Nghiên cứu bệnh do streptococcus iniae gây ra trên cá chẽm (lates calcarifer) và biện pháp phòng bệnh tt tiếng anh

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Nghiên cứu bệnh do streptococcus iniae gây ra trên cá chẽm (lates calcarifer) và biện pháp phòng bệnh tt tiếng anh

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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Major: Aquacuture Code: 9620301 TRUONG THI HOA STUDY ON Streptococcus iniae INFECTION IN BARRAMUNDI (Lates calcarifer) AND PREVETION METHODS Can Tho, 2019 THIS STUDY WAS ACHIEVED AT CAN THO UNIVERSITY Supervior: Assoc Prof Dr Dang Thi Hoang Oanh The dissertation will be defended at the Doctoral Dissertation Assessment Committee at the University Level At:……………………………………….…………… Time & Date:………………………………………… Reviewer 1: …………………………………………… Reviewer 2: …………………………………………… The dissertation is available at: Learning Recource Center of Can Tho University National library Vietnam PUBLISHED PAPERS OF THE AUTHOR Truong Thi Hoa, Nguyen Ngoc Phuoc, Dang Thi Hoang Oanh, 2018 Isolation and characterization of lactic acid bacteria from brackish fish species antagonistic to Streptococcus iniae isolated from haemorrhagic barramundi (Lates calcarifer) Journal of Agriculture and Rural Development 338:99-106 Truong Thi Hoa, Nguyen Ngoc Phuoc, Dang Thi Hoang Oanh, 2018 Characteristics of Streptococcus iniae infected in barramundi (Lates calcarifer) Can Tho University Journal of Science 54(3B):99-106 i Chapter OVERVIEW OF THE DISSERTATION 1.1 Introduction On barramundi, the diseases caused by S iniae were first reported in 1999 in Australia (Bromage et al., 1999), after that, the diseases were also reported in 2006 in Australia (Creeper and Buller, 2006), in 2010 in Thailand (Suanyuk et al., 2010) The diseases caused by S iniae showed signs of haemorrhagic on skin and fins, popped eyes (Bromage et al., 1999) There are other signs of diseases such as septicemia on skins, haemorrhagic on skin, fins, gills and anus, which were reported in S iniae infected barramundi and tilapia (Suanyuk et al., 2010) The accumulative mortality of this diseases can be as high as 70% in the barramundi fingerlings (Creeper and Buller, 2006) In Vietnam, the diseases caused by S iniae were first reported in 2013 on barramundi raised in Khanh Hoa (Tran Vi Hich, 2014) In Thua Thien Hue, from 2007, in order to prevent the diseases caused by intensive culture of white shrimp, the provincial committee encourage the culture of marine and brackish water fish species such as grouper, red snapper, rabbit fish, barramundi…, as alternatives for shrimp in polutted area In fact, seabas was used as a popular species by local farmer and was proven as a high value fish (Ton That Chat et al., 2010) However, the rapid increase in barramundi culture lead to environmental poluttion and diseases, including the haemorrhagic diseases on barramundi For treatment, antibiotics are widely used to treat bacterial pathogens on aquactic animals The use of antibiotics had brought some advantages, as well as disadvantages, such as the antibiotic-resistance problems (Weston, 1996; Tu Thanh Dung et al., 2008) Therefore, the studies on the bacteria which have antagonistic ability against bacterial pathogens are widely popular In fact, lactic acid bacteria, which have antagonistic ability against bacterial pathogens and provide advantages to the aquatic animal health, such as digestive enzymes (Nirunya et al., 2008) Lactic acid bacteria produces antimicrobial peptides and antifungal substances, bacteriocin which inhibit the development of pathogens (Ringo et al., 2005; Gatesoupe, 2007) Lactic acid bacteria can be isolated from different sources such as fermentative products, intestieve tracts of terrestrial animals and aquatic animals Several studies on instestial tract bacteria of fish from different living environment showed that lactic acid bacteria exist in the instestial tract of fish but not dominate other bacteria in the tract (Ringo et al., 2010) Hence, the addition of lactic acid bacteria to the diet of fish can improve the ability against bacterial pathogens and improve the natural immune defense of fish (Lauzon