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Canadian Journal of Veterinary Research logoLink to Canadian Journal of Veterinary Research
. 2009 Jan;73(1):21–24.

Retrospective serological survey of Porcine circovirus-2 infection in Mexico

Humberto Ramírez-Mendoza 1,, Héctor Castillo-Juárez 1, Jesús Hernández 1, Pablo Correa 1, Joaquim Segalés 1
PMCID: PMC2613592  PMID: 19337391

Abstract

Postweaning multisystemic wasting syndrome (PMWS) is considered a multifactorial emerging disease of which Porcine circovirus-2 (PCV-2) is the necessary infectious cause. However, retrospective studies have shown that PMWS is not a new disease and that PCV-2 has been circulating in pig farms for years. Most of these studies were performed in Europe and Asia; only a few were performed in North or South America.

A PCV-2 retrospective serological survey was carried out with 659 serum samples collected from pigs in Mexico between 1972 and 2000. Serological analyses were performed with an immunoperoxidase monolayer assay (IPMA). The overall prevalence of PCV-2 antibodies was 59% (387/659); the prevalence was 27% (24/90) for the period from 1972–1979; 44% (74/169) from 1980–1989, and 72% (289/400) from 1990–2000. Antibodies to PCV-2 were detected in at least 1 pig from all tested years since 1973. This study shows evidence of enzootic PCV-2 infection in Mexico for many years before the first description of PMWS in the country (in 2001), further supporting results obtained in other parts of the world. To date, this study provides the earliest evidence of PCV-2 infection in the North and South American continents.


Porcine circovirus-2 (PCV-2), a single-stranded DNA virus that infects pigs, is classified in the family Circoviridae and genus Circovirus. This virus was initially linked to an emerging clinical and pathological condition of pigs from Canada (1,2) that was named postweaning multisystemic wasting syndrome (PMWS). Now it is known that PMWS is a multifactorial disease in which PCV-2 is the necessary infectious agent but is usually not sufficient to trigger the clinical condition (3). The virus also is known to be ubiquitous among domestic and wild swine (49). Moreover, evidence of PCV-2 infection was established as early as 1969 in Europe (10) and 1985 in North America (11). Retrospective studies have provided evidence of the existence of PMWS a decade before the disease was first described (1015). Thus, it can be concluded that PMWS is not a new disease and that PCV-2 has been circulating in pig farms for years.

In Mexico, PMWS has been reported in domestic swine (16) and is considered to be of economic importance, yet no studies on its prevalence and associated production losses had been performed. Moreover, it is believed that PCV-2 is ubiquitous in pigs in Mexico, according to results for backyard pigs in Mexico City (4), as has been described in other countries (3). However, no retrospective studies on PCV-2 infection and PMWS had been performed in Mexico. The aim of the research described herein was to study PCV-2 infection by testing of serum samples collected from Mexican pigs between 1972 and 2000.

The 659 samples corresponded to 22 unrelated farms, and the pigs were of various ages. The samples, stored at −20°C, at the Centro Nacional de Investigación Disciplinaria en Microbiología (CENID-Microbiología), in Mexico City, had originally been collected to monitor the results of vaccination and eradication programs against classical swine fever throughout the country. Samples were available for all years in the 1972–2000 period, except for 1974–1976, 1978, 1982, 1986, and 1993.

Antibodies to PCV-2 were detected by an immunoperoxidase monolayer assay (IPMA) (17). Briefly, trypsinized PK-15 cells free of PCV-2 and Porcine circovirus-1 (PCV-1) were seeded in 96-well plates and incubated for 5 d at 37°C in 5% CO2. The cells were then fixed with methanol containing 1% hydrogen peroxide for 5 min at room temperature. After 3 washes with phosphate-buffered saline (PBS) containing 0.05% Tween 80 (PBS-T), the cells were incubated for 1 h with serum samples diluted 2-fold from 1:20 to 1:20 480 in PBS-T with 1% bovine serum albumin (BSA). Serum 211-P-PCRV (VMRD, Pullman, Washington, USA), a porcine hyperimmune serum against PCV-2 was used as a positive control with a known PCV-2 antibody titer of 1:1280. The plates or dishes were washed again with PBS-T and, after the addition of peroxidase-labelled protein A (0.6 μg/mL) in PBS-T-BSA, were incubated for 1 h at 37°C. Finally, the plates were washed with PBS-T, and aminoethylcarbazole was added to visualize the reaction. Porcine serum 211-N-PCRV (VMRD) was used as a negative control. With IPMA, which is widely cited in literature (3), there is apparently a cross-reaction between PCV-1 and PCV-2 antibodies (17).

The serological results were grouped as negative or as and positive with a low titer (1:20 to 1:80), intermediate titer (1:320 to 1:1280), or high titer (1:5120 or greater). The data were grouped, and the average prevalence in the 3 decades (1972–1979, 1980–1989, and 1990–2000) was compared with a chi-squared test under the hypothesis that the prevalence did not change over time, compared with the average for the complete data set. We used the arcsine transformation of the percentages in a linear regression with no intercept to test for changes in seroprevalence over time. Statistical analyses were performed with the use of SAS, version 9.1 (SAS Institute, Cary, North Carolina, USA).

