| The meningitis was regarded as the characteristic clinical symptoms in Nile tilapia (Oreochromis niloticus) infected by Streptococcus agalactiae. It is important and significant to establish an intraperitoneal infection model for S.agalactiae in Nile tilapia and develop a new clinical and histopathological scoring system to reflect the degree and extent of inflammation as well as the presence of necrosis in the brain.1. Establish a Nile tilapia(Oreochromis niloticus) model of the meningitis in S.agalactiae infection:43 healthy Nile tilapias,were randomly divided into 4 groups:1 control group (n=10) and 3 experimental groups(n=33).These three experimental groups were seperated with different infection doses:1.0×106cfu group (n=11), 1.0×10 cfu group (n=11),1.0×10 cfu group (n=11).We set up a model of meningitis by intraperitoneal injection of experimental tilapia at the doses of 1.0× 107、1.0×10、 1.0×109 colony-forming units (cfu)/mL S.agalactiae,0.1mL/fish. the control group also used the same dose of 0.85% normal saline by intraperitoneal injection.Together with the clinical symptoms, variation of anatomy, the cerebral histopathological changes(cerebrum, optical lobe, cerebellum and medulla oblongata included), and bacteriological screening, we scored these three groups and obtained the best one until each tilapia of these three experimental groups is moribund or dead.The results showed as followed:1.Macropathological changes showed that the infected tilapia died in the second day post infection, however, neurological symptoms were not apparently observed in the dead fish. Survivors developed constant aberrant swimming, exophthalmia and corneal opacity days after the third day.2:Histopathological changes showed that large amount of bacteria stained bluish violet colonized the subarachnoid space and leptomeninges,according to microscopical examination of four regions of the brain. Histopathology revealed the changes of leptomeningitis and meningoencephalitis,including congestion/hemorrhage,edema,loose and thickening in meninges, inflammatory cell neutrophilic leukocytes infiltration, superficial ground substance of brain observed inflammatory cell infiltration and lysis spongy-structure,gliocyte hyperplasiain.and so on.Compared with the scores,we found that the group of 107cfu was the best,which showed significant clinical symptoms the minimum diversity value and the condition of the sick fish contributed to the observational study. We chose the dose of 107cfu to inject tilapia intraperitonealy to develop a model of S.agalactiae meningitis with the percrentage of 100% from the third day post infection.2. The dynamic study on the injury of Streptococcus agalactiae meningitis in Nile tilapia (Oreochromis niloticus):Based on the model of S.agalactiae meningitis pathology in Nile Tilapia, we chose the dose of 107cfu to infect the tilapias.152 healthy tilapias,80 fish used the dose of 0.5×108cfu/mL by intraperitoneal injection,0.2mL/fish.72 control fish also used the same dose of 0.85% normal saline by intraperitoneal injection.After injection, respectively 4h、8h、12h、16h、20h、24h、 28h、32h、36h、40h、44h、48h、52h、56h、60h、64h、68h、72h,4 sampling of the experimental and the negative control group in each set were collected randomly for anatomic observation and the dynamic pathological changes upon the tissue sections.ln the early stage of the meningitis(4-20h), coinciding with the congestion and the hemorrhage of meningeal blood vessels in the tissue, we observed cerebral hemorrhage on postmortem. In the second stage of the meningitis(24-40h), coinciding with swelling of neuron and slight floccule appeared in cerebral ventricle, we observed cerebral edema and flakiness cerebrospinal fluid on postmortem.ln the later stage of the meningitis(44-64h), coinciding with infiltration of neutrophil in the subarachnoid space, we observed cerebrospinal fluid jellied on postmortem.In the moribund stage of the meningitis(68h-72h), coinciding with spongiform degeneration of encephalic ground substance, some led to a liquefactive or suppurative focus, nerve fiber(especially in the medulla oblongata)fracture and lysis, besides, vascular reaction, gliocyte hyperplasiain and neuronophagia,we observed cerebromalacia on postmortem. A little S.agalactiae can be found in 40h, moreover, large amount of S.agalactiae appeared in the subarachnoid space in 72h. According to the time of necrosis occurred in these five parts of the brain, chronologically, S.agalactiae invaded as follows:medulla oblongata (4h)——mesencephalon (52h)——telencephalon (52h)——diencephalon (after 72h)——cerebellum (after 72h).In addition, macrophages occurred as follows:cerebrum(24h) thalamencephalon(32h)——meninges(52h)——medulla oblongata(72h).3. A Preparation on Polyclonal Antibody of Streptococcus agalactiae Hyaluronidase.A pair of specific primers were designed based on the hylB gene of Streptococcus agalactiae GD201008-001 isolated from tilapia (GenBank accession No. CP003810.1 published on the GenBank.First,we cloned the hylB gene,then the cloning vector pMD19-T-hylB was successfully obtained.Expression vector pET32a(+)-hylB was built sequentially and recombinant protein was expressed using BL21(DE3). The results demonstrated recombinant Streptococcus agalactiae hyaluronidase was a protein which has a length of 98.89KDa and the expression condition was at 37℃ for 4h, with 0.1mmol/L IPTG. The protein was then purified with affinity chromatography and biofunctional protein was gained after protein renaturation. The purified protein was used to immune two healthy rabbits,by this way,we obtained the antiserum. After agar diffusion testing, the titer of the anti-serum was 1:16.The antiserum of Streptococcus agalactiae hyaluronidase will be used for immunohistochemical staining in our future research,we will discuss how can S.agalactiae and hyaluronidase of the S.agalactiae contribute to the meningitis. |
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