Prevalence of infection with porcine circovirus-2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) in an integrated swine production system experiencing postweaning multisystemic wasting syndrome

Abstract

The objective of this study was to evaluate the prevalence of infection with porcine circovirus-2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) through a longitudinal study in an integrated swine production system (7 farms) experiencing postweaning multisystemic wasting syndrome (PMWS). Risk factors for PCV-2 infection and for PCV-2 and PRRSV coinfection were also evaluated. Fifteen sows from each herd and 4 non-cross-fostered piglets from each sow were randomly selected at farrowing and ear-tagged at birth. Serum samples were analyzed for antibodies to PCV-2 and for detection of the PCV-2 and PRRSV genomes. Statistical analyses involved 2 approaches. The 1st approach characterized the dynamics of PCV-2 infection and their relationship with PRRSV infection. The 2nd approach analyzed the probability of being infected by PCV-2 or by both PCV-2 and PRRSV through a generalized linear mixed model incorporating sow and farm characteristics. At the 1st sampling time (1 wk of age), there was a significant relationship between sow PCV-2 infection and piglet PCV-2 infection (P < 0.0001). The risk of PCV-2 and PRRSV coinfection was 1.85 times greater in piglets from a sow with low titers of PCV-2 antibodies than in piglets from sows with medium to high titers (P = 0.03) and was 2.54, 2.40, and 2.02 times greater, respectively, in piglets from primiparous sows, PCV-2-infected sows, and farms in an area of high pig density than in piglets from sows of higher parity (P = 0.004), noninfected sows (P = 0.04), and farms in a low-density area (P = 0.09).

Porcine circovirus-2 (PCV-2) is considered the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS) (1). This ubiquitous infectious agent circulates in virtually all farms, with or without the syndrome (2–4). Therefore, PMWS is considered a multifactorial disease in which the virus is necessary but not sufficient to trigger the clinical condition. This situation makes diagnosis of the disease impossible through virus or antibody detection alone (5).

The clinical signs and gross lesions of PMWS are relatively nonspecific, but characteristic microscopic lesions associated with the presence of PCV-2 occur in lymphoid tissues (6). Experimental inoculations with PCV-2 have sporadically reproduced PMWS in pigs (7). However, coinfection with porcine parvovirus (PPV) or porcine reproductive and respiratory syndrome virus (PRRSV) elicits more severe lesions (8,9), sometimes with clinical signs comparable to those observed under field situations. In fact, under field conditions, PMWS is seldom found with PCV-2 infection alone, and coinfection with many different pathogens has been widely described (6,10,11). Among potential triggers for PMWS in PCV-2-infected pigs, PRRSV infection is considered a major risk factor (3).

The main objective of the study described herein was to evaluate, through a longitudinal study, the prevalence of PCV-2 and PRRSV infections in 7 conventional farms of an integrated swine production system experiencing PMWS in the nursery, the fattening units, or both. A 2nd objective was to identify factors associated with the risk of PCV-2 infection and PCV-2 and PRRSV coinfection in pigs of this production system.

The study was performed in 4500 Landrace (50%) × Large White (25%) × Duroc (25%) sows at 7 sow farms (nos. 1 to 7; site 1) with 300 to 700 sows per farm. The boar breed used in the production system was Pietrain. Briefly, pigs born at the 7 sow farms were moved to nurseries (site 2) at a weaning age of 21 d: the nurseries were multi-origin by site but single origin by room and were managed all-in/all-out by room. The pigs were moved to the fattening units (site 3) at 8 to 10 wk of age. Pigs from sow farms 1 and 6 were not mixed with pigs of other origins, whereas pigs from farms 2, 3, 4, 5, and 7 were mixed such that there were 2 to 3 origins per fattening unit. Fattening pigs were managed all-in/all-out by building. All animals were fed, housed, and handled with due concern for their welfare. The facilities operated under the guidelines of the animal care and use committee of the Universitat Autónoma de Barcelona.

The production system was experiencing mortality rates more than 2 times the historic average in the late-nursery or early-fattening pigs, or both, despite medication with wide-spectrum antibiotics in feed, by water, and by injection. The diagnosis of PMWS was based on the clinical picture at the farm, histopathological study of lymphoid tissue from affected pigs, and in situ hybridization to detect PCV-2 in those tissues. Therefore, the criteria for herd and individual case definitions of PMWS were fulfilled (1,12). The production system was known to be enzootic for PRRSV.