Table I presents the grouped results and overall PCV-2 serological prevalence for each year and the entire study period. The number of PCV-2-positive samples was 24 out of 90 (27%) for the period from 1972–1979, 74 out of 169 (44%) for 1980–1989, 289 out of 400 (72%) for 1990–2000, and 387 out of 659 (59%) for the entire study period. Significant differences (P < 0.0001) in seroprevalence among the 3 decades were observed (X2 = 82.55 with 2 degrees of freedom). The linear regression coefficient of the seroprevalence increase across the years was positive (adjusted R2 = 74.27%, P < 0.0001) and significant.

Table I.

Results of testing serum samples from Mexican pigs for antibodies to Porcine circovirus-2 (PCV-2)

Number of samples; PCV-2 antibody titera
Year Total number of samples Negative Low Intermediate High Seroprevalence (%)
1972 15 15 0 0 0 0
1973 31 12 19 0 0 61
1977 24 20 3 1 0 17
1979 20 19 0 1 0 5
1980 19 18 1 0 0 5
1981 11 1 0 5 5 91
1983 10 1 8 1 0 90
1984 1 0 1 0 0 100
1985 6 1 4 0 1 83
1987 24 9 11 2 2 62
1988 51 39 7 3 2 24
1989 47 26 11 10 0 45
1990 3 3 0 0 0 0
1991 48 6 8 24 10 88
1992 32 21 5 4 2 34
1994 32 10 12 10 0 69
1995 32 6 5 17 4 81
1996 33 0 1 17 15 100
1997 32 1 13 14 4 97
1998 119 47 21 36 15 60
1999 37 16 6 11 4 57
2000 32 1 8 19 4 97
Total 659 272 144 175 68 59
a

Low — titer of 1:20 to 1:80; intermediate — titer of 1:320 to 1:1280; high — titer of 1:5120 or greater.

The data compiled in this retrospective study indicate that antibodies to PCV-2 have been present in the Mexican swine population at least since 1973, 28 years before the first description of PMWS in the country (16). To date, this study provides the earliest evidence of PCV-2 infection in the American continents. The data agree with those from serological retrospective studies performed in other countries, in which PCV-2 antibodies were also detected before knowledge of the existence of PMWS — as early as 1969 in Belgium (10), 1973 in Northern Ireland (18), and 1985 in Canada (11) and Spain (13).

An interesting contribution of the present study is the time-period comparisons that showed the increase in PCV-2 seroprevalence over time. This situation might reflect a significant increase in virus circulation among Mexican farms during the studied period, but specific yearly results (and the fact that the exact ages of the studied pigs were unknown) prevent us from establishing this conclusion. In fact, a huge variation in PCV-2 seroprevalence was observed among each range of years; for instance, 1973 had a very high rate compared with the rest of the years within the 1972–1979 study period. This suggests a year effect or, more specifically, farm or age effects within a year. It is well known that PCV-2 seroprevalence may significantly vary among age groups within a farm and between farms (6,8). The overall increase in PCV-2 titer across the time periods is probably linked to an increase in virus dissemination or infectious pressure, or both, over time. The results allow one to speculate that an increased PCV-2 infectious pressure over time could have resulted in the emergence of PMWS by 2000–2001 or even before.

From the results of this study, it is possible to hypothesize that 1973 could be the year of PCV-2 introduction into the Mexican swine population, since no pigs had positive results in 1972. However, several facts prevent us from coming to this conclusion. First, a low number of animals were tested in 1972. Second, the original serum samples were selected for other study purposes (monitoring classical swine fever vaccination and eradication programs). And, third, most of the breeding pig population in Mexico during the early 1970s was imported from the United States, suggesting potential introduction of the virus with such genetic stock. If this last hypothesis is correct, it would not modify the hypothesis that our results represent the oldest evidence of PCV-2 infection in the American continents. Older samples would need to be tested to determine if 1973 was really the year of introduction of PCV-2 into Mexico.

It would be interesting to know if clinical signs compatible with PMWS were eventually observed in the studied PCV-2 positive pigs. However, given the main purpose of the sample collection and the lack of specificity of PMWS clinical signs (3), it is not possible to establish the existence of PMWS in Mexico during the period from 1973–2000. The proper way to assess the presence of the disease during said period would be by retrospective histopathological analyses of formalin-fixed, paraffin-embedded lymphoid tissues from pigs suspected of having the disease.

The serological technique used in this study may detect antibodies elicited by PCV-1 infection; titers of antibodies to PCV-2 and PCV-1 are usually strongly associated, the former being much higher than the latter (17). In fact, in another study using IPMA (19), only pigs with high titers of antibody to PCV-2 showed reactions indicating antibody against PCV-1. Moreover, the serological data are supported by virus detection in pigs: rates of infection with PCV-2 are high, reflecting this virus’s ubiquity (3), whereas rates of infection with PCV-1 are very low (20). Therefore, although infection with PCV-1 might not be excluded as the potential cause of seropositivity in a low proportion of pigs, the available data indicate that positive IPMA results are really attributable to PCV-2 and hardly ever to PCV-1.

The results of the present study confirm that PCV-2 is a relatively old virus that was circulating among Mexican pigs for several decades before the first description of PMWS (16). The findings represent the earliest evidence of PCV-2 infection in pigs in the Americas. Although the results do not represent the seroprevalence of PCV-2 in all of Mexico (only 22 farms were tested), they suggest that PCV-2 infection is probably widely distributed among the commercial farms of this country, as has been assessed from backyard pigs in Mexico City (4).

Acknowledgments

This study was financed with funds from the National Autonomous University of Mexico (PAPIIT IN-226105-3) and from the Mexican government (SAGARPA-CONACYT 2004-C01-61). We are thankful to Marcia Castillo-Mendoza for her comments on the manuscript.

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