Summary

Porcine Circovirus 2 is the etiological agent of many associated diseases that impact performance and increase mortality. Vaccination is costly and time-consuming.

In this study, 81 barrows of two commercial genetic lines were infected with a PCV2b strain and nine barrows receiving no inoculation were controls. Blood was drawn weekly and tested for levels of IgG, IgM, and viraemia.

Infected pigs showed three patterns of IgM response: early, late, and limited response. Pigs showing no response generally had faster growth and lower viraemia, most likely due to an inhibition of virus replication. Early response individuals tended to have lower viraemia and faster growth compared to individuals with late response. This important variation in immune response has a potential economic value and could be used in management practices and breeding programmes.

Future research will be focused on dissection of genetic and non-genetic factors explaining variation in immune response and disease susceptibility.

Introduction

Economic losses associated with susceptibility to Porcine Circovirus Associated Diseases (PCVAD) continue to have an impact on swine industry. Pigs affected by PCVAD display characteristics of wasting, diarrhea, interstitial pneumonia, dermatitis, lymphoid depletion leading to decreased immune responses, and susceptibility to other pathogens. Porcine Circovirus 2 (PCV2) is the causative source of PCVAD, but additional factors influence disease progression, of which host genetics and secondary immune stressors are highly important. Secondary infection with swine influenza, Mycoplasma hyopneumoniae and Porcine Reproductive and Respiratory Syndrome (PRRS) can influence the severity and progression of PCVAD.

Vaccines for PCVAD exist, but they increase production costs, and producers who utilize the vaccine may still experience outbreaks. Even though only 5 to 15 per cent of pigs infected with PCV2 display clinical symptoms, the whole herd must be immunized, which is a costly practice. Recent research has indicated that host genetics could influence susceptibility to disease. For example, several reports suggest that Landrace pigs are more susceptible to PCVAD than Pietrain and Duroc pigs. These breed differences mean that genetic variation within breeds likely exists and that selection for resistance may be possible. However, disease resistance is difficult to improve using traditional selection methods that require regular disease challenges or uniform and continued exposure to the pathogen. Selection based on DNA markers may be more effective. As long as marker panels with known relationships with immune response variables are available, selection can be practiced in any population without exposure to the pathogen. Before DNA selection can be implemented, comprehensive disease phenotypes and DNA samples need to be collected from pigs uniformly exposed to PCV2b to determine the relationships between DNA markers and disease susceptibility.

This study investigated individual response to experimental PCV2 infection by profiling major indicators of immune response in pigs from two crossbred lines. This study represents initial efforts to establish a large collection of samples and PCVAD phenotypes that will be used to identify genes and DNA markers associated with PCVAD resistance.

Materials and Methods

PCV2b isolate

Isolate UNLVBMS was recovered from a pig that had symptoms characteristic of PCV2b infection. Viral DNA was isolated using QIAamp DNA Minikit (Qiagen). Two pairs of primers were used for amplification of the entire PCV2b genome. DNA amplification was performed using GoTaq Flexi DNA Polymerase (Promega and PCR products were purified using ExoSAP-IT (USB Corporation). The viral DNA was sequenced from both directions using dye terminators and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Animals and facility

Animal use and experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Nebraska– Lincoln (UNL). The experiment included 91 crossbred barrows originated from 24 litters that were either Large White × Landrace (W×R, n=72) or a three-way cross with Duroc sires and W×R dams [D (W×R), n=19]. Pigs were born at the UNL Swine Farm and at approximately 35 days of age were transported to the animal science research facility where the disease challenge was conducted. Pigs at the farm are routinely tested for major pathogens and are known to be negative for PRRSV. The pigs were housed in one room and randomly allocated to 18 identical pens with a combination of slatted and solid surface flooring. The pens provided approximately 0.65 square metres of floor space per pig. All pigs were fed ad libitum using a standard balanced diet.

Experimental infection

The objective was to infect pigs with virus after maternal antibodies had waned and before natural infection had occurred. Blood was drawn from pigs before infection and analyzed for IgG (maternal antibodies) and IgM (self-antibodies produced in response to infection) to determine when to initiate infection with PCV2b. The sample-to-positive (S/P) ratios of the maternal antibodies (IgG) in all individuals selected for the experiment were less than 0.3 at the time of inoculation, the level specified by the Ingezim ELISA protocol, which indicates that protection from maternal antibodies had waned. S/P ratios of IgM were less than 0.4, indicating that natural infection had not occurred.

The virus inoculum contained the titer of 10^4.0 50 per cent tissue culture infection dose (TCID50)/mL in minimum essential media with 50µ/mL gentamicin and five per cent foetal calf serum. The original experimental protocol was to infect each pig with 2mL of inoculum intramuscularly (IM) and 3mL intranasally (IN). However, approximately 20 minutes after inoculation of the first 20 pigs, anaphylactic shock occurred and eight of the pigs died.

