Pasteurellosis is widely distributed all over the world and can spread across species and regions . As the number of rabbits used as food, household pets, and laboratory animals increases, as well as the increase of the number and activities of wild rabbit populations, the risk of interspecific pathogen transmission has also increased. In this study, we examined the clinical symptoms, histopathological changes, colonization of P. multocida, and host innate immune responses in P. multocida-infected rabbits.
The clinical symptoms of pasteurellosis in rabbit include rhinitis with purulent nasal discharge, pneumonia, abscesses, otitis media, and septicemia [22,23,24]. In current study, clinical symptoms were observed at 1 dpi. The infected rabbits presented primarily with respiratory symptoms, with severe dyspnoea and serous nasal fluid. At the end of the experiment, the mortality rate was 60%. Our results were similar with previous study, 11/12 rabbits were seriously ill. All the sick rabbits developed acute septic syndrome with respiratory failure and shock . Previous research showed that the inflammation caused by P. multocida due to the infiltration of neutrophils and macrophages in lungs of buffalo calves . In this study, rabbits showed respiratory symptoms after infection, with the obvious necropsy lesions and histopathological changes in the lungs. Consolidation, hemorrhage, and neutrophil infiltration were observed in the lung. Hemorrhage was observed in trachea and thymus. The thymus is the largest immune organ in rabbit. In thymus, the number of lymphocytes decreased and cell necrosis occurred. The pathogenicity of P. multocida is directly related to its colonization in rabbit tissues. Our results showed that, with the exception of the thymus and kidney, the proliferation of P. multocida in tested tissues reached a maximum at 2 dpi and then declined at 3 dpi. The mortality rate of rabbits was also the highest at 2 dpi. In addition, the number of P. multocida in lung and thymus remained high level at 3 dpi, which was consistent with the results of pathological changes. According to the above results, it was revealed that P. multocida can rapidly replicate in various tissues and organs of rabbit and cause bacteremia after infection.
Innate immunity system is the first line of defense against the infection of pathogenic microorganisms, which is mediated by phagocytes such as dendritic cells and macrophages. TLR4 binds to the co-receptors MD-2 and CD14 of lipopolysaccharide of Gram-negative bacteria to recognize it in soluble form or on bacteria . TLR2, together with TLR1 or TLR6, identifies pathogen-associated molecular patterns as homodimers or heterodimers . In this study, the expression of Tlr2 and Tlr4 was significantly induced in thymus after challenge with P. multocida. The expression of Tlr2 significantly up-regulated by 16.63-fold in lung. Our results indicated that TLR2 signaling pathway was activated after P. multocida infection. According to previous study, P. multocida infection promote the expression of pattern recognition receptors, inflammatory cytokines, and chemokines in murine lungs. Especially, up-regulation of TLR2 and NLRs mediates P. multocida infection . However, other studies reported that the association of TLRs in P. multocida infection is mainly that TLR4 mediated signal pathway recognizes the toxin and LPS [29, 30]. Our results showed that the expression of Tlr4 significantly increased in thymus, but inhibited in lung. Therefore, we speculated that P. multocida can activate TLR4-mediated signaling pathways to induce inflammatory responses, but in a tissue-dependent manner. On the other hand, excessive proinflammatory cytokine production can lead to severe immunopathological changes, even life-threatening. For this reason, in the development of inflammation, a feedback system is required to regulate the restriction and balance between the proinflammatory and anti-inflammatory.
In mice and cattle, the inflammatory mediators were highly expressed in lung after P. multocida infection. The number of neutrophils and the expression of proinflammatory cytokines (TNF-α, IL-6, IL-1, IL-8, and IL-12) increased in lung . These inflammatory responses play important roles in P. multocida infections. After activation of TLRs, NF-κB signaling was stimulated to resist bacterial infection by regulating production of cytokines [32, 33]. Recently, previous study has been demonstrated that the expression of pro-IL-1β induced by P. multocida is partially dependent on TLR4 in macrophages. The expression of pro-IL-1β was significantly up-regulated after P. multocida infection, but partially decreased in TLR4 knockout macrophages . In current study, the expression of Il1β was significantly increased in lung and thymus. IL-6 and IL-8 are important cytokines in the inflammatory response, and cooperate with other cytokines to regulate the inflammatory response process. TNF-α is an endogenous pyrogen that induces fever and stimulates endothelial cells and leukocytes to release various inflammatory mediators that promote neutrophil phagocytosis. It has been reported that P. multocida highly induced the secretion of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in mouse . Similarly, our results showed that the expression of Il6, Il8, and Tnf-α was significantly increased, indicating that the TLRs pathway was activated after P. multocida challenge. The expression of most cytokines peaked at 2 dpi and decreased at 3 dpi. This trend was consistent with the colonization of P. multocida in rabbit tissues. We speculated that the high expression of proinflammatory cytokines (Il1β, Il6, Il8, and Tnf-α) in lung might be the main cause of respiratory inflammation and septicemia.
In conclusion, the whole disease process of P. multocida infection in rabbit were investigated. The clinical symptoms were mainly respiratory symptoms, with severe dyspnoea and serous nasal fluid. The pathological changes are directly related to colonization of P. multocida in rabbit tissues. TLRs signaling pathways were activated after P. multocida infection, the overexpression of inflammatory factors aggravates tissue damage and mortality, which is also the main cause of respiratory inflammation and septicemia. Altogether, this study has pioneered the systematic exploration of the colonization, innate immune responses, and etiology in P. multocida-infected rabbits. This study also provide new insights into the relationship between P. multocida pathogenicity and defense response in rabbits.