Update on Porcine Circovirus

Patrick G. Halbur, DVM, PhD
Professor of Veterinary Diagnostic and Production Animal Medicine

Tanja Opriessnig, DVM
Post Doctoral Research Associate
Veterinary Diagnostic and Production Animal Medicine

College of Veterinary Medicine, Iowa State University

Introduction
The first objective for this presentation is to update the audience on the clinical manifestations of PCV2-associated diseases observed in the U.S.  The second objective is to update the audience on the main findings of our past and current research and the potential direction of future research on PCV2-associated diseases at Iowa State University.  The final objective is to discuss current strategies for control of PCV2-associated diseases based on experiences from our research and field experiences. 

PCV2-associated disease manifestations in U.S. field cases
PCV2 is associated with several disease manifestations including postweaning multisystemic wasting syndrome (PMWS), respiratory disease as part of the porcine respiratory disease complex (PRDC), reproductive disorders, enteritis, and porcine dermatitis and nephropathy syndrome (PDNS). Based on submissions to the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL), the overall incidence of PCV2-associated disease in pigs increased dramatically in the U.S. in the 5 year period from 1998-2002 but may have peaked (Figure 1).

PCV2-associated pneumonia is the most common manifestation of PCV2 infection in cases submitted to the ISU-VDL (Table 1). Harms et al. (11) recently described three cases of PRDC typical of submissions to the ISU-VDL. In these cases, PCV2 was associated with lung lesions in pigs coinfected with PRRSV, or SIV, and/or Mycoplasma hyopneumoniae (M. hyopneumoniae), and a variety of opportunistic bacteria. The basis for concluding that PCV2 is involved in respiratory disease is that PCV2 antigen is associated with characteristic lung lesions. Lesions characteristic of PCV2-associated pneumonia include necrotizing and ulcerative bronchiolitis, bronchiolitis obliterans fibrosa, mixed inflammation and fibroplasia in the lamina propria and peribronchiolar areas, and granulomatous inflammation in the alveolar septa.

PMWS is the second most common manifestation of PCV2-associated disease in our submissions. Diagnosis of PMWS is based on a history of wasting, the confirmation of lymphoid depletion and/or granulomatous inflammation in the lymphoid tissues by histopathological examination, and demonstration of PCV2 antigen associated with the lymphoid lesions.

The third most common diagnosis is PCV2-associated systemic infection. This diagnosis is used on those cases with a history of wasting and in which lymphohistiocytic or granulomatous inflammation is observed in a variety of organs in association with PCV2 antigen. Lymphoid depletion either was not observed or lymphoid tissues may not have been submitted in these cases.

Cases of PCV2-associated enteritis are relatively uncommon. Most of the PCV2-associated enteritis cases are from grow-finish pigs. PCV2-associated enteritis cases often clinically and grossly resemble subacute or chronic ileitis. The intestinal mucosa is grossly thickened and mesenteric lymph nodes enlarged. Microscopic examination confirms the presence of granulomatous enteritis, and the absence of crypt hyperplasia typical of Lawsonia intracellularis-induced proliferative enteritis. Confirmation is by immunohistochemical demonstration of abundant PCV2 antigen associated with the granulomatous enteritis lesions.

PCV2-associated reproductive failure is uncommonly diagnosed. When confirmed it is typically characterized by fetal lymphohistiocytic myocarditis and/or myocardial fibrosis and confirmed by immunohistochemical demonstration of abundant amounts of PCV2 antigen associated with the myocardial lesions. PCV2-associated abortions are typically sporadic in occurrence, occur in gilts and are characterized by increased numbers of mummified fetuses.  We have recently observed a case where boars in a boar stud were coinfected with PCV2 and M. hyo. and P. multocida type D resulting in infertility and shedding of PCV2 in the semen.

