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Tongue tips exudate: An alternative specimen for PRRSV RNA detection

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Population-based sampling schemes do not cover all pig production phases, such as stillborn and newborn pig groups.

Porcine reproductive and respiratory syndrome virus remains one of the most challenging pathogens for swine production with a total cost of $664 million annually to swine production in the United States (1). Characterizing the activity of PRRSV in herds over time is critical to guiding biosecurity and disease management practices. The proportion of population-based samplings, e.g., processing fluids (2) and family oral fluids (3), compared to individual-based sampling methods, e.g., blood samples, for PRRSV monitoring in the U.S. swine industry has significantly increased (4). However, these population-based sampling schemes do not cover all pig production phases, e.g., stillborn and newborn pig groups.

Recently, an alternative method using tongue tips from dead pigs was shown to detect PRRSV RNA (5). The objective of this study was to characterize tongue tip sampling, comparing PRRSV detection by qPCR to other conventional methods. Validating an additional sample type allows the industry to expand options for monitoring and surveillance. Tongue tips may be a practical way to sample for risk-based sampling, as it targets dead animals. It also allows sampling of the stillbirth and neonatal losses population

Methods
Three PRRSV RNA-positive unstable breeding herds based on Holtkamp et al. (6) were selected for sampling (farms A, B and C). On each farm, tongue tips, blood samples, processing fluids and family oral fluids were collected according to Figure 1. Tongue tips (≈2 cm) were held from the dead piglets with forceps, severed with a scissor, and placed in disposable bags (Figure 2) from three age groups: newborn (<24h of age), processing (2-7 days of age), and weaning (18-21 days of age). Following the sampling, tongue tips were frozen at -20°C.

Within the same room and age group from which tongue tips were collected, blood samples (n≈45) from one piglet per litter were randomly collected. Additionally, processing fluids (n≈3) and family oral fluids (n≈25) were collected from processing and weaning age pigs, respectively. Tongue tips, processing fluids, and family oral fluids were tested individually, and serum samples were tested in pools of five, for PRRSV RNA detection by RT-rtPCR, and the probability of RNA detection in tongue tips was compared to serum samples, processing fluids and family oral fluids for each age group.

Iowa State UniversityISU Fig 1 080222.JPG

Iowa State UniversityISU Fig 2 080222.JPG

Results
Tongue tips were successfully obtained from all age groups, with an average number of eight tongue tips per bag. In farms A and B, PRRSV RNA was detected in 100% and 95.2%, respectively, of total collected tongue tips, and pooled serum samples were positive in all age groups in different percentages for both farms. In particular, in the newborn age group, the average Ct value from tongue tips (29.4 and 26.4, respectively) was numerically lower than the average Ct value from serum samples (34 and 26.8, respectively).

PRRSV RNA was detected in processing fluids from farms A and B (100% and 50%, respectively), and family oral fluids were positive in all sampled farms, in different percentages (farm A 11.7%, farm B 22.8% and farm C 43.4%). At farm C, no PRRSV RNA was detected in tongue tips, and pooled serum samples were PRRSV RNA-positive only in the weaning age. Further, no PRRSV RNA was detected in tongue tips when pooled serum samples from the same age group tested PRRSV RNA-negative in all farms.

Discussion and conclusion
Collecting tongue tips from dead piglets was practical, suitable and time-efficient for farm personnel under field conditions, and pig welfare-friendly as it does not involve live pigs. Furthermore, tongue tips can also be used as a sampling method in farms not performing castration, i.e., when processing fluid sampling is not an option.

Additionally, the use of risk-based samplings, such as tongue tips, provides a good estimate of the population's health status, of the fact that it is derived from dead animals, which were potentially more likely to harbor PRRSV. Given that approximately 50% and 75% of the piglets' losses happen during the first three and seven days of life, respectively (7,8), collecting tongue tips from dead piglets for monitoring and surveillance can be an effective way to sample a large number of pigs in the farrowing room, even in low mortality rates. Taken together, these results demonstrated the diagnostic value of tongue tips for PRRSV monitoring and surveillance.

References

  1. Holtkamp DJ, Kliebenstein JB, Neumann EJ, Zimmerman JJ, Rotto HF, Yoder TK, et al. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. J Swine Heal Prod. (2013) 21:72–84. doi: 10.31274/ans_air-180814-28
  2. Lopez WA, Angulo J, Zimmerman JJ, L Linhares DC. Porcine reproductive and respiratory syndrome monitoring in breeding herds using processing fluids. J Swine Heal Prod. (2018) 26:146–50. Available online at: https://www. aasv.org/jshap/issues/v26n3/v26n3p146.pdf.
  3. Almeida MN, Rotto H, Schneider P, Robb C, Zimmerman JJ, Holtkamp DJ, et al. Collecting oral fluid samples from due-to-wean litters. Prev Vet Med. (2020) 174:104810. doi: 10.1016/j.prevetmed.2019.104810
  4. Trevisan G, Linhares LCM, Crim B, Dubey P, Schwartz KJ, Burrough ER, Main RG, Sundberg P, Thurn M, Lages PTF, Corzo CA, Torrison J, Henningson J, Herrman E, Hanzlicek GA, Raghavan R, Marthaler D, Greseth J, Clement T, Christopher-Hennings J, Linhares DCL. Macroepidemiological aspects of porcine reproductive and respiratory syndrome virus detection by major United States veterinary diagnostic laboratories over time, age group, and specimen. PLoS One. 2019 Oct 16;14(10):e0223544. doi: 10.1371/journal.pone.0223544.
  5. Baliellas J, Novell E, Enric-Tarancón V, Vilalta C, Fraile L. Porcine Reproductive and Respiratory Syndrome Surveillance in breeding Herds and Nurseries Using Tongue Tips from Dead Animals. Vet Sci. 2021 Nov 2;8(11):259. doi: 10.3390/vetsci8110259.
  6. Holtkamp DJ, Torremorell M, Corzo CA, L Linhares DC, Almeida MN, Yeske P, et al. Proposed modifications to porcine reproductive and respiratory syndrome virus herd classification. J Swine Heal Prod. (2021) 29:261–70
  7. Koketsu Y, Takenobu S, Nakamura R. Preweaning mortality risks and recorded causes of death associated with production factors in swine breeding herds in Japan. J Vet Med Sci. 2006 Aug;68(8):821-6. doi: 10.1292/jvms.68.821.
  8. Shankar BP, Madhusudhan HS, DB H. Pre-Weaning Mortality in Pig-Causes and Management. Veterinary world. 2009 Jun 1;2(6).

Source: Isadora F. Machado, Edison S. Magalhaes,Ana Paula Serafini Poeta Silva,Daniel A. Moraes, Guilherme Ceza, Mafalda Pedro Mil-Homens, Onyekachukwu Henry Osemeke, Rodrigo Paiva, Phillip Gauger, Giovani Trevisan, Gustavo Sousa Silva, Daniel C. L. Linhares and Cesar A. A. Mourawho are solely responsible for the information provided, and wholly own the information. Informa Business Media and all its subsidiaries are not responsible for any of the content contained in this information asset. 

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