| Epidemiological investigations in different countries indicated that dual infection of porcine circovirus type 2 (PCV2) and Haemophilus parasuis (HPS) are so pervasive, particularly PCV2 and Haemophilus parasuis serovar 4 (HPS4) dual infections combined with other pathogen widespread popularity in China. It is considered to be a significant problem on the economic and public health in many parts of the world. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. Moreover, studies investigating the relationship of PCV2/HPS4 coinfection are lacking in the literature, which is urgently needed us further study the mechanism of it. Therefore the objective of this study was to use the natural birth pigs to study the pathgenesis of PCV2 alone or in combination with HPS4.1. Effect of experimental co-infection with PCV2 and HPS4 on pigletsIn this study, Twenty-four four-week-old natural birth piglets were randomly allocated to four groups (6 pigs/each group). Group 1 piglets were intratracheally with HPS serotype 4 (MD0322 strain) (2ml/pig,) (5.5 X108CFU); Group 2 piglets were inoculated with PCV2-WH by intranasally and intramuscularly(2.5 ml/pig) (107.3TCID50/ml), group 3 (n=6) piglets were coinfected with HPS strain MD 0322 and PCV2, the dose and route of microbe inoculation was the same as the first two groups, and group 4 (n=6) piglet of the noninfected group was treated similarly with an identical volume of PBS as control. All the groups were inoculated synchronously. Groups were individually housed in separate rooms. For 28 days following challenge, pigs were under continuous observation to evaluate clinical symptoms, total white blood cell counts, viremia, virus distributions in different tissues.(1) The results showed that all the coinfection piglets developed PMWS, characterized by moderate dyspnea, anorexia, prostration, reduced weight gain severely in the meantime.(2) Our results showed that the PCV2/HPS4 coinfected group had a greater reduction in WBC and lymphocytes than the PCV2-infected group. We also observed apparently lymphnoditis, indicating that coinfection inhibits the host defense more severely. The levels of granulocytes in PCV2/HPS4 coinfected group were dramatically reduced at seven DPI in comparison with that in singular HPS4-infected pigs. The phagocytic and bactericidal activities of neutrophil granulocytes were obviously suppressed, leading to severe pathological processes, that is why coinfected group developed interstitial pneumonia with small amount of fibrinous exudates and arthritis exacerbation. Therefore, the PCV2/HPS4 coinfected group likely causes more severe immunosuppression than either PCV2 or HPS4 infection alone.(3) From the detection of virus level in serum we got to know that coinfection with PCV2 and HPS4 increased the amount of PCV2 genomic copy numbers in the sera, resulting in the slower generation and lower levels of antibodies against HPS4 and PCV2. Potentiates PCV2 replication may cause more severe immunologicmjury.(4) The distributions of PCV2 were different in various tissues, especially increased the amount of PCV2 viral load in lymphoid tissue and immune organs in coinfected pigs including lymphatic enlarging and congestion, interstitial pneumonia and gastrohelcosis, enlargement. The distribution of PCV2 in different tissue was detected by qRT-PCR, the results showed PCV2 was mainly distributed to the immune system including tonsil and lymph nodes and tonsil has the highest viral load, the tonsil in singular PCV2-infected pigs exhibited the highest PCV2-genome equivalent copy number (2.04×1010 GECN per gram of tissue) comparion with the coinfected pigs (3.17×1010 GECN per gram of tissue). Hence HPS4 could support PCV2 replication at the site of infection. We assume that coinfection with PCV2 andHPS4 enhances the pathogenicity of PCV2 in pigs.2. Transcript analysis of Primary alveorlar marcrophages (PAMs) in porcine infected with PCV2 and HPS serovar 4 in vivoWe used Affymetrix GeneChip Porcine Genome Array to analyse the differentially expressed (DE) transcripts of Primary alveorlar marcrophages (PAMs) in porcine infected with PCV2 and HPS serovar 4 in vivo. All pigs were slaughtered at 28 dpi which reproducted the lesions of PMWS by infection of NFCD Pigs with PCV2 alone or in combination with HPS4 above. PAMs were lavaged from the lungs and the total RNA were extracted by trizol reagent. The RNA labelling and hybridization were conducted by a commercial Affymetrix array service.With a filter of false-discovery rate less than 0.05 and fold change greater than 2, at 28 d.p.i. A total of 753 differentially expressed (DE) transcripts were identified in the PAM after HPS4, PCV2 infection separately and PCV2-HPS4 coinfection.178 genes were up-regulated,80 genes were down-regulated in PCV2 single infection.195 genes were up-regulated,108 genes were down-regulated in PCV2 and HPS4 coinfection.149 genes were up-regulated,43 genes were down-regulated in HPS4 group.19 genes were classified into the pathway related to Cytokine-cytokine receptor interaction.There also have 10 genes about T cell receptor signaling pathway, which involved in the immune response. Differentially expressed genes of the host immune response against pathogens related to innate immune, which included Ficolin and A3F.5 DE genes TCR-a, TCR-β,CD3E, CD3G and LCK are related to the pathway of The Complement System. A large set of DE genes in PAMs are involved in the, we could see C1s, C1qR, CR2, C4bBP, DAF are all up-regulated. These are genes related to anti-virus,ISG20L2, GBP1 and MCP2, all these three genes in PCV2 and HPS4 coinfection are up-regulated significantly. DE genes related to immunosuppression which included S1P1,GATA-3,DPPⅣand TGF-βare regulated significantly. Results of this study revealed that a series of genes involved in immune responses are activated following HPS4 and PCV2 invasion, particularly genes of the inflammatory. This finding could contribute to explaining the complicated mechanism of that underlies the pathogenesis of the disease. |
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