1 THAI NGUYEN UNIVERSITY THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN MANH CUONG INVESTIGATING SOME BIOLOGICAL CHARACTERISTICS OF STREPTOCOCCUS SUIS CAUSING PIG DISEASES IN THAI NGUYEN PROVINCE AND MANUFACTURING AUTOVACCINE FOR PREVENTION Major: Parasitology & Veterinary Microbiology Code : 9.64.01.04 SUMMARY OF DOCTORAL THESIS THAI NGUYEN - 2019 This thesis was completed at: THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY Scientific advisors: Assoc Prof Dr To Long Thanh Dr Nguyen Van Quang Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the university examination committee At: Thai Nguyen University of Agriculture and Forestry Thai Nguyen University At hour, on date month year The thesis can be found at Libraries: - National library of Viet Nam - Thai Nguyen University of Learning Resource Center Library of Thai Nguyen University of Agriculture and Forestry LIST OF PUBLICATION RELATED TO THE THESIS Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Do Hong Anh (2018), "Determination of some biological characteristics of Streptococcus suis caused disease in pigs in Thai Nguyen province", Veterinary Sciences and Techniques Magazine, 25(6), pp 36 - 42 Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Nguyen Quang Tuyen, Do Hong Anh (2019), "Isolation, serotype determination and toxicity test of Streptococcus suis strains caused disease in pigs in Thai Nguyen province", Veterinary Sciences and Techniques Journal, 26(2), pp 67 - 73 Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, Nguyen Quang Tuyen, Do Hong Anh (2018), "Results of experimental inactivated vaccine preventing pneumonia, athritis diseases for pigs in Thai Nguyen province", Science and Technology Journal of Agriculture and Rural Development, November's volume, pp 130 135 INTRODUCTION Urgency of the thesis Pig production in Thai Nguyen province in recent years has developed rapidly quantity and improved quality, meeting the demand of pig product consumption in the province and export-orientation, contributing to job creation and increasing income for animal producers in the province, therefore it plays an important role in the economic development of the province and has become a leading livestock industry to be interested in developing Pig production in Thai Nguyen is mostly small scale farms, so it faces many difficulties, especially in the field of disease control, which greatly affects the efficiency of livestock production In addition to infectious diseases that occur in pigs such as foot and mouth disease, swine fever, PRRS, pneumonia, arthritis in pigs caused by Streptococcus suis (S suis) also occurs quite commonly leading to economic loss for farmers The disease is caused by S suis in pigs with pathological manifestations such as hemorrhage, pneumonia, arthritis, encephalitis resulting in death, especially in the period of post-weaning piglets, affecting weight gain, animal breed quality and increasing the rate of breeding program S suis plays an important role in respiratory disease and arthritis in pigs Investigation of Nguyen Thi Noi and Nguyen Ngoc Nhien (1993) on the respiratory bacterial flora of 162 pigs with respiratory infection accompanied with coughing showed that Streptococcus accounted for 74.0% Investigation results of Do Ngoc Thuy et al (2009b) from healthy pigs raised at pig farms, which were taken to slaughter houses which expressed symptoms of pneumonia, PRRRS or arthritis, encephalitis, S suis was isolated from all of them with different rates Among the S suis strains isolated, important and common pathogenic serotypes ,7 and were found S suis not only causes infection in pigs, it also causes dangerous diseases in humans In Vietnam, many cases and deaths have been recorded due to streptococcal infection According to statistics of the general department of Preventive Medicine (Ministry of Health) in 2017, in the country 171 cases of S suis in pigs were recorded in the whole country, 14 of which died In particular, in june -2018 in Thai Nguyen province, there were cases suffering from S suis infection (Phuong Nam, 2018) The affected