The effect of vaccination against Porcine reproductive and respiratory syndrome virus (PRRSV) on the Porcine circovirus-2 (PCV-2) load in porcine circovirus associated disease (PCVAD) affected pigs

Abstract

A diagnostic project was initiated across the United States in 2006 to improve the understanding of porcine circovirus associated diseases (PCVAD) as well as to identify co-factors in PCVAD-affected farms. A Porcine circovirus-2 (PCV-2) DNA real-time polymerase chain reaction quantitation (qPCR) was established according to a published method and sera from a total of 23pig farms across the United States were examined for viral loads for PCV-2 and analyzed for any possible effects of Porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on this parameter. Vaccination against PRRS resulted in significantly lower viral loads for PCV-2 in animals 13 wk or older compared with nonvaccinated animals, but vaccination of pigs against PRRS had no effect on qPCR results for PCV-2 in 4- to 12-week-old pigs. Interestingly, PRRS vaccinates had significantly lower viral loads when peak wasting disease was observed in the herds. The qPCR method for PCV-2 proved to be an important tool for help in the antemortem diagnosis of PCVAD as well as in the monitoring of infection dynamics.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine and is widespread in global pig populations. It has been estimated that the costs associated with PRRS are around $560 million in the United States, 88% of which are attributable to increased mortality rates and decreased growth performance in post weaning pigs (1). A small, enveloped, positive-stranded RNA virus, PRRSV belongs to the family Arteriviridae that replicates predominantly in alveolar macrophages. Porcine circovirus-2 (PCV-2) has been identified as the causative agent for postweaning multisystemic wasting syndrome (PMWS) in pigs (2). Recently, the term porcine circovirus associated diseases (PCVAD) has been accepted by the American Association of Swine Veterinarians (AASV) to more fully represent the range of disease symptoms that are attributed to PCV-2. Porcine circovirus-2 is a small, nonenveloped, single-stranded DNA virus with a circular genome (3). Infection with PCV-2 is characterized by lymphoid depletion and lymphohistiocytic inflammation in various organs. Although PCV-2 can be a primary cause of disease, it is rarely found as the sole pathogen in affected pigs (4). Co-infection with other viral and bacterial pathogens severely worsens the clinical outcome of this disease syndrome (5,6). Experimental infection of pigs with PRRSV and PCV-2 in combination has shown to induce more severe clinical symptoms and lesions than those associated with infection by either agent alone (7). However, the mechanism for the interaction of PRRSV and PCV-2 in the pathogenesis of PCVAD remains unclear.

A collaborative diagnostic project was initiated by Boehringer Ingelheim Vetmedica Inc. and the Iowa State University Veterinary Diagnostic Laboratory in the United States in 2006 to improve the understanding of PCVAD as well as to identify co-factors in PCVAD-affected farms. Data were analyzed to discern if vaccination against PRRSV would have any impact on viral load for PCV-2 using a quantitative polymerase chain reaction (qPCR) method in field cases of PCVAD.

Materials and methods

Diagnostic projects

In order to elucidate the features of PCVAD in clinically affected farms, a diagnostic investigation was carried out in 59 randomly selected farms across the United States. A criterion for enrolment was an increased mortality of 50% due to PCVAD. A total of 15 pigs from each farm were necropsied at peak mortality (n = 5), 3 to 4 wk before peak mortality (n = 5), and at 5 to 6 wk before peak mortality (n = 5). In addition, 10 serum samples were collected from each live pig in each of the following age groups: gilts, sows, and pigs at 4 wk, 10 wk, 14 wk, 18 wk, and 24 wk of age. Thus a total of 85 serum samples were collected as well as tissues samples from 15 pigs per farm for diagnostic investigation. Serum samples were analyzed for PRRSV, Mycoplasma hyopneumoniae and Salmonella antibodies as well as for PRRS viremia by PCR. The serum samples were also analyzed by quantitative PCR for PCV-2. Each diagnostic project was accompanied by a questionnaire designed to elucidate details on housing, management practices, and medications and vaccinations that were routinely applied.