PCV2-associated PDNS is rarely observed in the case load submitted to the ISU-VDL.  PDNS is typically sporadic within the group of pigs involving less that 0.5% of the group.  PMWS is inconsistently seen in groups with PDNS.  When we diagnose PDNS we almost always demonstrate PCV2 infection in the skin, or kidney, and/or lymphoid tissues of the affected pigs. 

Key Points from Past PCV2 Research Projects at ISU
We have repeatedly used segregated early weaning (SEW) to derive PCV2 virus-free and PCV2 antibody-free pigs from PCV2 positive sows (21).  Segregated early weaning appears to be a useful technique to derive PCV2-free pigs from positive breeding herds. Variability in maternal antibody levels within herds may account for variability in manifestation and onset of PCV2-associated disease and allow for endemic infection of the population. 

Our group developed and characterized the pathogenicity of a molecular clone of PCV2 (5). Specific pathogen free (SPF) pigs infected with the cloned genomic PCV2 developed the hallmark gross and microscopic lymphoid lesions of PMWS. Evidence of wasting was not observed in the 35-day duration of the DNA clone study; however, the infectious DNA clone work further established PCV2 as the cause of the hallmark lymphoid lesions of PMWS. Although essential for the development of PMWS, PCV2 may require other factors or agents to induce the full spectrum of clinical signs and lesions associated with advanced cases of PMWS.

We recently reported on the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2 (6). The chimeric PCV1-2 clone contained the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1. The chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genome backbone induced a strong and specific antibody response to the pathogenic PCV2 capsid antigen and was attenuated (minimal-to-no lesions, low level and reduced length of viremia, low or nondetectable levels of viral antigen in lymphoid tissues) when inoculated into pigs. The fact that the lymphoid depletion induced by the chimeric virus was markedly reduced in incidence, and that the lymphoid depletion observed was significantly less severe than that induced by the PCV2 infectious DNA clone, further confirms the association of PCV2 with the hallmark lymphoid lesions of PMWS.

Pallares et al. (26) recently summarized the most common coinfections that occur in pigs with PCV2-associated PMWS.  PRRSV was detected in 52% of the cases, Mycoplasma hyopneumoniae in 36% of the cases, bacterial septicemia and/or pneumonia in 22%, swine influenza virus (SIV) in 5.4%, and singular PCV2 infection in only 2% of the cases.  We recently coinfected conventional SEW pigs with PCV2 and PPV and tested the effect of PPV vaccine on reducing disease and lesions associated with this coinfection (22).  Clinical signs consistent with PMWS (fever, respiratory disease, jaundice, weight loss) were seen only in pigs in the PCV2/PPV coinfected groups, PPV vaccinated as well as non-vaccinated.  PCV2/PPV-coinfection of SEW pigs increases the incidence of clinical disease and gross and microscopic lesions characteristic of PMWS.

PCV2 and M. hyopneumoniae coinfections are common in field cases of PRDC (11, 26). We recently developed a model to investigate the interactions between PCV2 and M. hyopneumoniae and to test intervention strategies for PCV2/M. hyopneumoniae coinfection. PCV2/M. hyopneumoniae coinfected pigs had more severe clinical respiratory disease, significantly reduced average daily weight gain, significantly higher PCV2 genomic DNA copy numbers in serum, longer PCV2 viremia length, more severe macroscopic lung lesions, a significantly higher incidence and higher amount of PCV2 antigen in lymphoid and lung tissues, and a higher incidence and more severe microscopic lung and lymphoid lesions compared to the single-M. hyopneumoniae or single-PCV2-inoculated groups.  The results of this study indicate that M. hyopneumoniae infection increases the severity and duration of PCV2-induced lung and lymphoid lesions, PCV2 replication in tissues, and the incidence of PMWS in conventional pigs (23).

