Yan Chuanjie (3) Xu Zhen (1) Xiao Zhimeng (1) Chen Changfu (1,2) Abstract This paper reports the results of studies on the non-specific immune function and disease resistance of HB-101 Spermophilus elegans on pelodiscus sinensis. A total of 6 groups were set up in the experiment. The amount of HB-101 Essential Ingredients in each group of feeds was 0 mg/kg (control group), 10.0 mg/kg.BW (test group I), and 20.0 mg/kg. BW ( Test II), 30.0 mg/kg.BW (Trial III), 40.0 mg/kg. BW (Trial IV) and 50.0 mg/kg. BW (Trial V). After 30 days of feeding, blood was collected from 10 Chinese sturgeon cubs in each group. The phagocytic activity of leukocytes, serum lysozyme, and complement activity in the blood of Trionyx sinensis were measured. Thirty Chinese were also taken from each group. Indole larvae were artificially infected with Aeromonas hydrophila injection to test their resistance to disease. The results showed that the phagocytic activity of leukocytes in the blood of the Chinese sturgeon juveniles in each experimental group was significantly higher than that in the control group (t-test, P<0.05), whereas the lysozyme activity in the serum of the juvenile Chinese sturgeon was measured in the test groups III, IV and V. All were significantly higher than the control group (t test, P<0.05). Feeding HB-101 can enhance the resistance of the Chinese sturgeon larvae to artificially infected A. hydrophila viable bacteria. The results of the study proved that the amount of HB-101 added to the Chinese sturgeon larvae bait is about 20.0 mg/kg.BW. Preliminary studies by the authors and other authors have confirmed that feeding the right amount of HB-101 Sterile Trionyx sinensis to Chinese sturgeon (Pelodiscus sinensis) can enhance its non-specific immune function [1], suggesting that HB-101 Sterile has an immunostimulant Characteristics, but also the immune system of Chinese sturgeon, like other aquatic animals can be activated by immune stimulants [2 ~ 9]. However, it is unclear whether the nonspecific immune function of Chinese sturgeon induced by HB-101 Sterile Gourd has no effect on its disease resistance. After Robertsen et al. administered β-glucan extracted from yeasts to Salmon salar, the live bacteria challenged the test fish to Aeromonas salmonicida and Vibrio anguillarum. Resistance to infections with pathogenic bacteria such as Yersinia ruckeri was significantly increased [4]. After Anderson et al. used injection and soaking methods to administer β-glucan to Salvelinus fontinalus, the fish's resistance to artificial infection with live A. salmonicida was also significantly enhanced [5]. After Brattgjerd et al. injected yeast glucan into the Atlantic salmon, although the macrophages of experimental fish were found to have increased phagocytic activity and H2O2 production in vitro, the challenge test results showed that the test and control fish There was no significant difference in anti-infectiveness [6]. Jeney et al. used dextran as an adjuvant in combination with the inactivated vaccine of A. salmonicida. The results only proved that it could significantly increase some of the non-specific immune indices of the tested rainbow trout (Oncorhynchus mykiss), but did not treat the tested fish against disease. Determination of force [7]. 1 Materials and methods 1.1 Test Trials and Breeding Conditions of Trionyx sinensis The breeding trials of Trionyx sinensis in this study were carried out in the wintering greenhouse in Liangzihu special aquatic seedlings base of Wuhan Duofu Science and Technology Farm Co., Ltd. In each 24.0 M2 cement rearing pond, there were 600 to 650 Chinese sturgeon cubs weighing an average of about 63.0 g. The temperature in the greenhouse varied between 24°C and 33°C, and the light was artificial light and natural light. Every day, feeds are fed at 9:00 am and 6:00 pm, and the bait is fed with a small amount of water to synthesize the dough and then fed. The amount of feeding is such that the Chinese sturgeon cubs can reach satiation with a small amount of surplus. 4 h after feeding, remove the remaining bait. The bait used was the full-price juvenile bait produced by Wuhan Gaolong Bait Co., Ltd. The control group used no other nutrients except HB-101. 1.2 HB-101 Sterile Essentials The HB-101 Essentials for this study is a powder manufactured by Pacific Life Engineering (Wuhan) Co., Ltd. 1.3 Experimental design Six stocking ponds with the same conditions were randomly selected as test pools in all 60 stocking ponds of the same specifications. The experimental Chinese sturgeon cubs were randomly divided into 6 groups. Since the Chinese sturgeon cubs are difficult to distinguish between male and female in appearance, the sex of the experimental Chinese sturgeon cubs was not distinguished in this study. The amount of HB-101 Sterile Need for each group of feed was 0 mg/kg (control), 10.0 mg/kg.BW (test I), 20.0 mg/kg. BW (test II), and 30.0 mg/ kg.BW (Trial III), 40.0 mg/kg.BW (Trial IV) and 50.0 mg/kg.BW (Trial V). The prepared bait is stored in a cool place, and the bait is prepared 4 hours before feeding. Feed for 30 days continuously. 