie a été fortement compromise par une mortalité massive due à des microvariants du virus herpétique Ostreid herpesvirus 1. Dix ans après l'émergence de cette maladie, on observe une forte concentration du secteur autour d'entreprises moins nombreuses mais de plus grande envergure qui ont remplacé l'ancien tissu d'exploitations artisanales et occasionné un déclin de l'emploi dans les communautés littorales. Au lieu de rechercher des solutions techniques, le secteur a eu recours à des stratégies de compensation axées sur la production, par exemple en augmentant le nombre de naissains mis en place dans les fermes, en augmentant le prix des huîtres de consommation ou en développant la vente directe, stratégies dont l'impact sur la rentabilité semble avoir été positif. En revanche, les mesures de biosécurité mises en place et les réponses apportées aux foyers, y compris celles introduites par le secteur lui-même ont eu des conséquences imprévues pour les écloseries et les éleveurs des zones indemnes de maladie, principalement en raison des restrictions imposées aux transferts d'animaux, sans pour autant prévenir la propagation de la maladie à l'échelle mondiale. Une meilleure coordination des réponses sectorielles et publiques face à l'émergence des maladies épizootiques affectant l'aquaculture devrait être possible. Il sera également indispensable de recourir davantage aux avancées techniques mises au point par la recherche dès que ces solutions auront été dûment validées.

La producción de moluscos es la tercera actividad acuícola más productiva del mundo, y la ostra japonesa (o del Pacífico) (Crassostrea gigas) ocupa un lugar destacado entre las principales especies cultivadas. Desde 2008, la viabilidad de esta industria en Europa, Nueva Zelanda y Australia está amenazada por episodios de mortandad masiva de C. gigas causados por microvariantes del herpesvirus de los ostreidos 1 (ostreid herpesvirus 1). Diez años después de la aparición de la enfermedad, lo que se observa es que la industria se ha ido concentrando en unas pocas empresas de grandes dimensiones, que han desplazado a las pequeñas empresas ostrícolas y causado la pérdida de numerosos empleos en las comunidades costeras. En lugar de buscar soluciones técnicas, la industria ha optado más bien por estrategias de producción compensatorias (como aumentar el número de semillas de ostra por explotación, subir los precios de mercado de las ostras de mesa o recurrir a la comercialización directa) que parecen haber deparado rentabilidad. Las políticas de seguridad biológica y la respuesta a los brotes, incluida la del propio sector, han tenido consecuencias imprevistas para los viveros y acuicultores situados en zonas libres de la enfermedad, debido sobre todo a las restricciones impuestas a los desplazamientos de animales, sin que ello haya servido para impedir la diseminación mundial de esta patología. Puede haber margen para coordinar más eficazmente las respectivas respuestas de la industria y de los poderes públicos ante la aparición de enfermedades epizoóticas en la acuicultura. Lo que sin ninguna duda es necesario es incorporar en mayor medida los adelantos técnicos resultantes de la investigación, una vez contrastada debidamente cada solución.

The immunogenicity and tissue reactivity of Mycobacterium avium subsp paratuberculosis inactivated whole cell vaccine is dependent on the adjuvant used.

Johne's disease (JD) is a chronic enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current commercial vaccines are effective in reducing the occurrence of clinical disease although vaccinated animals can still become infected and transmit MAP. Many vaccinated sheep develop severe injection site lesions. In this study a range of adjuvants (Montanide TM ISA 50V, ISA 50V2, ISA 61VG, ISA 70 M VG, ISA 71 VG, ISA 201 VG and Gel 01 PR) formulated with heat-killed MAP were tested to determine the incidence of injection site lesions and the types of immune profiles generated in sheep. All the novel formulations produced fewer injection site lesions than a commercial vaccine (Gudair®). The immune profiles of the sheep differed between treatment groups, with the strength of the antibody and cell mediated immune responses being dependant on the adjuvant used. One of the novel vaccines resulted in a reduced IFN-γ immune response when a second "booster" dose was administered. These findings have significance for JD vaccine development because it may be possible to uncouple protective immunity from excessive tissue reactivity, and apparently poorly immunogenic antigens may be re-examined to determine if an appropriate immune profile can be established using different adjuvants. It may also be possible to formulate vaccines that produce targeted immunological profiles suited to protection against other pathogens, i.e. those for which a bias towards cellular or humoral immunity would be advantageous based on understanding of pathogenesis.

Sheep and cattle exposed to Mycobacterium avium subspecies paratuberculosis exhibit altered total serum cholesterol profiles during the early stages of infection.