There has been considerable interest and concern about the effect of adjuvanted vaccines on enhancement of PCV2-induced disease.  We conducted a study to determine if vaccination of pigs with commercial bacterins that are commonly used in the U.S. enhances PCV2 replication and the incidence and severity of clinical signs and lesions characteristic of PMWS.  There was a significantly (<0.05) longer length of viremia (2.14±0.26 versus 4.44±0.23 weeks), a wider range of tissue distribution of PCV2 antigen, an increased incidence and severity of lymphoid depletion, and an increased incidence of lymphoplasmacytic hepatitis in pigs vaccinated with a commercial APP and M. hyopneumoniae vaccine and inoculated with PCV2 compared to PCV2-inoculated unvaccinated pigs (20).  This work adds to the body of evidence in the literature to support the hypothesis that common vaccine regimens may contribute to enhancement of PCV2-associated disease (2, 4, 15, 16). However, on a herd basis, we still feel that the risk of elimination of effective vaccines may be greater than the risk of inducing PMWS in a low percentage of the population.

In addition to the type of vaccine used, other factors such as timing of vaccination and age of pig in relation to PCV2 infection and viremia may also be very important. The objective of another recent study was to determine if the timing of vaccination with a commercially available M. hyopneumoniae vaccine had an effect on PCV2 replication and PCV2-associated lesion severity. Seventy-eight pigs were randomly assigned to 8 groups. Group 1 pigs (n=10) were vaccinated at 2 and 4 weeks of age, group 2 pigs (n=9) were vaccinated at 4 and 6 weeks of age, group 3 pigs (n=10) were vaccinated at 6 and 8 weeks of age, group 4 pigs were vaccinated at 8 and 10 weeks of age with 1 ml of a commercial oil-in-water adjuvanted vaccine. Group 5 pigs (n=9) were vaccinated once with a “double dose” (2 ml) at 4 weeks of age, and group 6 pigs (n=10) were vaccinated once with a double dose at 8 weeks of age. Group 7 (n=10) and 8 (n=10) pigs remained non-vaccinated. At 8 weeks of age, pigs in group 1-7 pigs were inoculated with PCV2. The results of this study indicate no or minimal PCV2-associated lesions when pigs are vaccinated 2-4 weeks prior to expected PCV2 exposure (24).

Comparison of the effect of different adjuvants on PCV2-induced disease and lesions
Field trials and experimental studies suggest that the use of common adjuvanted commercial vaccines may enhance the severity of PCV2-associated disease. Based on this information, some veterinarians advise their clients to discontinue the use of commercial vaccines in herds with recurrent PCV2-associated PMWS and PCV2-associated respiratory disease. This may minimize the risk of triggering progression of PCV2 infection to PCV2-associated disease but it may also allow for recrudescence of other diseases that in the past were effectively controlled by vaccination.  The first objective of this study was to determine if the adjuvants (as opposed to the antigen) in commercial swine vaccines induce increased replication of PCV2 and increased incidence of PCV2-associated disease and lesions. The second objective was to determine if there is a difference among adjuvants in this effect.  Ninety, 12-14 day-old pigs, were randomly assigned to five groups of 17-19 pigs per group. Group 1 (n=19) pigs were vaccinated with a commercial M. hyopneumoniae vaccine with an oil-in-water adjuvant. Group 2 (n=17) pigs were vaccinated with a commercial M. hyo vaccine with an aqueous adjuvant. Group 3 (n=18) pigs were vaccinated using an oil-in-water adjuvanted vaccine containing the same amount and type of M. hyopneumoniae antigen as in group 2. Group 4 (n=18) pigs were vaccinated using an aluminum hydroxide adjuvanted vaccine containing the same amount and type of M. hyopneumoniae antigen as in group 2. Group 5 (n=18) pigs served as the controls and were sham-vaccinated with saline. Pigs were injected with 2 mL of one of the four M. hyopneumoniae vaccines at four and again at six weeks of age. PCV2 was inoculated intranasally on the day of the second vaccination at 6 weeks of age. Half of the pigs were necropsied at 21 days post inoculation (DPI). The remaining pigs were necropsied at 35 DPI. There were no differences among groups in clinical disease scores. At 21 DPI all vaccinated groups had significantly (p<0.05) more severe lymphoid depletion than in the saline group; however, there were no differences among vaccinated groups. At 35 DPI, group 1 pigs had significantly (p<0.05) higher amounts of PCV2 DNA in serum than pigs in groups 2, 4, and 5 as determined by quantitative real-time PCR. There was a significant (p<0.05) increase in severity of lymphoid depletion in the lymph nodes, tonsil, and spleen in groups 1 and 3 compared to groups 2, 4, and 5. Group 3 had significantly (p<0.05) higher amounts of PCV2 antigen in lymph node, tonsil and spleen compared to groups 2, 4, and 5.

