Effects of swab pool size and transport medium on the detection and isolation of avian influenza viruses in ostriches.

BACKGROUND: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. RESULTS: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (106.7 - 103.0 vs. 106.6 - 103.1 EID50 for H5N8 HPAIV and 105.5 - 101.4 vs. 105.1 - 101.3 EID50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. CONCLUSION: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.

Continuing evolution of H6N2 influenza a virus in South African chickens and the implications for diagnosis and control.

BACKGROUND: The threat of poultry-origin H6 avian influenza viruses to human health emphasizes the importance of monitoring their evolution. South Africa's H6N2 epidemic in chickens began in 2001 and two co-circulating antigenic sub-lineages of H6N2 could be distinguished from the outset. The true incidence and prevalence of H6N2 in the country has been difficult to determine, partly due to the continued use of an inactivated whole virus H6N2 vaccine and the inability to distinguish vaccinated from non-vaccinated birds on serology tests. In the present study, the complete genomes of 12 H6N2 viruses isolated from various farming systems between September 2015 and February 2019 in three major chicken-producing regions were analysed and a serological experiment was used to demonstrate the effects of antigenic mismatch in diagnostic tests. RESULTS: Genetic drift in H6N2 continued and antigenic diversity in sub-lineage I is increasing; no sub-lineage II viruses were detected. Reassortment patterns indicated epidemiological connections between provinces as well as different farming systems, but there was no reassortment with wild bird or ostrich influenza viruses. The sequence mismatch between the official antigens used for routine hemagglutination inhibition (HI) testing and circulating field strains has increased steadily, and we demonstrated that H6N2 field infections are likely to be missed. More concerning, sub-lineage I H6N2 viruses acquired three of the nine HA mutations associated with human receptor-binding preference (A13S, V187D and A193N) since 2002. Most sub-lineage I viruses isolated since 2015 acquired the K702R mutation in PB2 associated with the ability to infect humans, whereas prior to 2015 most viruses in sub-lineages I and II contained the avian lysine marker. All strains had an unusual HA0 motif of PQVETRGIF or PQVGTRGIF. CONCLUSIONS: The H6N2 viruses in South African chickens are mutating and reassorting amongst themselves but have remained a genetically pure lineage since they emerged more than 18 years ago. Greater efforts must be made by government and industry in the continuous isolation and characterization of field strains for use as HI antigens, new vaccine seed strains and to monitor the zoonotic threat of H6N2 viruses.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

The Molecular Epidemiology of Clade 2.3.4.4B H5N1 High Pathogenicity Avian Influenza in Southern Africa, 2021-2022.

In southern Africa, clade 2.3.4.4B H5N1 high pathogenicity avian influenza (HPAI) was first detected in South African (SA) poultry in April 2021, followed by outbreaks in poultry or wild birds in Lesotho and Botswana. In this study, the complete or partial genomes of 117 viruses from the SA outbreaks in 2021-2022 were analyzed to decipher the sub-regional spread of the disease. Our analysis showed that seven H5N1 sub-genotypes were associated with the initial outbreaks, but by late 2022 only two sub-genotypes still circulated. Furthermore, SA poultry was not the source of Lesotho's outbreaks, and the latter was most likely an introduction from wild birds. Similarly, SA and Botswana's outbreaks in 2021 were unrelated, but viruses of Botswana's unique sub-genotype were introduced into SA later in 2022 causing an outbreak in ostriches. At least 83% of SA's commercial poultry cases in 2021-2022 were point introductions from wild birds. Like H5N8 HPAI in 2017-2018, a coastal seabird-restricted sub-lineage of H5N1 viruses emerged in the Western Cape province in 2021 and spread to Namibia, causing mortalities in Cape Cormorants. In SA ~24,000 of this endangered species died, and the loss of >300 endangered African penguins further threatens biodiversity.

Identification of bacteria in the tracheal swabs of farmed ostriches and their effect on the viability of influenza A virus.

Avian influenza surveillance is a requirement for commercial trade in ostrich products, but influenza A viruses (IAVs) have proven difficult to isolate from ostrich tracheal swabs that test positive using molecular methods. We hypothesized that microbes unique to the ostrich trachea propagate in the transport medium after sampling and affect viral viability. We cultured tracheal swabs from 50 ostriches on 4 farms in South Africa, and recovered and identified 13 bacterial, 1 yeast, and 2 fungal species. Dietzia sp. had not been identified previously in the oropharyngeal tract of a bird, to our knowledge. The bacteria were tested for antimicrobial susceptibility, and most aerobic species, except for Streptococcus sp. and Pseudomonas sp., were sensitive to enrofloxacin; all were susceptible to sulfonamide. Virus inhibition experiments determined that ostrich-source Streptococcus sp., Pantoea sp., and Citrobacter freundii produced extracellular metabolites that caused a substantial reduction in the IAV titers of 99.9%. Streptomyces, Corynebacterium, Staphylococcus, Arthrobacter gandavensis, Pseudomonas putida, and Acinetobacter spp. similarly reduced the viability of IAV from 77.6% to 24.1%. Dietzia appeared to have no effect, but Rothia dentocariosa, Rhodotorula spp., and Clostridium spp. slightly increased the viability of IAV by 25.9, 34.9, and 58.5%, respectively.

Complete Genome Sequence of a Class I Avian Orthoavulavirus 1 Isolated from Commercial Ostriches.

A hemagglutinating virus isolated during routine surveillance in ostriches was sequenced, identified as avian orthoavulavirus 1 (AOaV-1), and classified as a class I genotype 1.2 virus, with recent common ancestors in Eurasian wild ducks. This is the first class I AOaV-1 isolate from Africa and the first identified in ostriches.

Evolutionary consequences of a decade of vaccination against subtype H6N2 influenza.

The evolutionary dynamics of chicken-origin H6N2 viruses isolated in South Africa between 2002 and 2013 were investigated. Sub-lineages I and II continued to co-circulate under vaccination pressure, but sub-lineage I, from which the inactivated vaccine was derived, displayed a markedly higher mutation rate and a three-fold increase in the emergence of potential antigenic sites on the globular head of HA compared to sub-lineage II. Immunological pressure culminated in a critical phenotypic change as four of the five isolates from 2012 to 2013 had lost the ability to haemagglutinate chicken erythrocytes, correlating with a pattern of predicted O-glycosylation sites at residues 134, 137 and 141 within the critical 130 loop of the receptor binding domain site. Coassortment of the HA, NA and M genes in the respective sub-lineages contrasted reassortment of the other internal protein genes, and the vaccine seed strain itself was the probable donor of segments to sub-lineage II field strains.