Iowa Livestock Health Advisory Council

FINAL REPORT

Title: Interaction and disease potentiation of PCV2 and PRRSV isolates in the conventional pig model

Principle Investigator: / Tanja Opriessnig, Dr. med. vet., Ph.D.
Assistant Professor
Veterinary Diagnostic and Production Animal Medicine
College of Veterinary Medicine
Phone: 294-1137
E-mail:
Co-Investigator: / Phillip Gauger, DVM, MS
Clinician
Veterinary Diagnostic and Production Animal Medicine
College of Veterinary Medicine
E-mail:
Collaborator: / Kay S. Faaberg, PhD
Research Microbiologist, USDA-ARS B-14
Virus and Prion Diseases of Livestock
National Animal Disease Center
E-mail:

Date of Completion of the Project: June 30, 2009

Industrial Summary

1. Statement of the problem

Porcine circovirus associated disease (PCVAD) has re-emerged in the United States in recent years. The outbreaks have raised concerns over introduction of a new more virulent PCV2-variant (PCV2b) into North America. However, both the resident U.S. cluster (PCV2a) and the recently introduced PCV2b cluster are capable of inducing clinical disease in gnotobiotic pigs whereas no clinical disease was observed in conventional pigs inoculated with either PCV2a or PCV2b. Recently, a marked increase in submissions of PRRSV-associated pneumonia to the Veterinary Diagnostic Laboratory at Iowa State University was observed. PRRSV is an RNA virus and is capable of continuous genomic changes. Isolates that are currently present in the US are quite different from those characterized in the early 90’s. The sudden increase of PCV2b in North American swine herds and the concurrent increase of PRRSV-associated pneumonia provide evidence of an interaction between certain PRRSV strains and recent PCV2 genotypes. This observation merits further investigation.

2. Objective

The main objective was to determine the differences in interaction among PCV2 strains and PRRSV isolates measured by the severity of clinical disease, macroscopic and microscopic lesions, and amount of PRRSV and PCV2 RNA/DNA/antigen associated with the lesions.

3. Results

Under the conditions of this study, there were no significant differences in the clinical signs, severity of macroscopic or microscopic lesions, level of viremia or onset of immunity when conventional pigs were coinfected with either PCV2a or PCV2b and two pathogenically different strains of PRRSV when necropsied 12 days post inoculation. Similar to previous reports that describe no differences between PCV2a and PCV2b in conventional pigs singularly inoculated with the either virus,3 we did not observe clinical differences when PCV2 cluster viruses were coinfected with different isolates of PRRSV.Therefore, it appears clinical differences between PRRSV isolates, regardless of their emergence in the swine population or level of virulence, do not influence the severity of lesions, onset of disease or create more severe clinical signs when coinfected with PCV2 in an experimental setting.

4. Impact on the industry

The information from this study can help producers and stakeholders in the industry, who influence the health decisions of their clients, understand the pathogenesis of PCV2/PRRSV coinfections and how it relates to their specific swine operation. The apparent lack of pathogenic differences between the coinfected groups in this study provides no evidence that would support changing current vaccine protocols or biosecurity measures in existence nor does it require alternate serological measures to monitor exposure or progression of disease in a herd.

Previous reports have observed adequate PCV2 vaccine efficacy in PCV2-PRRSV coinfection models.1 Due to the similarity in clinical disease between the different coinfections observed in this report, it can be assumed PCV2 vaccine efficacy should remain adequate in coinfections that involve diverse strains of PRRSV. In addition, biosecurity measures and principles can be maintained without alteration or concern for the specific viruses that are currently circulating within a herd. Although new introductions of viruses should be diagnosed and monitored with the same vigilance as previously implemented during routine diagnosis of disease.

Scientific Report

a. Materials and Methods

Experimental design

Forty-five SPF pigs were derived from sows free of PRRSV, SIV, PCV2 and Mycoplasma hyopneumoniae. At arrival in the research facility the pigs were randomly divided into 5 groups and rooms of 8 pigs: A group inoculated with PCV2a and PRRSV-old, one group inoculated with PCV2a and PRRSV-new, one group inoculated with PCV2b and PRRSV-old, one group inoculated with PCV2b and PRRSV-new, and one negative control group.

Table 1. Experimental Design

Group / Group
Abbreviation / Na / Doseb
PRRS PCV2 / PRRS Amount
INc / PCV2 Amount
IN IMd
Control
NC 6845/PCV2a
NC16845/PCV2b
IA 12/PCV2a
IA 12/PCV2b / Control / 8 / - / - / -
NC PRRS/2a / 9 / 105.0 104.3 / 2ml / 3ml 2ml
NC PRRS/2b / 9 / 105.0 104.3 / 2ml / 3ml 2ml
IA PRRS/2a / 9 / 105.0 104.3 / 2ml / 3ml 2ml
IA PRRS/2b / 9 / 105.0 104.3 / 2ml / 3ml 2ml

a Number of pigs in each group

b Tissue culture infectious dose (TCID50)/ml

c Intranasal

d Intramuscular

Inoculation

All groups were inoculated intranasally (2ml) with their respective inoculums described in the experimental design. Pigs inoculated with PCV2 isolates received a dose approximately 104.5 TCID50/ml and the pigs inoculated with PRRSV isolates received approximately 105 TCID50/ml. We used PCV2a isolate 40895 (1998), PCV2b isolate NC-16845 (2006), PRRSV-old isolate ISU-12 (1992), and PRRSV-new isolate NC-16845 (2006). PCV2a and PRRSV-old have previously both been well characterized in the pig model. PCV2b and PRRSV-new were isolated from the same farm with a history of severe PCVAD in 2006.

