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.

Evidence for Multidrug Resistance in Nonpathogenic Mycoplasma Species Isolated from South African Poultry.

One hundred seventy-eight mycoplasma strains isolated from South African poultry flocks between 2003 and 2015 were identified by full-genome sequencing and phylogenetic analysis of the 16S rRNA gene and were classified as follows: Mycoplasma gallisepticum (25%), M. gallinarum (25%), M. gallinaceum, (23%), M. pullorum (14%), M. synoviae (10%), and M. iners (3%), as well as one Acheoplasma laidlawii strain (1%). MIC testing was performed on the axenic samples, and numerous strains of each species were resistant to either chlortetracycline or tylosin or both, with variable sensitivity to enrofloxacin. The strains of all species tested remained sensitive to tiamulin, except for one M. gallinaceum sample that demonstrated intermediate sensitivity. The mutation of A to G at position 2059 (A2059G) in the 23S rRNA gene, which is associated with macrolide resistance, was found in the South African M. gallisepticum and M. synoviae strains, as well as a clear correlation between macrolide resistance in M. gallinarum and M. gallinaceum and mutations G354A and G748A in the L4 ribosomal protein and 23S rRNA gene, respectively. No correlation between resistance and point mutations in the genes studied could be found for M. pullorum Only a few strains were resistant to enrofloxacin, apart from one M. synoviae strain with point mutation D420N, which has been associated with quinolone resistance, and no other known markers for quinolone resistance were found in this study. Proportionally more antimicrobial-resistant strains were detected in M. gallinaceum, M. gallinarum, and M. pullorum than in M. gallisepticum and M. synoviae Of concern, three M. gallinaceum strains showed multidrug resistance to chlortetracycline, tylosin, and oxytetracycline.IMPORTANCE Nonpathogenic poultry Mycoplasma species are often overlooked due to their lesser impact on poultry health and production compared to the OIE-listed pathogenic strains M. gallisepticum and M. synoviae The use of antimicrobials as in-feed growth promoters and for the control of mycoplasmosis is common in poultry production across the world. Here, we provide evidence that certain nonpathogenic Mycoplasma species are acquiring multidrug resistance traits. This would have significant implications if these species, for which no vaccines are applied, are able to transfer their antibiotic resistance genes to other mycoplasmas and bacteria that may enter the human food chain.