This work suggests that at the early stages of infection (21 DPI) all adjuvants tested (oil-in-water, aqueous, aluminum hydroxide) increased the severity of lymphoid depletion associated with PCV2. In the later stages of infection (35 DPI) the oil-in-water adjuvants (group 1 and 3) increased the length of PCV2 viremia, increased the amount of PCV2 in serum and tissue, and increased the severity of lymphoid depletion compared to the aqueous and aluminum hydroxide products (12). Practitioners need to weigh the risk of not using vaccines and thereby potentially allowing coinfections (i.e. M. hyopneumoniae) to enhance PCV2-associated diseases, versus the risk of using certain vaccines (i.e. M. hyopneumoniae ) on PCV2-infected pigs and thereby potentially increasing the severity of PCV2-associated diseases.

Differences in Host susceptibility to PCV2-induced disease
A high percentage of pigs are infected with PCV2 and have no evidence of clinical disease, decreased performance, or microscopic lesions. Genetic analysis to date has failed to demonstrate major differences between PCV2 isolates recovered from clinically healthy pigs and pigs suffering from PMWS. Host genetic differences have not been explored in a controlled manner. The overall objective of this study was to determine if different pig breeds have differences in susceptibility to PCV2-associated disease and lesions. Three breeds were compared in this study: Duroc (n=23), Landrace (n=19), and Large White (n=21). The selection of the pigs on the source farm was done randomly with the intention to have representatives from four sires and no more than 2-5 piglets from each litter (Duroc [7 litters], Landrace [6 litters], Large White [5 litters]) within each breed. The pigs (all female) were segregated early weaned at 10-12 days of age and housed offsite where the different breeds were randomly mixed together.  The PCV2 inoculation was done at 5-7 weeks of age. The pigs were infected intranasally (1 ml) and intramuscularly (0.5 ml) with PCV2 isolate ISU-40895 at a dose of 10^4.3 TCID₅₀.

One Landrace pig developed clinical PMWS characterized by persistently elevated body temperature and wasting and had to be euthanized early. At the 21 DPI necropsy, two additional pigs (both Landrace) with gross lesions characteristic of PMWS (generalized lymphadenopathy, non-collapsed, mottled-tan lungs) were identified. Clinical and/or gross evidence of PMWS was not detected in the other breeds. There were no significant differences in average daily gain or genomic PCV2 DNA copy numbers between pigs of different breeds. The majority of the pigs seroconverted to PCV2 by 35 DPI. At 21 DPI, tonsils were significantly (p<0.05) more depleted in the Landrace pigs compared to the other breeds. The three Landrace pigs with gross lesions typical of PMWS also had severe lymphoid depletion, severe granulomatous inflammation of tonsil, lymph nodes, and spleen, and high amounts of PCV2 antigen associated with the lesions. At 35 DPI, lymph node depletion was significantly (p<0.05) more severe in the Landrace pigs. There was no influence of sires and no influence of dams on the results. The incidence of PMWS based on gross and microscopic lesions was 0% in Durocs, 15.8% in Landrace, and 0% in Large White. The three PMWS affected pigs originated from different litters; two were from the same sire. These results suggest that differences in host susceptibility to PCV2-induced disease exist and that Landrace pigs are predisposed to PCV2-associated lymphoid depletion and PMWS (25).  Further investigation of host susceptibility to PCV2-associated disease is warranted.