Quantification of PCV2 DNA and PRRSV RNA

The DNA and RNA of all serum samples obtained at the day of inoculation and at 3, 6, 9, and 12 days post inoculation was extracted and assessed for PCV2 DNA and PRRSV RNA using quantitative real-time PCR as we have previously described.2,5

Clinical evaluation

Pigs were evaluated daily for respiratory disease and icterus as previously described.4 Pigs were weighed upon arrival, on the day of PCV2 infection, and every 3 days thereafter.

Necropsy

All pigs will be necropsied at 14 days post inoculation. At necropsy the percentage of lung affected by pneumonia (0-100%) was scored using a previously described scoring system.4 Enlargement of lymph nodes will be scored as well.

Microscopic examination and immunohistochemistry

Severity of lymphoid depletion and the presence and severity of lymphohistiocytic inflammation were scored as previously described in our model.4 Immunohistochemistry (IHC) for PCV27 will be conducted on selected tissue sections (lymph nodes, tonsil, spleen).

Statistical analysis

Analysis of variance (ANOVA) was used to detect significant differences among treatment groups with a p-value of 0.05 as being considered significant.

b. Results and Discussion

Clinical presentation

Mild, transient lethargy and inappetance was observed in all inoculated groups, although coughing or sneezing was not a feature. There was no significant time by group interaction in rectal temperatures after PRRSV and PCV2 inoculation between all four coinfected groups. In addition, significant differences were not identified between pigs inoculated with PRRSV or PCV2 regardless of the isolate administered (Fig 1). A significant difference (P <0.05) was observed between the NC PRRSV/2a group and un-inoculated controls 3 days post infection (dpi). A four inoculated groups had temperatures significantly higher than the controls at 6 dpi; whereas at 9 dpi, NC PRRSV/2a pigs had significantly higher temperatures (P <0.05) than all other groups in the study. By 12 dpi, NC PRRSV/2a, IA PRRSV/2a and IA PRRSV/2b groups were significantly different (P <0.05) than the controls and NC PRRSV/2b.

Macroscopic lesions

Macroscopic lesions were characterized by mottled-tan lungs of varying degrees of severity and mild-to-moderate enlargement of lymph nodes (Fig 2). Significant differences (P <0.0001) were only observed between the coinfected groups and the controls; trends were not identified that indicated more severe lesions in one group over the others.

Microscopic lesions and incidence of PCV2 and PRSV antigen in tissues

Lung tissues had focal-to-diffuse, mild-to-severe, lymphohistiocytic interstitial pneumonia. The mean microscopic lung lesion scores were lowest in the controls (0.75 ± 0.25) being significantly different (P <0.0001) than the four coinfected groups (Fig 3). Differences were not observed between the coinfected groups where lung lesion scores ranged from 4.44 to 4.78. Significant differences were not observed between comparisons of the NC- and IA-PRRSV inoculated groups or the PCV2a- and PCV2b-inoculated groups.

Lymphoid lesions were characterized by no-to-mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups. Significant differences were not observed between all groups, including controls, when lymph nodes, spleen, tonsil and thymus were evaluated. The incidence of PCV2-antigen in lung sections as determined by IHC stains was 16/18 in PCV2a inoculated pigs and 11/18 in PCV2b inoculated pigs. The incidence of PRRSV-antigen in lung sections was 16/18 in NC-PRRSV inoculated pigs compared to 18/18 in IA-PRRSV inoculated pigs. The incidence of PCV2 antigen in lung sections was significantly higher (P <0.05) in the IA-PRRSV/2a group compared to NC-PRRSV/2b and controls. IA-PRRSV/2b and NC-PRRSV/2a had significantly higher quantities of PCV2 antigen in lungs compared to only controls. Conversely, there were no differences in the incidence of PRRSV antigen in lungs between all inoculated groups although the incidence was significantly higher (P <0.05) in all inoculated groups compared to controls.

The incidence of PCV2 antigen in lung sections was significantly higher (P <0.05) in all IA-PRRSV inoculated pigs compared to NC-PRRSV inoculated pigs or controls. Conversely, there were no differences in the incidence of PCV2 antigen in lungs between the PCV2a inoculated pigs and PCV2b inoculated pigs. The incidence of PRRSV antigen in lungs was not significantly different between NC- and IA-PRRSV inoculated pigs or PCV2a- and PCV2b-inoculated pigs. The incidence of PCV2 antigen was consistently higher in lymphoid tissues in the IA-PRRSV/2a, IA-PRRSV/2b and NC-PRRSV/2a compared to NC-PRRSV/2b pigs or controls although significant differences did not exist between the inoculated groups.