The dose administered to the remaining pigs was therefore altered. Most of the pigs (n=58) received 1mL IM and 3mL IN. The rest of the pigs received a variation of this dose: 1mL IM with 0.5 mL IN (n=4) or 2mL IM with 0.5 mL IN (n=8). The pigs used for these treatments were chosen at random. Ten pigs were selected as negative controls, assigned to separate pens, and not inoculated.

Serology

Blood samples were collected before inoculation and at 7, 14, 21, and 28 days post innoculation (d.p.i.). Levels of PCV2 specific antibodies, IgG and IgM, were measured from serum using ELISA assays (Ingenasa). Samples were considered positive if the calculated S/P ratio was greater than 0.3 for IgG and 0.4 for IgM. Antibody data were normalized based on positive control values obtained for each plate.

Clinical evaluation and necropsy

Pigs were observed daily for clinical signs of infection, and weighed at 0, 7, 14, 21 and 28 d.p.i. Necropsy was performed at 28 d.p.i. Lung, spleen, and mesenteric and bronchial lymph nodes were collected for histology examination and gene expression analyses.

PCV2 quantification

Viral DNA was extracted from serum collected at 7, 14, 21, and 28 d.p.i. using QIAamp DNA Minikit (Qiagen). Estimates of the number of viral copies were obtained by quantitative real-time PCR using TaqMan Master Mix and ABI 7900 Real Time PCR System (Applied Biosystems). The area under the curve (AUC) was calculated to estimate total viral load throughout the 28-day experiment.

Statistical analysis

Least Square Means (LSM) were obtained using mixed-model procedures including the immune response pattern with crossbred lines as fixed effects and pen and litter as random effects. Analysis of the IgM antibody profiles during the challenge revealed that infected individuals are characterized by three patterns of immune response and could be separated in three groups:

1. individuals that responded immediately to infection with the highest change in specific PCV2 IgM antibodies from 7 to 14 days post infection( d.p.i.) (n=33)
2. individuals that responded late with the greatest change from day 14 to 21 (n = 40); and 3) individuals that did not respond to infection (n=7) (Figure 1).

Most pigs clearly fit into one of these groups, but in some cases pigs were placed in a group based on a subjective judgment. Correlations among traits were calculated from variances and covariances adjusted for line effects.

Results and Discussion

Genetic characterization of the PCV2b isolate

The PCV2b strain utilized in this experiment was isolated from a pig showing clinical symptoms of PCVAD. The viral DNA was isolated and sequenced having the highest genetic similarity with the PCV2b strain FMV-05-6507. This strain was first identified in 2005 in Quebec, Canada, and is known to induce clinical signs of postweaning multisystemic wasting syndrome (PMWS, a form of PCVAD) and increased mortality rate.

Anti-PCV2-IgM and –IgG antibodies

All candidate pigs for the PCV2b challenge were produced by dams that were vaccinated for PCVAD. The offspring receive anti-PCV2 antibodies via colostrum that provides immunity against this pathogen. Its effectiveness depends on the rate of antibody decay that varies from five to 21 weeks. Seventy-five per cent of the 120 five-week-old candidate pigs for experimental challenge had levels of maternal antibodies (IgG) below the threshold (S/P ratio lower than 0.3) that differentiate PCV2 negative from positive pigs. Eighty-two per cent of the W×R pigs (n=89) had IgG levels below the threshold compared to 58 per cent of the D (W×R) pigs (n=31). The levels of IgG in the two crossbred groups did not differ (P>0.10) before infection.

With the exception of day 7, pigs in Group 3 had the lowest IgM values during the challenge. Observed IgM values calculated as sample to positive ratios for each group at 0, 14, 21, and 28 d.p.i. are presented in Figure 2. At day 0 and 7, IgM values were similar, and at day 14, the controls had the lowest value at 0.77, whereas the Group 1 pigs had the highest at 1.38. The IgM values of the pigs from Group 1 remained elevated at day 14, and then the response began to wane at day 21. The IgM values of Group 2 pigs reached the peak at day 21 and declined thereafter. At day 21, the controls and Group 3 pigs had the lowest IgM values, 0.92 and 0.89, respectively. At necropsy on day 28, the controls and Group 3 pigs continued to have the lowest IgM values, whereas Group 1 pigs had the highest and Group 2 had intermediate values.

Recent reports showed that rate of maternal IgG decay varies considerably between individuals and breeds, which affects infection outcome and disease progression. There were no differences between groups in IgG level at day 0 (Table 1). With the exception of day 7, Group 3 had the lowest IgG values during the challenge. Interestingly, the IgG values of all individuals from Group 3 were below the positive threshold.

