Investigation of association between bovine viral diarrhoea virus and cervid herpesvirus type-1, and abortion in New Zealand farmed deer.

This study tested for association between bovine viral diarrhoea virus (BVDv) and cervid herpesvirus type-1 (CvHV-1) exposure and abortion in New Zealand farmed red deer. Rising two-year-old (R2, n = 22,130) and mixed-age (MA, n = 36,223) hinds from 87 and 71 herds, respectively, throughout New Zealand were pregnancy tested using ultrasound early in gestation (Scan-1) and 55-89 days later (Scan-2) to detect mid-term abortion. Sera from aborted and non-aborted hinds at Scan-2 were tested for BVDv and CvHV-1 using virus neutralisation tests. Available uteri from aborted hinds and from hinds not rearing a calf to weaning were tested by PCR for herpesvirus DNA. In herds with aborted hinds, 10.3% of 639 R2 and 17.2% of 302 MA hinds were sero-positive for BVDv and 18.6% of 613 R2 and 68.5% of 232 MA hinds were sero-positive for CvHV-1. There was no association between BVDv sero-status and abortion at animal level (R2 p = 0.36, MA p = 0.76) whereas CvHV-1 sero-positivity was negatively associated with abortion in MA hinds (p = 0.01) but not in R2 hinds (p = 0.36), MA). Eleven of 108 uteri from aborted R2 hinds but no MA hinds were positive for herpesvirus DNA. Vaginal samples from four R2 and one MA aborted hinds tested were negative for herpesvirus DNA. A Cervid Rhadinovirus type-2 (CRhV-2) was identified in seven PCR positive uteri samples. Findings suggest that BVDv and CvHV-1 may not be associated with abortion in R2 hinds, but association needs to be tested further in MA hinds. The role of CRhV-2 requires clarification.

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand.

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons. LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D752 that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak. DIAGNOSIS: Equine herpesvirus myeloencephalopathy. CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.

Surveillance for avian influenza virus subtypes H5 and H7 in chickens and turkeys farmed commercially in New Zealand.

AIM: To determine the status of avian influenza (AI) virus sub-types H5 and H7 of New Zealand's commercial chicken and turkey farms. METHODS: A cross-sectional serological survey, stratified by production sector, used a sample frame defined by those farms registered with the Poultry Industry Association of New Zealand (PIANZ) or the Egg Producers Federation of New Zealand (EPF). Sectors included were chicken broiler, caged/barn layer, free-range layer, pullet rearer and turkey broiler. The survey used a between- and within-farm design prevalence of 5% (95% confidence for chickens, 99% confidence for turkeys) and 30% (95% confidence), respectively, of AI virus subtypes H5 and H7. The epidemiological unit was the farm for the free-range layer sector, and the individual shed/barn for the other sectors. Serum samples were screened using a commercial generic influenza A indirect ELISA; positive samples were subjected to haemagglutination-inhibition (HI) testing for AI virus subtypes H5 and H7. A comprehensive investigation, that included widespread serological and antigenic screening, was carried out on all farms identified with serum reactors to either the H5 or H7 virus subtype. RESULTS: A total of 4,180 blood samples from 167 chicken and 10 turkey farms were collected and tested using ELISA. Positive ELISA results were returned from 26 farms, comprising 10 caged/barn layer, 14 free-range layer and two turkey (shed-raised) broiler farms. HI testing of ELISA-positive sera for the H7 subtype virus identified no positive sera in any sector. Reactors to the H5 subtype virus were limited to three free-range layer chicken farms; each farm returned a single serum reactor. Follow-up investigations on these free-range farms identified evidence of historic exposure to the H5 subtype virus on one farm, and concluded that the serum reactors identified in the initial sampling round on the other two farms were non-specific (false-positive) reactions. CONCLUSIONS: The survey found no evidence of active infection with notifiable AI viruses, and provided evidence of absence of exposure to AI virus subtypes H5 and H7 in the chicken broiler, caged/barn layer, turkey broiler and pullet-rearer sectors at a between- and within-farm prevalence of 5% and 30%, respectively, with 95% confidence. The results established commercial free-range layer farms as a risk sector for exposure to notifiable AI virus.

A cross-sectional survey of influenza A infection and management practices in small rural backyard poultry flocks in New Zealand.

