Combination of ELISA and RT-PCR in serum

Commercial ELISAs and conventional or quantitative RT-PCR are the most routinely used diagnostic tests. In combination, they can be used not only to detect an infection, but also to determine approximately when it occurred and to monitor the immune status at individual or at farm level. If sequencing is also performed, we can even establish the possible sources of the virus.

¹ Excluding the effects related to low sensitivity and/or low specificity.
² In adults, viraemia is short; so, this pattern may be already observed after 14 days post-infection. In young pigs, viraemia is longer; so, this pattern may be observed after 14-28 days post-infection.

LABORATORY CONFIRMATION IN REPRODUCTIVE FAILURE.

Seroconversion measured by ELISA can be used to demonstrate the involvement of PRRS virus in reproductive failure. However, seroconversion can be only observed in a previously negative animal/farm. Thus, in some circumstances, serum from sows should not be considered as one of the first choices to diagnose reproductive failure due to PRRS virus: 1) it is difficult to detect the virus in blood by RT-PCR, as viraemia in sows is usually of short duration; 2) seroconversion by ELISA cannot be demonstrated in a previously positive or vaccinated animal/herd.
 

Nor should samples from aborted, mummified or autolysed foetuses be used as the main sample in reproductive failure, since PRRS virus is relatively labile and it rapidly disappears from these samples.

In reproductive failure where PRRS virus is one of the main pathogens suspected to be involved, best samples to use are: blood samples from sows (to determine seroconversion by ELISA, when possible) and/or blood samples from weak-born piglets (to determine PRRS virus viraemia by RT-PCR). 

LABORATORY CONFIRMATION IN A RESPIRATORY OUTBREAK.

Seroconversion measured by ELISA can be used to demonstrate the involvement of PRRS virus in a respiratory outbreak. However, seroconversion can be only observed in a previously negative animal/farm. Series of samples from various ages for detection of antibodies by ELISA is useful to determine the age of the infection and, consequently, the best time to analyse for the presence of virus in the blood. RT-PCR is useful because viraemia is longer in young pigs. Through IHC or ISH combined with histopathology, the link between PRRS virus and lesions can be analysed in tissues, especially in lungs.

The presence of concurrent infections can considerably complicate the diagnosis of PRRS in a respiratory outbreak. PRRS virus infection can have no effect, but it can also cause a serious respiratory outbreak. 

© Laboratorios Hipra, S.A. All Rights Reserved.
No part of this website or any of its contents may be reproduced, copied, modified or adapted, without the prior written consent of HIPRA.

REFERENCES:

• Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, Walker I, Kennedy S. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication. Arch Virol. 2000, 145:2421-9.

• Bautista EM, Meulenberg JJ, Choi CS, Molitor TW. Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus. Arch Virol. 1996:1357-65.

• Benfield DA, Nelson E, Collins JE, Harris L, Hennings JC, Shaw DP, Goyal SM, McCullough S, Morrison RB, Joo HS, Gorcyca D, Chladek D. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J Vet Diagn Invest. 1992. 4, 127-133.

• Benfield D, Nelson J, Rossow K, Nelson C, Steffen M, Rowland R. Diagnosis of persistent or prolonged porcine reproductive and respiratory syndrome virus infections Vet Res. 2000, 31:71.

• Brockmeier SL, Palmer MV, Bolin SR. Effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, Bordetella bronchiseptica, or a combination of both organisms in pigs. Am J Vet Res. 2000, 61:892-9.

• Brockmeier SL, Halbur PG, Thacker EL. Porcine respiratory disease complex. In KA Brogden, JM Guthmiller, eds, Polymicrobial Diseases. Washington, DC: ASM Press. 2002, 231-58.

• Calzada-Nova G, Schnitzlein W, Husmann R, Zuckermann FA. Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists. Vet Immunol Immunopathol. 2010, 135:20-33.

• Christopher-Hennings J, Nelson EA, Hines RJ, Nelson JK, Swenson SL, Zimmerman JJ, Chase CL, Yaeger MJ, Benfield DA. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars. J Vet Diagn Invest. 1995, 7:456-64.

• Darwich L, Díaz I, Mateu E. Certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology. Virus Res. 2010, 154:123-32.

• Díaz I, Darwich L, Pappaterra G, Pujols J, Mateu E. Different European-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs. Virology. 2006, 351:249-59.

• Díaz I, Gimeno M, Darwich L, Navarro N, Kuzemtseva L, López S, Galindo I, Segalés J, Martín M, Pujols J, Mateu E. Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection. Vet Res. 2012, 19:43:30.