Fifteen sows from each of the 7 sow herds were randomly selected from groups that farrowed the same week. This number was chosen because it was the maximum number of available sows at the smallest farm. Sow identification and parity were recorded, and blood samples were drawn from the sows at farrowing. Sows were blocked neither by parity nor by litter size owing to the small number of animals selected from each farm. Four piglets that had not been cross-fostered were selected from each sow, ear-tagged at birth, and followed up until 6 mo of age. Blood samples were taken from the 60 piglets per farm at 1, 4, 8, 12, 16, and 22 wk of age in tubes without anticoagulant. Serum was obtained after blood clotting and analyzed for the PCV-2 and PRRSV genomes by polymerase chain reaction (PCR) (4) and reverse-transcription (RT)-PCR (13), respectively. The sensitivity of the 2 techniques was about 10⁴ plaque-forming units and 10² TCID₅₀ (median tissue culture infective dose) per milliliter of serum, respectively. Extraction and amplification of DNA or RNA was performed in pools of 4 serum samples from sibling piglets. When pools were positive for PCV-2 the serum samples were processed again individually by means of the same PCR. When pools were positive for PRRSV it was not possible to process the samples individually with the resources available for laboratory analysis. The sow serum samples were analyzed for antibodies to PCV-2 by an immunoperoxidase monolayer assay (IPMA) and for the PCV-2 and PRRSV genomes as described for the piglets’ samples. The IPMA had 6 possible values for PCV-2 antibody titer: 1/20, 1/80, 1/320, 1/1280, 1/5120, and 1/20480. Low antibody titers were defined as those 1/80 or lower (2).

The piglets were followed up until 6 mo of age, and the date of death was recorded. Necropsy was performed only on fresh specimens: to avoid misinterpretation of pathological findings no autolytic carcasses were examined. The main purpose of the pathological examination was to state whether PMWS was a significant contributor to farm disease and death rates. Tissue samples (lung, inguinal superficial lymph node, spleen, kidney, and liver) underwent histopathological study, in situ hybridization to detect PCV-2 nucleic acid (6), and immunohistochemical study to detect PRRSV antigen (14).

Statistical analyses involved 2 approaches. The 1st approach characterized the PCV-2 infection dynamics and their relationship with PRRSV infection. The 2nd approach analyzed the probability of being infected by PCV-2 or by both PCV-2 and PRRSV with a generalized linear mixed model that incorporated sow and farm characteristics.

The PCV-2 infection dynamics were analyzed in terms of the number of infected piglets over time. Therefore, a PCV-2-infected piglet was defined as a PCV-2 PCR-positive animal at a given sampling time. A new counting variable, the total number of infected sampling times per piglet, allowed us to describe the distribution of piglets (expressed as a percentage) according to the number of times there was a positive PCV-2 PCR result. On the other hand, the PRRSV infection dynamics were analyzed in terms of the number of positive serum pools over time.

Contingency tables with the corresponding chi-squared statistics were used to evaluate the relationship between PCV-2 and PRRSV infection, and the same statistical test was used to determine whether sow characteristics [parity group, PCV-2 infection status, and PCV-2 antibody titer (classified as low or high)] could have any influence on piglet PCV-2 infection. Briefly, a random distribution of PCV-2-infected piglets was compared with the observed distribution. For this task, a binomial distribution of the expected number of infected piglets per sow was calculated with the use of a random variable and the SAS system, version 9.1 (SAS Institute, Cary, North Carolina, USA).

Chi-squared statistics were used to analyze differences in PCV-2 infection dynamics among the farms with respect to prevalence of infection as well as farm size and location. The prevalence of infection was defined as the ratio between the number of PCV-2-positive pigs or PRRSV-positive serum pools and the total number of studied piglets or pools in each period. The significance level was established as 0.05, and all the analyses were carried out with the SAS system, version 9.1.

With the main goal of explaining the infections over time, taking into account the hierarchical structure of the data, generalized linear mixed models (15) were established for the analysis of infections: one for PCV-2 infection (a binary response variable of 1 for infected piglets and 0 otherwise) and the other for PCV-2 and PRRSV coinfection (a binary response variable of 1 for coinfected piglets and 0 otherwise). Both models included covariates related to the sow [parity group (recategorized as 1 if parity = 1 and 0 if parity ≥ 2), PCV-2 infection status, and PCV-2 antibody titer] and farm [herd size (1 if number of sows at farm is ≥ 500; and 0 if number of sows at farm is < 500) and location (0 or 1 according to pig density (16): 0 if low, < 250 pigs/km²; 1 if high, ≥ 250 pigs/km²)]. These models took into account the hierarchical structure of data and random factors (sow and farm effects). The analyses were extensions of the ordinary logistic model for modelling the binary response variable. In that sense, odds ratios (ORs) for the covariates (sow and farm characteristics) were obtained to compare the risk of being infected by PCV-2 or PCV-2 and PRRSV according to each of the categorical covariates. The analysis was carried out with the GLIMMIX procedure of the SAS system, version 9.1, with the significance level fixed at 0.05.

