Transmission of porcine reproductive and respiratory syndrome virus in domestic pigs via oral ingestion of feed material

Scott Dee Pipestone Research, Pipestone, MN

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 DVM, PhD, DACVM
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Travis Clement Animal Disease Research and Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD

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Eric Nelson Animal Disease Research and Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD

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Abstract

OBJECTIVE

The purpose of this case study was to describe the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) under field and experimental conditions via the consumption of PRRSV-positive swine feed.

ANIMALS

1 domestic swine breeding herd and 20 laboratory-maintained experimental domestic pigs.

CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES

A 2,500-sow PRRSV-naïve biosecure breeding herd became infected during the autumn months. It experienced a feed outage involving a specific bin on October 23 (day 0), with the bin refilled on October 24 (day 1). From October 28 to 30 (days 5 to 7), signs of anorexia and hyperemia were observed in 30 gestating sows after consuming feed from this bin. On November 1 (day 9), blood samples from 10 affected sows were PRRSV positive by reverse transcriptase PCR. In contrast, sows in the same room that had consumed feed from other bins were clinically normal and PRRSV negative. To investigate whether the feed delivery introduced PRRSV to the herd, on November 2 (day 10) 4 samples of feed material from the interior walls of the index bin were collected and tested by reverse transcriptase PCR.

TREATMENT AND OUTCOME

All 4 samples were positive for PRRSV RNA with cycle threshold values ranging from 26 to 29. Nucleic acid sequencing indicated that the open reading frame 5 region of the PRRSV in feed samples was 100% homologous to PRRSV from index cases. To assess viability of the virus, PRRSV-naïve pigs were allowed to consume the index feed bin samples and became infected with PRRSV based on viral RNA in oral fluid samples, clinical signs, and postmortem lesions.

CLINICAL RELEVANCE

These results suggest that feed was a likely source of PRRSV introduction to the herd. This is the first report of PRRSV transmission through feed.

Abstract

OBJECTIVE

The purpose of this case study was to describe the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) under field and experimental conditions via the consumption of PRRSV-positive swine feed.

ANIMALS

1 domestic swine breeding herd and 20 laboratory-maintained experimental domestic pigs.

CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES

A 2,500-sow PRRSV-naïve biosecure breeding herd became infected during the autumn months. It experienced a feed outage involving a specific bin on October 23 (day 0), with the bin refilled on October 24 (day 1). From October 28 to 30 (days 5 to 7), signs of anorexia and hyperemia were observed in 30 gestating sows after consuming feed from this bin. On November 1 (day 9), blood samples from 10 affected sows were PRRSV positive by reverse transcriptase PCR. In contrast, sows in the same room that had consumed feed from other bins were clinically normal and PRRSV negative. To investigate whether the feed delivery introduced PRRSV to the herd, on November 2 (day 10) 4 samples of feed material from the interior walls of the index bin were collected and tested by reverse transcriptase PCR.

TREATMENT AND OUTCOME

All 4 samples were positive for PRRSV RNA with cycle threshold values ranging from 26 to 29. Nucleic acid sequencing indicated that the open reading frame 5 region of the PRRSV in feed samples was 100% homologous to PRRSV from index cases. To assess viability of the virus, PRRSV-naïve pigs were allowed to consume the index feed bin samples and became infected with PRRSV based on viral RNA in oral fluid samples, clinical signs, and postmortem lesions.

CLINICAL RELEVANCE

These results suggest that feed was a likely source of PRRSV introduction to the herd. This is the first report of PRRSV transmission through feed.

History

In 2013, the annual cost of porcine reproductive and respiratory syndrome (PRRS) to the US swine industry was estimated to be $664 million1; however, a recent report estimated the current cost of the disease at approximately $1 billion/y (Personal communication: G. Spronk DVM, Pipestone Veterinary Services, AASV annual meeting invited lecture, March 6, 2023). Published routes of PRRS virus (PRRSV) transmission include infected breeding stock and contaminated semen, along with fomite-based risks (ie, contaminated transport; personnel boots, coveralls, and supplies; and aerosols).2 Currently, there is no published evidence of PRRSV transmission via feed. This case describes the transmission of PRRSV under field and experimental conditions following consumption of PRRSV-positive swine feed. An ethical review of the field phase of the case study was not needed, as the authors had received the farm owner’s permission to investigate and publish the case. An ethical review of the experimental phase was conducted and is described in the Treatment and Outcome section.