AIM: To obtain baseline data on the management of small non-commercial backyard poultry flocks, in two rural regions of New Zealand, to investigate potential transmission pathways for avian influenza (AI), and to investigate the presence of AI in these flocks. METHODS: During August-October 2006 a questionnaire was sent to 105 farms in the Bay of Plenty and Wairarapa with poultry flocks comprising fewer than 50 chickens, located near wetlands where AI virus had been detected previously in wild ducks. Information was collected on the number and species of poultry reared, opportunities for interaction between wild birds and poultry, farm biosecurity measures, and health status of poultry. Between September and November 2006, blood and tracheal/cloacal swabs were collected from poultry on a subset of 12 high-risk farms in each location. Influenza A-specific antibodies in sera were assayed using ELISA, and positive sera were further tested for the presence of H5 and H7 subtype-specific antibodies, using haemagglutination inhibition (HI) assay. The presence of influenza A virus in swabs was detected using real-time reverse transcriptase-PCR (RRT-PCR). RESULTS: Returned questionnaires were received from 54 farms. Overall, 80% had only chickens, 13% chickens and ducks, and 7% had chickens and other galliform species. Nearly all (96%) kept backyard chickens for personal consumption of eggs, with a small proportion (19%) preparing birds for the table. On surveyed farms wild waterfowl were seen on pastures (70%) and/or farm waterways (46%). Waterfowl were recorded as visiting areas where domestic birds were kept on 31% of farms. Bird litter and manure were composted (94%) or buried (6%) on-farm, as were most (82%) dead birds. During the targeted cross-sectional survey of 24 farms clinical disease was not recorded in any poultry flock. Of 309 chicken sera tested, 11 (3.6%) from five farms across both regions tested positive for influenza A antibodies. In contrast, 16/54 (30%) duck sera from three farms in the Wairarapa were positive. Avian influenza H5 and H7 subtype-specific antibodies were excluded in ELISA positive sera using HI testing, and influenza A virus was not detected using RRT-PCR. CONCLUSIONS: The study confirmed that small backyard poultry flocks located near waterfowl habitats were exposed to non-notifiable low-pathogenic AI viruses. Findings indicate a number of potential risk pathways for the transmission of AI viruses between wild birds and non-commercial poultry, and hence the need for continued surveillance for AI in backyard flocks and wild birds in New Zealand.

Surveillance for highly pathogenic avian influenza in migratory shorebirds at the terminus of the East Asian-Australasian Flyway.

AIM: To determine if migratory birds arriving in New Zealand in the Southern Hemisphere spring of 2004 were infected with the highly pathogenic avian influenza (AI) virus, H5N1. METHODS: Cloacal and faecal samples were collected from migratory red knots following their arrival in New Zealand in October 2004. Two species of resident sympatric birds, wrybill and mallard duck, were sampled prior to, and following, the arrival of migratory birds. RESULTS: No AI viruses were isolated from migratory or resident shorebirds. Non-pathogenic AI viruses were isolated from six resident mallard ducks, comprising the endemic subtypes H4 (n=2), H7 (non-pathogenic), H10, and H11 (n=2). CONCLUSIONS: Highly pathogenic AI H5N1 virus was not detected in migratory shorebirds or sympatric water birds in the Firth of Thames, New Zealand, in 2004-2005, despite the possible proximity of migratory birds to outbreaks of the disease in East Asia in 2004.

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand.

A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to > or =1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00-0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France.

A survey for paramyxoviruses in caged birds, wild birds, and poultry in New Zealand.

AIMS: To determine the presence of avian paramyxovirus (APMV) types 1, 2, and 3 in caged and wild birds, and APMV-2 and -3 in poultry in New Zealand. METHODS: Blood samples collected from caged (231) and wild birds (522) from various regions of New Zealand in 1997-99 were tested by haemagglutination inhibition (HI) test for antibodies to APMV types 1, 2, and 3. Blood samples collected from 1778 commercial poultry in 1996-99 were tested for APMV-2 and APMV-3 antibodies and the samples that reacted with APMV-3 antigen were tested for antibodies to APMV-1. Isolation of APMV was attempted from cloacal swabs collected from 116 of the caged birds and 175 of the wild birds sampled. RESULTS: Antibodies to APMV types 1, 2, and 3 were detected in 4.8, 1.7, and 2.6%, respectively, of caged bird samples. The majority of these caged birds were 'exotic' or 'fancy' poultry breeds. Amongst wild birds, 4.2% had titres to APMV-2 and over half of these were passerine birds; 1.7% of the samples had titres to APMV-1 and 0.8% to APMV-3 antigen. No virus was isolated from any of the cloacal swabs tested. Of the 1778 poultry serum samples tested, only 5 reacted with APMV-3 antigen and these were later found to be cross-reactions to APMV-1. No reactions were detected with APMV-2 antigen. CONCLUSIONS: APMV-1 is present in caged birds, wild birds, and poultry of New Zealand. There is no conclusive evidence of the presence of APMV-2 and APMV-3 in poultry or APMV-3 in wild birds. The results do not provide conclusive evidence for the presence of APMV-2 in wild birds in New Zealand.

Pathological studies of wobbly possum disease in New Zealand brushtail possums (Trichosurus vulpecula).

Following an outbreak of wobbly possum disease in a colony of brush tail possums (Trichosurus vulpecula), the disease was established experimentally in captive possums by inoculating the animals intraperitoneally with tissue homogenates. Crude tissue homogenates of liver remained infectious after freezing at -75 degrees C or filtration through a 0.22 micron filter. The disease was characterised by docility, incoordination, loss of balance and wasting. Fifteen of 16 infected animals had to be euthanased owing to the severity of clinical signs. Cachexia was the only change observed postmortem. Histology revealed widespread perivascular infiltrations with plasma cells and lymphocytes which were severe in the liver and kidney and moderate to mild in a variety of other tissues, including skeletal and cardiac muscle. Changes in the brain consisted of a mild to moderate mononuclear perivascular cuffing. Most of the animals had small to large numbers of circulating nucleated red blood cells and eosinopenia when they were euthanased. There was a consistent decrease in serum albumin concentration and an increase in serum globulins, which resulted in a decreased albumin:globulin ratio. Virus-like particles were observed in preparations of liver from two animals; they appeared to be spherical or icosahedral and were 45 nm in diameter.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep.

