In situ hybridization and histopathological observations during ostreid herpesvirus-1-associated mortalities in Pacific oysters Crassostrea gigas.

In a previous longitudinal study conducted during a mortality investigation associated with ostreid herpesvirus-1 (OsHV-1) microvariant in New Zealand Pacific oysters in 2010-2011, temporality of OsHV-1 nucleic acid detection by real-time PCR assay and onset of Pacific oyster mortality was observed. The present study further elucidated the role of OsHV-1 using an in situ hybridization (ISH) assay on sections of Pacific oysters collected from the same longitudinal study. Hybridization of the labelled probe with the target region of the OsHV-1 genome in infected cells was detected colorimetrically using nitro blue tetrazolium (NBT). OsHV-1 presence and distribution in spat indicated by the ISH signal was then compared with the existence of pathological changes in oyster tissues. Dark blue to purplish black NBT cell labelling was seen predominantly in the stroma of the mantle and gills at Day 5 post introduction to the farm. The distribution and location of ISH signals indicated the extent of OsHV-1-infected cells in multiple tissues. Histopathological abnormalities were mostly non-specific; however, a progressive pattern of increasingly widespread haemocytosis coincided with the appearance of OsHV-1-infected cells in spat collected at different time-points. The visualisation of an increasing number of OsHV-1-positive cells in spat prior to a marked increase in mortality indicated the strong likelihood of an on-going and active viral infection in some oysters. Further studies are recommended to elucidate OsHV-1 pathogenesis in Pacific oysters in association with other potentially causal variables, such as elevated temperature and interaction with Vibrio spp. bacteria.

Detection of Foot-and-Mouth Disease Virus in the Absence of Clinical Disease in Cattle and Buffalo in South East Asia.

Foot-and-mouth disease virus (FMDV) is widespread throughout much of the world, including parts of South East Asia. Surveillance is often limited in endemic areas, relying predominantly on passive outbreak reporting. As part of the World Organisation for Animal Health (OIE)'s South East Asia and China Foot-and-Mouth Disease Project (SEACFMD), field sampling was performed to help understand evidence of widespread virus exposure observed in previous studies. Serum and dry mucosal swabs were collected to evaluate the presence of FMDV RNA on the nasal, oral, and dorsal nasopharyngeal mucosal surfaces of 262 healthy cattle (n = 84 in Laos; n = 125 in Myanmar) and buffalo (n = 48 in Laos; n = 5 in Myanmar) immediately following slaughter in three slaughterhouses. Swabs and serum were tested by the OIE/FAO World Reference Laboratory for foot-and-mouth disease (WRLFMD) using pan-serotypic real-time reverse transcription-PCR (rRT-PCR) and serum was evaluated using the FMD PrioCHECK non-structural protein (NSP) ELISA. In total, 7.3% of animals had detectable FMDV RNA in one or more of the three sites including 5.3% of nasopharyngeal swabs, 2.3% of oral swabs, and 1.5% of nasal swabs. No FMDV RNA was detected in serum. Overall, 37.8% of animals were positive for NSP antibodies, indicating likely past natural exposure to FMDV. Results were comparable for Laos and Myanmar, and for both cattle and buffalo, and were not significantly different between age groups. Detectable FMDV RNA present on the oral and nasal mucosa of clinically-healthy large ruminants in Laos and Myanmar demonstrates the importance of sampling asymptomatic animals as part of surveillance, and may indicate that subclinical infection plays a role in the epidemiology of FMD in these countries.

Atypical scrapie/Nor98 in a sheep from New Zealand.

In a consignment of sheep brains from New Zealand, to be used in Europe as negative control material in scrapie rapid screening test evaluations, brain samples from 1 sheep (no. 1512) gave the following initially confusing results in various screening tests: the brainstem repeatedly produced negative results in 2 very similar screening kits (enzyme-linked immunosorbent assay [ELISA]-1, ELISA-2), a macerate made from brainstem and cerebellum returned a clearly positive result in ELISA-2, and the macerate and a brainstem sample gave negative results in a third screening test (ELISA-3). In subsequent testing, cerebellum tissue alone tested strongly positive in ELISA-1 and produced a banding pattern very similar to atypical scrapie/Nor98 in a confirmatory Western blot (WB). The macerate showed weak staining in the confirmatory WB but presented a staining pattern identical to atypical scrapie/Nor98 in the scrapie-associated fibril WB. The latter test confirmed conclusively the first case of atypical scrapie/Nor98 in a sheep from New Zealand. Other parts of the brain either tested negative or very weak positive in ELISA-2 and in WBs, or tested with negative results by histopathology and immunohistochemistry. It appears that sheep no. 1512 is a case of atypical scrapie/Nor98 in which the abnormal prion protein was detected mainly in the cerebellum. This case emphasizes the need to retain brainstem, and cerebral and cerebellar tissues, as frozen and fixed materials, for conclusive confirmatory testing. Furthermore, consideration should be given to which screening method to use.

Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle.

In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.

Comparative evaluation of four competitive/blocking ELISAs for the detection of influenza A antibodies in horses.

New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n=365), and Australian field infected (n=99) and experimentally infected horses (n=3). Diagnostic specificities (DSP) and diagnostic sensitivities (DSE) were determined as follows: ELISA-1=98.1%/99.0%; ELISA-2=90.1%/99.0%; ELISA-3=98.1%/96.0%; ELISA-4=95.3%/99.0%. For ELISA-1, DSP and DSE results were comparable to previously published data on a larger sample number from Australian horses (Sergeant et al., 2009). Receiver operating characteristics (ROC) and frequency histogram analysis were also performed. The area under the curve (AUC) ranged from 0.996 to 0.979, with ELISA-1 possessing the highest AUC, followed by ELISA-2, ELISA-4 and ELISA-3. Separation of the negative and the positive serum panel was best for ELISA-4, followed by ELISA-2, ELISA-1 and ELISA-3. In three experimentally infected horses, sero-positivity was detected between 7 and 9 days post-infection, with ELISA-4 being most sensitive, followed by ELISA-1, ELISA-2 and ELISA-3. Overall, the four ELISAs performed well in this evaluation but some differences were observed.