and Ringo, 2011) However, there was no report about the effect of the addition of lactic acid bacteria to the fish diet to the inspecific immune factors of barramundi From these perspectives, I carried out the thesis entittled: “Study on the diseases caused by Streptococcus iniae on barramundi (Lates calcarifer) and potential prevention solutions” to supply novel information about diseases caused by Streptococcus iniae on barramundi in order to support the sustainable development of barramundi culture 1.2 Objective of the study General aims: to supply novel information about diseases caused by Streptococcus iniae on barramundi in order to support the sustainable development of barramundi culture Specific aims: to determine the pathogenic characteristics of Streptococcus iniae on barramundi and provide potential diagnosis and prevention solutions for this diseases 1.3 Scope of the study This study focus on the haemorrhagic diseases caused by Streptococcus iniae on barramundi in Thua Thien Hue province, to isolate the lactic acid bacteria isolates from the intestinal tracts of rabbit fish, barramundi, tilapia which demonstrated the antagonistic activities to S iniae, as well as determinating the haematology profile and the antagonistic activities of barramundi serum to the pathogen S iniae on barramundi fingerlings 1.4 Contents of the study To determine the pathogenic characteristics of Streptococcus iniae on barramundi To isolate the lactic acid bacteria isolates from the intestinal tracts of rabbit fish, barramundi and tilapia To determine the haematology profile and the antagonistic activities of barramundi serum to the pathogen S iniae on barramundi fingerlings 1.5 Scientific significance of the dissertation This study will supply the information of the pathogenesis characteristics of haemorrhagic diseases caused by Streptococcus iniae on barramundi To assess the effect of the addition of lactic acid bacteria to the fish diet to the inspecific immune factors of barramundi The results of this study can be used to make probiotics for prevention of haemorrhagic diseases caused by Streptococcus iniae on barramundi Besides the applied meaning, this study will help the researcher to improve the research ability and provide a valuable reference for research institutes, universities and students 1.6 New findings of the dissertation The harmorrhagic diseases caused by S iniae was studied and the histology of this diseases in barramundi in Thua Thien Hue province was described Three isolates of Lactobacillus fermentum from the intestinal tracts of barramundi and rabbit fish were recovered, and identified which showed strong antagonistic acitivity to the S iniae L fermentum isolate (C21) was used as supplement to the commercial diet to improve some innative immune system against the S iniae pathogens on barramundi Chapter METHODOLOGY 3.1 Research objects Streptococcus iniae isolates from barramundi (Lates calcarifer) have signs of haemorrhagic diseases Lactic acid bacteria isolates from the intestinal tracts of rabbit fish (Siganus guttatus), barramundi (Lates calcarifer) and tilapia (Oreochromis niloticus) Barramundi (Lates calcarifer) 3.2 Time and date of study This study was carried out from 12/2014 to 8/2018 at the wet lab and microbilogy lab of Department of Fish Pathology, University of Agriculture and Forestry, Hue University 3.4 Methodology 3.4.1 Methodology of determination of pathogenic characteristics of S iniae on barramundi 3.4.1.1 Isolate and identify the S iniae on barramundi Sampling method A total of 87 haemorrhagic fish were sampled at 10 barramundi monoculture farms in Thua Thien Hue province from 01/2016 to 12/2016 Fish sampled showed clinical signs of skin haemorrhagic, popped eyes and septicemia Fish were stored in styryofoam carton and shipped to the Lab for analysis Method of culture, identification and biochemistry tests of bacteria Bacteria was cultured and identified based on the method of Frerichs and Millar, (1993) 16S rRNA sequencing The S iniae identification was also done by 16S rRNA sequencing with primers Sin 1: CTAGAGTACACATGTAGCT(AGCT)AAG and Sin 2: GGATTTTCCACTCCCATTAC according to Zlotkin