people confirmed that they had contact with sick pigs in feeding, slaughtering, eating pork or blood pudding infected with Streptococcus suis Derived from the practical situation of production and food safety at present, aiming at better understanding of pneumonia and arthritis in pigs associated with S suis, which is a scientific basis for thesising measures of effective prevention and treatment, an urgent requirement, making contribution to improvement of production efficiency, increasing income for pig producers and protecting public health, we carried out the project: "Investigating some biological characteristics of Streptococcus suis causing pig diseases in Thai Nguyen province and manufacturing Autovaccine for prevention" Objective of thesis - Identifying some biological characteristics of S suis causing pneumonia and arthritis in pigs raised in Thai Nguyen province - Study on production of Autovaccine using S suis strains isolated for prevention of pneumonia and arthritis in pigs and trials of effective treatment regimens for pneumonia and arthritis caused by S suis, making contribution to promoting pig production in Thai Nguyen province The scientific and practical significance of the thesis 3.1 Scientific significance - It is a systematic investigation of biological characteristics of S suis strains in pigs raised in Thai Nguyen - Investigation of manufacturing Autovaccine using S suis strains isolated for effective prevention of pneumonia, arthritis of pigs raised in Thai Nguyen and recommendation of effective treatment regimens of pneumonia, arthritis associated with S suis - Supplement and enrichment of scientific data, using as reference in teaching and thesising of pathogenic S suis in pigs 3.2 Practical significance - Trials of autovaccination of pigs raised in Thai Nguyen province has contributed to decreasing the rate of pigs suffering from pneumonia, arthritis caused by S suis - Applying the treatment regimens for pneumonia, arthritis in pigs reaches high efficiency, contributing to increased income for animal producers and sustainable development of pig husbandry in Thai Nguyen province - The results of the thesis are a scientific basis to formulate effective measures for prevention and treatment of diseases caused by S suis in pigs New contributions of the thesis: - A systematic and complete investigation of S suis causing pig diseases in Thai Nguyen province - Autovaccine that has been successfully manufactured by isolated S suis strains can be effectively used for prevention pigs from pneumonia arthritis; the treatment regimens of pigs can be applied in practice of pig production in Thai Nguyen Chapter REVIEW OF LITERATURE Chapter CONTENTS, MATERIALS AND METHODS 2.1 Objects and location 2.1.1 Objects of the thesis - S suis isolated from pigs that infected by pneumonia and arthritis - S suis Autovaccine to prevent pigs from pneumonia and arthritis 2.1.2 Study location - Pig farms and farmer household in Thai Nguyen province which were suspicious of being infected by S suis - Division of Microbiology, National Veterinary Diagnostic Center, Department of Animal Health - Institute of Life Science, Thai Nguyen University 2.1.3 Study period -From 2015 to 2018 2.2 Contents of the thesis 2.2.1 Investigating the situation of pneumonia and arthritis in pigs raised in Thai Nguyen - Identifying prevalence of affected pigs and motarlity of pigs with pneumonia and arthritis over the years in some districts and cities of Thai Nguyen province from 2015-2017 - Investigating the rate of prevalence and mortality of pigs caused by pneumonia, arthritis in pigs raised in Thai Nguyen from 2015-2017 at different ages and seasons 2.2.2 Investigating some biological characteristics of isolated S suis strains - Determining some biological and chemical characteristics of S suis strains isolated - Testing susceptibility of S suis strains isolated to some antibiotics - Serotyping isolated S suis strains - Determining virulent factors of isolated S suis strains 2.3.3 Investigating exprimental inactivated Autovaccine manufacture with alumminium adjuvant from