Using Environmental Sampling Techniques to Conduct Influenza A Virus Surveillance in Poultry and Waterfowl at Ohio Agricultural Exhibitions

SUMMARY.

The outbreak of highly pathogenic H5Nx influenza A viruses (IAVs) in the United States during 2014–2015 caused devastating economic losses; therefore, several measures were established to control and eliminate highly pathogenic H5Nx from U.S. poultry flocks. One such measure was a temporary ban on poultry exhibitions during 2015, and this decision dramatically affected youth raising poultry as part of agricultural education programs. During the summer of 2016, surveillance of the environment was conducted at 20 Ohio agricultural fairs to estimate the prevalence of IAV in exhibition poultry to determine the baseline during nonoutbreak exhibition seasons. Of the 400 total samples collected, two were positive by real-time reverse transcription–PCR; however, virus isolation attempts with both embryonating chicken eggs and cell culture were unsuccessful. The detection of nucleic acid highlights the risk exhibition poultry could play in the transmission and spread of IAVs between humans, swine, wild birds, and domestic poultry during low or highly pathogenic IAV outbreaks. Additional surveillance at agricultural fairs and biosecurity education for youth exhibitors in this setting are warranted to reduce risk.

Wild waterfowl are a natural reservoir for influenza A viruses (IAVs) and are commonly infected with diverse lineages of low pathogenic strains with minimal health detriment (1,2). However, on occasion, IAVs from wild birds are introduced into commercial poultry, and they can become highly pathogenic, causing significant morbidity and mortality among the infected poultry populations. The occurrence of highly pathogenic H5Nx IAVs in the United States during 2014–2015 caused devastating economic losses, with 248 reported cases costing the industry and government nearly US$5 billion in response efforts (3). Therefore, several measures were implemented to control and eliminate highly pathogenic H5Nx IAVs from U.S. poultry flocks (4).

One such measure was a temporary, 1-yr ban on poultry exhibitions, which dramatically affected youth raising poultry as part of agricultural education programs. It has been well documented that swine at agricultural fairs are commonly infected with IAVs and occasionally infect humans (5,6,7,8). These exhibitions are also a primary location for backyard poultry to encounter humans and other mammals; however, the risks of this behavior for IAV transmission in and from avian species is undocumented. It was hypothesized that although IAVs are possibly circulating in exhibition poultry during agricultural fairs, the prevalence was likely low among clinically healthy birds. Estimating the effect of poultry exhibition bans on IAV elimination requires knowledge of typical IAV prevalence in these animal populations during the exhibition period.

MATERIALS AND METHODS

During the summer of 2016, surveillance was conducted at 20 Ohio agricultural fairs to estimate the prevalence of IAV in exhibition poultry. All fairs participating in this study also participated in swine IAV surveillance. Samples were collected from five market chicken cages, five nonmarket chicken cages, five turkey cages, and five duck cages, totaling 20 samples per fair. Environmental sampling techniques were used to avoid handling of exhibition birds. Dry, sterile cotton gauze was used to wipe the inside surfaces of cages, waterers, and feeders of both market and nonmarket chickens and turkeys. In addition, fecal material was collected from the floors of both market and nonmarket duck cages by using a dry, sterile polyester-tipped swab. All sample types were transported in brain-heart infusion broth containing penicillin and streptomycin (9). All samples were screened by real-time reverse transcription–PCR (RRT-PCR), as previously described (9). Following RRT-PCR screening, virus isolation attempts were completed in both embryonating chicken eggs and Madin-Darby canine kidney cell (MDCK) culture, as previously described (9,10).

RESULTS AND DISCUSSION

In all, 400 samples were collected and screened for IAV by using RRT-PCR. Two samples, one collected from a chicken cage and one from a turkey cage at two different agricultural fairs, were RRT-PCR positive. Virus isolation attempts with both embryonating chicken eggs and MDCK culture were unsuccessful. Samples were tested in MDCK culture to identify swine linage IAVs that may not be efficiently propagated in embryonating egg culture. The cycle threshold values of the RRT-PCR–positive samples were 38 and 39, which were too high to attempt genomic sequencing from the original samples.

Although viable IAV was not detected in exhibition poultry at the selected Ohio agricultural fairs in 2016, the presence of nucleic acid suggests at least contamination in the exhibition area, if not actual infection. Swine at both fairs were negative for IAV; therefore, they were not responsible for the nucleic acid detection in the poultry exhibition environment. Poultry cages at agricultural fairs are supplied by the fairgrounds and could have been used for a poultry exhibition prior to the fair when sampling occurred. If cages were not cleaned and disinfected in between exhibitions, the contamination could have been residual from prior events. This detection highlights the risk exhibition poultry could play in the transmission and spread of IAV between humans, swine, wild birds, and domestic poultry of low pathogenic IAV or highly pathogenic avian influenza outbreaks.

Environmental sampling has been used by multiple investigators to identify the presence of IAV in commercial poultry barns, wild birds, and live bird markets, providing justification for using similar techniques at poultry exhibitions (11,12,13,14). Avoidance of handling individual birds is imperative at a fair setting, because a different youth exhibitor who may not consent to bird handling owns each individual animal. Environmental testing is ideal in this setting; however, there are inherent limitations with theses sampling techniques. Using sterile cotton gauze at a fair setting to collect samples from swine is reported to be 83% as effective as the polyester swab, the gold standard (15). In addition, several components included in environmental samples are known to inhibit PCR reactions by lysing cells, degrading nucleic acid, and inhibiting polymerase activity (16,17). These issues need to be taken into consideration when interpreting negative results found in poultry at fairs, and alternative sampling methods should be considered in future surveillance efforts at agricultural fairs.

Agricultural education programs, such as 4-H and the National FFA Organization, are ideal venues to increase knowledge in the area of zoonotic diseases, as individuals involved in rearing and exhibiting animals have increased exposure to host species. A study conducted among 4-H members and non–4-H members found that the 4-H members reported significantly higher scores for measured life skill categories (working with groups, understanding self, communicating, making decisions, and leadership). However, disease prevention was determined to be the life skill least represented in 4-H youth development programming, making this an important addition to youth agricultural programs (18,19). These youth programs are imperative for attracting the interest of and training the next generation of agriculturalists, which the world will rely on for safe and secure food sources. However, contracting IAV from agricultural exhibitions is not only detrimental for the health of these youth but could also deter them from pursuing careers in agriculture. Cancelling poultry exhibitions to prevent disease transmission may or may not have had an effect on IAV spread in commercial poultry but did have an effect on the youth involved. There were many negative feelings toward the lack of shows; however, there was increased opportunity to teach youth regarding disease prevention, biosecurity, and epidemiology. The knowledge learned regarding an IAV outbreak can be applied in many situations, as youth continue in agricultural endeavors. These youth are likely the future of agriculture and therefore will be critical leaders in controlling forthcoming zoonotic diseases. Further investigation is warranted to fully understand the posed risk and to develop mitigation strategies to minimize transmission events.

Abbreviations:

IAV

influenza A virus

MDCK

Madin-Darby canine kidney cell

RRT-PCR

real-time reverse transcription–PCR