Ostriches (Struthio camelus) are highly versatile production animals raised for their low-cholesterol, lean red meat; fine leather, and feathers used in the fashion industry. Namibia, Australia, Botswana, France, Indonesia, and some other territories like China, the USA, and the Middle East to a lesser extent raise ostriches commercially, but South Africa remains the lead export producer with 75% of the global market share, and the European Union (EU) as the main market . The number of slaughter birds in South Africa has been on a constant decline from 250,000 per annum in 2011 to < 150,000 in 2020. Drought in the region has played a role, but the most significant driver of the decline is virus identification-related long delays to regain freedom of avian influenza (AI) infection status. Ostrich producers aiming to export ostrich meat must be registered with the competent authority and comply with strict bio-security requirements, amongst other measures. The fresh meat of birds originating from a holding that was exposed to notifiable avian influenza (i.e., viruses of the H5 or H7 subtypes) within 6 months prior to slaughter may not be exported; therefore, routine screening of ostriches to prove freedom of infection from influenza A virus (IAV) infection is central to maintaining export status .
The extensive nature of the farming system predisposes ostriches to frequent contact with wild birds. Wild waterfowl species are the natural reservoirs of all low pathogenicity influenza A viruses (LPAIV) as well as clade 126.96.36.199 H5Nx high pathogenicity influenza A viruses (HPAIV) [3, 4], thus any IAVs excreted in sufficient titres in their faeces and oral secretions into a shared environment may be ingested or inhaled by the ostriches. Atypically for a terrestrial species, ostriches normally show no clinical signs with LPAIV or HPAIV infection [4, 5] yet like gallinaceous birds, ostriches can facilitate mutation of LPAIV to HPAIV after a period of intra-host virus circulation. The emergence of H5N2 HPAIV in ostriches caused three unrelated epidemics in 2004, 2006 and 2011 in South Africa’s Eastern and/or Western Cape provinces , and ostriches also contracted clade 188.8.131.52B H5N8 HPAIV strains introduced by migratory waterfowl in the 2017–2018 epidemics [4, 5]. Regardless of the implications for the export market, early detection and preventing notifiable IAVs from circulating in ostriches is very important, as any spill-over to the mainstream poultry industry could threaten regional food security or even human health.
In South Africa, serological surveillance for IAV infection of all registered ostrich farms is compulsory every 6 months. When H5 or H7-specific antibodies are detected, immediate disease investigation and control measures are implemented, including intensified sampling for serological testing and agent detection using real-time reverse transcription polymerase chain reaction (RT-rtPCR). Repeated sampling events must be done to detect the presence or absence of AI-specific antibodies and the virus antigen at a > 5% prevalence with 95% confidence in each epidemiological group. Any RT-rtPCR positive results must be tested further to identify the IAV subtype and pathotype [2, 4]. Where the presence of HPAI is confirmed, strict quarantine and movement restrictions come into effect and suspension of export from the infected zones, province and/ or the country. Only when the infected zone or compartment is free from infection for at least 6 months from the date of the last infected farm or compartment is the outbreak declared over to the World Animal Health Organization (OIE), and exports can resume .
Ostrich tracheal swabs contain higher viral titres than cloacal swabs , therefore the standard practice in South Africa is to collect tracheal swabs, pooling a maximum of five into a single tube containing 50% v/v phosphate-buffered saline (PBS): glycerol medium that, notably, does not contain antimicrobials. Virus isolation is the gold standard for agent detection  but is not performed routinely with RT-rtPCR-positive ostrich samples for reasons such as cost and the limited supply of Specific Antigen Negative or Specific Pathogen Free (SPF) embryonated chicken eggs (ECEs). Furthermore, the success rates where egg isolations were attempted on ostrich tracheal swab fluids with RT-rtPCR cycle threshold values < 30 have been inexplicably poor, certainly lower than that for chickens for which the same sampling methods are used . For example, from March to November 2019 a national laboratory tested 977 ostrich swab pools of which 109 (11.2 %) were IAV positive by RT-rtPCR. None were typed as H5/H7 positive on RT-rtPCR, and only one virus was isolated and later identified as the H11N1 subtype (A. Olivier personal communication; C. Abolnik, unpublished diagnostic case reports). During the 2011 H5N2 HPAI outbreak, only three viruses were isolated from swab pools of twenty ostrich farms with multiple RT-rtPCR-positive pools each  and in the 2017–2018 H5N8 HPAI outbreaks, 38 ostrich tracheal swab pools were H5N8 HPAIV RT-rtPCR-positive, but only two viruses were isolated whereas 39 viruses were isolated from the oropharyngeal and cloacal swabs or tissue samples of other avian species .
To reduce testing costs but still meet statistical sampling requirements to detect the presence of IAV infection, the OIE and other sources recommend that up to eleven swabs from chickens or turkeys may be pooled without a loss in sensitivity of the RT-rtPCR assay [7, 9, 10], but since there may be differences between shedding patterns for different hosts , the pooling of more than 5 ostrich swabs is not permitted by the national veterinary authority. Therefore, in this study we used spiking experiments to evaluate whether increasing ostrich tracheal swabs from five to ten per pool would affect the sensitivity of H5N8 HPAIV or H7N1 LPAIV detection by quantitative RT-rtPCR. We also compared the efficacy of H5N8 HPAIV and H7N1 LPAIV isolation from ostrich tracheal swabs in the standard PBS: glycerol medium without antimicrobials to a protein-based viral transport medium containing antimicrobials, since our recent study  identified specific bacteria in ostrich tracheal swabs that directly affect the viability of IAVs.