Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Veterinary Microbiology and Preventive Medicine


Veterinary Microbiology

First Advisor

Kyoung-jin Yoon


Surveillance and monitoring are key components for the early detection of avian influenza virus (AIV) in both migratory wild birds and commercial poultry. Recent outbreaks of newly emerged Eurasian-North American H5N2 highly pathogenic avian influenza virus (HPAIV) of migratory wild bird origin have led to huge financial loss for the US poultry industry. This epidemic further emphasized the importance of on-going surveillance. This dissertation presents results of four independent studies to characterize AIV from migratory wild birds (MWBs) of US flyways, evaluate the utility of nucleic acid-based testing for direct subtyping of AIV in samples from MWBs, evaluate the role of feed in the spread of AIV to commercial poultry facilities, and explore alternative sampling for surveillance and monitoring of AIV in commercial poultry facilities.

The first study was to characterize subtypes and sequences of AIV circulating in non-mallard MWBs in comparison to ones in mallards and poultry. A total of 1,158 non-mallard swabs positive for AIV RNA were tested by virus isolation (VI). The VI attempts yielded 87 AIV isolates (7.5% success rate), which were then sequenced for matrix, hemagglutinin, and neuraminidase genes. Based on the VI result, the Central flyway registered the highest AIV positivity rate while the lowest was in Atlantic flyway. AIV was isolated more from Blue-winged teal species-wide and from male ducks. Age-wise, samples from hatch-year birds resulted in more AIV isolates than ones from after-hatch-year birds. The common subtypes among non-mallard isolates were H3N8 (23.0%), H4N6 (18.4%), and H4N8 (18.4%), which differed from subtypes detected from mallard isolates. Genetic comparison of the HA gene between non-mallard and mallard MWBs revealed 85.5-99.5% H3 identity and 89.3-99.7% H4 identity, suggesting interspecies mingling of AIVs. Comparisons between MWBs (both mallard and non-mallard) and poultry isolates revealed 79.0-99.0% H3 identity and 88.7-98.4 % H4 identity, emphasizing the need for continued AIV surveillance in MWBs.

The second study involved development of a PCR-based subtyping assay for detecting all AIVs. The assay was developed with the objective to detect AIVs directly from MWB samples. Another objective was to evaluate the utility of this newly developed assay as compared to the current USDA surveillance testing strategy (virus isolation followed by sequencing of isolates). A SYBR® Green-based real-time reverse transcription-PCR (rRT-PCR) panel for 16 known hemagglutinin subtypes was developed and validated. When the panel was evaluated on 90 AIV RNA-positive oropharyngeal cloacal swabs from MWBs, the PCR panel was able to detect new AIVs subtype missed by VI-based testing method, besides demonstrating a significantly fast turnaround of results. Moreover, the PCR panel was able to detect concurrent infection. The newly developed SYBR® Green rRT-PCR can be a useful tool for monitoring MWBs for AIVs. The third study was to assess the role of feed in the spread of AIV to a poultry facility. Feed ingredients and complete layer mash samples were collected from commercial poultry barns (n=3) and feed mills (n=4) in the Midwest that experienced HPAI H5N2 2014-2015 and tested by a quantitative real-time RT-PCR (RT-qPCR) and, if positive, by VI. Additionally, AIV-negative feed materials were spiked with various concentrations of an LPAIV (H5N2). Virus-spiked cell culture media were also prepared in the same manner. Both spiked feed and media samples were tested by RT-qPCR either immediately or after incubation at -20°C, 4°C, 22°C, and 37°C for 24, 48, and 72 hours. Some of the feedstuffs collected from poultry facilities or feed mills were positive for AIV RNA but negative by VI test. In spiked feeds, the AIV titer was 1-3 logs lower in feed than the corresponding media, even when tested immediately after spiking, suggesting feed might have a negative impact on the virus or PCR detection. The half-life of AIV RNA was shorter at a higher temperature. The viral RNA in feeds decayed rapidly over time at 37°C (p<0.05), suggesting that feedstuffs should be maintained in the cold chain for testing. Furthermore, any of the PCR-positive feed samples were negative by VI, suggesting heat treatment of feeds could be an alternative to chemical treatment when contamination is suspected. Collectively, study observations indicate that AIV survivability in feed is relatively low, thus rendering it a low risk for virus spreading.

In the fourth study, various environmental samples were evaluated for their utility in monitoring AIV at commercial poultry facilities. The study was comprised of three experiments. In the first experiment, environmental samples collected from commercial layer facilities in the US Midwest that experienced HPAI H5N2 2014-2015 were evaluated for the effect of sampling locations on AIV detection. In the second experiment, environmental samples collected by Swiffer® Sweeper dry pads (Swiffer® pads) and drag swab, as well as water samples collected from AIV-negative commercial layer facilities, were spiked with the LPAIV and evaluated for detection of AIV RNA immediately or after incubation at -20°C, 4°C, 22°C, and 37°C for 24, 48, and 72 hours, respectively. In the third experiment, Swiffer® pads, drag swabs, and boots swabs were evaluated for their efficiency of collecting feces and water spiked with the LPAIV under a condition simulated for a poultry facility floor. Various sampling locations within a poultry house, such as cage, egg belt, floor, manure belt and pit, and ventilation equipment, were equally useful for environmental sampling to detect AIV RNA. The half-life of AIV in environmental samples was comparable but decreased with increasing temperatures. Additionally, collection devices did not differ significantly in their ability to detect AIV RNA in environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as non-invasive samples for monitoring AIV.

Overall these studies indicated that AIV subtypes differ across flyways and between migratory wild bird species. Alternative approaches such as PCR-based direct subtyping may aid current VI-based AIV surveillance system in migratory wild birds. Commercial poultry feed is less likely to act as vector for AIV, however, the presence of AIV in raw feed ingredients warrants strict biosecurity measures. Finally, environmental sampling of commercial poultry facilities using various collection devices could be used as an alternative AIV surveillance tool.


Copyright Owner

Shahan Azeem



File Format


File Size

235 pages

Available for download on Friday, January 07, 2022