et al., (1998) The PCR protocol was followed the protocol of Zlotkin et al (1998) with 1X buffer 10X; 1,5mM MgCl2; 200μM dNTPs; 2,5 U Taq DNA polymerase; 0,5μL forward primer (Sin1); 0,5μL reversed primer (Sin 2) and 500 ng DNA sample Thermal cycles PCR is 94°C in 3’; 94°C in 60’’; 55°C in 60’’; 72°C in 90’’; with 35 cycles; 72°C in 10’ The PCR product length is approximately 300 bp The PCR product was then purified by kit KIT Isolate II PCR and Gel (BioLine, Germany) and send to the Macrogen Laboratory – South Korea for sequencing 3.4.1.2 Barramundi challenged with S iniae Fish preparation Barramundi fingerlings, average length of 6.6±0.4 cm/fish, average weight of 7.2±0.5 g/fish The healthy fish were acclimated for 14 days prior to challenge and were fed Nanolis C food (Ocialis, Vietnam) times/day at and 17 hours, at 8% bodyweight each Experimental design Experiment 1: Determination of toxicity of bacteria The experiment on the determination of toxicity of bacteria based on the method of Dilok (2012) The experiment consisted of 11 treatments, including 10 challenged treatments at 109 CFU/mL to the cavity of fish The S iniae bacteria which caused mortality within days of experiment to be considered as highly toxic strains of bacteria for experiment Experiment 2: Determination of 50% lethal dose (LD50 Lethal dose 50) The experiment on the determination of 50% lethal dose based on the method of Reed and Muench (1938) The experiment was conducted in 14 days, fish was checked times/week, fish was checked times a week for cumulative mortality The 50% lethal dose was determined based on the cumulative mortality in each treatment after experiment Experiment 3: The determination of challenge dose of bacteria The challenged experiment of bacteria was determined based on the method of Dilok (2012), (Table 3.4) Table 3.4: The determination of challenge dose of bacteria isolated HTA1 and HTA3 on barramundi Isolates Experiments HTA1 HTA3 Control Fish (fish/tank) 10 10 10 Bacterial dentity (CFU/mL) LD50 LD50 Saline solution Replicates 3 During 14 days of experiment, fish was checked times/week for fish health and the fish with signs of diseases to isolate the bacterial strains and histopathology study 3.4.1.3 The determination of histopathology characteristics of barramundi infected S iniae The barramundi showed signs of diseases and uninfected fish from the experiment for histopathology study The tissue of liver, spleen, kidney, brain of infected diseases and healthy fish was fixed in formalin solution 4% The fixation tissue was then analysed based on the method of Mohamed (2009) The samples were then observed under microscopes 3.4.2 Identification and selections of lactic acid bacteria from gut of marine fishes which have antagonistic activities against S iniae 3.4.2.1 Identification of lactic acid bacteria from gut of seabsss, rabbit fish and tilapia Sampling Barramundi, tilapia and rabbit fish were caught from nature, with adult size At the point of sampling, fish was healthy and show no signs of diseases Fish was then carried to the Laboratory and dissected Bacterial samples from gut was then used for identification Lactic acid bacteria identification method Lactic acid bacteria identification followed the method of Nirunya et al (2008) Lactic acid bacteria identification based on physical and biochemical characteristics Biochemical tests were conducts: Gram test, catalase, oxidase, mobility, spore production, CaCO3 degration and gelatin liquidation Bacteria was then identified based on the methods of Cowan and Steels (Barrow and Feltham, 1993) 3.4.2.2 The determination of antagonistic activities of lactic acid bacteria against S iniae The determination of antagonistic activities of lactic acid bacteria against S iniae was followed the method of Hamza et al (2012) The wells-agar distribution method was used to determine the antagonistic activities of lactic acid bacteria against S iniae The antagonistic activities of lactic acid bacteria against S iniae was followed the method of Galindo (2004) 3.4.2.3 The identification lactic acid bacteria which had antagonistic activities of against S iniae The lactic acid bacteria