Differences in IgG levels between groups occurred on day 14, 21, and 28 (P<0.05). IgG values for infected and control pigs did not differ at day 0, averaging approximately 0.77. At day 7, the controls had the highest values at 0.94, whereas the other three groups remained steady around 0.75. At 14 d.p.i, the controls were lowest at 0.70 and the Group 2 pigs were the highest at 1.83. The same trend continued at 21 d.p.i.

The IgG response in pigs in Group 3 was markedly different. Values remained low until day 14, increased to a value of 0.91 at day 14, and then returned to baseline levels at day 28. There were no significant differences in IgM and IgG between the two genetic lines, W×R and D (W×R), at any time point (Figures 3 and 4).

Amount of PCV2 DNA in serum samples

Following the experimental infection, quantitative PCR results for viral copy count showed that most of the barrows at day 7, 14, 21, and 28 d.p.i. tested positive for PCV2 DNA (Table 1). Differences in viral copy number between W×R and D (W×R) existed at day 7 (P<0.006) and day 14 (P<0.01). DNA copy number increased in both lines through day 21 and then declined (Figure 5).

Pigs in Group 3 had fewer viral copies than pigs in the other groups. At day 7, Group 1 had the highest response at 4.56, whereas the response in Group 3 was 3.85. At day 14, 21, and 28, Group 2 had the highest viral copy count at 5.24, 6.17, and 4.7, respectively; Group 3 remained the lowest at 4.27, 4.67, and 3.96, respectively. For viraemia AUC, the Group 3 pigs were the lowest, with Group 1 being intermediate and Group 2 being the highest. viraemia of the control pigs remained baseline throughout the study.

The amount of PCV2 administered to pigs did not influence disease progression, with the exception of viraemia results at day 7 (P<0.05). However, there were only four pigs in the group that differed from the others, and they received the lowest viral dose of all 81 pigs infected.

Clinical evaluation

Only one death occurred during the four weeks of experimental challenge. This individual died during the last week of challenge, displayed wasting disease and had the highest viral load (AUC) in the first three weeks of the challenge. This individual displayed an early immune response and was inoculated with the lowest viral dose. The only difference in growth occurred between Groups 1 and 2 for average daily gain during the 28-day period (P<0.05). However, consistent with viraemia and IgM responses, pigs in Group 3, that had a limited immune response, had the greatest ADG.

Correlations

Correlations between average daily gain and viraemia results are summarized in Table 2. Moderately strong, negative correlation was estimated between average daily gain during the entire challenge and viral load (AUC) (r=-0.29).

Conclusion

The results of this preliminary research provide strong evidence of significant variation in immune response in experimental challenges with PCV2b. Analysis of the profiles of viral load and antibody response revealed three groups of individuals that display important variation in response to infection. This variation is the source of the differences in growth between pigs during the challenge. The main difference between individuals from Group 1 and 2 was in the time of response, whereas the difference between these two groups and Group 3 was in the magnitude of response. The source of limited immune response in individuals from Group 3 is most likely a mechanism that inhibited virus replication. Individuals that responded immediately to infection (Group 1) had greater ADG than those that responded late (Group 2). Pigs in Group 3 had the greatest rate of growth during the 28-day period following infection, but the observed differences were not statistically significant.

The objective of our future research is to identify if the source of the differences is genetic and to uncover potential genes and genetic variants responsible for PCVAD susceptibility. This research will generate knowledge that can be applied to other viral diseases such as PRRSV. The long-term objective is to generate a panel of genetic markers that can be used in breeding programs to improve genetic resistance to PCVAD. The benefits of improving PCVAD susceptibility using genetic markers are obvious: lower production costs associated with improved robustness and a decrease in the number of vaccinated pigs, fewer welfare issues, and increased international competitiveness of the US swine industry.

¹Theresa P. Bohnert is a graduate student, and Autumn M. McKnite is a research technician in the UNL Animal Science Department; Judith W. Galeota is research manager, Timothy Moural, research technologist, and Seth Harris, assistant professor in the UNL School of Veterinary Medicine and Biological Sciences; Rodger K. Johnson is a professor, Thomas E. Burkey and Daniel C. Ciobanu are assistant professors in the UNL Animal Science Department.

Reference

Bohnert T.P., A.M. McKnite, J.G. Galeota, T.W. Moural, S.P. Harris, T.E. Burkey, R.K. Johnson and D.C. Ciobanu. 2011. Variation in response to infection in experimental challenges with porcine circovirus 2b. Nebraska Swine Report 2011, p31-35.

Further Reading You can view other papers from the 2011 Nebraska Swine Report by clicking here. Find out more information on PMWS by clicking here.