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The Iflunces of Aeromonas Hydrophila Strain T0608on the Immune Indexes of Blood of Pelodiscus Sinensis

Author: WangJinJin
Tutor: ZhaoHaiQuan
School: Anhui Agricultural University
Course: Microbiology
Keywords: Pelodiscus sinensis Aeromonas hydrophila strain T0608 blood cellclassification Diff-Quik stain index of serum enzyme
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Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract


Chinese soft-shelled turtle (Pelodiscus sinensis) is very inmportant one in China’seconomic aquatic crops, and China has the most production of Chinese soft-shelled turtlein the world. But in recent years, pathogens of the the turtles are emerging and large-scaleoutbreaks of diseases and death of the turtle happen because the turtle are breeding in largescale and with high density. The common disease of turtle are the skin fester disease, redshell disease, white shell disease, red neck disease, scabies disease, mumps disease,perforation disease, hemorrhage, etc. The reason of these diseases are included pathogenicfungi, bacteria, viruses, parasites and other non-biological factors and unknown ones.These diseases are often disorders, contagious, wide range infecting, with high mortality,and cause serious economic losses to the breeding industry.Hu Dayan obtained a isolate of Aeromonas hydrophila strain T0608from liver, spleenand kidney of the dead turtles. The strain is not the original outbreak reason of viral disease,but is a strong poison one which can attact Chinese soft-shelled turtles. The strain T0608was used in three gradient to infect healthy turtle. Then strain T0608could causesoft-shelled turtle to dead, and the strain could be re-isolated from the dead turtles.In order to establish the sub-lethal dose of infection of T0608strain to turtle, theconditions of breeding Chinese soft-shelled turtles were monitored and minimize thefactors affecting the environments; the different blood sampling methods affecting serumenzymes were discussed, and results showed that EDTA-Na2and heparin could be used forserum enzyme indicators experiment.Three blood smears stain methods for Chinese soft-shelled turtle, Diff-Quik stain kit,Giemsa, and Wright-Giemsa method, were used for hematologic observation. The bloodcell images were captured and analyzed with DP-71digital imaging system and Image-proExpress5.1for the size measuring and blood cells sorting. The blood cells of Chinesesoft-shelled turtle could be clearly distinguished by Diff-Quik stain both for nuclear andcytoplasm and for unique cytoplasmic particles of white blood cells. The size oferythrocyte, neutrophils, eosinophils, basophils were17.86μm×12.43μm,16.26μm×13.67μm,12.28μm×10.98μm and14.63μm×12.83μm, respectively. The percentage ofneutrophils,eosinophils, monocytes, lymphocytes were54.7%,13.72%,6.22%,24.72%and respectively. No significant diffrents were observed among Diff-Quik stain, Giemsastain and Wright-Giemsa stain in blood cell characterization and leukocyte counting. Diff-Quik stain and Giemsa stain showed better results than Wright-Giemsa stain both innuclear and cytoplasm staining and in easier distinguished particles for different whiteblood cells. The Diff-Quik stain, giving better definition for nucleic and particles withmore convenient and faster processing, could be considered a better method for blood cellstain for Chinese soft-shelled turtle, specially for large specimens staining.The AKP stain and PAS stain were also used stain blood cells of Chinese soft-shelledturtles. Alkaline phosphatase-positive particles could be found in mature neutrophil of theturtle, and neutrophils were decreased somewhat wich could be stained with MPO stain;positive granules were all observed in both health and sick turtles.After attacked by strain T0608with sub-death dosage, the number of white bloodcells of turtle increased, in which neutrophils and mononuclear cells increased obviously.Serum alkaline phosphatase, acid phosphatase, aspartate aminotransferase, alanineaminotransferase, superoxide dismutase and lysozyme arose. After attack for96h variousactivities of enzymes in serum of the turtles declined, but still higher than those of thecontrol group. The group attacked by T0608for96h had the highest GPT activity of72.74Carmen-style units having a significant difference with the control group. the activities ofAKP, ACP, SOD and LZM were highest after48hours. The inactivated strains T0608(7.5×108cuf/ml) was used to immune turtles, and the results showed that the AKP activitywas higher than that of the control group after24h. GOT activity of the immuned grouparrived at the highest point in96h, being59.37%higher than that of the control group;GPT got44.87Carmen-style units after24h, and LZM reached to2.74U in96h, all a littlehigher than those of the control group but without significant differences.

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