By Daniel Moraes, Phillip Gauger, Cesar A. Moura, Isadora Machado, Guilherme Cezar, M. Mil- Homens, Onyekachukwu Osemeke, Rodrigo Paiva, Alex Ramirez, Gustavo Silva, Daniel Linhares

Influenza A virus is consistently within the top three most prevalent etiologies in swine respiratory disease cases within the United States¹. IAV is a major contributor to the porcine respiratory disease complex along with porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae² and porcine circovirus type 2. Active surveillance is highly significant for detecting emerging strains of IAV and assessing the threat to both swine and public health³.

The most common specimens used for molecular testing in the United States are nasal swabs, nasal wipes and oral fluids. Recently, udder wipes have been reported with promising diagnostic sensitivity⁴-⁶. However, sampling options used to detect IAV vary based on the herd sensitivity and prevalence of the sample type and the convenience of performing sample collection.

Family oral fluids specimens were demonstrated to be an effective population-based sample type for PRRSV RNA detection⁷. FOF allows testing more animals using fewer samples, thus offering an economic advantage over individual pig sampling⁸. However, studies have not yet evaluated the efficacy of FOF for the detection of IAV RNA in breeding herds where IAV prevalence is low. Also, information is lacking regarding the successive use of FOF over time as a method to monitor the presence and epidemiology of IAV in commercial herds.

Therefore, the objective of this study was to compare the probability of detection of IAV RNA by PCR-based assays between selected individual and population-based samples. The following sample types were compared: sow nasal wipes, nasal wipes from all pigs within a litter, udder wipes, FOF and drinker wipes.

Weaning-age piglets (17-21 days) from three breeding herds located in the midwestern United States were screened for IAV positivity using udder wipes to ensure evidence of IAV circulation. One herd tested positive and study samples were collected within 48 hours of screening test completion. Samples were collected at the farm using matched sets of FOF, udder wipes and nasal wipes from sows and all 3-week-old piglets within the respective litter. Fifty-seven litter-matched FOF, udder wipes and nasal wipes were used to compare the probability of detecting IAV RNA by RT-rtPCR in a breeding farm. 

One of the three breeding herds tested IAV RNA-positive (6/35, 17.1%) at screening utilizing udder wipes and was selected for study sample collection 48 hours later. A total of 57.9% (33/57) FOF samples tested positive, as well as 49.1% (28/57) of the udder wipe samples, and 28.1% (16/57) of the sow nasal wipe samples. A total of 15.8% (9/57) of the drinker wipe samples and 66.6% (38/57) of the pig nasal wipes tested positive for IAV RNA. Overall, the RT- rtPCR cycle threshold values for positive samples ranged from 24.4 to 37.9, with FOF having the lowest medium value amongst all sample types, followed by pig nasal wipes and udder wipes (Figure 1).

Figure 1. IAV RT-rtPCR cycle threshold (Ct) value by sample type.

There was a wide variation in percentage of positive piglets between the three rooms sampled (90.9%, 70.8% and 9.1% for piglet nasal wipe samples in Rooms A, B and C, respectively (Figure 2). This finding was in agreement with that described by Almeida et al. (2021) for PRRSV, highlighting the importance of sampling as many rooms as possible to reflect the herd status for IAV activity.

Figure 2. Proportion of IAV-positive piglets within litters, as measured by PCR from nasal wipes. A, B and C denote each room in one breeding herd.

FOF and udder wipes presented higher IAV positivity compared to other sample types, using the individual pig nasal wipes as the reference. Based on the results of this study, FOF is a resourceful alternative population-based sample type for IAV in the breeding herd, in addition to udder wipes. The room-level piglet PCR positivity ranged from 2 to 90% within the same breeding herd and same day. This emphasizes the danger of extrapolating PCR results between rooms. Instead, efforts should be made to increase coverage to multiple rooms when the purpose of sampling is to understand IAV activity within the herd.

The take home messages for producers and veterinarians were:

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