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10(3.2) egg lethal doses (ELD50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40 degrees C) was indicative to the likelihood of detectable antigenaemia. After day 5 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

Observations on the relationship in chickens between the virulence of some avian influenza viruses and their pathogenicity for various organs.

Comparative histological and immunocytochemical studies were conducted on formalin-fixed tissues from chickens infected with avian influenza viruses of varying virulence. Results showed a distinct pattern of disease that depended on the virulence of the virus and the susceptibility of the birds. At 3 days post-intranasal inoculation with a highly virulent H7N7 virus, all 6-to-8-week-old specific-pathogen-free (SPF) birds were affected, and all developed pancreatic necrosis and encephalitis associated with specific immunoperoxidase staining. Other same-aged SPF birds were only occasionally affected 6 to 8 days after intravenous inoculation with almost avirulent H4N4, H6N2, or H3N8 virus. Specific lesions and immunoperoxidase staining were noted in the kidneys only. The H7N7 virus in older commercial birds and an H7N3 virus in young SPF and older commercial birds caused intermediate mortality rates at 4 to 11 days postinoculation, and there was a broad range of lesions and specific immunoperoxidase staining in the pancreas, brain, kidney, heart, and skeletal muscle. Two exceptional birds had immunostaining of small blood vessels throughout their bodies with or without lesions or staining in the tissues, which may have represented a transitory pre-localizing phase occurring in many birds. There was necrosis without virus antigen detection in the bursae, thymuses, and cecal tonsils, possibly secondary to stress or only transitory infection of virus. These data indicate that rapid, retrospective diagnosis of avian influenza in fixed tissues is possible by using an immunoperoxidase test on pancreas, brain, and kidney.

Isolation of chicken anaemia virus from broiler chickens in New Zealand.

Chicken anaemia virus was isolated for the first time in New Zealand from the New Zealand domestic chicken population. The virus was recovered from diseased birds in five separate flocks of broiler chickens aged between 14 and 33 days of age. Six isolates were obtained from bone marrow and lymphoid tissues using the MDCC-MSB1 cell line derived from Marek's disease lymphoma. All isolates were resistant to chloroform and survived exposure to 70 degrees C for 5 minutes. The main clinical features consistently associated with the disease outbreaks were increased mortality, yolk sac infections, sub-cutaneous haemorrhages and atrophy of the thymus. Fungal pneumonia occurred in two flocks, and gangrenous dermatitis as a result of bacterial infection in another flock. Microscopic examination showed atrophy of the thymus, reduced medullary haematopoiesis and inflammation resulting from secondary infections.

EDS 76 HI antibodies in a flock of free-range layers.

An investigation of poor laying performance in a flock of free-range hens revealed high levels of serum antibodies to EDS 76 in the flock initially examined and in another, older flock on the same farm. These flocks had contact with ducks on a farm dam and were supplied with untreated drinking water from the dam. Serological evidence indicated that another flock supplying the same egg packing station had been infected with EDS 76 virus. Little serological evidence of EDS infection was detected from five other flocks supplying the packing station, parent breeders or the ducks resident on the dam. Therefore, the source of the EDS 76 virus remains conjectural.

Polymerase chain reaction amplification for direct detection of chicken anemia virus DNA in tissues and sera.

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of chicken anaemia agent (CAA) DNA. The assay used a single set of 20-base primers complementary to sequences located in the coding regions of the CAA replicative form (RF) DNA genome at positions 485 to 504 and 1048 to 1067. The observed amplification product had the expected size of 583 bp and was confirmed to derive from CAA RF DNA by a unique Hind III restriction enzyme cleavage pattern. The amplified fragment was shown to be specific for CAA RF DNA after chemiluminescence dot blot hybridisation with a digoxigenin-labelled 25-base internal probe. The optimised PCR assay was specific for CAA and highly sensitive, being able to detect a single CAA-infected MDCC-MSB1 cell and at least 100 fg of CAA RF DNA. Preliminary results also showed that the PCR assay can detect CAA DNA in clinical specimens from chicks experimentally infected with CAA.

Pasteurella multocida infections in rockhopper penguins (Eudyptes chrysocome) from Campbell Island, New Zealand.

During an investigation into the population decline of rockhopper penguins (Eudyptes chrysocome) on Campbell Island, New Zealand, avian cholera (Pasteurella multocida) was found in dead adults and chicks. An RNA enveloped virus was isolated from Ixodes uriae, a tick which commonly parasitizes rockhopper penguins on the island. It is not known whether this virus is virulent for penguins. No evidence was obtained to suggest that avian cholera was the principal cause for the decline in the rockhopper penguin population.