Pathogenic mycobacteria such as Mycobacterium tuberculosis are capable of utilising cholesterol as a primary carbon-based energy source in vitro but there has been little research examining the significance of cholesterol in vivo. Johne's disease is a chronic enteric disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). This study sought to evaluate the levels of total serum cholesterol in the host following exposure to MAP. Blood samples were collected from both sheep and cattle prior to experimental challenge with MAP and at monthly intervals post-challenge. Total serum cholesterol levels in sheep challenged with MAP were significantly elevated at 9 weeks post-inoculation (wpi) in comparison to controls. When stratified based on disease outcome, there was no significant difference in serum cholesterol at the timepoints examined between MAP exposed sheep that were susceptible and those that were resistant to Johne's disease. There was a similar elevation in serum cholesterol at 9 wpi in cattle with histopathological gut lesions associated with disease or those with an early high IFN-γ response. Total serum cholesterol in exposed cattle was significantly lower when compared to controls at 13 wpi. Taken together, these results demonstrate changes in serum cholesterol following MAP exposure and disease progression which could reflect novel aspects of the pathogenesis and immune response associated with MAP infection in both sheep and cattle.

Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

Case definition terminology for paratuberculosis (Johne's disease).

Paratuberculosis (Johne's disease) is an economically significant condition caused by Mycobacterium avium subsp. paratuberculosis. However, difficulties in diagnosis and classification of individual animals with the condition have hampered research and impeded efforts to halt its progressive spread in the global livestock industry. Descriptive terms applied to individual animals and herds such as exposed, infected, diseased, clinical, sub-clinical, infectious and resistant need to be defined so that they can be incorporated consistently into well-understood and reproducible case definitions. These allow for consistent classification of individuals in a population for the purposes of analysis based on accurate counts. The outputs might include the incidence of cases, frequency distributions of the number of cases by age class or more sophisticated analyses involving statistical comparisons of immune responses in vaccine development studies, or gene frequencies or expression data from cases and controls in genomic investigations. It is necessary to have agreed definitions in order to be able to make valid comparisons and meta-analyses of experiments conducted over time by a given researcher, in different laboratories, by different researchers, and in different countries. In this paper, terms are applied systematically in an hierarchical flow chart to enable classification of individual animals. We propose descriptive terms for different stages in the pathogenesis of paratuberculosis to enable their use in different types of studies and to enable an independent assessment of the extent to which accepted definitions for stages of disease have been applied consistently in any given study. This will assist in the general interpretation of data between studies, and will facilitate future meta-analyses.

Variation in susceptibility of different breeds of sheep to Mycobacterium avium subspecies paratuberculosis following experimental inoculation.

Exposure to Mycobacterium avium subspecies paratuberculosis (MAP) does not always lead to Johne's disease. Understanding differences in disease susceptibility of individual animals is a key aspect to controlling mycobacterial diseases. This study was designed to examine the susceptibility or resistance of various breeds of sheep to MAP infection. Merino, Suffolk first cross Merino, Border Leicester, and Poll Dorset sheep were orally inoculated with MAP and monitored for 14 months. Clinical disease occurred more frequently in the Merino (42%) and Suffolk first cross Merino (36%) compared to the Border Leicester (12%) and Poll Dorset (11%) breeds. Infection risk, as determined by culture of gut and associated lymphoid tissues, ranged from 75% for the Suffolk first cross Merino to 47% for the Poll Dorset sheep. Significant differences were identified in the site in the intestines of the most severe histopathological lesions and the immune responses to infection between the breeds. However, there was no difference in faecal MAP shedding by clinical cases between breeds. All breeds tested were susceptible to MAP infection, as determined by infection and clinical disease development, although there were differences in the proportions of diseased animals between the breeds. Poll Dorset and Border Leicester sheep were more resilient to MAP infection but there was evidence that more animals could have developed disease if given more time. These findings provide evidence of potential differential disease susceptibility between breeds, further our understanding of disease pathogenesis and risks of disease spread, and may have an influence on control programs for paratuberculosis.

Applying the One Health Concept to Mycobacterial Research - Overcoming Parochialism.

Mycobacterial infections remain a public health problem. Historically important, globally ubiquitous and with a wide host range, we are still struggling to control mycobacterial infections in humans and animals. While previous reviews have focused on individual mycobacterial infections in either humans or animals, a comprehensive review of the zoonotic aspect of mycobacteria in the context of the One Health initiative is lacking. With the purpose of providing a concise and comprehensive resource, we have collated literature to address the zoonotic potential of different mycobacterial species and elaborate on the necessity for an inter-sectorial approach to attain a new vision to combat mycobacterial infections.

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.

Pathogenesis of Frog Virus 3 ( Ranavirus, Iridoviridae) Infection in Wood Frogs ( Rana sylvatica).