• García-Nicolás O, Quereda JJ, Gómez-Laguna J, Salguero FJ, Carrasco L, Ramis G, Pallarés FJ. Cytokines transcript levels in lung and lymphoid organs during genotype 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. Vet Immunol Immunopathol. 2014, 160:26-40.

• Gimeno M, Darwich L, Diaz I, de la Torre E, Pujols J, Martín M, Inumaru S, Cano E, Domingo M, Montoya M, Mateu E. Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates. Vet Res. 2011, 42:9.

• Gómez-Laguna J, Salguero FJ, Pallarés FJ, Carrasco L. Immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs. Vet J. 2013, 195:148-55.

• Harms PA, Sorden SD, Halbur PG, Bolin SR, Lager KM, Morozov I, Paul PS. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet Pathol. 2001, 38:528-39.

• Haynes JS, Halbur PG, Sirinarumitr T, Paul PS, Meng XJ, Huffman EL. Temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (PRRSV) by in situ hybridization in pigs infected with isolates of PRRSV that differ in virulence. Vet Pathol. 1997, 34:39-43.

• Hill H. Overview and history of Mystery Swine Disease (swine infertility/respiratory syndrome). Proceedings of the Mystery Swine Disease Committee Meeting, Livestock Conversation Institute, Denver, CO. 1990, 29–31.

• Horter DC, Pogranichniy RM, Chang CC, Evans RB, Yoon KJ, Zimmerman JJ. Characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection. Vet Microbiol. 2002, 86:213-28.

• Jung K, Renukaradhya GJ, Alekseev KP, Fang Y, Tang Y, Saif LJ. Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections. J Gen Virol. 2009, 90:2713-23.

• Keffaber KK. Reproductive failure of unknown etiology. AASP. 1989, 1: 2–10.

• Kitikoon P, Vincent AL, Jones KR, Nilubol D, Yu S, Janke BH, Thacker BJ, Thacker EL. Vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of SIV vaccination. Vet Microbiol. 2009, 139:235-44.

• Labarque GG, Nauwynck HJ, Van Reeth K, Pensaert MB. Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs. J Gen Virol. 2000, 81:1327-34.

• Loula T. Mystery pig disease. Agri-practice. 1991, 12:23–34.

• Lunney JK, Benfield DA, Rowland RR. Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine. Virus Res. 2010, 154:1-6.

• Mateu E, Tello M, Coll A, Casal J, Martín M. Comparison of three ELISAs for the diagnosis of porcine reproductive and respiratory syndrome. Vet Rec. 2006, 159:717-8.

• Mateu E, Diaz I. The challenge of PRRS immunology. Vet J. 2008, 177:345-51.

• Meier WA, Galeota J, Osorio FA, Husmann RJ, Schnitzlein WM, Zuckermann FA. Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination. Virology. 2003, 309:18-31.

• Nelson EA, Christopher-Hennings J, Benfield DA. Serum immune responses to the proteins of porcine reproductive and respiratory syndrome (PRRS) virus. J Vet Diagn Invest. 1994, 6:410-5.

• OIE. Appendices IV and V of the REPORT OF THE OIE AD HOC GROUP ON PORCINE REPRODUCTIVE RESPIRATORY SYNDROME Paris, 9 - 11 June 2008 http://www.oie.int/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/PRRS_guide_web_bulletin.pdf.

• Pol JM, van Leengoed LA, Stockhofe N, Kok G, Wensvoort G. Dual infections of PRRSV/influenza or PRRSV/Actinobacillus pleuropneumoniae in the respiratory tract. Vet Microbiol. 1997, 55:259-64.

• Renukaradhya GJ, Alekseev K, Jung K, Fang Y, Saif LJ. Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs. Viral Immunol. 2010, 23:457-66.

• Rodríguez-Gómez IM, Gómez-Laguna J, Carrasco L. Impact of PRRSV on activation and viability of antigen presenting cells. World J Virol. 2013, 2:146-51.

• Rovira A, Cano JP, Muñoz-Zanzi C. Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA. Vet Microbiol. 2008, 130:60-8.

• Rovira A, Clement T, Christopher-Hennings J, Thompson B, Engle M, Reicks D, Muñoz-Zanzi C. Evaluation of the sensitivity of reverse-transcription polymerase chain reaction to detect porcine reproductive and respiratory syndrome virus on individual and pooled samples from boars. J Vet Diagn Invest. 2007, 19:502-9.