Anti-PCV2-IgG antibody levels

All pigs were negative for PCV2-specific anti-IgG antibodies at 0 dpi. The group anti-PCV2-IgG antibody levels are summarized in Fig. 4. Three coinfected groups (NC PRRS/2a, NC PRRS/2b, and IA PRRS/2a) seroconverted to PCV2 at 12 dpi. One group, IA PRRS/2b, was within the suspect range for seroconversion at the end of the study. Significant differences in antibody response were lacking between all coinfected groups. IA PRRS/2a elicited a significantly different (P <0.05) antibody response compared to controls by 9 dpi. NC PRRS/2a, NC PRRS/2b and IA PRRS/2a were significantly different (P <0.05) than controls at 12 dpi. The NC PRRS/2b group did not generate an immune response that was considered different from the controls throughout the trial.

Anti-PRRSV-IgG antibody levels

All pigs in all groups were negative for PRRSV-specific antibodies at 0 dpi (Fig 5). On 9 dpi, 22/44 pigs had seroconverted to PRRSV. By 12 dpi, all of the IA PRRSV inoculated pigs had seroconverted whereas 14/18 NC PRRSV had seroconverted. Significant differences (P <0.05) were observed at 9 dpi between the IA PRRSV and controls; by 12 dpi, all PRRSV inoculated pigs had significantly different (P <0.05) S/P ratios compared to controls.

Incidence and amount of PCV2 in sera

All pigs were negative for PCV2-DNA in serum at 0 dpi (Fig 6). 28/36 PCV2 inoculated pigs were positive for PCV2-DNA at 3 dpi; all pigs were positive for PCV2-DNA by 6 dpi. There was no significant time by group interaction in quantity of PCV2 DNA throughout the trial. However, the IA-PRRSV inoculated pigs contained consistently higher amounts of PCV2 DNA in serum throughout the trial.

Incidence and amount of PRRSV in sera

All pigs were negative for PRRSV-RNA in serum at 0 dpi (Fig 7). All PRRSV-inoculated pigs were positive for PRRSV-RNA at 3 dpi. There was no significant time by group interaction in quantity of PRRSV RNA throughout the trial. However, NC-PRRSV inoculated pigs contained consistently higher quantities of PRRSV RNA in their serum by 9 and 12 dpi being significantly higher (P <0.0001) than the IA-PRRSV/2b inoculated group at both time points.

Evaluation of the clinical effect between coinfections of PCV2a and PCV2b with a recently isolated PRRSV and PRRSV isolated ten years previously did not reveal significant differences between clinical signs, macroscopic and microscopic lesions, amount of PCV2 and PRRSV antigen in tissue, level of viremia or onset of immunity. The lack of clinical differences between these coinfection models may be contributed to several factors. Conventional pigs used in this trial were inoculated with a variant PCV2 or PRRSV isolate on the same day, which does not parallel infections in field situations. Often, PRRSV infections precede exposure to PCV2 in the field which may cause aberrant effects on the immune system, cytokine responses, or susceptibility to other pathogens allowing for more severe clinical disease. In addition, the PRRSV isolates chosen for this study, although characterized and isolated from recent field cases of high mortality, do not clinically represent all PRRSV isolates. Differences in pathogenicity, virulence, or effects on the immune system between strains of PRRSV may exist and could cause different clinical responses than what was reported in this study.

It would have been ideal to have included additional control groups that would have represented the PCV2 variants and PRRSV isolates as single infections. Differences in clinical disease and lesions may have been observed between coinfected groups versus single infected pigs giving an impression for the severity of disease that can occur when more than one pathogen is involved in a disease process. Unfortunately, limitations in acquiring PCV2 negative pigs, funding and pig space for research did not allow for additional groups.

The length of the study was precisely calculated to maximize PRRSV lung lesions that characteristically dissipate after 12 dpi and dictated the duration of the study. Reported literature has noted that PRRSV-RNA persisted for longer periods of time in serum in pigs dually infected with PRRSV and PCV2.6 In this particular study, clinical disease and lesion severity may have been negated due to the short duration of this study, presuming clinical differences may have been noted had the experiment lasted longer.

c. Tables and Figures

Tables included in part 3.

d. Reference List

1. Opriessnig, T., D. M. Madson, J. R. Prickett, D. Kuhar, J. K. Lunney, J. Elsener, and P. G. Halbur. 2008. Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection. Vet. Microbiol. 131:103-114.

2. Opriessnig, T., N. E. McKeown, K. L. Harmon, X. J. Meng, and P. G. Halbur. 2006. Porcine circovirus type 2 infection decreases the efficacy of a modified live porcine reproductive and respiratory syndrome virus vaccine. Clin. Vaccine Immunol. 13:923-929.