Wood frogs ( Rana sylvatica) are highly susceptible to infection with Frog virus 3 (FV3, Ranavirus, Iridoviridae), a cause of mass mortality in wild populations. To elucidate the pathogenesis of FV3 infection in wood frogs, 40 wild-caught adults were acclimated to captivity, inoculated orally with a fatal dose of 104.43 pfu/frog, and euthanized at 0.25, 0.5, 1, 2, 4, 9, and 14 days postinfection (dpi). Mild lesions occurred sporadically in the skin (petechiae) and bone marrow (necrosis) during the first 2 dpi. Severe lesions occurred 1 to 2 weeks postinfection and consisted of necrosis of medullary and extramedullary hematopoietic tissue, lymphoid tissue in spleen and throughout the body, and epithelium of skin, mucosae, and renal tubules. Viral DNA was first detected (polymerase chain reaction) in liver at 4 dpi; by dpi 9 and 14, all viscera tested (liver, kidney, and spleen), skin, and feces were positive. Immunohistochemistry (IHC) first detected viral antigen in small areas devoid of histologic lesions in the oral mucosa, lung, and colon at 4 dpi; by 9 and 14 dpi, IHC labeling of viral antigen associated with necrosis was found in multiple tissues. Based on IHC staining intensity and lesion severity, the skin, oral, and gastrointestinal epithelium and renal tubular epithelium were important sites of viral replication and shedding, suggesting that direct contact (skin) and fecal-oral contamination are effective routes of transmission and that skin tissue, oral, and cloacal swabs may be appropriate antemortem diagnostic samples in late stages of disease (>1 week postinfection) but poor samples to detect infection in clinically healthy frogs.

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus).

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South-East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV-infected fish sharing a common water source.

Detection of infectious spleen and kidney necrosis virus (ISKNV) and turbot reddish body iridovirus (TRBIV) from archival ornamental fish samples.

Although infections caused by megalocytiviruses have been reported from a wide range of finfish species for several decades, molecular characterisation of the viruses involved has been undertaken only on more recent cases. Sequence analysis of the major capsid protein and adenosine triphosphatase genes is reported here from formalin-fixed, paraffin-embedded material from 2 archival ornamental fish cases from 1986 and 1988 in conjunction with data for a range of genes from fresh frozen tissues from 5 cases obtained from 1991 through to 2010. Turbot reddish body iridovirus (TRBIV) genotype megalocytiviruses, previously not documented in ornamental fish, were detected in samples from 1986, 1988 and 1991. In contrast, megalocytiviruses from 1996 onwards, including those characterised from 2002, 2006 and 2010 in this study, were almost indistinguishable from infectious spleen and kidney necrosis virus (ISKNV). Three of the species infected with TRBIV-like megalocytiviruses from 1986 to 1991, viz. dwarf gourami Trichogaster lalius (formerly Colisa lalia), freshwater angelfish Pterophyllum scalare and oscar Astronotus ocellatus, were infected with ISKNV genotype megalocytiviruses from 2002 to 2010. The detection of a TRBIV genotype isolate in ornamental fish from 1986 represents the index case, confirmed by molecular sequence data, for the genus Megalocytivirus.

Specific faecal antibody responses in sheep infected with Mycobacterium avium subspecies paratuberculosis.

Many studies have examined the serum antibody response to Mycobacterium avium subspecies paratuberculosis (MAP) infection in cases of Johne's disease (JD), but there are no reports on the mucosal antibody response. Faecal immunoglobulin (Ig) G and IgA ELISA responses were examined from sheep experimentally inoculated with MAP for up to 23 months post inoculation (PI). Corresponding serum IgG responses and the presence of viable MAP shed in faeces were also examined. The sheep were divided into three groups: (i) "un-inoculated controls" (n=10), (ii) "clinical cases" (n=8) which were inoculated animals that developed clinical disease and had moderate to high levels of MAP shedding and (iii) "survivors" (n=11) which were inoculated animals from which MAP could not be cultured from tissues at the conclusion of the trial. Serum IgG responses gradually increased in all inoculated animals, peaking at 12-16 months PI. A significant increase in the levels of MAP-specific faecal IgG and IgA was measured in the survivors at 16 and 17 months PI, while levels in the un-inoculated controls and clinical cases remained at baseline levels. The detection of faecal Ig in the survivors coincided with the removal of sheep that developed clinical disease. The data suggest that some sheep produced MAP-specific IgG and IgA in the intestinal mucosa, which was released into their faeces. We hypothesise that the survivors produced faecal Ig as a direct response to ingestion of MAP associated with environmental contamination from clinical cases. Thus MAP specific mucosal antibodies may play a previously unreported role as part of a protective response triggered by environmental exposure.

Histopathological Characterization of Cutaneous Delayed-type Hypersensitivity and Correlations with Intestinal Pathology and Systemic Immune Responses in Sheep with Paratuberculosis.