Diagnostic data are available from a total of 59 farms including data from 23 farms that have qPCR results obtained using the method described in the following text. The sera of the remaining 36 farms were tested using different qPCR methods for PCV-2 and, therefore, have been excluded from this evaluation. Harding et al (8) have recently shown that qPCR method differences have a real effect the respective results or that they cannot be directly compared. For that reason, we chose to include only data that were obtained with the assay published by Brunborg et al (9) run on a Light-Cycler 480.

PCV-2 DNA real-time PCR quantitation

A TaqMan-based real-time PCR for quantitation of PCV-2 in serum/plasma was established according to the method published by Brunborg et al (9). The PCV-2 DNA is extracted from serum samples using a QIAamp 96 DNA Blood Kit (QIAGEN, Valencia, California, USA). Samples are lysed and a series of buffers are added. The DNA is bound to the filter membrane and is eluted and stored until use. Real-time PCR is performed on the extracted DNA using a Roche LightCycler 480 instrument. The PCV-2 is serially diluted and 8 different concentrations are included in each run to derive a standard curve. Extracted DNA samples are transferred to a 96-well plate containing a mixture of water, enzymes, probes, and primers from Roche Diagnostics Inc. A fluorescent probe is bound to the DNA, and a signal is produced as it is amplified. Thermal-cycling is carried out for 45 cycles to create copies of the target DNA sequence and amplification is detected on each cycle in real-time by light emitted from the TaqMan probe.

Serial dilutions of plasmid containing a known concentration (from 1.0 × 10⁴ to 1.0 × 10¹⁰ plasmid copies/mL) of PCV-2 DNA are used to create a standard curve. The concentration of DNA found in the sample is plotted against the standard curve. By determining where the sample falls on the standard curve, the instrument determines the initial concentration of PCV-2 DNA in that particular sample.

For quality assurance purposes, every PCR run includes a positive control (PCV-2 tissue culture) and a negative control. A run is considered valid when the following requirements are met: 1) the correlation coefficient of the serially diluted standards must be 0.95 or higher; and 2) the negative control does not cross the baseline and the positive control is detected.

Results are reported in scientific notation as genomic equivalents (viable or nonviable viral DNA) per mL of serum. Scientific notation is written as log base 10 (such as 10⁵) which is often stated as “5 logs” of genomic equivalents/mL.

As stated in the sensitivity section previously, any samples with values < 10 000 genomic equivalents/mL of is recorded as < 10⁴. This means that either the animal is free of PCV-2 virus or that the animal carries PCV-2 virus at levels too low for this test to detect.

Statistical analysis

The effects of vaccination of growing pigs against PRRSV on qPCR results for PCV-2 were statistically analyzed by means of the Wilcoxon Rank-Sum test, using computer software (SAS, version 8.2; SAS, Cary, North Carolina, USA). Results were considered significant if P ≤ 0.05. The null hypothesis was that vaccination against PRRSV would have no impact on qPCR PCV-2 results.

Results

Pigs from 41 herds were not vaccinated against PRRS, pigs from 13 herds were vaccinated with US modified live virus (MLV) PRRS vaccines, and the vaccination status of 5 projects remained unknown. In all vaccinated herds, the PRRS vaccine was applied to piglets at time of weaning. Results of the published qPCR method for PCV-2 are available for a total of 23 herds, 6 herds of which were using PRRS MLV vaccine in pigs; 12 did not use PRRS vaccine, and the PRRS vaccination status in 5 cases is unknown. The results of the herds that had an unknown PRRS vaccination status, therefore, were not included in the statistical analysis.

