Unlocking pooling strategies for Influenza A Virus RNA detection

Surveillance essential to monitor virus activity, help swine veterinarians, pork producers make management, biosecurity and disease control decisions.

May 7, 2024

6 Min Read
National Pork Board

By Daniel C A Moraes, Onyekachukwu Osemeke, Phillip Gauger, Cesar A. Moura, Onyekachukwu Osemeke, Giovani Trevisan, Gustavo Silva, Daniel Linhares, Iowa State University

Influenza A virus infection has a significant negative economic impact on pig production due to increased mortalities and decreased growth performance. A study reported that IAV infection caused economic losses as high as $3.23 per pig. Co-infection of IAV and porcine reproductive and respiratory syndrome virus or Mycoplasma hyopneumoniae costs $10 per pig [1].

IAV infection in swine breeding herds may be at low prevalence (<15%), with the clinical impact being more evident downstream in the nursery population [2]. Therefore, IAV active surveillance is essential to understand and monitor virus activity to help swine veterinarians and producers make management, biosecurity, and disease control decisions.

The most common sample types used for IAV molecular testing in the United States are oral fluids and lung tissue samples. Family oral fluids are an effective population-based sample type for PRRSV RNA detection by RT-rtPCR [3]. In addition, a recent study that assessed the effect of pooling FOF on the probability of PRRSV RNA detection demonstrated that PRRSV RNA was detected at 4% prevalence using up to five pools of 10 FOF [4].

Recently, udder wipes have been described for IAV RNA detection with promising diagnostic sensitivity [5]. A recent study tested the effect of pooling UW and showed that pooling 3 UW was an appropriate alternative as a population sample for IAV RNA detection [6]. However, the effect of pooling samples on the probability of IAV detection may vary based on the sample type, pooling size, and prevalence of IAV in the population.

To achieve greater confidence in the results from population-based samples and determine the level of pooling that would demonstrate optimal detection sensitivity, a comparison of the different sample types pooled at different levels and different concentrations of the target (IAV) was needed. Under this study, we hypothesized that pooling population-based sampling increases the probability of IAV detection, assuming a fixed budget for diagnostic testing (i.e., the same sample size across all sampling schemes).

The previous study using FOF significantly improved monitoring and surveillance protocols for IAV in breeding herds, adding important alternatives for IAV detection, but it is still cost-prohibitive for most producers [7]. Therefore, this study aimed to compare different sample types, FOF samples, UW, and nasal swipes at different pooling levels on the probability of IAV detection in swine breeding herds.


Forty-five PCR-positive IAV samples (15 FOF x 15 UW x 15 NW) were selected and pooled with PCR-negative samples in the following proportions: undiluted, 1:3, 1:5 and 1:10 (Figure 1). The negative samples for the dilutions originated from a breeding herd that conducted an IAV elimination and had repeatedly tested IAV PCR negative in the same sample types. Each pooling level for each sample type was tested in six replicates to confirm IAV status and establish cycle threshold values, according to the method described by Osemeke et al. [4]. PCR-positive samples were categorized into Ct groups based on the preliminary experiment results (i.e., three categories: Category A - Ct value lower than 30; Category B - Ct value between 30-34; Category C – Ct value between 34-38).

In summary, there were 45 positive samples, each having four dilution levels and six replicates, making 1,080 RT-rtPCR tests. In addition, data from the ISU VDL from 2018 to 2023 was analyzed to understand the cycle threshold value distribution by Category A - Ct < 30; Category B - Ct between 30-34; Category C - Ct between 34-38 for the sample types FOF, UW, and NW.

For FOF and UW, the probability of IAV detection in Ct categories A and B did not decrease when the dilution level increased to 1/10. In category C of both sample types, the PD was observed to decrease as the dilution level increased, reaching a value of 18% at 1/10 dilution (Figure 2). For NW, the PD did not decrease for all dilution levels, only in category A. In addition, for NW for Category B, the dilution level was not statistically different until dilution level 1:5 (Figure 2).


This study assessed the pooling effect on the PD between different IAV surveillance samples. From data from the ISU VDL from 2018 to 2023, 80% of all the IAV RT-qPCR positive oral fluid samples from breeding herds were in Categories A and B, while 20% were in Category C. For UW, 79% of positive samples were in categories A and B, and 21% in category C. Considering NW and nasal swabs, 84% were in Categories A and B, and 16% were in Category C.

These results showed that about 80% of the IAV-positive samples had Ct values < 34; thus, practitioners may want to consider pooling samples to improve coverage on a fixed testing budget, testing more samples with the same budget. However, the selection of pooling size should also consider clinical history, costs, the diagnostic question, and the expected prevalence of IAV in the population.

Take home

The take-home messages for producers and veterinarians were:

  • Family oral fluids and udder wipes: the probability of IAV detection at Ct values < 34 did not decrease at dilutions 1:3, 1:5, and 1:10;

  • Low prevalence farm: practitioner should consider smaller pools: 1:3 or 1:5;

  • Practitioners should consider pooling samples to improve coverage and reduce costs.


  1. Haden, C., Painter, T. and Fangman, T., HD 2012. Assessing production parameters and economic impact of swine influenza, PRRS and Mycoplasma hyopneimoniae on finishing pigs in a large production system In. In Proceedings of AASV Annual Meeting (pp. 75-76).

  2. Garrido-Mantilla, Jorge, Juan Sanhueza, Julio Alvarez, Marie R. Culhane, Peter Davies, Matthew W. Allerson, and Montserrat Torremorell. "Impact of nurse sows on influenza A virus transmission in pigs under field conditions." Preventive Veterinary Medicine 188 (2021): 105257.

  3. Almeida, M.N., Corzo, C.A., Zimmerman, J.J. et al. Longitudinal piglet sampling in commercial sow farms highlights the challenge of PRRSV detection. Porc Health Manag 7, 31 (2021). https://doi.org/10.1186/s40813-021-00210-5.

  4. Osemeke, O. H., de Freitas Costa, E., Almeida, M. N., Trevisan, G., Ghosh, A. P., Silva, G. S., & Linhares, D. C. L. (2022). Effect of pooling family oral fluids on the probability of PRRSV RNA detection by RT-rtPCR. Preventive veterinary medicine, 206, 105701. https://doi.org/10.1016/j.prevetmed.2022.105701.

  5. Garrido-Mantilla, J et al. Comparison of individual, group, and environmental sampling strategies to conduct influenza surveillance in pigs. BMC Vet Res 15, 61 (2019). https://doi.org/10.1186/s12917-019-1805-0.

  6. de Lara, A. C., Garrido-Mantilla, J., Lopez-Moreno, G., Yang, M., Barcellos, D., & Torremorell, M. (2022). Effect of pooling udder skin wipes on the detection of influenza A virus in preweaning pigs. Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 34(1), 133–135.

  7. Moraes, D.C.A. et al. 2023. Use of Family Oral Fluid to detect Influenza A Virus in the breeding herd. 54th AASV Annual Meeting 2023, United States. In Proceedings of AASV Annual Meeting. https://doi.org/10.54846/am2023/125.

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