isolated S suis strains - Selecting S suis strains used for experimental Autovaccine manufacture - Investigating Autovaccine produced from selected S suis strains - Determining immune response of pigs to experimental Autovaccine 2.2.4 Investigation of prevention and treatment of arthritis, pneumonia in pigs caused by S suis - Experimenting Autovaccine produced from isolated S suis for prevention of pneumonia and arthritis in pigs - Using treatment regimens of pneumonia and arthritis in pig caused by S suis 2.3 Materials - Samples were collected from lung, retropharyngeal fluid, synovial fluid, cerebrospinal fluid of sick or dead pigs with symptoms and lesions suspicious of pneumonia or S suis infection such as fever, anorexia , dyspnea, cough, swollen joints, nerve signs - Healthy white mice (18-20g/mouse); - Healthy piglets at the age of 5-6 weeks were not vaccinated with Autovaccine against pneumonia, arthritis caused by S suis - Media and chemicals used according to the thesis procedure of the division of Microbiology of the Veterinary Institute and the National Veterinary Diagnostic Center and the Animal Health Department - API 20 Strep kit system for determining biochemical characteristics and nomenclature of S suis (BioMérieux, Marcy l’Etoile, France) - Antibotic assay paper for testing antibiotic sustibility produced by Oxoid argency (UK) - Chemicals and bio-products used for agglutination reactions, serology and PCR technique, IHA reaction 10 - Antibiotics: Cefanew-LA, Marflo-45%, Marphamox-LA and Gluco-K-CNamin produced by Marphavet Veterinary phar maceutical Company - Standard strains of S suis and corresponding standard sera were provided by the National Veterinary Diagnostic Center, department of Animal Health, used for reference of strains isolated using PCR and serology - Common laboratory instruments, inoculation chamber, autoclaves, centrifuges, shaking machines, PCR technique used for investigation of bacteria belonging to the Central Veterinary Diagnostic Center, Animal Health Department and Institute of Life Sciences of Thai Nguyen University 2.4 Methods of the thesis 2.4.1 Method of epidemiological thesis Using methods of descriptive epidemiology and practical epidemiology of Nguyen Nhu Thanh (2001) 2.4.2 Sampling method - Samples were collected from retropharyngeal fluid, syncvial fluid: Using a sterile cotton swab to place deeply in the throat, and the inflamed joint of sick pigs then place in cotton swab tube, stored at 0C and transferred to the laboratory for cultture and isolation of bacteria - Lung samples from sick or dead pigs that expressed signs and lesions suspicious of S suis infection were placed in sterile nylon bags, stored at 40C and brought to the laboratory immediately for culture and isolation of bacteria 2.4.3 Methods of the thesis - Methods of culture, isolation and testing of bacteria were carried out according to the routine methods of Department of Microbiology, National Veterinary Institute, National Veterinary Diagnostic Center and Department of Animal Health - PCR technique was used to identify genes encoding some virulence factors and S suis pathogenic serotypes according to Wisselink H.J et al (2002b), Silva L.M et al (2006), Do Ngoc Thuy and Le Thi Minh Hang (2009a) - LD50 calculation method according to Reed L.J & Muench H (1938) - Determination of virulence of S suis isolated in experimental animals of Sawade (1985) - Determination of susceptibility to antibiotics of S suis isolated from laborratory animals according to Bauer A.W (1966) 10 18 The results of our thesis were in agreement with those of some authors such as Cu Huu Phu (2011), identified S suis serotype strains in pigs with PRRS at the rate of 58.33 % and Le Van Duong et al (2013) when determinating serotype of S suis isolated from samples of pigs with PRRS in Bac Giang, serotype accounted for the highest rate (56.29%), followed by serotype (17.03%) and the lowest was serotype (5.18%) 3.2.6 The results of determination of gene encoding virulent factors of isolated S suis The thesis results showed that among the genes encoding the virulence