1.4 At the 2nd d after the end of scheduled continuous feeding for 30 days, blood was collected from each test and control pool randomly by 10 Chinese sturgeon cubs and the blood was collected by the method of decapitation. The blood of each quail was divided into 2 parts. One part was anticoagulated with heparin to prepare anticoagulant for phagocytic activity of phagocytic cells; the other blood was centrifuged at 4°C to prepare serum, which was stored in a -20°C freezer to prepare the following indicators. 1.5 Anatomical Observation and Organ Measurements The larvae of the Chinese sturgeon after blood collection were dissected, and the similarities and differences between the naked eye and the control group were observed. The separated organs and tissues were weighed and measured separately to compare the test. The body index (referred to as "body index") is similar to that of the control group. 1.6 Preparation of phagogalacturin Staphylococcus aureus was inoculated in liquid broth, cultured at 37°C for 48 h, collected by centrifugation, and formalin at a concentration of 0.5% was added to the broth. Inactivated for 24 h at 37 °C, washed three times with sterile saline, and adjusted to a concentration of 1.0108 cells/mL, which was formalin killed S. aureus (F. formalin killed S. aureus, F -SA), used as a phagocytose to detect the phagocytic activity of blood cells. 1.7 Determination of phagocytic activity of blood cells 0.2 mL of F-SA was added to 0.3 mL of anticoagulated blood, shaken, incubated for 45 minutes in a constant temperature water bath at 25°C, and shaken every 10 minutes. Blood smears were pipetted with a pipette. Five samples of each were smeared, fixed with methanol for 10 min, stained with Giemsa staining solution for 1 h, washed with water and dried, examined by microscopy, and the percentage of phagocytic percentage (PP) and phagocytosis of white blood cells were calculated according to the following formula. Phagocytic index (PI). 1.8 Determination of serum lysozyme activity Using lyophilized powder of Micrococcus lysoleikticus as a substrate, the substrate was mixed with a 0.1 mol/L potassium phosphate buffer solution of pH 6.4 to form a suspension of a certain concentration (OD570). ≈ 0.35~0.5). Take 3.0 mL of this suspension and 0.05 mL of the test serum in a test tube and mix. Measure the optical density (A0) at 570 nm. Then incubate at 37°C in a water bath for 30 min. Immediately after removal, place it in an ice bath. The reaction was stopped at 10.0 min and its optical density at 570 nm (A) was measured. The bacteriolytic activity is calculated as follows: Uk=[(A0-A)/A]1/2 1.9 Determination of serum complement C3 and C4 activity was performed using a kit manufactured by Iriscon Biotech Co., Ltd., which was required to add each reagent and serum, respectively, at 37°C in a water bath for 10 min, and using a transmission turbidimetric method in a 722 UV spectrophotometer 340 The optical density (A) of the assay tube and the standard tube was read at the nm wavelength to calculate the activity of complement C3 and C4. 1.10 Resistance to Disease Testing Aeromonas hydrophila BS-1 strain [10] isolated from diseased Chinese sturgeon was inoculated in BHI (Difco) medium and cultured at 28°C for 36 h. The bacteria were collected by centrifugation and adjusted to a concentration of 2.3107 cfu/mL with sterile saline. 30 Chinese sturgeon cubs were randomly collected from each experiment and control group. After being injected with 0.4 mL of A. hydrophila live broth on each sturgeon's litter, the larvae were continuously reared for 21 days. The dead Chinese sturgeon larvae were dissected and the pathogenic bacteria were isolated. The serum of the mouse anti-A. hydrophila strain and the isolated pathogens were used for slide agglutination test to determine whether it was caused by A. hydrophila infection. The mortality rate of each group was calculated, and Relative Percentage survival (RPS) was calculated according to the following formula. 