Development of a mouse model for PCV2-associated diseases
Our hypothesis was that mice can be infected with our PCV2 DNA clone and that there are differences in PCV2 replication and PCV2-associated lesions in different lines of mice. We used BALB/c mice, C57BL/6 mice and C3H/HeN mice. These lines are frequently used in research but exhibit markedly different immune and inflammatory responses to certain pathogens as well as to self antigens. One-hundred-forty-five, male, 4-week old mice were used in this study. The mice were randomly assigned to treatment groups. Preliminary results suggest that there are differences between the lines.  A summary of results will be presented at the conference.

Use of PCV1-2 chimeric clones as vaccines for PCV2-associated diseases
We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs (6). Since then we have demonstrated that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs (7). A total of 48 specific-pathogen-free piglets were randomly assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscularly with the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection of the chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated intramuscularly the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1-3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.

Effect of PCV2 passive antibody levels on vaccination with chimeric PCV1-2 vaccine and challenge with wild-type PCV2
Nearly all conventionally-raised piglets have passively acquired antibodies against PCV2.  The passive antibodies vary greatly among pigs, and profiles of rate of decay are variable within swine populations.  The objective of this experiment was to determine what levels of passively-acquired antibodies may or may not interfere with vaccine-induced protective immunity.  A total of 46 specific-pathogen-free pigs were randomly assigned to seven groups. Pigs in group 7 (n=6) were challenged with wild type PCV2 at 9 weeks of age.  Pigs in groups 1 (n=6), 2 (n=6), and 3 (n=8) were vaccinated with the live chimeric vaccine at 3 weeks of age and then experimentally-challenged with wild type PCV2 at 9 weeks of age.  Pigs in groups 4 (n=6), 5 (n=6), and 6 (n=8) were infected with wild type PCV2 at 3 weeks of age, and then re-challenged at 9 weeks of age.  Pigs in groups 1 and 4 were negative for PCV2 antibodies (S/P <0.2), pigs in groups 2 and 5 had low PCV2 antibody levels (S/P 0.2 – 0.5), and pigs in groups 3 and 6 had high PCV2 antibody levels (S/P >0.5) at initiation of the study.  Blood was collected weekly for PCR and serology testing.  At 63 days post inoculation (DPI) all pigs were necropsied and gross and microscopic lesions were scored. 

Individual pigs within all groups had periodically elevated temperatures.  Comparison of the mean rectal temperatures found no significant (p=0.4397) difference between groups.  Respiratory scores were significantly higher (p<0.05) for groups 4 and 5 compared to groups 1, 2, 3, and 7 for DPI 7-28, and the higher scores persisted in group 5 until 42 DPI.  There was no significant difference in average daily gain (ADG).  Antibody levels decayed for groups 3 and 6 throughout the study.  Antibody levels decayed in groups 1-3 until 42 DPI.  Groups 1 and 2 appeared to have an anamnestic response by day 56, while group 3 did not.  Group 4 seroconverted (from PCV2 challenge at 0 DPI) before challenge with PCV2 at 42 DPI.  Pigs in group 7 had significantly more severe lymphoid depletion than pigs in groups 1 (p=0.025), 2 (p=0.0259), 5 (p=0.033), and 6 (p=0.003).  This suggests that immune protection was induced by the chimeric PCV2 vaccine for pigs with passive antibody levels <0.5 at the time of vaccination, whereas, pigs with passive antibody levels >0.5 at the time of vaccination did not develop vaccine-induced protective immunity.  These results indicate that the novel PCV2 chimeric vaccine is effective when given to young pigs with maternal antibody levels <0.5 S/P.  This should allow swine producers to use this type of PCV2 vaccine well ahead of expected exposure to PCV2 at 10-16 weeks of age.