factors, arcA gene carried by S suis strains accounted the highest percentage (75.5%), followed by mrp gene (71.1%), sly gene (62.2%) and epf gene (57.7%) types of gene combinations encoding virulence factors were identified including: arc/sly, arcA/mrp, arcA/ epf, arcA/mrp/sly, arcA/mrp/sly and arcA/mrp/sly/epf in 45 S suis strains studied Our thesis results were similar to those of some authors such as Do Ngoc Thuy and et al (2009) from healthy pigs and pigs with pneumonia symptoms, PRRS arthritis symptoms, or encephalitis from which S suis were isolated at different rates In 211 S suis strains studied 18 serotypes of which were identified, most commonly were serotype 2, 7, and combinations of virulence genes Investigation of Cu Huu Phu et al (2013) showed that serotype strains carried the most gene combinations which were scatteredly distributed in combinations: arcA, arcA/sly; arcA/mrp; arcA/mrp/sly and arcA/mrp/sly/ epf Results of PCR used for determining genes encoding virulence of S suis strains isolated were shown in Figure 3.2 Notes: Marker: 100 bp 18 Well 1: arcA Gene Well 2: mrp Gene 19 Well 3: sly Gene Well 4: epf Gene Figure 1: Results of PCR used for determining genes encoding virulence of S suis strains isolated 3.2.7 Result of investigating some biochemical characteristics of S suis strains selected after passage cultures From different kinds of pigs such as sucking piglets, postweaning piglets, porkers, gilts and sows, we selected S suis strains representing the pathogenic serotypes in pigs, including: serotype strains (S.TN2-1, S.TN2-2, S.TN2-3); strains of serotype (S.TN7-4, S.TN7-5, S.TN7-6) and strains of serotype (S.TN9-7, S.TN9-8, S.TN9-9) distributed in districts/cities of Thai Nguyen province The selected S suis strains selected after passage cultures, were found to be stable in biochemical characteristics and in accordance with the evaluation criteria according to the S suis disease diagnosis procedure in pigs (TCVN 8400-2: 2010) These are important characteristics of a master seed in propagation for working seed because they can ensure and retain their biological characteristics through different working seed batches 3.2.7 Results of virulent examination of some selected S suis strains after passage cultures Table 3.7 showed that after passage cultures of S suis strains belong to serotype 2, 100% of the experimental mice died after 24 hours, of which 66.67% of mice died from 12 -24 hours and 33.33% strains from 18-24 hours The strains of S suis serotype also killed 100% of experimental mice in the period of 12-24 hours, of which 33.33% of strains caused 12-24 hours of rat death and 66.67% of strains 1824 hours Meanwhile, only 33.33% of tested serotype strains killed 100% of experimental mice after 24-36 hours of injection and 66.67% of strains killed 50% of mice within the previous 36-48 hours after injection Table 3.7: Results of virulent examination of some selected S suis strains after passage cultures Strain letter S-TN2-1 19 Numbe Injectio r of Serotyp n dose mice e (ml/ injected mouse) (mouse) 2 0.5 Results Numbe Time r of period Percent dead for death (%) mice (hour) (mouse) 2/2 18 - 24 100 Reisolation + 20 S-TN2-2 S-TN2-3 S-TN7-4 S-TN7-5 S-TN7-6 S-TN9-7 S-TN9-8 S-TN9-9 2 7 9 2 2 2 2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 2/2 2/2 1/2 2/2 1/2 2/2 2/2 2/2 12 - 24 12 - 24 < 48 24 - 36 < 36 12 - 24 18 - 24 18 - 24 100 100 50 100 50 100 100 100 + + + + + + + + All of dead mice slaughtered showed that the lesions were similar, for example presence of abscess at the injection sites, pneumonia and lung congestion, swollen soft, heart and pericardial effusion When the bacteria were isolated again, it was possible to obtain pure S suis from heart blood Thus, it can be seen that serotype 2, and strains were the three serotype types that dominated among S suis strains isolated from pigs with pneumonia and arthritis in Thai Nguyen that were high virulent strains These strains can cause death of mice shortly after passage cultures This was a very important feature for screening and selection of bacterial strains to produce