2 results 2.1 Effect of HB-101 Robust Acne on Body Fingers of Trionyx sinensis The results of the measurement of the body weight and body index of Chinese sturgeon clams were shown in Table 1. Control and test groups during the trial Table 1 Weight gain of control and experimental Chinese sturgeon larvae 1) There was no significant difference in weight gain, body weight and visceral ratio, and visceral to hepatic ratio of Chinese sturgeon cubs (t test, P>0.05). In addition, after visual observation, there was no significant difference in body shape and visceral morphology and color between the control and experimental groups. It shows that various doses of HB-101 are robust and have no significant adverse effects on the growth of Chinese sturgeon larvae and the normal physiological functions of the body tissues and organs. Table 1 The phagocytic activity of leukocytes in the blood of experimental Trionyx sinensis 2.3 Changes in Lysozyme and Complement Activity The results of the measurement of lysozyme and complement activity in the serum of test sturgeon juvenile were shown in Table 3. It can be seen from Table 3 that the lysozyme activity in the serum of the Chinese sturgeon cubs fed with the HB-101 Sterile Needs is higher than that in the control group, and the highest lysozyme activity is HB-101 Sterile Gourd Requirement 40.0 mg/ Group VI of kg.BW followed by Group V. Statistical analysis showed that there was a significant difference between the lysozyme activity in serum of Group III, Group VI, and Group V pups of Trionyx glauca and the control group (t test, P<0.05). Table 3 Activity of lysozyme and complement C3, C4 in the serum of test sturgeon juvenile Table 4 Mortality and relative protection rate of Chinese sturgeon larvae after challenge with live bacteria Out of the larvae treated with HB-101 Sterile Needle 1 had higher RPS, while in the diets HB-101 was added to groups 3, IV, and V above 30.0 mg/kg.BW. The mortality rate of juvenile Chinese sturgeon was significantly lower than that of groups I and II with additions of 10.0 mg/kg.BW and 20.0 mg/kg.BW. 3 Discussion As with other animals, the non-specific immune function of Chinese sturgeon also includes humoral immunity and cellular immunity [11]. In the cellular immunity of aquatic animals, the role of leukocytes in the blood is crucial again [12]. The neutrophils and monocytes of fish have been shown to have strong phagocytic ability, which is more important for specific immune function than animals with lower vertebrates.[13] ]. The phagocytosis of leukocytes is an important component of non-specific immunity in animals. Therefore, by measuring the phagocytic function of leukocytes in the blood, it can reflect the body's non-specific cellular immune state [14]. Studies have shown that there is a β-glucan receptor on the macrophages of Atlantic salmon, so it can be stimulated by β-glucan to enhance its phagocytic activity [15]. The results of this study demonstrate that the addition of HB-101 Sterility to the diet of Chinese sturgeon cubs can effectively enhance the phagocytic activity of leukocytes, suggesting that this substance has a stimulating effect on the immune system of Chinese sturgeon clams. However, further studies are needed to determine whether this substance acts on the kind of immune cells of the Chinese sturgeon larvae and initiate the cellular immune process. references 1 Chen Changfu, Yao Yi, Wu Fan et al. Effects of immunopolysaccharide (yeast cell wall) on non-specific immune function of Chinese sturgeon. Chinese Journal of aquatic biology, 2004,28(6): Effect of HB-101 on Non-specific Immune Function and Disease Resistance in Juvenile Soft-shelled Turtles (Pelodiscus sinensis) by Oral Administration Qin Chuanjie1) Xu Zhen1) Xiao Zhimeng1) Chen Changfu1,3) The Therms were divided into 6 groups and were fed with feed supplemented with different ratio of HB-101 , that was 0 mg/kg (control group), 10.0 mg/kg.BW (group I), 20.0 mg/kg. BW (group II), 30.0 mg/kg. BW (group III), 40.0 mg/kg. BW(group IV)and 50.0 mg/kg.BW(group V), respectively. 30 days later, the turtles were blooded, and the phagocytic activity of the leucocytes, serum lysozyme activity, complement activity and disease resistance were monitored. The results The highest complement activity of the serum in group II, III, IV and V were significantly higher than the control group (t. -test, p0.05). The results demonstrated that the best supplementation of the of activite yeast cell in the soft-shelle d turtle is about 20.0 mg/kg. 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HB-101 Striking Need for Chinese Sturgeon
(1) College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;
(2) State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Prof. Chen Changfu Phone u.edu.cn
Key words Trionyx sinensis; HB-101 stunt; lysozyme; complement; disease resistance; Aeromonas hydrophila
In this study, after using the HB-101 Robust Cucumber produced by Wuhan Taifu Industry Co., Ltd. to quantify and feed the Chinese sturgeon cubs, based on the determination of several non-specific immune index changes, the live bacterium was used to test the Chinese sturgeon. The disease resistance of young cubs is reported below.