Diagnostic Findings and Interpretation

In November, a 2,500-sow PRRSV-naïve breeding herd in west-central Minnesota was confirmed to be infected with PRRSV. The herd had been populated with PRRSV-naïve breeding stock, used PRRSV-free semen, and had been PRRSV naïve for 5 years based on the absence of clinical signs of PRRSV along with monthly testing of sows and piglets via blood and oral fluid sampling. Regarding biosecurity protocols in place at the time of the infection, the herd had quarantined and tested all incoming breeding stock from a PRRSV-naïve source, used PRRSV-free semen, was air filtered, and practiced protocols validated to reduce the risk of mechanical transmission of the virus, including transport sanitation, personnel shower in/out and use of a designated supply entry area.2 The herd did not employ a feed additive to mitigate viral transmission via this route. All protocols and practices were audited monthly via unannounced visits by trained inspectors. Prior to the outbreak, the farm experienced an unexpected feed outage in a specific feed bin, which sourced feed to a distinct subpopulation of 500 gestating sows on October 23 (day 0), requiring an emergency delivery on October 24 (day 1). On October 28 to 30 (days 5 to 7), clinical signs of anorexia and pyrexia (40 to 41 °C) were observed in 30 animals that had consumed this feed, with no signs noted in other animals consuming other feed from other bins. The manager contacted the herd veterinarian, who collected blood samples from 10 of the 30 affected sows on November 1 (day 9), and all 10 samples were determined to be PRRSV positive by reverse transcriptase PCR (RT-PCR). A random sample of 10 blood samples from clinically normal sows in the same room were blood tested as well and found to be RT-PCR negative. On November 2 (day 10), an investigation regarding potential routes of viral entry to the herd was conducted. These events are summarized (Table 1).

Table 1

Chronological summary of events during the field investigation phase of the case study.

Event Date (study day)
Feed outage October 23 (day 0)
Feed delivery October 24 (day 1)
Consumption period prior to observation of clinical signs October 25–29 (days 2–6)
Observation of clinical signs October 28–30 (days 5–7)
PRRSV diagnosis November 1 (day 9)
Feed samples collected November 2 (day 10)

PRRSV = Porcine reproductive and respiratory syndrome virus.

Based on the history of the outbreak, 1 segment of the investigation focused on the possibility of PRRSV entry to the farm through feed delivery. Upon inspection of the interior walls of the index bin, it was observed that clusters of feed material (feed particles and feed dust) from the recent delivery were adhered to the interior walls. To access this material, a published method for sampling contaminated feed bins for porcine epidemic diarrhea virus RNA was implemented.3 Specifically, synthetic woven paint roller pads (23 cm in length, 0.95 cm nap length; Sherwin Williams) were attached to 3.6-m extension poles (REA-C-H No. 44016; Ettore Products Co) to access the surface area of interior bin walls at multiple heights. A total of 4 samples were collected. To minimize environmental contamination of the roller prior to placement within the bin interior, a 4.4-L plastic bag (Ziploc; SC Johnson & Son Inc) covered the roller during ascension of the bin ladder. Following insertion of the roller into the bin, the bag was removed and the roller was drawn across the inner walls, forcing the adhered feed material to attach to the pad. In addition, the pad was drawn across the top layer of the existing feed that remained in the bin to collect more material. Upon completion of sampling, the bag was replaced over the roller and the apparatus was removed from the bin. Once on the ground, 50 mL of 7.2% phosphate buffered saline was poured into the bag, soaking the pad and promoting absorption of liquid. Using manual pressure, liquid was then forced from the pad into the bag and a 10-mL aliquot was decanted into a 15-mL plastic Falcon tube (Becton, Dickinson and Co) for diagnostic testing. In addition, 6 control feed bins were also sampled. These bins were near the index bin (3 to 10 m away) and had not received any recent feed deliveries, and animals consuming feed from these bins were not clinically affected. Four samples were collected from each control bin for a total of 24 control bin samples. On the day of feed sample collection, the ambient temperature at the farm averaged 5 °C, with a low temperature of 4 °C and a high of 6 °C. All samples were tested for the presence of PRRSV RNA using RT-PCR (Tetracore Inc) at the South Dakota State University Animal Disease Research and Diagnostic Laboratory.4 A sample with a cycle threshold of < 38 was considered PRRSV positive. If positive, the samples were nucleic acid sequenced and assessed for the presence of infectious virus by swine bioassay. Due to their proprietary nature, details regarding the primers and probes cannot be reported here.