identification was also done by 16S rRNA sequencing with primers LacF: AGCAGTAGGGAATCTTCCA and LacR: ATTCCACCGCTACACATG according to Nikolaou et al (2011) The PCR protocol was followed the protocol of Nikolaou et al (2011) The master mix included: 50 ng DNA, 10 pmol forward primer, 10 pmol reverse primer, µL PCR buffer (10X), 10 pmol dNTP, 5U Enzyme pfu and PCR water for 50 µL Thermal cycles PCR is 95°C in 5’; 95°C in 45’’; 48°C in 30’’; 72°C in 60’’; with 30 cycles; 72°C in 7’ The PCR product length is approximately 345 bp PCR product was then purified by KIT Isolate II PCR and haematology profile and the antagonistic ability to S iniae NT Number of fish (individual) 20 NT 20 Treatments L fermentum C21 S iniae HTA1 - 1.9x105 CFU/mL/fish 1.9x105 CFU/mL/fish NT 20 10 CFU/g feed NT 20 109 CFU/g feed Replicates 3 3 Identification: S iniae was injected to fish cavity after 14 days of experiment The experiment run for 28 days, the number of red blood cells, total white blood cells, lymphocytes, monocytes, leukocytes, thrombocytes of fish on day 1, 14, 21 and 28 to determine the haematology profile and the antagonistic ability to S iniae 3.4.3.4 Method for the determination of the haematology profile Red blood cells count: according to the method of Natt Herrick (1952) Total white blood cells count: Sample was prepared on lame and was dyed based on Humason, 1979 (cited by Rowley, 1990) Total white blood cells count and other haemolymphs were determined based on Chinabut et al (1991) and Hrubec et al (2000) 3.4.3.5 The antagonistic ability of barramundi serum to S iniae The antagonistic ability of barramundi serum to S iniae was determined based on method of Phuong et al (2007) The S iniae inhibited (Suc (%)) by barramundi serum was calculated as followed: Suc (%) = OD sample - OD blank OD control - OD bank x 100 With: Suc (%): Ratio (%) S iniae inhibited by barramundi serum; OD- Optical Density 3.4.3.6 Determining lysozyme activity in serum The lysozyme activity in serum was determined based on the method of Kumar et al (2007) The lysozyme activity in serum was determined by unit/min/mg protein of serum One unit of lysozyme (U) was determined by the amount of lysozyme absorbed at 450 nm with 0.001 absorb unit/min/mg (U/min/mg) 3.4.3.7 Survivals determiniation Fish survivals was conuted at 14 days post challenge of S iniae, based on the method of (Kumar et al., 2007): Survival (%) = Survival fish after challenge with S iniae Total fish challenged with S iniae x 100 The effective level of L fermentum in diet was calculated based on the protection level (relative percentage of survival - RPS) (%) with method from (Ellis, 1988) 3.5 Data analysis Crude data was input and analysed on Microsoft Excel 2016, and the analysed by ANOVA based on General Linear Model on SPSS version 20 Chapter RESULTS AND DISCUSSION 4.1 Ppathogenic characteristics of Streptococcus iniae on barramundi 4.1.1 Isolate and identify the S iniae on barramundi 4.1.1.1 Result of isolate the S iniae on barramundi A total of 87 haemorrhagic barramundi were collected to isolate bacteria The clinical signs showed that of haemorrhagic barramundi showed include erratic swimming, intra-ocular, corneal opacity and hemorrhage in the eye, at the base of the fins and in the opercula, abdominal sinuses contain mucus, hemorrhagic viscera, kidney swelling, hemorrhagic brain (Figure 4.1) Bacterial isolation from 87 moribund barramundi isolated 42 isolates of Streptococcus spp from the brain, kidney, spleen and liver of fish (20 isolates from the brain 10 (63%), isolates in the kidney (21.4%), isolates in the spleen (16.7%) and isolates in the liver (14.3%) Figure 4.1 Infected fish used for bacterial isolate (A- cornea opacity and hemorrhage in the eye; B- intra-ocular and corneal opacity in the eye, abdominal sinuses contain mucus) 4.1.1.2 Morphological, physiological and biochemical characteristics of the bacterial isolates on barramundi The result of sampling, isolation and culture of bacteria showed that on TSA + plate, after 24 to 48 hours of culture at 28oC, bacteria developed into small colonies with diameters of 0.7 - mm, opaque white, even edges, no pigmentation The result