Effect Of Coinfection Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) And Haemophilus Parasuis Infection On Inflammation Response In Pigs

Haemophilus parasuis（HPS）is a conditional pathogen present in the upper respiratory tract.There are 15 serotypes in total,among them,types 1,5,12,13 and 15 can cause death in piglets.Cross-immunity protection between these types is weak,Serotypes 5,12 and 13 are widely distributed in China,resulting in huge economic losses in the pig industry.Porcine reproductive and respiratory syndrome virus（PRRSV）is mainly replicated in alveolar macrophages（PAMs）and some monocyte cell lines,it can decrease the immunity of its host,thus the host is susceptible to secondary infections such as Porcine circovirus type 2（PCV2）,Swine fever virus（CSFV）,HPS,Streptococcus suis,parasitic pigs,Mycoplasma hyopneumoniae,and pleuropneumonia.Coinfection with PRRSV and other pathogens exacerbated clinical symptoms and increased mortality in sick pigs.The phenomenon of secondary HPS infection after PRRSV infection is very common in clinic,but there are few studies on PRRSV and HPS coinfection.In this study,a strain of bacteria was isolated from the pericardial fluid and synovial fluid of suspected HPS-infected pigs.After sequencing and serotyping,it was identified as serotype 13 HPS.The culture conditions and growth curves of serotype 13 HPS were studied.The animal model of 13 HPS-infected piglets and the effect of PRRSV and HPS coinfection on the pathogenicity of piglets.The main contents and results are as follows:（1）Study on the isolation and culture characteristics of Haemophilus parasuis:In clinical practice,we isolated a bacterial strain,which was identified by 16SrRNA genome and PCR serotyping.By optimizing the concentration of serum and nutritional factor necessary for HPS growth,it was found that 5%newborn bovine serum,10μg/mL nicotinamide adenine dinucleotide（NAD）was the most economical for bacterial proliferation.Then,the growth curve was plotted and found to have an OD₆₀₀00 value from0.2 to 0.6 for the logarithmic growth phase of this strain,and a large amount of acid was produced during the proliferation process.（2）Establishment of a method for the detection of Haemophilus parasuis:First of all,the SYBR GreenⅡfluorescence quantitative PCR method was established to detect the load of HPS.The way was specific,sensitive and stable.And then to establish the animal model of HPS infection alone.The clinical symptoms of each group were observed.The nasal swabs,blood and lung tissue samples were collected.The load of HPS were detected in the samples.The results showed that HPS was infected by intranasal inoculation with an infection dose of 2 mL（1×10⁶ CFU/mL）HPS.At this time,the piglets in the experimental group had obvious clinical symptoms.（3）Establishment of the animal model of PRRSV and HPS coinfection:The model of PRRSV and HPS coinfection was established,according to the infection dose of the above single infection of HPS.Then the clinical symptoms of appetite,respiration,cough,runny nose and diarrhea were observed.Clinical scores were recorded according to clinical phenomena.The load of PRRSV and HPS were detected in the samples,which were collected such as blood,nose swabs and lung.Three piglets in each group were euthanized on the 10th day of infection.Finally tested the serum TNF-ɑ,IL-1β,IL-6 and IL-8inflammatory factor changes.The body temperature of piglets in the PRRSV-infection group,HPS-infection group and HPS-PRRSV group were higher than those in the control group.The mean body temperature of the HPS+PRRSV group was significantly higher than that of the other infection groups from day 3 to day 14 after PRRSV infection.Clinical score results showed that the infection group had corresponding clinical symptoms,and the HPS+PRRSV group had more obvious clinical symptoms than the other single infection group（P<0.05）.Anatomical and pathological showed that the more severe pathological changes in the viscera（heart,liver,spleen,lung,kidney,lymph node）of piglets in the co-infection group were found.These results suggested that coinfection of PRRSV with HPS had produced more severe clinical symptoms.However,we also found that the titers of HPS and PRRSV in blood,nasal swabs and lung tissues of the co-infected group were significantly higher than those of the separately infected group（P<0.05）during a period of time after PRRSV infection.Specific antibody results of PRRSV and HPS in serum showed that the HPS+PRRSV group had significantly higher HPS and PRRSV antibody levels from the 6th day after coinfection than the single infection group（P<0.05）.From the above results,we speculated that PRRSV promote the proliferation of HPS in lung tissues and blood,and HPS had a certain impact on the pathogenicity of PRRSV.In the lung tissue TNF-ɑ,IL-1β,IL-6 and IL-8 inflammatory factor mRNA expression,according to the results of HPS+PRRSV group of inflammatory because of TNF-ɑ,IL-1β,IL-6 and IL-8 mRNA level obviously higher than that of single infection group（P<0.05）.HPS+PRRSV group of inflammatory cytokines in serum TNF-ɑ,IL-1β,and the concentration of IL-8 compared with single infection group had obvious difference（P<0.05）,the concentration of IL-6 and inflammatory factors between each group was no significant difference.From the above results,the conclusion that coinfection of HPS and PRRSV could promote the secretion of some inflammatory factors was drawn.In summary,the optimal OD₆₀₀ value range for harvesting the strain was obtained by optimizing the culture conditions of the serotype 13 HPS and plotting the growth curve.The clinical symptoms of group W2-10⁶ were found to be the most obvious after the harvested bacteria infected piglets.We found that PRRSV can promote the proliferation of HPS in pigs,HPS has a certain impact on the pathogenicity of PRRSV,and PRRSV and HPS coinfection can Promote the secretion and expression of some inflammatory factors in the body,through the successful construction of PRRSV and HPS coinfected animal models.