Avian influenza viruses in New Zealand wild birds, with an emphasis on subtypes H5 and H7: Their distinctive epidemiology and genomic properties.

The rapid spread of highly pathogenic avian influenza (HPAI) A (H5N1) viruses in Southeast Asia in 2004 prompted the New Zealand Ministry for Primary Industries to expand its avian influenza surveillance in wild birds. A total of 18,693 birds were sampled between 2004 and 2020, including migratory shorebirds (in 2004-2009), other coastal species (in 2009-2010), and resident waterfowl (in 2004-2020). No avian influenza viruses (AIVs) were isolated from cloacal or oropharyngeal samples from migratory shorebirds or resident coastal species. Two samples from red knots (Calidris canutus) tested positive by influenza A RT-qPCR, but virus could not be isolated and no further characterization could be undertaken. In contrast, 6179 samples from 15,740 mallards (Anas platyrhynchos) tested positive by influenza A RT-qPCR. Of these, 344 were positive for H5 and 51 for H7. All H5 and H7 viruses detected were of low pathogenicity confirmed by a lack of multiple basic amino acids at the hemagglutinin (HA) cleavage site. Twenty H5 viruses (six different neuraminidase [NA] subtypes) and 10 H7 viruses (two different NA subtypes) were propagated and characterized genetically. From H5- or H7-negative samples that tested positive by influenza A RT-qPCR, 326 AIVs were isolated, representing 41 HA/NA combinations. The most frequently isolated subtypes were H4N6, H3N8, H3N2, and H10N3. Multivariable logistic regression analysis of the relations between the location and year of sampling, and presence of AIV in individual waterfowl showed that the AIV risk at a given location varied from year to year. The H5 and H7 isolates both formed monophyletic HA groups. The H5 viruses were most closely related to North American lineages, whereas the H7 viruses formed a sister cluster relationship with wild bird viruses of the Eurasian and Australian lineages. Bayesian analysis indicates that the H5 and H7 viruses have circulated in resident mallards in New Zealand for some time. Correspondingly, we found limited evidence of influenza viruses in the major migratory bird populations visiting New Zealand. Findings suggest a low probability of introduction of HPAI viruses via long-distance bird migration and a unique epidemiology of AIV in New Zealand.

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.

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 three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus.

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.