The analysis of the surveys revealed that, in most cases, peak wasting disease occurred between 12 and 16 wk of age. No peak in disease was found before 8 wk of age. There was no significant difference in the time point of peak disease between PRRS vaccinated and unvaccinated herds. Based on the Idexx PRRS HerdChek^® enzyme-linked immunosorbent assay (ELISA) 2XR more than 76% of the weaned animals were negative at weaning with titers of <0.4. The effect of vaccination against PRRS with a US-modified live vaccine on qPCR PCV-2 results is presented in Table I.

Table I.

Effect of porcine reproductive and respiratory syndrome (PRRS) vaccination on Porcine circovirus-2 (PCV-2) viral load (qPCR PCV-2)

Age in weeks (range)	PRRS vaccination	Number of animals	Mean rank qPCR score	P value
4 (3–7)	No	131	79.8	0.2 (NS)
	Yes	30	86.2
10 (8–12)	No	109	64.0	0.4 (NS)
	Yes	20	70.4
14 (13–16)	No	111	75.1	0.02 (S)
	Yes	30	56.0
18 (17–20)	No	110	74.1	0.04 (S)
	Yes	39	57.4
22–24	No	110	67.7	0.03 (S)
	Yes	19	49.5

NS — not significant; S — significant.

As evident from Table I, vaccination of pigs against PRRS had no effect on qPCR results for PCV-2 in 4–12-wk old pigs (P > 0.05). However, vaccination against PRRS resulted in significantly lower PCV-2 viral loads (based on qPCR results) in animals 13 wk or older compared with nonvaccinated animals. Interestingly, PRRS vaccinates had significantly lower viral loads when peak wasting disease was observed in the herds.

Discussion

Diagnosis of PCVAD is based mainly on detection of the virus in lesions, predominantly in the lymphatic tissues of the affected animals. However, this information alone is not enough to support the diagnosis of PCVAD, since many animals harbor PCV-2 without developing clinical disease (9,10). Studies have shown that clinically sick pigs have significant amounts of virus in serum, usually at least 1.0 × 10⁷ PCV-2 genomes per mL of serum. Therefore, quantitation of PCV-2 in serum can help in the antemortem diagnosis of PCVAD. Recently, a real-time PCR method was found to be useful in quantifying PCV-2 in clinical samples from affected animals (11).

The results herein were derived from a field diagnostic project in which only PCVAD-affected farms were included, and lacks the inclusion of control herds. However, it has been postulated that controlling co-factors like PRRSV in herds affected with PCVAD will help to improve the clinical outcome of PCV-2 infection (12). Our findings contrast with an experimental study by Allan et al (13) in which the effects on PCV-2 infection in an experimental model following the use of a PRRSV MLV were reported. In that study, none of the pigs developed PCVAD, but PRRSV-vaccinated animals showed a higher PCV-2 viral load than those that were not vaccinated. The main difference between the findings of Allan et al (13) and the findings herein are that in our study only herds with PCVAD clinical signs were included, whereas in the experimental setting no overt clinical signs of PCVAD in the animals were expressed. Our study is the first observation that vaccination against PRRSV with a MLV decreases the magnitude of PCV-2 viremia as measured by the described qPCR in PRRS-positive and PCVAD-affected herds. Rovira et al (14) reported that PCV-2 viral loads are higher in dually infected pigs with PRRSV and PCV-2 than in pigs that had been inoculated with PCV-2 alone. It has been shown by Cano et al (15) that PRRSV vaccination with a US MLV reduces the duration of heterologous viral shedding, which may help explain our observation. The PRRSV vaccinated animals have a shorter duration of PRRSV viremia; therefore, it can be postulated that a less severe disease will occur in PRRSV- and PCV-2-infected, but PRRSV vaccinated pigs. This has been reported also by Alexopoulos et al (12) who observed a significant reduction of clinical signs of PCVAD in PRRS vaccinated pigs. The qPCR method may be an important tool for help in the antemortem diagnosis of PCVAD as well as in the monitoring of infection dynamics.