Autovaccine Based on the formula of LD50 calculation of Reed L.J & Muench H (1938) we determined LD50 of S suis isolated as follows: LD50 of serotype = 4.5 x 10 7; LD50 of serotype = 4.9 x 10 and LD50 of serotype = 4.8 x 107 3.3 Study results of experimental manufacturing Autovaccine from S suis strains isolated 3.3.1 Selection of S suis strains to produce experimental Autovaccine From the thesis results on biochemical characteristics, S suis serotyping and virulence of S suis strains isolated in pigs in Thai Nguyen were determined, we selected serotypes 2, and to produce inactivated Autovaccine with aluminum adjuvant Selected serotypes accounted for high percentage of all serotypes isolated that expressed gene encoding the virulent extracellular factor (EF-epf), the protein that was muramidase releasing protein (MRP), hemolytic factor Suilysin (SLY-sly) and arginine deiminase enzyme (ARC-arcA) that were genes producing toxins that play an important role in pig disease In addition, 20 21 the serotypes above were all highl virulent ones, causing death of laboratory mice within 12-36 hours 3.3.2 Autovaccine manufacturing for preventing pigs from S.suis 3.3.2.1 Results of cultivating S suis in broth to produce experimental Autovaccine The serotypes 2, and were cultured and shaken (300-500 times/minute) separately for 24 hours, the minimum density was 1.64 x 108 bacteria in ml of broth After that, it was inactivated with formalin at the percentage of 0.3% and aluminum adjuvant was added at the rate of 1/5 (20%) according to the procedure of manufacturing inactivated vaccine with aluminum adjuvant of the Veterinary Institute In order to test experimental Autovaccine procedure of testing inactivated vaccine with aluminum adjuvant was used according to TCVN 8684: 2011 and QCVN 01: 187-2018 Table 3.8: Results of bacteria counting in S suis broth used for experimental Autovaccine production Autovaccine batch Batch I (1.5 liters) Batch II (1.5 liters) Batch III (1.5 liters) Number of bacteria /1 ml of broth Total Average Broth Broth Broth Average number Average number Average number number of of bacteria /1ml of of bacteria /1ml of of bacteria /1ml of bacteria/1m l of broth broth broth broth 1.65 x 108 1.63 x 108 1.64 x 108 1.64 x 108 1.71 x 108 1.68 x 108 1.69 x 108 1.69 x 108 1.72 x 108 1.70 x 108 1.71 x 108 1.71 x 108 Table 3.8 showed that after culture period, there were average bacteria concentration of 1.64 x 10 to 1.71 x 108 bacteria/ml in broth from Autovaccine batches which were produced experimentally from the S suis strains isolated According to QCVN-01-187: 2018, number of bacteria that present in ml of vaccine to produce the vaccine must reach at least 1.5 x 108 bacteria/ml, so the above batches reached the criteria Based on the thesis results of Torremorell M (1997), we calculated the Autovaccine injection dose for pigs to be 2.5 ml / pig, equivalent to the average bacterial density of about 4.2 x 108 CFU/ml 3.3.2.2 Results of testing culture purity of S suis broth used to produce Autovaccine Testing culture purity was carried out simultaneously with the individual culture steps of each S suis strain from master seeds to working seeds The results showed that the broth used to produce 21 22 Autovaccine batches met the criteria of purity In media such as meat broth, liver broth, blood agar, MacConkey agar on which were cultured different S suis strains (S-TN2-2, S-TN7-5 and S-TN9-7) in all experimental Autovaccine batches: I, II and III were found only S suis growth 3.3.2.3 Results of sterility testing of the experimental Autovaccine The results showed that the experimental Autovaccine batches met the criteria for sterility at all stages after inactivation with 0.3% formalin, after adding 20% aluminum adjuvant and after bottling 3.3.2.4 Results of safe testing for experimentally manufactured Autovaccine: The results showed that Autovaccine was twice as high as the recommended injection dose after 21 days, all experimental pigs survived and did not result in any local or systemic reactions to the pigs, none of pigs showed any local signs such as