Comparison of PCV2-isolates from clinical PMWS cases with and without hallmark microscopic lesions of lymphoid depletion
The overall objective of this study is to investigate possible differences in virulence among PCV2-isolates which may account for differences in clinical manifestation of PCV2-associated diseases. Veterinarians were encouraged to submit a complete and specific set of tissues from cases with a clinical history consistent with PMWS (wasting/weight loss with or without respiratory disease, diarrhea, jaundice and/or anemia) over a time period of one year as part of a PMWS surveillance project funded by the National Pork Board and Iowa Pork Producers Association. A total of 100 cases were included in this study.  All cases were tested for the presence of concurrent bacterial infection by routine bacterial culture and for the presence of PRRSV, SIV, and Mycoplasma hyopneumoniae by either immunohistochemistry (IHC) or PCR. Further in vitro and in vivo characterization of selected cases is in process.

Influence of PCV2 infection on efficacy of other vaccines
Studies investigating the effect of PCV2-infection on vaccine efficacy are lacking. Vaccine failures result in major economic losses to the swine industry. In healthy pigs vaccines such as Mycoplasma hyopneumoniae, swine influenza virus, or PRRSV vaccines appear to be effective but their efficacy in pigs with PCV2-associated lymphoid depletion and lymphopenia has not been evaluated.  The goal of this study is to investigate the impact of experimental infection with PCV2 on the efficacy of a modified live PRRSV vaccine.  Preliminary results indicate that PCV2 infection at the time of PRRSV vaccination significantly decreased the efficacy of the vaccine.  Results will be summarized at the conference.

Future Projects
We will be attempting to experimentally treat and prevent PCV2-associated diseases using serotherapy as has reportedly been done successfully in field trials in Europe.  We expect there will be at least one commercial PCV2 vaccine available for field and experimental use in the next year and we will test these vaccines in our experimental models.  We will compare the efficacy of serotherapy to commercial and/or experimental PCV2 vaccination.  We will also be using both our pig and mouse models to more closely look at the effect of PCV2 on macrophage function.  We also hope to further pursue the host genetic susceptibility to PCV2-associated disease as well as better understand the virus-dependent differences in PCV2 virulence.

Control of PCV2-Associated Diseases
Not a lot has changed in approached to control of PCV2-associated diseases.  Rose et al. (27) recently reported on the risk factors for PMWS in French farrow-to-finish herds and found that things such as PPV or PRRSV coinfection of finishers, large pen size versus small pen size for weaners, and increased levels of cross-fostering increased the risk for PMWS; whereas long empty periods in the pig flow, regular treatment against external parasites, pen versus crated gestation, and internal versus external gilt replacement decreased the risk for PMWS. Successful treatment and control of PMWS has primarily focused on assuring good production practices that minimize stress (17, 18), eliminating coinfections or minimizing their effect (10, 11, 17, 22, 23, 27, 28) and eliminating potential triggering factors that induce immune stimulation (9, 20, 22, 24). Sero-therapy has been used with success in Europe but has not been widely used in other parts of the world (30). Until a PCV2 vaccine becomes available, our current recommendations for control of PCV2-associated diseases include the following:

• First confirm that you have a PCV2-associated problem by necropsy and histopathology and immunohistochemistry or in situ hybridization.

• Identify farm, site, or system specific concurrent infections through good quality diagnostic submissions.

• Eliminate or minimize the effects of PRRSV coinfection if present by breeding herd stabilization, pig flow changes, and/or vaccination.

• Eliminate or minimize the effects of SIV coinfection if present with breeding herd and possibly pig vaccination.

• Determine if PPV is present in tissues of affected pigs (by FA or PCR) and in the population by demonstration of seroconversion to PPV during the period of time where PCV2-associated disease occurs. Consider implementing PPV vaccination of growing pigs if PPV and PCV2 coinfection is confirmed.

• Minimize the effect of mycoplasmal pneumonia if present with vaccination and/or strategic pulse medication.