Treatment and Outcome

All 4 samples from the index bin were positive for PRRSV RNA by RT-PCR with cycle threshold values ranging from 26 to 29 (mean, 28). Nucleic acid sequencing indicated that the open reading frame 5 region of the virus was 100% homologous to viral samples obtained from blood from the index cases and had a restriction fragment length polymorphism pattern of 1-8-4. All control bin samples were PCR negative. To assess viability of the virus in the index bin samples, a swine bioassay was conducted. For this phase, PRRSV-naïve pigs housed in the Pipestone Research biosafety level 2 facility consumed the samples from the index or the control bins via natural feeding. All procedures involving animals throughout this phase of the study were performed under the guidance and approval of the Pipestone Research IACUC (protocol No. 2021-13). Animals (n = 12 three-week-old pigs) were sourced from a PRRSV-naïve herd and tested on arrival via oral fluid testing. The treatment group consisted of 6 piglets housed in 1 pen, while the remaining 6 served as the control group, housed in a separate room. The study encompassed a 14-day period with challenge through natural consumption of the designated feed material occurring on day 0, followed by oral fluid (pen-based) sample collection and clinical assessment on days 7 and 14 postconsumption, with necropsies conducted on day 14. Following IACUC approval, feed was withheld for 12 hours prior to challenge. For the preparation of challenge, 30 g of feed material from the 4 PCR-positive index bin samples was pooled and diluted in 30 mL of sterile phosphate buffered saline and placed in a feeding trough in the pens, and pigs were allowed to consume this sample via natural feeding behavior. Samples from the control bins were processed in a similar manner and pooled into one 30-g sample and fed to pigs in a similar manner. On day 7, oral fluid samples from the pen of pigs consuming samples from the index bin were PRRSV positive and pigs exhibited clinical signs of anorexia, pyrexia (40 to 42 °C), dyspnea, and weight loss. At necropsy on day 14, interstitial pneumonia and lymphadenomegaly were observed in 4 of the 6 pigs and PRRSV antigen was detected in lung and lymph node tissues from the same 4 pigs by immunohistochemistry. In contrast, no evidence of PRRSV infection, clinical signs, or lesions was observed in pigs fed feed from the control bins. A summary of these data is provided (Table 2).

Table 2

Summary of diagnostic data following challenge in the natural feeding bioassay.

Index bin feed samples Day 0 Day 7 Day 14
Oral fluids (pen)1 Negative Positive Positive
Clinical signs (pig) 0/62 4/6 4/6
Lesions and immunohistochemistry (+)3 NT NT 4/6
Control bin feed samples
Oral fluids (pen)1 Negative Negative Negative
Clinical signs (pig) 0/62 0/6 0/6
Lesions and immunohistochemistry (+)3 NT NT 0/6

Clinical signs (pig) = The number of pigs exhibiting clinical signs (anorexia, pyrexia, dyspnea, and weight loss)/number of pigs in the pen. Lesions and immunohistochemistry (+) = The number of pigs demonstrating lesions of interstitial pneumonia and lymphadenopathy observed at necropsy along with the detection of PRRSV by immunohistochemistry/number of pigs in the pen. NT = Not Tested. Oral fluids (pen) = One oral fluid sample was collected per pen of 6 pigs, tested for the presence of PRRSV RNA by PCR, and reported as positive or negative.

Comments

The results of this case suggest that feed may have been the source of PRRSV introduction to the herd and clearly indicate that feed samples from the index bin contained viable PRRSV. The virus was only detected in 1 feed bin, despite the sampling of 6 surrounding bins. The index sows only consumed feed from the index bin, and no other index cases were noted on the farm. The outbreak investigation provided no evidence of viral entry via other routes, as all incoming breeding stock and semen used were validated to be PRRSV naïve, there were no identified biosecurity breaches in the mechanical and aerosol biosecurity protocols, and testing of a subset of filters at a third-party laboratory (LMS Technologies Inc) indicated no loss of fractional efficiency or air flow. A limitation of the case was that unfortunately the local feed mill refused to allow testing of the facility environment, retained feed samples had been disposed of, and the truck used to deliver the feed to the index bin had been used several times to deliver to other sites. Regarding whether exhaust air from infected animals could have indirectly contaminated the feed in the index bin, external photographs indicated that the index bin was not adjacent to any exhaust fans. In contrast, several of the control bins that contained negative feed were found to be in close proximity to exhaust fans; therefore, it is unlikely that feed was contaminated in this manner. While it is true that the PRRSV-positive feed samples from the index bin could have been composed of feed from multiple deliveries, this was no different than the PRRSV-negative samples collected from the control bins. Another limitation of the case was that the results do not indicate the frequency of the event.

In conclusion, this is the first report of the potential for PRRSV transmission through feed, documented by both field observations and a controlled experiment. Therefore, while it may not be a high-risk point of entry, these data suggest that feed should be considered a possibility of viral entry when investigating PRRS outbreaks.

Acknowledgments

None reported.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

Funding

The authors have nothing to disclose.

References

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