inflammation or red ness and swelling at the injection site or systemic symptoms such as fever and anorexia The experimental pigs were healthy, eating normally, none of pigs expressed local reactions such as inflammation, swelling, and redness Thus, S.suis inactivated Autovaccine with aluminium adjuvant reached safety standards as stipulated 3.3.2.5 Results of effective experimental Autovaccine testing for laboratory animals - Challenged with S suis serotype 2: 21 days after the first Autovaccine injection, all experimental and control mice were challenged with S suis serotype at dose of 10LD50 (LD50 = 4.5 x 107 CFU) The results showed that there were10/10 mice surviving in all groups vaccinated with Autovaccine after 10 days, and protection reached 100% whereas in the control group, mortality was 100% 18 to 24 hours after challenge - Challenged with S suis serotype 7: 21 days after the first Autovaccine injection, all experimental and control mice was challenged with S suis serotype at dose of 10LD50 (LD50 = 4.9 x 107 CFU) The results showed that after 10 days there were 9/10 mice surviving in groups immunized with Autovaccine (group I and II), reaching the protection rate of 90%, in the third group, 10/10 mice survived reaching protection rate of 100% In control groups, the mortality was 100% during 18-24 hours 22 23 - Challenged with S suis serotype 9: The experiment was conducted in the same way as the serotype above, 21 days after the first Autovaccine injection, both experimental and control mice were challenged with S suis serotype at dose of 10LD50 (LD50 = 4.8 x 107 CFU) The results showed that after 10 days, there were 10/10 mice surviving in groups immunized with Autovaccine (group I and III), reaching the protection rate of 100%, in group II there were of 10 mice surviving reaching protection rate of 90 % In control group, the mortality was 100% during 18-24 hours Thus, all batches of experimental Autovaccine manufacture were sterile, safe and efficacious for laboratory mice 3.3.3 Results of determination of experimental Autovaccine immune response for preventing pigs from S suis 3.3.3.1 Results of antibody titre determination of the experimental pigs 30 days after autovaccination Table 3.9: Results of antibody titre determination of the experimental pigs 30 days after autovaccination Antigen used for testing S suis serotype S suis serotype S suis serotype Number of pigs tested by serums (pig) Exprimental Control Exprimental Control Exprimental Control 35 35 35 Number of serum samples tested 35 35 35 1/16 Number of positive samples (+) 35 35 35 Percen t (%) 100 100 100 Antibody titre 1/32 Number of Percen positive t samples (%) (+) 27 77.1 0 26 74.2 0 27 77.1 0 Number of serum samples tested 35 35 45 Number of serum samples tested 35 35 35 1/64 Number of positive samples (+) Percen t (%) 22.8 17.1 20.0 Table 3.9 showed that there was a good immune response in all pigs immunized with Autovaccine to the antigens of three bacterial strains used for vaccinne production including S suis serotype 2, and antigens Agglutination occured in 100% of serum samples at dilution of 1/6 and aglutination was determinated in serum samples of many pigs at dilution of 1/32 and 1/64 Specifically: With serotype antigen: At dilution of 1/16, agglunating antibody titre was determinated in100% of pigs , at dilution of 1/32, in 77.1% and at dilution of 1/64 in 22.8% With serotype antigen: At dilution of 1/16, agglutinating antibody titre was determinated in 100% of pigs tested; at dilution of 1/32, in 74.2% and at dilution of 1/64, in 17.1% With serotype antigen: At the dilution of 1/16, agglutinating antibody titre was determinated in 100% 23 24 of pigs tested , at dilution of 1/32, in 77.1% and at at dilution of 1/64 in 20.0% 3.3.3.2 Results of antibody titre determination of the experimental pigs 60 days after autovaccination Table 3.10: Results of antibody titre determination of the experimental pigs 60 days after autovaccination Antigen used for testing Number of pigs tested by serums (pig) S suis serotype Exprimental S suis serotype Exprimental S suis serotype