• If herd evidence suggests an association between vaccination practices and PCV2-associated disease, re-evaluate the necessity and timing of the vaccines in use. It may be beneficial to change the brand of vaccine used and/or the timing of administration of the vaccine.

• Treat specific bacterial coinfections with appropriate antimicrobials.

• Consider the use of anti-inflammatory drugs and enhanced diets on pigs that are slow to respond.

• Remove pigs that don’t respond to treatment.

• Adhere to all-in-all-out pig flow rules.

• Minimize mixing and moving of pigs whenever possible.

• Decrease pig density.

• Use disinfectants in buildings and transport vehicles that have been demonstrated to be efficacious against PCV2 (28). 

• If it is an option, consider changing pig source/pig genetics if the problem occurs repeatedly.

Acknowledgements
We recognize and appreciate the financial support from National Pork Board Pork Check Off Dollars, Iowa Livestock Health Advisory Council, Pfizer Animal Health Inc., Schering Plough Animal Health Inc., Fort Dodge Animal Health, Inc., and Merial towards PCV2 research at Iowa State University. We recognize and appreciate the collaboration on PCV2 experiments by Martin Fenaux and others from Dr. Xiang-Jin Meng’s group at Virginia Polytechnic Institute and State University, and Dr. Eileen Thacker and her laboratory at Iowa State University. We also appreciate the help with animal care provided by Pete Thomas, Chaiyan Kasorndorkbua, Diane McDonald, and the staff from Laboratory Animal Resources at Iowa State University.

References

1.       Allan GM, Kennedy S, McNeilly F, Foster JC, Ellis JA, Krakowka SJ, Meehan BM, Adair BM. 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J Comp Path. 121:1-11.

2.       Allan GM, McNeilly F, Kennedy S, Meehan B, Ellis J, Krakowka S. 2000. Immunostimulation, PCV-2 and PMWS. Vet Rec. 147:170-171.

3.       Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, Walker I, Kennedy S. 2000. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication. Arch Virol. 145:2421-2429.

4.       Allan GM, McNeilly F, McNair I, O’Connor M, Meehan B, Gilpin D, Ellis J, Townsend H, Lasagna C, Boriosi G, Krakowka S. 2001. Neonatal vaccination for Mycoplasma hyopneumoniae and post-weaning multisystemic wasting syndrome: a field trial. Pig J. 48:34-41.

5.       Fenaux M, Halbur PG, Haqshenas G, Royer R, Thomas P, Nawagitgul P, Gill M, Toth TE, Meng XJ. 2002. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions. J Virol. 76:541-551.

6.       Fenaux M, Opriessnig T, Halbur PG, Meng XJ. 2003. Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs. J Virol. 77:11232-11243.

7.       Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ.  2004. A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV2 type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs.  J Virol 78(12):6297-6303. 

8.       Halbur PG, Opriessnig T, Hoogland M, Thacker E, Yu S. 2003. PCV2-associated diseases: Research and diagnostic update. In: Proc Swine Disease Conf Swine Pract. 11:152-160.

9.       Harding J. 2002. PCV2 in Canada: prevalence, diagnostic advances, prevention, treatment, control. In: Proc Am Assoc Swine Vet.; PRRS & Type 2 Circovirus-advances in diagnostics, research and pathogenesis. pp 21-26.

10.   Harms PA, Sorden SD, Halbur PG, Bolin SR, Lager KM, Morozov I, Paul PS. 2001. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet Pathol. 38:528-539.

11.   Harms PA, Halbur PG, Sorden SD. 2002. Three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection. J Swine Health Prod. 10: 27-30.

12.   Hoogland M, Opriessnig T, Halbur P.  2004.  Comparison of the effect of different adjuvants on PCV2-associated diseases.  Manuscript submitted for publication.

13.   Kennedy S, Moffett D, McNeilly F, Meehan B, Ellis J, Krakowka S, Allan GM. 2000. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone and in combination with porcine parvovirus. J Comp Pathol. 122: 9-24.

