Designing Resilient Surveillance: Incorporating Oropharyngeal and Cloacal Sampling into Avian Influenza Monitoring Protocols

EXECUTIVE SUMMARY

Timely and accurate detection of avian influenza is crucial for curbing the spread of highly pathogenic strains like H5N1. Because shedding patterns vary by virus subtype and host species, surveillance strategies that incorporate both oropharyngeal and cloacal sampling significantly reduce the risk of false negatives. This dual-sampling approach ensures early detection across a broader range of infections. Ultimately, enabling faster interventions and stronger response outcomes.

• Shed/spread kinetics of H5N1 demand the earliest possible knowledge of infection if successful interventions to block farm to farm transmission are to be made.

• Viral tropism shifts to favor cloacal H5 shedding have been observed in wild birds and this may be a contributing factor towards the more efficient spread of the virus globally.
• The dynamic nature of the avian influenza genome generates variation that can affect shedding kinetics and hence detectability of the virus.
• The most effective monitoring programs require Oropharyngeal and Cloacal sampling in all bird species to minimize the risk of false negatives and hence missed intervention opportunities.
• Viruses of the H7 subtype may shed for longer periods of time from the cloaca and hence incorporating cloacal sampling is essential in geographical areas where H7 challenge is a risk.
• Elevated quantitative detections with H5 specific molecular protocols compared to Type A broad detection assays emphasizes the requirement for multiple specific H5 assay protocols – this is the reason for the inclusion of 3 individual assay targets for H5 in the Alveo Sense^TM Point of Need^TM test.
• Alveo Sense Poultry Avian Influenza Test can detect the presence of notifiable Avian Influenza RNA even in the relative absence of biologically active viruses.

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BACKGROUND

Avian influenza (AI) viruses in commercial poultry are often shed in larger quantities from the oropharyngeal (OP) route¹. This has historically driven sampling strategies due to the perceived respiratory tract tropism of the virus. However, viral tropism shifts of AI have been observed with more contemporary isolates tending towards cloacal (C) shedding in certain duck breeds². Such shifts are a possible contributing factor to increased rates of AI virus spread, allowing longer persistence in the wild bird reservoir. It is possible that increased virus mutation rates in AI-infected commercial chickens may be related to a higher replicate rate in that species³, hence increasing the chance of a change in virus tropism, the more extensive commercial poultry infections become. By this mechanism, the naturally high variability of the AI genome may indirectly lead to the risk

of reduced test sensitivity when OP swabbing is used exclusively. Certain H5 and H7 LPAIV strains in broiler breeders include smaller proportions of chickens actively shedding from the C tract only1. H7 viruses may shed for longer periods of time from the cloaca⁴. For all these reasons, the incorporation of testing protocols that feature both OP and C sampling to detect virus shedding in the design of any optimized avian influenza monitoring program can avoid the risk of a false negative result.

Experimental studies in ducks have shown that both OP and C samples are positive by real time PCR in birds challenged with

2.3.4.4.b H5 avian influenza virus by day two, with clinical signs visible at day 35. Real time PCR data indicated that quantities of RNA as defined by CT value may be higher with H5 specific assays compared to Type A specific assays1. This same effect is seen with the Alveo Sense LAMP assay with a more sensitive LOD recorded for H5 specific findings (Coopersmith et al 2025). Recent challenge data from PDRC demonstrates a strong and rapid detection of clade 2.3.4.4.b H5N1 from day one post challenge with cloacal pool positivity observed, even when up to four out of five samples within a pool are not biologically active, at day five post infection. Alveo Sense detected large quantities of target RNA at this final time point within 20 mins

of reaction start (consistent with a Real Time PCT CT equivalency of 27 and below), demonstrating robustness of detection of target in an environment of declining virus biological activity (Alveo Technologies/ Coopersmith/ Dijkman – internal communication and trial data).

Early detection capabilities of an assay are critically important as bird-to-bird transmission begins 24 hours after initial exposure⁵. The ability to detect nucleic acid targets when biological virus functions begin to fail is a practical requirement for the test to operate successfully under field conditions.

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REFERENCES

1. Slomka MJ, Reid SM, Byrne AMP, Coward VJ, Seekings J, Cooper JL, Peers-Dent J, Agyeman-Dua E, de Silva D, Hansen RDE, Banyard AC, Brown IH. Efficient and Informative Laboratory Testing for Rapid Confirmation of H5N1 (Clade 2.3.4.4) High- Pathogenicity Avian Influenza Outbreaks in the United Kingdom. Viruses. 2023 Jun 9;15(6):1344. doi: 10.3390/v15061344. PMID: 37376643; PMCID: PMC10304448.
2. Caliendo V, Leijten L, van de Bildt M, Germeraad E, Fouchier RAM, Beerens N, Kuiken T. Tropism of Highly Pathogenic Avian Influenza H5 Viruses from the 2020/2021 Epizootic in Wild Ducks and Geese. Viruses. 2022 Jan 28;14(2):280. doi: 10.3390/ v14020280. PMID: 35215873; PMCID: PMC8880460.
3. Kim G, Shin HM, Kim HR, Kim Y. Effects of host and pathogenicity on mutation rates in avian influenza A viruses. Virus Evol. 2022 Feb 21;8(1):veac013. doi: 10.1093/ve/veac013. PMID: 35295747; PMCID: PMC8922178.
4. Aamir UB, Naeem K, Ahmed Z, Obert CA, Franks J, Krauss S, Seiler P, Webster RG. Zoonotic potential of highly pathogenic avian H7N3 influenza viruses from Pakistan. Virology. 2009 Aug 1;390(2):212-20. doi: 10.1016/j.virol.2009.05.008. Epub 2009 Jun 16. PMID: 19535120; PMCID: PMC2710411.
5. Filaire F, Bertran K, Gaide N, Valle R, Secula A, Perlas A, Foret-Lucas C, Nofrarías M, Cantero G, Croville G, Majó N, Guerin JL. Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling. Vet Res. 2024 Aug 12;55(1):100. doi: 10.1186/ s13567-024-01357-z. PMID: 39135123; PMCID: PMC11318174.

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*Product Availability Disclaimer: This product is not available for sales or distribution in the United States. Availability of Alveo Sense Avian Influenza Test is subject to local regulatory approvals and may vary by country. For inquiries regarding the availability of Alveo Sense Avian Influenza Test in your country, please contact our Customer Service team at support@alveotechnologies.com

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