Percentage of phagocytosis (PP) = (number of cells involved in phagocytosis per 100 phagocytes/100) 100
Phagocytosis Index (PI) = total number of cells in the cell / number of cells involved in phagocytosis
Relative Protection Rate (RPS) = [1-(immunity mortality / control mortality)] 100%
Table 1 Growth of control and experimental juvenile soft-shelled turtle (Pelodiscus sinensis)
1) Mean value of 10 experimental juvenile soft-shelled turtle
2.2 Changes in leukocyte phagocytic activity The results of the PP and PI measurements of white blood cells in the test sturgeon juvenile blood were shown in Table 2. It can be seen from Table 2 that the PP and PI of leukocytes in blood of Chinese sturgeon fed with HB-101 are superior to that of the control group, and the highest of PP and PI is HB-101 with a healthy amount of 40.0 mg/kg.BW. Group IV. Statistical analysis showed that the PP and PI of white blood cells in the test group were significantly higher than those in the control group (t test, P<0.05).
Table 2 Phagocytic activity of leucocytes in the blood of juvenile soft-shelled turtle(Pelodiscus sinensis)
Group phagocytic activity Phagocytic activity
Groups PP PI
I 51.25.6 4.370.46
II 48.56.1 4.690.53
III 49.75.9 4.540.63
IV 50.44.8 5.030.66
V 48.65.3 5.120.74
Comparison with Control 38.84.9 3.590.52
The test results of the activity of complement C3 and C4 in the serum of the Chinese sturgeon cub showed that the activity of complement C3 was higher in the serum of the Chinese sturgeon fed with HB-101, and the highest activity of the complement C3 and C4 was observed. Group V was fed at 50.0 mg/kg.BW, followed by Group IV at 120.0 mg/kg.BW. Statistical analysis showed that there was a significant difference in serum complement activity between the II, III, IV and V groups of Chinese sturgeon puffer and the control group (t test, P<0.05).
Table 3 Lysozyme and complement activity in the serum of juvenile soft-shelled turtle (Pelodiscus sinensis)
Group lysozyme complement activity Complement activity
Groups Lysozyme C3 C4
I 0.2060.044 0.2830.062 0.2990.065
II 0.2280.056 0.3120.057 0.3140.065
III 0.3220.064 0.3350.062 0.3480.053
IV 0.3650.042 0.3720.051 0.3660.072
V 0.3660.054 0.3790.081 0.3720.049
Control Control 0.2040.037 0.2790.067 0.2930.039
2.4 After the artificial inoculation of pathogenic bacteria, the survival rate of the Chinese sturgeon larvae and the experimental group of the Chinese sturgeon larvae were artificially infected by viable bacteria. The deaths of the groups were as shown in Table 4. Can be seen from Table 4
Table 4 Mortality and relative percent survival (RPS) of juvenile soft-shelled turtle (Pelodiscus sinensis), after challenged with live Aeromonas hydrophila
The number of deaths and the number of deaths and the relative protection rate of the group to attack drug abuse
Number of fish Number of dead Mortality Relative percent
Groups Challenged Fish % Survival(RPS)%
I 30 11 36.7 60.6
II 30 10 33.3 64.3
III 30 7 23.3 75.0
IV 30 8 26.7 71.4
V 30 6 20.0 78.6
Compare Control 30 28 93.3
The activity of lysozyme is one of the material bases for determining whether phagocytic cells can be killed by phagocytized pathogens [12]. Therefore, determination of animal lysozyme activity can reflect the animal's non-specific humoral immune status to some extent. Lysozyme activity in fish serum can be increased by exposure of the body to antigenic substances or stimulated by environmental factors [12,16]. The results of this study demonstrated that the addition of HB-101 to the bait of Chinese sturgeon larvae could increase the activity of lysozyme in the serum and range from 10.0 mg/kg.BW to 50.0 mg/kg.BW, with HB. -101 Robust addition of the best quality, test oyster enzyme activity of the juvenile Chinese oyster lice also gradually increased. Since the highest dose used in this study was 50.0 mg/kg.BW, the higher dose of HB-101 Robust also had an inhibitory effect on the immune function of the juvenile Chinese sturgeon. It deserves further study.
