Tonsil biopsies and polymerase chain reaction assay for detection of breeding age gilts persistently infected with porcine reproductive and respiratory syndrome virus
Kris Fairbanks, DVM; Chris Chase, DVM, PhD; David A. Benfield, PhD
KF, CC, DAB: Department of Veterinary Science, South Dakota State University, Brookings, SD 57007; DAB present address: Associate Director, Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, OH 44691.
Practice tips are not peer refereed.
Fairbanks K, Chase C, Benfield DA. Tonsil biopsies and polymerase chain reaction assay for detection of breeding age gilts persistently infected with porcine reproductive and respiratory syndrome virus. J Swine Health Prod. 2002;10(2):87-88.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious RNA virus that has emerged as the most endemic and economically important disease of swine in the United States and other areas of the world. The syndrome is clinically characterized by poor conception rates, late-term abortions, and stillborn and weakborn pigs from sows of reproductive age. Respiratory distress, fever, lethargy, and high mortality may occurin suckling, weaned, and grow-finish pigs.1-4 While most herds return to normal production parameters within 4 to 6 months after an initial outbreak, the virus has been reported to persist in herds for up to 2 years, 5 and in individual pigs for extendedperiods of time. It is this persistence of PRRSV that appears to perpetuate the virus in nature and makes the management of PRRS outbreaks a difficult process.
A number of management protocols are available for control of PRRSV transmission in herds, all based on the predominant model of eliminating subpopulations of infected pigs from breeding herds.6 While these control strategies have been beneficial, results are not absolute, and many practitioners are now using test-and- removal to eliminate PRRSV from herds.
Test-and-removal depends on current diagnostic technology of serology (IDEXX HerdChek ELISA; IDEXX Laboratories, Westbrook, Maine), detection of infectious virus (sentinel pigs, swine bioassay, or virus isolation on cell culture), and detection of virus nucleic acid (polymerase chain reaction (PCR) assays). One problem confronting producers and practitioners is the difficulty in differentiating noninfected seropositive animals that have cleared the virus and persistently infected seropositive animals.
Several studies have indicated that PRRSV persists in tonsil, and viral nucleic acid has been detected in tonsilar tissue in one pig 252 days post-inoculation.7-10 As tonsil appears to be a prominent site for persistence of PRRSV, sampling of tonsil biopsies to detect viral nucleic acid by PCR is an appropriate antemortem diagnostic approachto identify persistently infected breeding swine. Removal of positive animals reduces viral load and the potential for transmission of the virus to naive animals.
Tonsil biopsy procedure
Our experience indicates that collection of tonsil biopsies
from alert pigs using a nose snare results in less than optimum
results and more stress and trauma to the pig. Biopsiesare collected
only from gilts positive for PRRSV antibodies by the commercial
HerdChek ELISA (sample:positive
ratio>=0.40). Selected animals are anesthetized using a combination of Telazol (5 mg; Fort Dodge Animal Health, Fort Dodge, Iowa), xylazine (250mg), and ketamine (250 mg). The xylazine and ketamine are used to reconstitute the lyophilized Telazol. The combination is given intramuscularly at a dose of 1 mL per 22.5 kg of body weight. This usually renders animals recumbentwithin 5 minutes, and the effect lasts approximately 30 minutes. If additional anesthesia is needed, the author uses xylazine given intravenously to effect.
The animal is positioned in lateral recumbencey, and the mouth is held open with a mouth gag (either a speculum or a 7- to 10-cm length cut from a 5 x 5-cm block of wood). An assistant, straddling the animal's neck and thorax, rotates the head and holds the tongue out of the mouth to allow for a better exposure of the tonsils. With a penlight illuminating the oral cavity, the operator takes the tonsil biopsy usinga sterile human uterine biopsy instrument (22 cm; Miltex Instrument Co, Inc, Bethpage, NY 11714).
The biopsy specimen is placed in a sterile plastic specimen bag, labeled, and put on ice. Care must be taken to inspect the sample, as the uterine biopsy instrument may slip off the tonsil, resulting in the collectionof oral or pharyngeal mucosa. Tonsil can generally be grossly identified as having a slightly rough appearance with visible craters or crypts.
Collected samples are submitted to a diagnostic laboratory for PCR testing. It is also highly recommended that a portion of the specimen or a second biopsy sample be fixed in 10% buffered formalin for microscopic examination to verify that the sample is tonsil. Using this technique, we succeed in sampling tonsil in at least 60% of biopsies: the remaining 40% usually contain adjacent epithelial tissue.
While the use of the combination anesthetic adds costs and time to the collection process, diagnostic results will be more accurateand satisfactory if the practitioner knows that proper biopsy material has been obtained. The described biopsy procedure reduces stress on the animal and the practitioner and allows for easier collection of the appropriate tonsil specimen. In preliminary experimental studies in ten gilts inoculated with PRRSV at 6 months of age, we detectedPRRSV nucleic acid in tonsil biopsiesfrom two of the gilts up to 84 days post inoculation.
References - refereed
2. Rossow KD. Porcine reproductive and respiratory syndrome. Vet Path. 1998;35:1-20.
3. Benfield DA, Collins JE, Dee SA, Halbur PG, Joo HS, Lager KM, Mengeling WL, Murtaugh MP, Rossow KD, Stevenson GW, Zimmerman JJ. Porcine reproductive and respiratory syndrome. In: Straw BE, D'Allaire S, Mengeling WL, Taylor DJ, eds. Diseases of Swine. 8th ed. Ames, Iowa: Iowa State University Press. 1999:201-232.
4. Wensvoort G, Terpstra C, Pol JMA, ter Laak JA, Bloemrad M, deKluyer EP, Kragten C, van Buiten
L, den Besten A, Wagenaar F, Broekhuijsen JM, Moonen PLJM, Zetstra T, de Boer EA, Tibben HJ, de Jong MF, van't Veld P, Groenland GJR, van Gennep JA, Voets MTh, Verheijden JHM, Braamskamp J. Mystery swine disease in the Netherlands: The isolation of Lelystad virus. Vet Q. 1991;13:121-130.
5. Chung, WB, Lin MW, Chang WF, Hsu M, Yang PC. Persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds. Can J Vet Res. 1997;61:292-298.
6. Dee SA, Joo HS, Henry S, Tokach L, Park BK, Molitor TW, Pijoan C. Detecting subpopulations after PRRS virus infection in large breeding herds using multiple serologic tests. Swine Health Prod. 1996;4:181-184.
7. Wills RW, Zimmerman JJ, Yoon KJ, Swenson SL, McGinley MJ, Hill HT, Platt KB, Christopher-Hennings J, Nelson. EAPRRS virus: a persistent infection. Vet Microbiol. 1997;55:231-240.
References - non refereed
1. Keffaber KK. Reproductive failure of unknown etiology. AASP Newsletter. 1989;2:1-10.
8. Kolb JR. Monitoring the risk of introducing PRRSV using tonsil biopsy, serum, buffy coat and ORF 7 PCR in seropositive replacement animals. Proc AASP.<>