The combination of PRRS virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs

Abstract

This paper reviews in vivo studies on the interaction between porcine reproductive and respiratory syndrome virus (PRRSV) and LPS performed in the authors' laboratory. The main aim was to develop a reproducible model to study the pathogenesis of PRRSV-induced multifactorial respiratory disease. The central hypothesis was that respiratory disease results from an overproduction of proinflammatory cytokines in the lungs. In a first series of studies, PRRSV was shown to be a poor inducer of TNF-alpha and IFN-alpha in the lungs, whereas IL-1 and the anti-inflammatory cytokine IL-10 were produced consistently during infection. We then set up a dual inoculation model in which pigs were inoculated intratracheally with PRRSV and 3-14 days later with LPS. PRRSV-infected pigs developed acute respiratory signs for 12-24h upon intratracheal LPS inoculation, in contrast to pigs inoculated with PRRSV or LPS only. Moreover, peak TNF-alpha, IL-1 and IL-6 titers were 10-100 times higher in PRRSV-LPS inoculated pigs than in the singly inoculated pigs and the cytokine overproduction was associated with disease. To further prove the role of proinflammatory cytokines, we studied the effect of pentoxifylline, a known inhibitor of TNF-alpha and IL-1, on PRRSV-LPS induced cytokine production and disease. The clinical effects of two non-steroidal anti-inflammatory drugs (NSAIDs), meloxicam and flunixin meglumine, were also examined. Pentoxifylline, but not the NSAIDs, significantly reduced fever and respiratory signs from 2 to 6h after LPS. The levels of TNF-alpha and IL-1 in the lungs of pentoxifylline-treated pigs were moderately reduced, but were still 26 and 3.5-fold higher than in pigs inoculated with PRRSV or LPS only. This indicates that pathways other than inhibition of cytokine production contributed to the clinical improvement. Finally, we studied a mechanism by which PRRSV may sensitize the lungs for LPS. We hypothesized that PRRSV would increase the amount of LPS receptor complex in the lungs leading to LPS sensitisation. Both CD14 and LPS-binding protein, two components of this complex, increased significantly during infection and the amount of CD14 in particular was correlated with LPS sensitisation. The increase of CD14 was mainly due to infiltration of strongly CD14-positive monocytes in the lungs. The PRRSV-LPS combination proved to be a simple and reproducible experimental model for multifactorial respiratory disease in pigs. To what extent the interaction between PRRSV and LPS contributes to the development of complex respiratory disease is still a matter of debate.

Fig. 1

Evolution of mean titers of PRRSV (a) and the cytokines IFN-α, TNF-α, IL-1 and IL-10 (b) in the lungs of PRRSV-inoculated pigs. Both virus titers in lung tissue (log₁₀ TCID₅₀/g) and BAL fluids (log₁₀ TCID₅₀/ml) are presented. IFN-α, TNF-α and IL-1 are expressed as bioactive units and IL-10 as pg/ml BAL fluid.

Fig. 2

Evolution of respiratory scores and lung TNF-α titers in pigs exposed to LPS at different days after PRRSV inoculation (a) and evolution of the amount of CD14 in lung tissue sections during PRRSV infection (no LPS exposure) (b). Each dot corresponds to one pig and the solid line represents the mean at each time point. The dotted line represents the detection limit. Respiratory scores range from 0 to 4, whereby 0 = normal; 1 = tachypnoea when stressed; 2 = tachypnoea at rest; 3 = tachypnoea and dyspnoea at rest; 4 = severe tachypnoea and dyspnoea with laboured, jerky breathing. Pigs inoculated with PRRSV only showed no respiratory signs (score 0) and had no detectable TNF-α, except at 14 DPI (32-109 U/ml) (see Fig. 1b).

Fig. 3

Effect of pentoxifylline, meloxicam and flunixin meglumine treatment on clinical scores of PRRSV–LPS inoculated pigs. Clinical scores are calculated as shown in Table 1. Values with an asterisk are significantly different from the untreated PRRSV–LPS group (Mann–Whitney test, P < 0.05).