Complements in animals are mainly synthesized by hepatocytes and macrophages, while complement in serum is mainly derived from hepatocytes [14]. The results of this study showed that the administration of HB-101 Roppongi can enhance the activity of complement C3 and C4 in the serum of Chinese sturgeon. In the test group, the activity of complement C3 and C4 in the serum of Chinese sturgeon was higher than that of the control group. The results of artificial infection test of A. hydrophila live strains in test and control groups of Chinese sturgeon larvae showed that oral administration of HB-101 robust can increase the RPS of A. hydrophila live bacteria infection and its RPS trend. The results were consistent with the phagocytosis activity of leukocytes in blood of Chinese sturgeon cubs and the rising tendency of lysozyme and complement activity in serum. This result indicates that the phagocytic activity of leukocytes, serum lysozyme, and complement activity in blood of Chinese sturgeon cubs were examined. It can reflect the disease resistance status of Chinese sturgeon clams.
In summary, the appropriate amount of HB-101 Sterility Accredited to promote the non-specific immune function of Chinese sturgeon clams has no significant effect on growth, body morphology and color of Chinese sturgeon clams. It shows that adding proper amount of HB-101 Robust Needs to the bait can increase the resistance of the Chinese sturgeon calf and prevent the occurrence of infectious diseases.
2 Chen Changfu, Chen Yu, Chen Chaoran et al. The characteristics of glucan and its regulation of animal immune function. Journal of Huazhong Agricultural University, 2003,22(1): 95~100
3 Chen Changfu, Chen Yu, Chen Chaoran et al. Research progress on immune defense function and immune prevention of aquatic crustaceans. Journal of Huazhong Agricultural University, 2003,22(2): 197~203
4 Robertsen B, Rorstad G, Raa R. Enhancement of non-specific disease resistance in Atlantic salmon, Salmon salar L., by a glucan from Saccharomyces carevisiae cell wall. J Fish Dis, 1990, 13(5): 391-400
5 Anderson DP, Siwicki A K. Duration of protection against Aeromonas salmonicida in brook trout immunostimulated with glucan or chitosan by injection or immersion. The Prog Fish Cult, 1996, 56(3): 258~261
6 Brattgjerd S, Evensen O, Lauve A. Effect of injected yeast glucan on the activity of macrophages in Atlantic salmon, Salmo salar L., as evaluated by in vitro hydrogen peroxide production and phagocytic capacity. Immunology, 1994, 83(4): 288~294
7 Jeney G, Anderson D P. Glucan injection or bath exposure given alone or in combination with bacterin enhancing the nonspecific defence mechanism in rainbow trout (Oncorhynchus mykiss). Aquaculture, 1993, (116): 315~329
8 Samuel M, Lam TJ, Sin Y M. Effect of laminaran [β(1,3)-D-glucan] on the protective immunity of blue gourami, Trichogaster trichopterus against Aeromonas salmonicida. Fish & Shellfish Immunol, 1996, 6(4) ): 443~454
9 Engstad RE, Robertsen B. Specificity of a beta-glucan receptor on macrophages from Atlantic salmon (Salmo salar L.). Dev Comp Immunol, 1994, 18(4): 397~408
10 Chen Changfu, Zhao Jianpei. Isolation and identification of pathogens of Chinese white peony disease. Journal of Huazhong Agricultural University, 1997, 25th: 101~107
11 Yang Xianle, Zhou Jianguang, Ai Xiaohui et al. Immune response of Chinese sturgeon to T3 bacterin antigen. Animal Journal of the Chinese Journal of Animal Science, 2001,47(2):163~169
12 Sakai M. Current research status of fish immunostimulants. Aquaculture, 1999,(172): 63~92
13 Samuel M, Lam TJ, Sin Y M. Effect of laminaran [β(1,3)-D-glucan] on the protective immunity of blue gourami, Trichogaster trichopterus against Aeromonas salmonicida. Fish & Shellfish Immunol, 1996, 6(4) ): 443~454
14 Ohno Shangren. Modification of the Bio-protection System of βグルカン. Journal of Bacteriology in Japan, 2000, 55(3): 527~537
15 Engstad RE, Robertsen B. Specificity of a beta-glucan receptor on macrophages from Atlantic salmon (Salmo salar L.). Dev Comp Immunol, 1994, 18(4): 397~408
16 Chen Changfu, Luo Yuliang, Cai Bing et al. Effects of raising water temperature on lysozyme activity of grass carp. Chinese Aquatic Sciences, 1996, 3(3):24~30
(1) Fishery College of Huazhong Agricultural University, Wuhan, 430070;
2) State Key Laboratory of Agricultural Microbiology of of Huazhong Agricultural University, 430070)
Keywods juvenile soft-shelled turtle, HB-101, lysozyme, complement, disease resistence, Aeromonas hydrophila