of Gram staining determined Gram (+) bacteria and cocci-shaped (Figure 4.2) Figure 4.2: Colonies morphology and Gram staining of S iniae (AColonies morphology of S iniae on TSA + plate; B- Gram staining of S iniae isolated from barramundi (100X)) The bacterial isolates were non-motile, catalase and oxidase negative, lysin decarboxylase negative, positive for hydrolysis of starch, no growth on the TSB plate containing 11 6.5% NaCl and the TSA plate did not supplement NaCl Results of slide agglutination test by using Slidex Strepto Plus revealed no reaction with A, B, C, D, F, G serotypes of Lancefield group (Table 4.2) On BA + plates, all isolates showed β-haemolys Using the API 20 Strep system test, the isolates tested were positive with hydrolysis of esculin and negative for hydrolysis of hippurate, positive for pyrrolidonyl arylamidase, β-Glucuronidase, alkaline phosphatase, arginine dihydrolase leucine arylamidase, negative for Voges-Proskauer, αGalactosidase, β-Galactosidase Results of determined biochemical characteristics and compared with S iniae isolate by Bromage et al (1999) and S iniae isolate of Rahmatullah et al (2017), 42 isolates of Streptococcus spp isolated from barramundi were identified as S iniae 4.1.2 Challenge of barramundi with Streptococcus iniae 4.1.2.1 Results of determine of bacterial virulence Results of determine of bacterial virulence showed that S iniae HTA1 and HTA3 isolates were fish 100% mortality in days of experiment Therefore, S iniae HTA1 and HTA3 isolates were chosen for from the isolates obtained from moribund barramundi for determine the LD50 4.1.2.2 Results of determine of the lethal dose 50% (Lethal dose 50 - LD50) HTA1 and HTA3 in intraperitoneal (i.p.) injection healthy barramundi were 1.9x105 CFU/mL and 1.5x105 CFU/mL, respectively 4.1.2.3 Results of the determination of challenge dose of bacteria The determination of challenge dose of bacteria isolated HTA1 and HTA3 on barramundi showed that After 48h challenged with the HTA1 or HTA3 isolate by intraperitoneal 12 (i.p.) injection, fish showed the typical clinical signs of diseases such as anorexia, haemorrhagic on body, and fins, exophthalmia eyes and viremia After 72h i.p injection challenged, the mortality was observed The cumulative mortality was the highest at days post challenged, accounted for 76.7% (HTA1 isolate) and 80% (HTA3 isolate), (Figure 4.6) Whereas, in the control group, uninfected fish showed no clinical sign, no death, and no S iniae were found Cumulative mortalities (%) 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 HTA1 HTA3 ĐC 10 11 12 13 14 Days post challenge Figure 4.6: The cumulative mortalities in barramundi in 14 days exposed to S iniae HTA1 and HTA3 isolates by intraperitoneal (i.p.) injection 4.1.2.4 Histopathological characteristics of barramundi infected with S iniae Histopathological analysis of S iniae infected barramundi revealed structural changed in liver, kidney, spleen and brain Necrosis and haemorhagic were observed in liver, kidney, spleen and brain in S iniae infected barramundi under experimental condition 13 Figure 4.7: Liver tissue of barramundi infected with S iniae (H&E staining), (100X); (S iniae in liver tissue of barramundi after days of challenge (arrows)) Figure 4.8: Kidney tissue of barramundi infected with S iniae (H&E staining), (100X); (a- melanomacrophage in the kidney tissue (arrows);b- structure of renal tubules changed in kidney tissue (arrows)) Figure 4.9: Spleen tissue of barramundi infected with S iniae (H&E staining), (100X); (melanomacrophage in the spleen tissue (arrows)) 14 Figure 4.10: Brain tissue of barramundi infected with S iniae (H&E staining), (100X); (S iniae in brain tissue of barramundi after days of challenge (arrows)) 4.1.2.5 The results of identification HTA1 and HTA3 isolates by polymerase chain reaction and the acid nucleic sequencing The isolated of HTA1 and HTA3 were identified via specific 16S rRNA amplification and sequencing The results showed that they are S iniae 4.2 Identification and selections of lactic acid bacteria from gut of brackish fishes which have antagonistic activities against S iniae 4.2.1 Result of isolation of