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Revista mexicana de ciencias pecuarias

versión On-line ISSN 2448-6698versión impresa ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.12 no.4 Mérida oct./dic. 2021  Epub 06-Jun-2022 

Technical notes

Detection of porcine reproductive and respiratory syndrome in porcine herds of Baja California, Mexico

Sergio Daniel Gómez-Gómeza 

Gilberto López-Valenciaa  * 

José Carlomán Herrera-Ramíreza 

Enrique Trasviña-Muñoza 

Francisco Javier Monge-Navarroa 

Kattya Moreno-Torresa 

Issa Carolina García-Reynosoa 

Gerardo Enrique Medina-Basultoa 

Miguel Arturo Cabanillas-Gámeza 

a Universidad Autónoma de Baja California. Instituto de Investigaciones en Ciencias Veterinarias. Km 3.5, Carretera San Felipe, Fraccionamiento Campestre, 21386, Mexicali, Baja California, México.


The objective of this study was to assess the presence of genotype 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) in Baja California (Baja), as well as the standardization of the qRT-PCR technique. A cross-sectional study was conducted from 2016 to 2017 in farms from Baja. It was obtained 97 blood samples from clinically healthy, not-vaccinated boars and sows. Primers were designed and standardize, in order to perform qRT-PCR tests from the buffy coat. Every positive results were confirmed by sequence studies. It was found that 9.3 % of the samples were positive. The positive samples came from 66.6 % of the sampled regions. This study demonstrates the presence of PRRSV-2 in Baja, therefore, it is necessary to conduct epidemiological studies in order to identify the magnitude of the problem and to establish preventive and control measures.

Key words Detection; Mexico; PCR; PRRS; Swine


El objetivo de este estudio fue evaluar la presencia del virus del síndrome reproductivo y respiratorio porcino genotipo 2 (VSRRP-2) en Baja California (Baja), así como la estandarización de la técnica qRT-PCR. Se realizó un estudio transversal de 2016 a 2017 en granjas de Baja. Se obtuvieron 97 muestras de sangre de verracos y cerdas clínicamente sanos y no vacunados. Se diseñaron y estandarizaron iniciadores con el fin de realizar pruebas qRT-PCR a partir de la capa leucocitaria. Todos los resultados positivos fueron confirmados por estudios de secuenciación. Se encontró que el 9.3 % de las muestras fueron positivas. Las muestras positivas provinieron del 66.6 % de las regiones muestreadas. Este estudio demuestra la presencia de VSRRP-2 en Baja, por lo tanto, es necesario realizar estudios epidemiológicos para identificar la magnitud del problema y establecer medidas preventivas y de control.

Palabras clave Detección; México; PCR; SRRP; Porcino

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by an RNA virus, with two currently known genotypes: 1 (European) and 2 (American)1. It is easily transmitted by saliva, nasal secretions, urine, semen, milk and colostrum2. It produces reproductive failure, weak-born piglets and respiratory diseases3. The main route of introduction of PRRS virus (PRRSV) into previously free countries is via pig movements and with introduction of semen; therefore, protocols must be in place to reduce the risk4. When first introduced into an immunologically naive herd, the virus spreads to pigs of all ages in about 2 to 3 wk2, with mortality rates in nursery pigs up to 69 %5. Most common diagnosis tests are commercial ELISA6 and in recent years, molecular techniques, particularly real time reverse transcriptase polymerase chain reaction (qRT-PCR)7.

In Mexico, PRRSV was first described in 1994 with an 8.1 % serological prevalence in imported pigs from USA and Canada8. Since 2002, it has been reported the presence of multiple variants of genotype 2 PRRSV (PRRSV-2) within pig farms throughout Sonora9-10, neighbor state of Baja California (Baja), and the second biggest pork producer in Mexico. Meanwhile, in Baja, the main pork production comes from small backyard producers, with installations made basically from rustic materials, with the same areas used for the different production stages; generally fed with swill and other food waste sub-products; usually with none preventive medicine programs or veterinary’s advice and in most cases not even adequate hygiene practices.