Among the 420 piglets studied, the number of individual PCV-2 PCR-positive samplings was variable: 2 animals from farm 1 had positive results at each sampling time, about 40 animals (mainly from farms 2, 3, 5, and 6) had positive results from 8 to 22 wk of age, and approximately 80 animals (mainly from farms 1, 3, 5, and 6) had positive results from 12 to 22 wk of age. Among the 105 sows studied, only 1 sow (from farm 1) was RT-PCR-positive for PRRSV, whereas 8 sows were PCR-positive for PCV-2. Overall, the proportion of positive results was lower for PRRSV than for PCV-2. Of the 105 serum pools studied, only 1 (from farm 2) was PRRSV-positive at 4 consecutive sampling times (from 4 to 16 wk of age), 10 pools (mostly from farms 1 and 2) were PRRSV-positive at 3 consecutive sampling times, 15 pools were positive at 2 consecutive sampling times, 40 pools were positive at only 1 sampling time, and the remaining 39 pools were negative at all sampling times.

Of the 420 piglets selected for study, 106 (25%) died after weaning. Necropsy was performed on the 63 (60%) that did not show obvious signs of autolysis. Of these 63, PMWS was diagnosed in 40 (63%) and PRRSV antigen was detected in the lungs of 4 (6%), all 4 of which had PMWS. Table I presents the details by farm.

Table I.

Mortality, necropsy, and histopathological results for the 60 piglets from each of 7 farms monitored for infection with porcine circovirus 2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV)

	Number (and %) of pigs on each farm
Farm no. (site 1)	Died during the study	Underwent necropsy	With PMWS diagnosisa	Positive for PRRSVb
1	22 (37)	17	14	0
2	13 (22)	7	3	0
3	23 (38)	11	10	3
4	16 (27)	6	2	0
5	11 (18)	10	5	0
6	13 (22)	9	4	1
7	8 (13)	3	2	0
Total	106 (25)	63	40	4

^aBased on characteristic microscopic lesions and detection of PCV-2 nucleic acid in the lesions by in situ hybridization.

^bBased on immunohistochemical detection of the PRRSV antigen.

Of the 420 piglets, 60 (14%) were never found to have PCV-2 infection during the study, whereas 13 (3%) were infected at 4 or more sampling times. Moreover, the percentage of PCV-2-infected piglets increased over time, from 35 (8%) at the 2nd sampling time to 200 (64%) at the last sampling time.

The random distribution of infected piglets was compared with the distribution observed from the data. The null hypothesis of no differences was rejected. This result suggests that PCV-2 infection in piglets depends on sow characteristics. At the 1st sampling time (1 wk of age) there was a significant relationship between sow PCV-2 infection and piglet PCV-2 infection (χ²₁ = 137.99; P < 0.0001). Size and location of the farm were also related to PCV-2 infection, the percentage of infected piglets being significantly higher (P < 0.05) on large farms (F = 26.06; P < 0.0001) and on farms in areas of high pig density (F = 12.42; P = 0.0005) (Table II).

Table II.

Proportion of PCV-2-infected piglets at various ages according to size and location of the farm

	% of PCV-2-infected piglets
	Number of sows in herd		Area swine density (pigs/km2)
Age (wk)	< 500	≥ 500	< 250	≥ 250
1	2	8	4	7
4	3	16	4	12
8	9	48	12	38
12	54	98	38	98
16	60	80	78	64
22	54	82	68	66

From a statistical point of view there was a positive relationship between PCV-2 and PRRSV infection per litter at the 4th and 5th sampling times (12 and 16 wk of age), whereas this relationship was not observed at the rest of the studied times (1, 4, 8, and 22 wk of age).

Size and location of the farm had significant effects on the risk of PCV-2 infection in the piglets. The risk of infection for piglets from small farms was only 22% of the risk for piglets from large farms [OR_{small vs large} = 0.216; 95% confidence interval (CI) = 0.127 to 0.367; P < 0.0001], and the risk was also lower for animals from farms in areas of low pig density than for animals from farms in areas of high pig density (OR_{low vs high} = 0.576; 95% CI = 0.412 to 0.806; P < 0.008).