• Rovira A, Reicks D, Muñoz-Zanzi C. Evaluation of surveillance protocols for detecting porcine reproductive and respiratory syndrome virus infection in boar studs by simulation modeling. J Vet Diagn Invest. 2007, 19:492-501.

• Salguero FJ, Frossard JP, Rebel JM, Stadejek T, Morgan SB, Graham SP, Steinbach F. Host-pathogen interactions during porcine reproductive and respiratory syndrome virus 1 infection of piglets. Virus Res. 2015, 202:135-43.

• Segalés J, Domingo M, Balasch M, Solano GI, Pijoan C. Ultrastructural study of porcine alveolar macrophages infected in vitro with porcine reproductive and respiratory syndrome (PRRS) virus, with and without Haemophilus parasuis. J Comp Pathol. 1998, 118:231-43.

• Shibata I, Yazawa S, Ono M, Okuda Y. Experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus. J Vet Med B Infect Dis Vet Public Health. 2003, 50:14-9.

• Solano GI, Segalés J, Collins JE, Molitor TW, Pijoan C. Porcine reproductive and respiratory syndrome virus (PRRSv) interaction with Haemophilus parasuis. Vet Microbiol. 1997, 55:247-57.

• Solano GI, Bautista E, Molitor TW, Segales J, Pijoan C. Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis by porcine alveolar macrophages. Can J Vet Res. 1998, 62:251-6.

• Sur JH, Cooper VL, Galeota JA, Hesse RA, Doster AR, Osorio FA. In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection. J Clin Microbiol. 1996, 34:2280-6.

• Terpstra C, Wensvoorst G, Pol JMA. Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch's postulates fulfilled. Vet Q. 1991, 13:131–36.

• Thacker EL, Halbur PG, Ross RF, Thanawongnuwech R, Thacker BJ. Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J Clin Microbiol. 1999, 37:620-7.

• Thacker EL, Thacker BJ, Young TF, Halbur PG. Effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia by Mycoplasma hyopneumoniae. Vaccine. 2000, 18:1244-52.

• Thanawongnuwech R, Brown GB, Halbur PG, Roth JA, Royer RL, Thacker BJ. Pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to Streptococcus suis infection. Vet Pathol. 2000, 37: 143-52.

• Tian K, Yu X, Zhao T, Feng Y, Cao Z, Wang C, Hu Y, Chen X, Hu D, Tian X, Liu D, Zhang S, Deng X, Ding Y, Yang L, Zhang Y, Xiao H, Qiao M, Wang B, Hou L, Wang X, Yang X, Kang L, Sun M, Jin P, Wang S, Kitamura Y, Yan J, Gao GF. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS One. 200, 2:e526.

• Tong GZ, Zhou YJ, Hao XF, Tian ZJ, An TQ, Qiu HJ. Highly pathogenic porcine reproductive and respiratory syndrome, China. Emerg Infect Dis. 2007, 13:1434-6.
  Van Alstine WG, Popielarczyk M, Albregts SR. Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs. J Vet Diagn Invest. 2002, 14:504-7.

• Vézina SA, Loemba H, Fournier M, Dea S, Archambault D. Antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus. Can J Vet Res. 1996, 60:94-9.

• Wensvoort G, Terpstra C, Pol JMA, Lask EA, Bloemraad M, de Kluyver EP, Kragten C, van Butten L, den Besten A, Wagenaar F, Broekhuijsen JM, Moonen PJM, Zetstra T, de Boer EA, Tibben AhJ, de Jong MF, van’r Veld P, Groenland GJR, van Gennep JA, Voets MTh, Verheijden JHM, Braamkamp J. Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet Q. 1991, 13:121–30.

• Wesley RD, Mengeling WL, Lager KM. Prior infection of nursery-age pigs with porcine reproductive and respiratory syndrome virus does not affect the outcome of transmissible gastroenteritis virus challenge. J Vet Diagn Invest. 1998, 10:221-8.

• Wills RW, Gray JT, Fedorka-Cray PJ, Yoon KJ, Ladely S, Zimmerman JJ. Synergism between porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis in swine. Vet Microbiol. 2000, 71:177-92.

• Wills RW, Doster AR, Galeota JA, Sur JH, Osorio FA. Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus. J Clin Microbiol. 2003, 41:58-62.

• Zimmerman JJ, Benfield DA, Dee SA, Murtaugh MP, Stadejek T, Stevenson GW, Torremorell M. Porcine reproductive and respiratory syndrome virus (porcine arterivirus). In: 10th ed. Diseases of swine, Ed. Wiley-Blackwell. 2012, 31:463-86.