Isolation And Identification Of Streptococcus Pneumoniae From Rhesus Monkey And Establishment And Application Of Species Specific FQ-PCR Assay For Studying The Invasion Mechanism Of Streptococcus Pneurnoniae In Vivo

1 Isolation and identification of bacteria from Rhesus monkeyThe Streptococcus pneumoniae disease occurred among rhesus monkeys(Macaca mulatta) suddenly and clusters of sudden deaths have been observed in a rhesus monkeys reproduce and breed farm in Sichuan province of China. According to the results of the clinical observation, necropsies, isolation and identification of bacteria and pathogenicity to mice, we conclude that the agent is Streptococcus pneumoniae.2 The development of species specific PCR and FQ-PCR for Streptococcus pneumoniaeStreptococcus pneurnoniae (SP) is one of the important zoonosis. Ply, encoding the cytolysin pneumolysin on the species specific DNA sequence of SP from GenBank, the specific primers which showed 818 bp fragment for PCR,96bp fragment for FQ-PCR, and the species specific PCR method and FQ-PCR method for SP detection was developed initially. Applied this assay to Streptococcus including Streptococcus anginosus ATCC 33397,Streptococcus mitis ATCC 49456, Streptococcus oralis ATCC 35037, and several common pathogenic bacterias of nasopharynx such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis,Enterococcus faecium indicated that it was highly specific to SP, there was no amplification with no SP genera. Also, a series of sensitivity experiments were performed and proved that the detection limit of this method was 6 copies/μL for genomic DNA,8CFU/μL for bacterial number and the standard deviation was 1.8%.The correlation coefficient for the associated standard curve was 1.000 and PCR efficiency was 98.5%, the formula was Y=-3.357X+48.485.Two artificial infected cases which were infected with SP were chosen to identify the accuracy and sensitivity of the PCR and FQ-PCR method after death.Results showed that the detection of heart,liver, brain and lung were identical between bacteria isolating and FQ-PCR. Used this assay to detect the brain indicated that the positive rates were significant difference(P<0.01),the FQ-PCR method was more sensitive. Thus, this FQ-PCR assay provides a more rapid and accurate method for identification of SP than traditional isolation methods. It will help to the isolation and identification of SP,detecting of SP infection suspected cases and investigation of molecular epidemiology.3 Study on the invasion mechanism of Streptococcus pneumoniae with artificial infected miceWe used this FQ-PCR assay to detect genomic DNA of SP for the artificially infected mice and the results showed that:After inoculated by intraperitoneally,the blood was positive at 2 h post inoculation, and the heart, lung, liver, spleen were positive at 4h. All the samples were positive at 8h, except duodenum, jejunum, ileum, cecum, rectum,thymus, pancreas, nose, throat, trachea,and brain. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, kidney, spleen, pancreas, brain and blood containing high concentrations of SP, with copies of SP being～1000-10000 times more than those in other regions. However, all the mice were dead at 36h.After inoculated by nose dripping, the copies of SP was lower than the former route. The lung was positive at 2 h post inoculation, and the blood was positive after 4h. All the samples were positive at 12h,except pancreas, duodenum, jejunum, ileum, cecum,rectum and brain,with copies of SP being-10-100 times more than former infected rout. The copy number of SP DNA in each tissue reached a peak at 24-36 h post inoculation, with the heart, lung, liver, spleen, kidney containing high concentrations of SP than those in other regions.It was still present up to 9 d post inoculation in the spleen, nose, throat, trachea and lung, without causing apparent symptoms.The common of the two infected routes were as followed, nose and lung had the highest copies number in the respiratory system, and it was still present up to 9 d; With the heart and spleen containing the highest concentrations of SP in the interal organs over the 9d period. Spleen was the primary site for invasion in immune organs of normal mice after challenge.The final organ to show a positive result was the brain at 24 h post inoculation, with low concentrations. This study will help to understand the mechanisms of action of SP infection in vivo.