The main objective of this study was to assess the presence of genotype 2 porcine reproductive and respiratory syndrome virus in the most representative pork production regions in Baja California, Mexico, besides the standardization of the qRT-PCR test for this disease.

The study was approved by the Institutional Committee for Animal Ethics, which is represented by the Academic Group of Animal Health and the Academic Group for Diagnosis of Infectious Diseases, both of which are part of the Institute of Research in Veterinary Sciences. The owners of pigs used in this research were informed about the study and they gave their consent.

A cross-sectional study was conducted in 26 farms within six regions of Baja: Ensenada, Mexicali, Tecate and Tijuana, as well as the Mexicali and San Quintin valleys. The farms were selected from a Baja California Pig Farmers Association Database and invited to participate according to their proximity and herd size. Only those who were interested were visited. Estimation of sample size was done for one disease detection11, considering a state swine population of 10,31512, a 99.5% diagnostic sensitivity, 4% expected prevalence and 95% confidence level.

n(1-(1-)1/D) (N-1/2(SeD-1))Se

where, n= sample size; N= population size; D= number of diseased; α= confidence level; Se= test sensitivity.

Accordingly, it was needed a sample size of at least 74; however, it was possible to collect a total of 97 blood samples from the jugular vein of apparently healthy boars and sows not involved in vaccination against PRRSV. It was used sterile Vacutainer® tubes with EDTA anticoagulant (BD, Franklin Lakes, NJ, USA). Samples were transported to the Molecular Biology Laboratory of the Institute of Research in Veterinary Sciences, then there were separated 200-300 µL of the buffy coat into sterile tubes for RNA extraction. RNA extraction was made using AurumTM Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The RNA was reconstituted to a final volume of 30 µL of prepared elution. RNA was stored at -70 °C until the qRT-PCR test were performed.

The RT-PCR primers were designed to amplify a fragment with a length of 87 bp of the nucleocapsid gene contained within the open reading frame 7 (ORF7) of PRRSV-2 (GenBank AF494042.1), since this is the most conserved viral protein in PRRSV-infected pig cells13. The primers were designed using Primer3Plus version 2.4, GenneRunner version 6.1 and OligoCalc version 3.2, generating the primers PRRS-USA-F 5’-CGATCCAGACTGCCTTTAAC-3’ and PRRS-USA-R 3’-CACTGTGGAGTTTAGTTTGC-5’.

The qRT-PCR conditions were optimized by testing primers in triplicate at 200, 400 and 800 nM with 1, 2 and 3 µL of RNA template in a total volume of 10 µL using a master mix with EvaGreen® dye (Biotium, Hayward, CA, USA). The best efficiency was achieved by using the primers at 800 nM in 2 µL of RNA template and was proved it through ten-fold dilutions to generate a melt curve analysis (Figure 1) and comparing it with agarose gel electrophoresis. Once obtained the best concentration, it was tested by triplicate 40 control positive samples and 40 negative samples so to achieve a 95% confidence of specificity14. The sensitivity was determined through ten-fold dilutions by generating a standard curve of 97.6 % efficiency, R2 of 0.996 and 3.25 slope (Figure 2).

Figure 1 Amplification peaks of different positive control dilutions in the melting temperature previously stablished (77.4-78.0 ºC) 

Figure 2 Standard curve of the designed primers 

Positive control RNA was extracted from Ingelvac PRRS® MLV (Boheringer Ingelheim) vaccine. Three different negative controls were used: master mix without RNA template, molecular grade water and air. All samples were tested in duplicate. The qRT-PCR reactions were executed in a CFX96 real-time thermocycler (Bio-Rad, Hercules, CA, USA). Test reactions consisted of 1 µl of RNA, 400 nM of each primer and master mix of iScript One Step RT-PCR with EvaGreen dye and molecular grade water in a total reaction volume of 10 μL. Thermocycler conditions were calculated using CFX96 software, resulting in an initial step of reverse transcription at 50 °C for 10 min, denaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 10 sec, annealing at 53.7 °C for 25 sec and extension at 72 °C for 20 sec. A melt curve analysis was performed after each run in order to confirm the melt temperature (Tm) of the amplified fragment, calculated between 77.4 and 78.0 °C. The positive samples were confirmed by sequencing at an external laboratory and these sequences, verified using BLAST tool (Figure 3).

