Effects Of Co-infection Of H9N2 Influenza Virus And Pseudomonas Aeruginosa On The Development Of Hemorrhagic Pneumonia In Mink

Hemorrhagic pneumonia is one of the important diseases that seriously endanger the mink breeding industry.It is traditionally thought to be caused by Pseudomonas aeruginosa(P.aeruginosa)infection.However,recent studies have shown that P.aeruginosa single infection is not easy to cause hemorrhagic pneumonia in mink,and there should be other reasons.Co-infection with Influenza A virus(IAV)and bacteria can affect the process and severity of host infection.In recent years,the co-infection of IAV and bacteria has become an important research front.Earlier studies in our laboratory showed that H9N2 AIV was prevalent in mink and caused mild respiratory diseases.This study was conducted to investigate whether co-infection with H9N2 IAV and P.aeruginosa could cause hemorrhagic pneumonia in mink.The H9N2 IAV(Mk/SD/F10/13)and P.aeruginosa(P.aeruginosa-CS-2)were isolated and stored in our laboratory and used to establish the pathogenicity test of co-infection of mink with H9N2 IAV and P.aeruginosa in different orders.Divided into simultaneous co-infection,P.aeruginosa co-infection following H9N2 IAV infection and H9N2 IAV co-infection following P.aeruginosa infection and the mink challenged with H9N2 IAV or P.aeruginosa served as single infection controls,and the mink inoculated with PBS as negative control.During the experiment,observe the clinical symptoms and pathological changes of mink,collect nasal wash liquid,serum,and tissue(heart,liver,spleen,lung,kidney,etc.)of mink for pathogenic or serological testing.As a result,mink infected with H9N2 IAV and P.aeruginosa showed more severe clinical symptoms than H9N2 IAV or P.aeruginosa infection alone.Moreover,the peak value of the clinical symptom score(CSS)of mink in the H9N2 IAV and P.aeruginosa infection group appeared earlier and the peak value was higher than that in the mink group infected with H9N2 IAV or P.aeruginosa alone(P < 0.05).And there was no significant difference in CSS peaks between co-infected groups in different orders.At the same time,studies have shown that co-infection exacerbates the pathological damage of mink lung tissue and leads to more pronounced apoptosis in lung tissue.The group of simultaneous co-infection with H9N2 IAV and P.aeruginosa has the highest AI.A real-time quantitative PCR(qRT-PCR)method for the HA gene was established todetect virus shedding of H9N2 IAV and its distribution and load in tissues.As a result,H9N2 IAV shedding level of mink co-infected with H9N2 IAV and P.aeruginosa was significantly higher than that of mink infected with H9N2 IAV alone at 1 and 3 dpi.At 3 dpi,the viral load in the lungs of co-infected mink was higher than that of mink infected with H9N2 IAV alone,and similar situation was found in trachea and other tissues.The bacterial load of lung tissue was examined by bacterial isolation and culture.The results showed that the clearance of P.aeruginosa in the lungs of mink co-infected with H9N2 IAV and P.aeruginosa was delayed.At 3 and 11 dpi,the bacterial load in co-infected mink lungs was significantly higher than in the mink infected with P.aeruginosa alone.In addition,the anti-H9N2 antibody titer of mink was measured using the HI test.The results showed that at 3 and 7 dpi,the HI titers of mink in P.aeruginosa co-infection following H9N2 IAV infection and H9N2 IAV co-infection following P.aeruginosa infection groups were significantly higher than those mink infected with H9N2 IAV alone,especially in P.aeruginosa co-infection following H9N2 IAV infection group,the anti-H9N2 antibody titer was always at the highest level.To detect the local cytokine response during co-infection with H9N2 IAV and P.aeruginosa,qRT-PCR was used to detect cytokine mRNA levels in mink lung and spleen.At3 dpi,the mRNA levels of IFN-α and IFN-γ in co-infected mink lungs were higher than those in other mink infected with H9N2 IAV or P.aeruginosa,while the mRNA levels of TNF-αwere also rapidly and strongly up expressed.In summary,co-infection of H9N2 IAV and P.aeruginosa in different orders leads to hemorrhagic pneumonia in mink,which deteriorates rapidly,and that P.aeruginosa should play a major role in the disease.This study enriches the research data of virus-bacterial co-infection and provides theoretical basis for the prevention and control of respiratory diseases in mink and even humans.