Studies On Synergetic Pathogenicity Of Co-infection With Porcine Circovirus Type 2 And Mycoplasma Hyorhinis

Porcine circovirus type 2(PCV2) is the main pathogen of porcine circovirus-associated diseases and causes tremendous economic losses to the porcine industry. Mycoplasma hyorhinis(Mhr) could infect porcines of various ages and cause pleuritis, peritonitis, arthritis, otitis and even swine enzootic pneumoniae(SEP), and reduce porcine growth performance seriously. It has high mobidity and low mortality, and always coinfected with PCV2 and Porcine reproductive and respiratory syndrome virus(PRRSV) clinically and improves the mortality of weaned piglets significantly. However, the pathogenicity and machenism of PCV2 and Mhr coinfection is still not clear up to now, and it is significant to deeply study the collaborative pathogenicity of PCV2 and Mhr to piglets. The methods for the detection of Mhr nucleic acid, antigen and antibody were established to clarify the two pathogens’ synergistic pathogencity to piglets, and Mhr(DL strain) and PCV2(PCV2b, YJ strain) were used in the present study.Firstly, an indirect ELISA was established for the detection of Mhr serum antibody. In order to detect the antibodies against Mhr, and indirect ELISA was established using the membrane proteins of Mhr as coating antigens. The antigens were extracted by 0.05% sodium deoxycholate(DOC). The sensitivey and specificy of the ELISA was determined to be 90.5% and 98.0% respectively. There was no cross-reaction with the reference sera of other several swine viruses and Haemophilus parasuis IV in the ELISA except for Mycoplasma hyopneumoniae(Mhyo),. The sera from the experimentally infected pigs with Mhr-DL strain were tested to be seroconversion at the third day after infection, and then the antibody titers reached 1: 6400 at the 35 th day after infection. One hundred and thirty eight sera from field farms were tested by the assay as well as IDEXX Mhyo antibody detection kit. The data showed that the positive rates of Mhr and Mhyo were 71.0% and 44.9% respectively. The positive rate of 457 pig sera collected from six provinces such as Heilongjiang, Jilin, Shandong was 70.3%. The results indicated that Mhr was highly prevalent in the pig herds of China.Secondly, monoclonal antibodies to Mhr membrane proteins were prepared and the antigenic epitopes were identified in the present study. DOC extracted membrane proteins of Mhr were used to immunize BALB/c mouse, and ten hybridomas were screened to stablely secerte antibodies to various membrane proteins of Mhr, which named as 1B4, 1D2, 1H8, 1H12, 2B6, 2H2, 3G2, 3G10, 4B7 and 4H2, respectively. All MAbs belonged to Ig G1 subclass and κ light chain except MAb 2H2, which belonged to Ig G2 b subclass. The supernatant titers of MAbs were from 1: 200 to 1: 400, and ascite titers were from 1: 1×105 to 1: 2×105. Five MAbs could react with one membrane protein at 40 k Da by Western blot and the membrane protein was identified as pyruvate dehydrogenase E1 complex α subunit(pd-α) by mass spectrometry, prokaryotic expression and Western blot assays. Then, three main eptopes of pd-α were identified by the methods of truncated expression and synthetic peptides, and they were 269SSDNPRIYRTEEEEK283, 299KDKKYITDEEIKQIW313 and 350LKEQKQHAKDY360, respectively.Thirdly, a real-time polymerase chain reaction(RT-PCR) with SYBR Green II was developed for rapid detection and quantification of Mhr in porcine tissue, and it had high specificity and sensitivity.Two specific primers were used in this method from the P37 gene of Gen Bank. Its amplificated effiecincy and the error value was 1.04 and 0.018, respectively. The inter-assay variable coefficient was less than 2% and the sensitivity of the established real-time PCR was at least 100-fold higher than that of the routine PCR assay. However, no cross reactions with Mhyo, PCV1, PCV2, CSFV, PRRSV and PPV were detected. To increase our understand of Mhr, the new method was used to quantify levels of Mhr genome in various tissues of pigs which were challenged in three different ways(intratracheal injection, intraperitoneal injection and the combination of the two former infected ways) and killed at 21 days post-inoculation. According to the results, Mhr genome mainly located in tonsils and lungs, and the Mhr genome level in both intraperitoneal injected piglets and intracheal and intraperitoneal injected piglets was higher than intratracheal injection piglets. The real-time PCR method described here should be useful for the study of Mhr infection and distribution in pigs.Lastly, To investigate the synergistic pathogenicity of Mhr with PCV2, thirty 30-day-old piglets were randomly distributed to six groups(n = 5 each): Mhr/PCV2(group 1, inoculated with Mhr and PCV2 1 week later), PCV2/Mhr(group 2, inoculated with PCV2 and Mhr 1 week later), Mhr–PCV2(group 3, inoculated with PCV2 and Mhr concurrently), singular PCV2 group(group 4), singular Mhr group(group 5), and a uninfected control group(group 6). Mild transient lethargy, fever, coughing, inappetence, and decreased daily weight gain were observed in all dual-infected groups and the singular Mhr-infected group. There were significantly higher levels of PCV2 and Mhr antibodies, larger amounts and wider range of tissue distribution of PCV2 antigens and nucleic acids in the dual-infected groups compared to the single-infected and control groups. PCV2 and Mhr dual-infection resulted in significantly more severe macroscopic and microscopic lung lesions and wider PCV2 DNA distribution compared with piglets infected with PCV2 alone. Cytokine detection showed a significant change in tumor necrosis factor-α, interleukin-2, and interleukin-6 levels in the infected groups, especially in the Mhr–PCV2 group, compared with the control group.