Panel A: Sequencing results of one of the positive samples, highlighting a sequence of 26 nucleotides. Panel B: BLAST database screenshot, showing the 26 nucleotides of the panel A, highlighted and matching a type 2 porcine reproductive and respiratory syndrome virus strain in the nucleocapsid protein gene. The database casts 100 results (not shown), all of them PRRSV-2 strains.

Figure 3 Sequence of positive samples and verified using BLAST tool 

It was found 9.3 % (9/97) positive samples to PRRSV-2, present in 66 % (4/6) of the regions where the study was carried out. The frequency of positive cases was 33 % (2/6) in Tijuana, 16.6 % (3/18) in San Quintin Valley, 11.1 % (3/27) in Mexicali and 3.7 % (1/27) in Ensenada (Figure 4). In Tecate (0/13) and Mexicali Valley (0/6) there was no positive samples. Signs of PRRS were not found or reported on any farm.

Figure 4 Geographical distribution of PRRSV in Baja California and the frequency in each tested area 

The main objective of this study was to assess the presence of type 2 porcine reproductive and respiratory syndrome virus in the most representative pork production regions in Baja California, therefore, considering the epidemiological design of the study can state that PRRSV-2 is present in Baja, with a prevalence of at least 4 %.

This study represents the first report of PRRSV-2 in Baja15, despite the previous report of its presence in the northwest area of the country16 which includes, besides the state of Baja, the state of Sonora and four other states. It is important to highlight the geographical proximity with Sonora as well as the characteristics of their pork industry, since Sonora introduces pork meat as well as live pigs and semen into Baja17, thus the possibility of PRRS contagion is present considering that vaccination against PRRSV has never been implemented in Baja since it is supposed to be free of the disease and furthermore, the biggest proportion of small backyard producers, who do not usually use primary preventive medicine measures.

In this study were also designed primers capable of detecting PRRSV-2 in the ORF7 region, and these results were confirmed by sequencing, proving the effectiveness of the test and the presence of PRRSV-2 in Baja, regardless of the lack of clinical signs18. This might be owed to the presence of low-virulence strains of PRRSV within Mexican territory19; or a low viral concentration within the samples20 given the amplification of the positive curves was observed after cycle 30.

This study demonstrates the broad presence of PRRSV-2 in Baja, even in absence of clinical signs that indicate the presence of the disease; therefore, it is necessary to make prospective epidemiological studies aiming at determining the prevalence and the possible associated risk factors in order to identify the magnitude of the problem as well as to establish preventive and control measures.


This study permitted to the first author to obtain the Doctor’s degree in Agricultural Sciences (Autonomous University of Baja California). The authors are also grateful to MVZ José Soto, Dra. Laura Kinejara, MC Arsenio Guzmán, MC Kelvin Espinoza and MC Ricardo Martínez.

Literature cited

1. Nan Y, Wu C, Gu G, Sun W, Zhang YJ, Zhou EM. Improved vaccine against PRRSV: Current progress and future perspective. Front Microbiol 2017;8:1635. [ Links ]

2. Pileri E, Mateu E. Review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination. Vet Res 2016;47:108. [ Links ]

3. Wang X, Marthaler D, Rovira A, Rossow S, Murtaugh MP. Emergence of a virulent porcine reproductive and respiratory syndrome virus in vaccinated herds in the United States. Virus Res 2015;210:34-41. [ Links ]

4. Organization of Animal Health. PRRS: the disease, its diagnosis, prevention and control. Paris: Office of International Epizootica, 2008:1-7. [ Links ]

5. Young B, Dewey C, Poljak Z, Rosendal T, Carman S. Clinical signs and their association with herd demographics and porcine reproductive and respiratory syndrome (PRRS) control strategies in PRRS PCR-positive swine herds in Ontario. Can J Vet Res 2010;74(3):170-177. [ Links ]

