Coinfection modulates inflammatory responses, clinical outcome and pathogen load of H1N1 swine influenza virus and Haemophilus parasuis infections in pigs

Abstract

Background: Respiratory co-infections are important factor affecting the profitability of pigs production. Swine influenza virus (SIV) may predispose to secondary infection. Haemophilus parasuis (Hps) can be a primary pathogen or be associated with other pathogens such as SIV. To date, little is known about the effect of coinfection with SIV and Hps on the disease severity and inflammatory response and the role of Hps in the induction of pneumonia in the absence of other respiratory pathogens. In the study we investigated the influence of SIV and Hps coinfection on clinical course, inflammatory response, pathogens shedding and load at various time points following intranasal inoculation. The correlation between local concentration of cytokines and severity of disease as well as serum acute phase proteins (APP) concentration has been also studied.

Results: All co-infected pigs had fever, while in single inoculated pigs fever was observed only in part of animals. Necropsy revealed lesions in the lungs all SIV-inoculated and co-inoculated pigs, while in Hps-single inoculated animals only 1 out of 11 pigs revealed gross lung lesions. The SIV shedding was the highest in co-inoculated pigs. There were no differences between Hps-single inoculated and co-inoculated groups with regard to Hps shedding. The significant increase in Hps titre in the lung has been found only in co-inoculated group. All APP increased after co-infection. In single-inoculated animals various kinetics of APP response has been observed. The lung concentrations of cytokines were induced mostly in SIV + Hps pigs in the apical and middle lobe. These results correlated well with localization of gross lung lesions.

Conclusions: The results revealed that SIV increased the severity of lung lesions and facilitated Hps (PIWetHps192/2015) replication in the porcine lung. Furthermore, Hps influenced the SIV nasal shedding. Enhanced Hps and SIV replication, together with stronger systemic and local inflammatory response contributed to a more severe clinical signs and stronger, earlier immune response in co-inoculated animals. We confirmed the previous evidence that single-Hps infection does not produce significant pneumonic lesions but it should be in mind that other strains of Hps may produce lesions different from that reported in the present study.

Keywords: Disease severity; Immunity; Pathogens shedding; Pigs; Respiratory co-infections.

Fig. 1

Rectal temperature (mean ±SD) in pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps). Number of pigs affected: Hps + SIV 11/11; SIV 7/11; Hps 3/11

Fig. 2

Clinical score (mean±SD) of pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps). The significant differences between groups are indicated with the same superscripts

Fig. 3

Lung score (mean±SD) of pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps). The significant differences between groups are indicated with the same superscripts

Fig. 4

Nasal virus shedding (mean ±SD) after inoculation of pigs with swine influenza virus (A/Poland/Swine/14131/2014) and or Haemophilus parasius. Mean virus titres (determined by cell culture) in nasal swabs collected during study period. The dashed line represents the detection limit. * - the significant differences compared to control group; a - the significant differences between inoculated groups; b - the significant differences between SIV and Hps + SIV inoculated groups; c – the significant differences between SIV, Hps + SIV and Hps

Fig. 5

Nasal bacteria shedding (mean ±SD) after inoculation of pigs with Haemophilus parasius. Mean colony forming units (CFU) (determined by quantitative PCR) in nasal swabs collected during study period. * - the significant differences compared to control and SIV group

Fig. 6

Swine influenza virus titre (mean ±SD) (determined by cell culture) in the lung at 2, 4 and 10 days after single or dual inoculation of pigs with swine influenza virus and/or Haemophilus parasius. The dashed line represents the detection limit. Columns with various superscripts within the same day of study differ significantly

Fig. 7

H. parasuis titres (mean ±SD) (determined by quantitative PCR) in the lung at 2, 4 and 10 days after single or dual inoculation of pigs with swine influenza virus and/or Haemophilus parasius. The dashed line represents the detection limit. * - significant differences compared to remaining groups

Fig. 8

Mean (±SD) HI antibody titres against A/Poland/Swine/14131/2014 SIV in of pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps). * - significant differences compared to groups not inoculated with SIV

Fig. 9

Mean (±SD) level of antibodies against Haemophilus parasius in of pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps) * - significant differences compared to control group; a - significant differences between co-inoculated pigs and Hps single-inoculated groups. ELISA ratio (ER) was calculated as directed by the manufacturer (Swinecheck®HPS, Biovet, Canada) according to the following formula: $\mathrm{Er}=\frac{\mathrm{OD}\text{of test serum from wells with}\mathrm{Ag}‐\mathrm{OD}\text{of test serum from wells without}\mathrm{Ag}}{\text{mean}\mathrm{OD}\left(+\right)\text{from wells with}\mathrm{Ag}‐\text{mean}\mathrm{OD}\left(+\right)\text{from wells without}\mathrm{Ag}}$ (+) - positive control, Ag - antigen

Fig. 10

Concentrations of SAA, Pig-MAP, Hp and CRP (mean ±SD) in serum of pigs before and on various time-point after single or dual inoculation with swine influenza virus (SIV) and/or Haemophilus parasius (Hps). CRP: * - significant differences between co-inoculated and control group; a - significant differences between Hps-inoculated and control group; Hp: a- significant differences between single and dual-inoculated groups; * - significant differences between co-inoculated and control pigs; b - significant differences between SIV and control groups, c - significant differences between Hps and control groups. SAA: * - significantly differences between co-inoculated and control pigs, a – significant differences between single-inoculated and control pigs. Pig-MAP: a – significant differences between co-inoculated and control pigs; * - significant differences between Hps and control group

Fig. 11

Quantification of cytokines in lung tissue of pigs single or dual inoculated with swine influenza virus (SIV) and/or Haemophilus parasius (Hps) (mean±SD). acc-accessory lobe; * - significant differences compared to control pigs (within the same day, at the same part of the lung)