lactic acid bacteria isolates The results showed that 61 lactic acid bacterial isolates were isolated from the gut of adult tilapia, barramundi and rabbit fish All isolates were Gram-positive, rod or coccishaped, non spore formation, non-mobile, negative for catalase and oxidase, CaCO3 degration and no gelatin liquidation (Table 4.7) Table 4.7: Result of isolation of lactic acid bacteria from tilapia, barramundi and rabbit fish Fish Barramundi Tilapia Rabbit fish Quantity (individual) 50 50 50 Isolated tissue Ruột Ruột Ruột 15 Lactic acid bacteria isolates 27 19 15 Figure 4.13 Lactic acid bacteria shape after Gram staining (100X), (A - Rod-shaped bacteria; B - Cocci-shaped bacteria) Table 4.8: Morphological, physiological and biochemical characteristics of the lactic acid bacterial isolated from tilapia, barramundi and rabbit fish Test Colony color Cell morphology Rods Cocci Gram-staining Mobile Spore formation Catalase Oxidase CaCO3 degration Gelatin liquidation Growth in MRS agar 1% NaCl Growth in MRS agar 1,5% NaCl Growth in MRS agar 2% NaCl Growth in TSA 1,5% NaCl Growth in TSA 2% NaCl Growth in TSA 1,5% NaCl Temperature, 4oC Temperature, 28oC Temperature, 37oC Test results of lactic acid bacteria Barramundi Tilapia Rabbit fish (n = 27) (n = 19) (n = 15) Opalescent Opalescent Opalescent white white white 18 + + + + + + + 14 + + + + + + + 13 + + + + + + + + + + + + + Identification: “+”: positive; “-”: negative 16 4.2.2 The results of determine of antagonistic activities of lactic acid bacteria against S iniae The results of determine of antagonistic activities of lactic acid bacteria against S iniae showed that within the collection of these 61 lactic acid bacterial isolates, 28 isolates antagonistic to S iniae, especially, the isolated of C21, D1 and D7 showed strong antagonistic activity to S iniae (inhibition zone larger than or equal to 20 mm in diameter) (Table 4.9) Table 4.9: The results of determine of antagonistic activities of lactic acid bacteria against S iniae Isolates Antagonistic Isolates Antagonistic Isolates Antagonistic activity activity activity D1 D2 D3 D4 D5 +++ ++ + - C7 C8 C9 C10 C11 ++ - D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 C1 C2 C3 C4 C5 C6 R1 R2 R3 R4 R5 + ++ ++ - C12 + R6 +++ C13 R7 C14 R8 C15 R9 + C16 R10 + ++ C17 + R11 C18 R12 C19 + R13 + C20 R14 + C21 +++ R15 + C22 R16 + C23 + R17 + + C24 R18 ++ + C25 R19 + + C26 + ++ C27 Identification: (-): no inhibition zone recorded or inhibition zone less than or equal to mm in diameter; (+): inhibition zone from to mm in diameter; (++): inhibition zone from to 20 mm in diameter; (+++): inhibition zone larger than or equal to 20 mm in diameter 17 4.2.3 The results of identify lactic acid bacteria which had antagonistic activities of against S iniae The results of electrophoresis PCR of C21, D1 and D7 isolates showed successful amplification of the gene with the size of 345bp The isolated of C21, D1 and D7 were identified via specific 16S rRNA amplification, sequencing and the phylogenetic tree The results showed that they are Lactobacillus fermentum 4.3 The effect of the addition of lactic acid bacteria to the fish diet to the the haematology profile and the antagonistic activities against S iniae of barramundi 4.3.1 The results of determined of the haematology profile Blood samples were colleted on day 1, 14, 21 and 28 for hamatological analysis and the ability of fish serum collected to identify the against of S iniae The results showed that, on day 21, the number of reb blood cells and total white blood cells of fish in NT (L fermentum supplement to the barramundi diet at 109 CFU/g in feed and no S iniae i.p injection) and NT (L fermentum supplement to the barramundi diet at 109 CFU/g in feed for 14 days before i.p injection to the barramundi with the dose of 1.9x105 CFU S iniae /mL/fish) were significantly higher than those from NT (no L fermentum supplement to the barramundi diet and no S iniae i.p injection) and NT (no L fermentum supplement to the barramundi diet and S iniae i.p injection to the barramundi cavity after 14 days with the dose of 1.9x105 CFU/mL/fish), (p

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