Screening And Application Of Probiotics From Intestinal Tract Of Chinese Softshell Turtle (Trionyx Sinensis)

The aim of present study is to isolate and identify the potential probiotics from the intestinal tract of Chinese softshell turtle (Trionyx sinensis), and then research the potential probiotics effect, including the effects on the growth and immune function after addition the probiotics into the basic diet. This experiment will provide the foundation for application of probiotics in Chinese softshell turtle farming. The main results are as flows:(1) Through spot inoculation as the first screening and Oxford cup method as the second screening, the bacterial strain Dec-43showed antagonism to the all three indicating strains including Aeromonas hydrophila, A. sobria and A. caviae. The results of bacterial morphological examination, physiological and biochemical experiments of Dec-43were consistent with the physiological and biochemical property of Enterococcus faecium. By extracting the total genomic DNA of the Dec-43, PCR amplifying of the16S rRNA gene with universal primers, obtained a gene segment of1448bp. The sequencing and BLAST analysis results of the segment showed the strains Dec-43was similar to E. faecium and the homology was99%. It indicated the Dec-43was E. faecium. After the single factor test, determined the optimum growth temperature was36℃, the optimum growth salinity was0.6%, the optimum growth initial pH was7, the optimum growth inoculum size was10%. Through the orthogonal experiment, determined the carbon (glucose) and nitrogen (peptone) content was15g/L and10g/L.(2) The Chinese softshell turtles were randomly divided into four groups:the control group (C) fed the basic diet; the experiment groups (E1, E2and E3) fed the basic diet that supplemented the10cfu/g,10cfu/g and10cfu/g probiotics Dec-43respectively. After raised for56days, the specific growth rate (SGR) of all three experiment groups and the relative weight gain (RWG) of the E2group were significantly higher than control group (C)(P<0.05). The activities of blood leukocyte phagocytic and serum lysozyme, myeloperoxiodase (MPO), complement (C3, C4) of the experiment groups were raised to different level compared with the control group. The results suggested that the probiotics Dec-43can encourage the growth and enhance the immune function of T. sinensis. (3) The Chinese softshell turtles of each group were divided into two groups and injected with A. hydrophila or stroke-physiological saline solution after fed56days. And then collected the liver, spleen, kidney and intestine tissues at different times (12,24,36,48,60and72h) and examined the expression level of the target gene (ATF4and CD9) by the method of RT-PCR. The result showed that the expression of ATF4in the liver of E2experiment group was significantly lower than control group (C) at24and48h, but higher than control group only at36h. The expression of ATF4in the spleen of E1experiment group was significantly higher than control group at12,36,48and60h. The expression of ATF4in the kidney of E1experiment group reached the maximum value before control groups, as same as the minimum value. The expression of ATF4in the intestine of E1experiment group was lower than control group and not significant different with the control group. These results showed that through supplemented the probiotics Dec-43in the diet, the inflammation response to outside pathogens infection in the liver and intestine had reduced, the immune response to infection of kidney had be accelerated, and the immune response to infection of spleen had enhanced, these changes contributed to host to eliminate the pathogens and recovery to homeostasis. The expression of CD9in the liver of E3experiment group was significantly higher than control group at12h, lower than control group at24h, and not significant different with the control group at other times. The expression of CD9in the spleen of E1experiment group was significantly higher than control group at12h, lower than control group at72h, and not significant different with the control group at other times. The expression of CD9in the kidney of three experiment groups was significantly higher than control group at all times except60h. The expression of CD9in the intestine of E1experiment group was higher than control group at48h, and not significant different with the control group at60h, and lower than control group at other times. The expression of CD9in the intestine of E2experiment group was significant higher than control group at48and60h, but significant lower or not significant different than control group at other times. The expression of CD9in the intestine of E3experiment group was significant higher than control group at48h but significant lower than control group at12and36h. There was no significant difference between cntrol group and experiment groups at other times. These resulted that supplementing of probiotics Dec-43would accelerate the immune response to pathogens of liver and spleen, and then recovery to homeostasis, as well as promote T cell activation in kidney, and maintain homeostasis of intestine in the initial infection, promote T cell activation in mid-infection and recovery to homeostasis after infection.