Sow parity, PCV-2 infection status, and PCV-2 antibody titer had a significant effect (P < 0.05) on PCV-2 and PRRSV coinfection in the piglets. The risk of coinfection for piglets from a sow that was primiparous, was PCV-2-infected, or had low titers of PCV-2 antibodies was 2.54, 2.40, and 1.85 times higher, respectively, than the risk for piglets from sows with greater parity (OR_{first vs other} = 2.546; 95% CI = 1.333 to 4.865; P = 0.004), no PCV-2 infection (OR_{positive vs negative} = 2.4; 95% CI = 1.040 to 5.71; P = 0.04), and medium to high PCV-2 antibody titers (OR_{low vs high} = 1.851; 95% CI = 1.050 to 3.264; P = 0.03).

Piglets from farms in an area of high pig density had a risk of coinfection 2.02 times greater than that of piglets from farms in a low-density area (OR_{high vs low} = 2.019; 95% CI = 0.882 to 4.619; P = 0.09).

The results of this study provide further epidemiologic insights into PMWS on pig farms. Since the first descriptions of PMWS in Europe (1,3,6) and North America (17), PRRSV has been considered one of the main triggers of the disease, but no studies on the prevalence of PCV-2 and PRRSV coinfection on farms over time had been carried out. The most significant conclusions of this field work are that piglets from primiparous sows, PCV-2-infected sows, and farms in an area of high pig density have a higher risk of coinfection than piglets from sows of greater parity, noninfected sows, and farms in low-density areas. Moreover, the risk of piglet PCV-2 infection is significantly higher on large farms in high-density areas than on small farms in low-density areas.

Data for this study came from farms belonging to a pig production company, and we did not have a standard process for selecting individuals; therefore, this was an exploratory study on infection dynamics on PMWS-affected farms. Cross-fostering of piglets in the first 24 to 48 h after birth was considered a confounding factor. In such a situation the PCV-2 and PRRSV serologic and virologic status of the farrowing sow could have differed from that of the nursing sow. Therefore, the study was performed only with non-cross-fostered piglets.

There were no reasons to consider a selection bias in the sampling, taking into account that the farms did not have different intrinsic characteristics. The number of piglets studied per sow (4) was selected according to the laboratory resources available. Theoretically, pooling might have resulted in false-negative PCR results if a low level of virus was present in only 1 of the 4 samples. However, this possible effect may be considered negligible in view of the good sensitivity of the PCR techniques used for PCV-2 and PRRSV detection and the high number of positive pools throughout the study.

Size and location of farms must be taken into account as explanatory factors in many infectious pig diseases. Several epidemiologic studies have shown that the bigger the farm, the easier the transmission of infectious agents, and location of the farm in an area of high pig density increases the probability of positive results for more infectious agents and the prevalence of infectious diseases (18,19). Although this investigation was not a case–control study, since all the studied farms were affected by PMWS, the results complement those of most reported epidemiologic case–control studies, which have found a higher risk of PMWS in large herds and on farms closer to other pig herds (20,21). In contrast, a Danish study did not find a relationship between PMWS and the distance to other affected farms (22).

Our results show that PCV-2 infection dynamics in piglets depend on their dam characteristics. In fact, at the 1st time studied (1 wk of age), there was a significant relationship between sow PCV-2 infection and piglet PCV-2 infection. In a recently published study on the same farms, in which mortality was taken into account instead of PCV-2 infection (23), the percentage of piglets dying was higher among those from viremic sows than for those from nonviremic sows and was also higher for piglets from sows that had low or undetectable titers of antibody to PCV-2 than for piglets from sows with high antibody levels. Considering the 2 studies together, greater amounts of maternally derived PCV-2 antibody seem to protect against piglet death as well as to modify the dynamics of PCV-2 infection. This result is logical since infection with PCV-2 is necessary for PMWS (1). Finally, low sow parity carried a significant risk for piglet coinfection with PCV-2 and PRRSV. This result agrees with the general knowledge that the age structure of a farm has an effect on health status: the higher the number of primiparous sows, the higher the probability of herd outbreaks of various pig diseases (24,25).

In conclusion, this investigation, performed in 7 PMWS-affected farms, allowed the study of PCV-2 and PRRSV infection dynamics and their interaction. Sow parity, PCV-2 infection status, and PCV-2 antibody titer had a significant effect on PCV-2 and PRRSV coinfection in the piglets. Moreover, the risk of PCV-2 infection in the piglets was linked with the size and location of the pig farm. A positive relationship was observed between PCV-2 and PRRSV infections per litter at several sampling times, highlighting the suggestion of many others that PRRS control can be a reliable way to decrease the clinical impact of PMWS on farms coinfected with PRRSV.