6. Henao-Diaz A, Giménez-Lirola L, Magtoto R, Ji J, Zimmerman J. Evaluation of three commercial porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISAs using samples of known status. Res Vet Sci 2019;125:113-118. [ Links ]

7. Yang Q, Xi J, Chen X, Hu S, Chen N, Qiao S, et al. The development of a sensitive droplet digital PCR for quantitative detection of porcine reproductive and respiratory syndrome virus. Int J Biol Macromol 2017;104(A):1223-1228. [ Links ]

8. Milián SF, Cantó AGJ, Weimersheimer RJ, Coba AMA, Anaya EAM, Correa GP. Estudio seroepidemiológico para determinar la presencia de anticuerpos contra el virus del síndrome disgenésico del cerdo en México. Tec Pecu Mex 1994;32(3):139-144. [ Links ]

9. Batista L, Pijoan C, Lwamba H, Johnson CR, Murtaugh MP. Genetic diversity and possible avenues of dissemination of porcine reproductive and respiratory syndrome virus in two geographic regions of Mexico. J Swine Health Prod 2004;12:170-175. [ Links ]

10. Burgara-Estrella A, Reséndiz-Sandoval M, Cortey M, Mateu E, Hernández J. Temporal evolution and potential recombination events in PRRSV strains of Sonora Mexico. Vet Microbiol 2014;174(3-4):540-546. [ Links ]

11. Cannon, RM. Sense and sensitivity-designing surveys based on an imperfect test. Prev Vet Med 2001;49(3-4):141-63. [ Links ]

12. State Information Office for Sustainable Rural Development. Livestock statistical notebook, 2011-2015. Baja California, México: Ministry of Agricultural Development, 2016:19. [ Links ]

13. King SJ, Ooi PT, Phang LY, Nazariah Z, Allaudin B, Loh WH, et al. Phylogenetic characterization of genes encoding for viral envelope glycoprotein (ORF5) and nucleocapsid protein (ORF7) of porcine reproductive & respiratory syndrome virus found in Malaysia in 2013 and 2014. BMC Vet Res 2016;13(1):3. [ Links ]

14. Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, et al. Guidelines for validation of qualitative real-time PCR methods. Trends in Food Science & Technology 2014;37(2):115-126. [ Links ]

15. National Secretary of Health Quality and Food Safety. Bulletin of the Secretary of Epidemiological Surveillance Inform, 2017. . Accessed Oct 10, 2018. [ Links ]

16. Martínez-Bautista NR, Sciutto-Conde E, Cervantes-Torres J, Segura-Velázquez R, Mercado-García MC, Ramírez-Mendoza H, et al. Phylogenetic analysis of ORF5 and ORF7 of porcine reproductive and respiratory syndrome (PRRS) virus and the frequency of wild-type PRRS virus in Mexico. Transbound Emerg Dis 2018;65(4):993-1008. [ Links ]

17. Extension and Territorial Innovation Group. Innovation program pig chain of GEIT livestock, Mexicali, in the State of Baja California. Baja California, Mexico: Rural Extension and Innovation Center. 2015:10. [ Links ]

18. Zimmerman JJ, Benfield A, Dee SA, Murtaugh MP, Stadejek T, Stevenson GW, et al. Porcine reproductive and respiratory syndrome virus (Porcine Arterivirus). In: Zimmerman JJ, et al, editors. Diseases of swine. 10th ed. Ames, Iowa: Wiley-Blackwell, 2012:461-486. [ Links ]

19. Weimersheimer R, Canto AJEE, Anaya EGJ, Coba AMA, Millán SF, Correa GP. Frecuencia de anticuerpos contra el virus del síndrome disgenésico y respiratorio en cerdos sacrificados en rastros de México. Tec Pecu Mex 1997;35:139-144. [ Links ]

20. Walker NJ. A technique whose time has come. Science 2002;296:557-559. [ Links ]

Received: August 24, 2020; Accepted: December 29, 2020

*Autor de correspondencia:

Conflict of interest statement

The authors have no financial or personal relationship with people or organizations that could inappropriately influence or bias the content of the paper.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License