Cell-mediated immunity has been exploited historically in the diagnosis of mycobacterial diseases through elicitation of a delayed-type hypersensitivity (DTH) reaction following intradermal injection of an antigen. Here we describe the histopathological features of the cutaneous DTH reaction and its association with intestinal pathology and systemic immune responses in sheep with Mycobacterium avium subspecies paratuberculosis (MAP) infection. A mixed mononuclear cellular infiltrate dominated the DTH reaction and was present in perivascular and periadnexal patterns. Multiple multinucleate giant cells were present in the cellular infiltrate in one sheep while plasma cells were an obvious feature in six others. Sheep with paucibacillary intestinal lesions had the greatest degrees of cutaneous induration, more severe cellular infiltration in DTH lesions and high systemic interferon (IFN)-γ production. In contrast, sheep with multibacillary intestinal lesions, and particularly those with dissemination of MAP to extra-intestinal tissues, had minimal cutaneous induration, nil to mild cellular infiltration in DTH lesions and high serum anti-MAP antibody levels. Systemic IFN-γ production generally was augmented following skin sensitization. In general, the gross and histopathological features of the cutaneous DTH response matched the stage of paratuberculosis reflected by intestinal pathology and systemic measures of humoral and cellular immunity.

Pathology of a Bohle-like virus infection in two Australian frog species (Litoria splendida and Litoria caerulea).

Gross and histopathological examination was performed on seven captive magnificent tree frogs (Litoria splendida) and one green tree frog (Litoria caerulea) that had died or been humanely destroyed while naturally infected with Mahaffey Road virus, a Bohle iridovirus-like ranavirus. Necropsy examination revealed skin lesions consisting of multiple small pale or haemorrhagic papules and ulcers in most frogs. Other common gross findings were perineural haemorrhage affecting the spinal nerves, hydrocoelom, hepatomegaly and splenomegaly with pinpoint pale foci throughout the parenchyma. On histological examination, vasculitis with prominent endothelial necrosis was found in a wide range of tissues. Widespread lymphoid necrosis and fibroblast necrosis were usual findings. Multifocal epithelial cell necrosis in the epidermis, liver and pancreas was found commonly. Non-suppurative meningoencephalitis, myelitis and ganglioneuritis were present variably. Intracytoplasmic basophilic inclusion bodies were found variably in hepatocytes, renal tubular epithelium and keratinocytes. Immunohistochemistry demonstrated ranavirus antigen in endothelial cells, fibroblasts, macrophages, lymphocytes and epithelial cells in a wide range of tissues. The finding of widespread venous and lymphatic endothelial necrosis and demonstration of abundant endothelial antigen suggests that endothelial tropism of the virus plays a significant role in the pathogenesis of the infection.

Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep.

An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p < 0.05). The sensitivities were 5.9% (95% CI: 1-26.9), 27.9% (95% CI: 14.7-45.7) and 35% (95% CI: 18.1-56.7), for low grade, paucibacillary and multibacillary lesion grades, respectively. The cost of the commercial assay kit was 2.7 to 5.2 times greater than that of the 316v ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

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.

Effectiveness of Gudair™ vaccine for the control of ovine Johne's disease in flocks vaccinating for at least 5 years.

OBJECTIVE: Examine the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (Mptb) at least 5 years after starting vaccination with Gudair™ in flocks of varying initial prevalence of ovine Johne's disease (OJD) and identify risk factors for variation in vaccine efficacy. METHODS: Pooled faecal culture (PFC) was conducted for 41 flocks from southern NSW and Victoria to determine estimates of current OJD prevalence. The data were compared to estimates of prevalence at or prior to commencement of vaccination at least 5 years earlier, based on available serological or PFC tests when vaccination commenced. A cross-sectional study was conducted to identify risk factors for differing prevalence levels in 36 of the flocks. RESULTS: Historical data enabled classification of 37 flocks as high (13; 35.1%), medium (10; 27.0%) or low (14; 37.8%) estimated initial OJD prevalence. Results of PFC in 2008-09 identified that 81.1% (30/37) of flocks had detectable shedders, with 48.6% (18/37) flocks still classified as medium or high OJD prevalence, including 50% (7/14) of flocks initially classified as low prevalence. Shedding was not detected in 18.9% (7/37) flocks. Flocks with OJD prevalence exceeding 1% at 5 years or more following the commencement of vaccination were associated with reports of sheep straying and introduction of new sheep. CONCLUSION: Despite significant declines in estimated OJD prevalence following vaccination for ≥5 years, 81.1% of flocks were shedding Mptb and considered at risk of spreading the disease or suffering recrudescence of losses if vaccination were to cease. Flock managers are advised to persist with vaccination.