14.   Krakowka S, Ellis JA, Meehan B, Kennedy S, McNeilly F, Allan G. 2000. Viral wasting syndrome of swine: Experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet Pathol. 37:254-263.

15.   Krakowka S, Ellis JA, McNeilly F, Ringler S, Rings DM, Allan G. 2001. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV2). Vet Pathol. 38:31-42.

16.   Kyriakis SC, Saoulidis K, Lekkas S, Miliotis CC, Papoutsis PA, Kennedy S. 2002. The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome. J Comp Path. 126:38-46.

17.   Madec F, Eveno E, Morvan P. et al. 2000. Post-weaning multisystemic wasting syndrome (PMWS) in pigs in France: Clinical observations from follow-up studies on affected farms. Livestock Prod Sci. 63:223-233.

18.   Madec F, Rose N, E. Eveno et al. 2001. PWMS: On-farm observations and preliminary analytic epidemiology. In: Proc ssDNA viruses of plants, birds, pigs, primates. pp 86-88.

19.   Nawagitgul P, Harms PA, Morozov I, Thacker BJ, Sorden SD, Lekcharoensuk C, Paul PS. 2002. Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV. Clin Diagn Labor Immun. 9:33-40.

20.   Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares F, Thacker EL, Brockus CW, Ackermann MR, Thomas P, Meng XJ, Halbur PG. 2003. Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol. 40:521-529.

21.   Opriessnig T, Yu S, Thacker EL, Halbur PG. 2004. Derivation of porcine circovirus type 2 negative-pigs from positive sow herds. J Swine Health Prod.  12(4):186-191.

22.   Opriessnig T, Fenaux M, Yu S, Evans RB, Cavanaugh D, Gallup JM, Pallares FJ, Thacker EL, Lager KM, Meng X-J, Halbur PG. 2004. Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus. Vet Microb. 98(3-4):209-220.

23.   Opriessnig T, Thacker EL, Yu S, Fenaux M, Meng XJ, Halbur PG. 2004. Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2.  Vet Pathol. 41(6):624-40.

24.   Opriessnig T, Halbur PG, Yu S, Thacker EL, Fenaux M, Meng XJ. 2004. Effect of timing of Mycoplasma hyopneumoniae bacterin administration on development of PCV2-associated lesions.  Vet Rec. Submitted for publication.

25.   Opriessnig T, Fenaux M, Thomas P, Evans RB, Rothschild M, Meng XJ, Halbur PG.  2004.  Evidence of host-dependent differences in susceptibility to porcine circovirus type 2-associated disease and lesions.  Submitted for publication.

26.   Pallares FJ, Halbur PG, Opriessnig T, Sorden SD, Villar D, Janke BH, Yaeger MJ, Larson DJ, Schwartz KJ, Yoon KJ, Hoffman LJ.  2002. Porcine circovirus type 2 (PCV-2) co-infections in U. S. field cases of postweaning multisystemic wasting syndrome (PMWS).  J Vet Diagn Invest 14(5):515-519.

27.   Rose N, Larour G, Le Diguerher G, Eveno E, Jolly JP, Blanchard P, Oger A, Le Dimma M, Jestin A, Madec F. 2003. Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in French farrow-to-finish herds. Prev Vet Med. 61:209-225.

28.   Rovira A, Balasch M, Segalés J, García L, Plana-Durán J, Rosell C, Ellerbrok H, Mankertz A, Domingo M. 2002. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J Virol. 76:3232-3239.

29.   Royer RL, Nawagitgul P, Halbur PG, Paul PS. 2001. Susceptibility of porcine circovirus type 2 to commercial and laboratory disinfectants. J Swine Health Prod. 9:281-284.

30.   Waddilove AEJ, Marco E. 2002. Assessing serotherapeutic control of PMWS in the field. In: Proc 17^th Congress of the International Pig Veterinary Society, Ames, IA. p 204.