Isolation, Identification Of Streptococcus Agalactiae From TIL Apia In Guangzhou And Pathobiologic Characterization

Streptococcus agalactiae was once considered a pathogen of only domestic animals and human, causing smastitis in cows and meningitis in human neonates. S agalactiae is now best known as a cause of meningoencephalitis and septicaemia in fish. On July,2010, a pandemic disease outbreaks in tilapia, which exhibited exophthalmos as the main symptoms in Guangdong. A total of19strains of5agalactiae were isolated from the tilapia. To investigate the genetic characteristics in detail, we detected six main virulence genes of19fish isolates, one isolate from cow and two human isolates of S agalactiae,, including surface immune protein (sip), C5a peptidase (scpB),laminin-binding protein (lmb), alpha protein (bca), beta protein (bac) and hyaluronate lyase (hylB) by PCR. The result showed that there were no differences among the19fish isolates but diversity in fish, cow and human isolates. Genes hylB and sip were detected in all strains, but genes lmb and scpB could be detected in human and cow isolates, and bca and bac were only detected in fish isolates. The results indicated that virulence genes were diverse in the isolates from diferent hosts.We experimentally infected BALB/c mice with S agalactiae GD-001to examine tissue distribution in different time point and to observe the histopathological changes and antigen localization by immunohistochemical staining in brain.6-week-old Balb/c mice were infected with100μL of5×104cfu/ml GD-001, while the control group was injected with phosphatebuffered saline (PBS).The result showed that GD-001was only detected in liver and spleen at3h after infection, but in blood, liver, spleen and brain at6h. Bacterial counts in liver, brain, spleen andblood increased to107～108cfu/g at24h after infection. Bacterial counts in liver were larger than those in other organ. Histopathological findings in brains of the infected mice exhibitted encephalitis with increasing of microglia.GD-001Ags were detected by immunohistochemistry in microglia and hippocampus and nerve fiber of brain.Brain micro vascular endothelial cell (BMEC) bEnd.3and astrocytes M1800 were cocultured in cell culture inserts to construct blood-brain barrier models, and apoptosis or necrosis of BMEC caused by S. agalactiae were examined using Annexin V-FITC/PI. In order to the permeability of S. agalactiae in the blood-brain barrier, were detected. The result of4h leak test and HRP permeability showed that GD-001could increase permeability of blood brain barrier model. The infection with S. agalactiae could cause early apoptosis and late necrosis of BMEC. It is speculated that apoptosis and necrosis of BMEC might be one of the factors that cause damage of the blood-brain barrier.