Preliminary Study On Pathogenic Bacteria Of Whitmania Pigra With Red Swollen Disease

Whitmania pigra Whitman is one of the base of the original animal medicinal leeches as the "Chinese pharmacopoeia"(2015),It is the mainstream varieties of medicinal leeches market in China and also the main raw material of traditional Chinese medicine leech was used to treat blood stasis and syndrome,that can remove blood stasi and cure cardiovascular diseases.At present,the market demand for W.pigra is large,and the wild resources of W.pigra are increasingly exhausted.Artificial breeding has become an inevitable trend.In recent years,a lot of research has been done on the W.pigra’s breathing and digestive physiology,feeding patterns,breeding density and feeding frequency.On this basis,the artificial breeding of W.pigra has achieved initial success.However,under artificial high-density intensive farming conditions,high temperature in summer is prone to bacterial diseases,affecting W.pigra production efficiency and market supply and demand.This experiment aims to identify the pathogenic bacteria of high temperature W.pigra disease in summer,to determine the damage of pathogenic bacteria to different tissues of W.pigra and its effect on the physical and chemical properties of different tissues of W.pigra.The prevention and control provides a scientific basis.The research results are as follows:(1)Bacterial isolation and determination of diseased W.pigra lesions and intestines using 16S rDNA technology and traditional culture methods.The 16S rDNA molecular technique showed that the relative abundance of bacteria in each treatment was the most abundant in the bacteria of Proteobacteria,Bacteroides and Firmicutes.Compared with healthy grass muscles and intestines,the relative abundance of the pathogenic W.pigra muscles,Bacteroides,Pseudomonas,and Desulfovibrio bacteria in the intestinal tract is significantly increased,and is abundant in the diseased W.pigra culture water and sediments.The results showed that the proliferation of Bacteroides,Pseudomonas and Vibrio was one of the causes of bacterial diseases of W.pigra.The bacteria isolated from the muscles and intestines of diseased W.by traditional separation methods were identified as Pseudomonas fluorescens.The return infection showed that the W.pigra infected with Pseudomonas fluorescens had the same symptoms as the natural disease of the W.pigra,and found low concentration.Pseudomonas fluorescens has a low lethal rate to W.pigra,and high concentrations of Pseudomonas fluorescens have a strong lethality against W.pigra,with a mortality rate of 100%.It shows that Pseudomonas fluorescens is one of the pathogens of W.pigra disease as high temperature.(2)The results of tissue sectioning indicate that the bacterial disease has a significant effect on the muscles and intestines of the W.pigra.The bacterial disease causes the intestinal tract to shrink,the inflammation of the inner wall of the intestinal tract causes inflammation,destroys the structure of the tissue,and causes the muscle cells of the W.pigra to be dispersed,the arrangement is not uniform,the intercellular substance is sparse,and the cell gap becomes large;the bacterial disease will be destroyed.The structure of the muscle cell of the W.pigra causes the cell membrane of the W.pigra muscle cell to rupture,the nucleus to dissolve,the cell wall to swell,and the cells to be arranged sparsely.(3)Bacterial diseases have significant effects on the physical and chemical properties of the muscles and intestines of the W.pigra.The antioxidant enzymes(SOD,CAT,POD)of the muscles and intestines of the diseased W.pigra are significantly lower than those of the healthy W,pigra and the intestinal enzymes.(P<0.05);the energy metabolism enzymes(LDH,SDH,MDH)in the muscles and intestines of diseased W.pigra were significantly lower than those in healthy W pigra and intestinal enzymes(P<0.05);diseased W.pigra muscles and intestines The expression levels of heat shock protein 70,90 and lectin were significantly higher than those of healthy W.pigra(P<0.05).The results of bacterial regression experiments showed that the activities of antioxidant enzymes and energy metabolism enzymes in the muscles and intestines of the infected W.pigra showed a trend of increasing first and then decreasing.The antioxidant enzymes and energy metabolism enzymes in the muscles and intestines of the W.pigra infected at the late stage were significantly Compared with the control group,the relative expression of heat shock protein and lectin in the muscles and intestinal tract of infected W.pigra also increased first and then decreased,but the relative expression levels in the late stage of infection were still significantly higher than those in the control group.