Exprimental Control Control Control 5 5 5 1/16 Number Number of of serum positive samples samples tested (+) Percen t (%) Antibody titre 1/32 Number Number of of Percen serum positive t samples samples (%) tested (+) 1/64 Number Number of of serum positive samples samples tested (+) Percen t (%) 35 35 100 35 32 91.4 35 12 0 0 34.2 35 35 100 35 30 85.7 35 25.7 0 0 0 35 35 100 35 31 88.5 35 10 28.5 0 0 0 Table 3.10 showed that the Autovaccine stimulated pigs to produce good immunity at the time of 60 days after immunization with antigens from bacterial strains including S suis serotype 2, and 9, which agglutination occurred in 100% of serum samples of pigs at serum dilution of 1/16 and aglutination reaction increased in serum samples of most pigs at dilution of 1/32 and 1/64 compared to the first test after 30 days Specifically: With serotype antigen: At dilution of 1/16, agglutinating antibody titre was determinated in 100% of pigs; at dilution of 1/32, in 91.4%, increasing by more than 14.3%; At dilution of 1/64 in 34.2% increasing by more than 14.4% With the serotype antigen: At dilution of 1/16 agglutinating antibody titre was determinated in 100% of serum samples; at dilution of 1/32, in 85.7% increasing more than 11.2%; at dilution of 1/64, in 25.7% increasing by 8.3% With the serotype antigen: At dilution of 1/16 agglutinating antibody titre was determinated in 100% of pigs; at dilution of 1/32, in 88.5% increasing by 11.4%; at dilution of 1/64, in 28.57%, increasing by 8.0% 3.3.3.3 Results of antibody titre determination of the experimental pigs 90 days after autovaccination Table 3.11: Results of antibody titre determination of the experimental pigs 90 days after autovaccination 24 25 1/8 Antigen used for testing S suis serotype S suis serotype S suis serotype Number of pigs tested by serums (pig) Exprimental Control Exprimental Control Exprimental Control 35 35 35 Number of positive samples (+) 35 35 35 Percent (%) 100 100 100 Antibody titre 1/16 1/32 Number Number of Percen of Percen positive t positive t samples (%) samples (%) (+) (+) 33 94.2 22 62.8 0 0 31 88.5 19 54.2 0 0 32 91.4 20 57.1 0 0 1/64 Number of Percent positive (%) samples (+) 17.1 0 11.4 0 14.2 0 Table 3.11 showed that at the time of 90 days after autovaccination of pigs, aglutination reaction occured in 100% of pigs tested at serum dilution 1/ and from dilution of 1/16 to 1/64 the agglutination rate of serum antibody in experimental pigs decreased gradually compared to the second test after 60 days Specifically: With serotype antigen: At serum dilution of 1/8, agglutinating antibody titre was determiated in 100% of pigs; at serum dilution of 1/16, in4.2%, decreasing by 5.8%; at the dilution of 1/32, in 62.8% decreasing by 28.6%; at the the dilution of 1/64, in 17.1% decreasing by 8.6% With serotype antigen: At serum dilution of 1/8, agglutinating antibody titre was determiated in 100% of pigs; at the dilution 1/16, in 88.5% decreasing by 11.5%; at the the dilution of 1/32, in 54.2% decreasing by 31.5%; at the dilution of 1/64, in 11.4%, decreasing by 14.3% With serotype antigens: At the dilution of 1/8 agglutinating antibody titre was determiated in 100%; at the dilution of 1/16, in 91.4% decreasing by 8.6%; at the dilution of 1/32, in 57.1% decreasing by 31.4%; at the dilution of 1/64 in 14.2% decreasing by 14.3% 3.3.3.4 Results of antibody titre determination of the experimental pigs 120 days after autovaccination Table 3.12 showed that 120 days after immunization with Autovaccine, the antibody titre in the experimental pigs from serum dilution of 1/16 to 1/64 aglutination rate of serum antibody in the experimental pigs decreased gradually compared to the third test 90 days after immunization with Autovaccine Specifically: With serotype antigen: At serum dilution of 1/8, agglutinating antibody titre was determinated in 100% of pigs; at the dilution of 1/16, in 68.5%, 25 26 decreasing 25.7%; At the dilution of 1/32, there were 28.5%, at the dilution of 1/64, negative titre of agglutinating antibody was determinated With serotype antigen: At the dilution of 1/8, agglutinating antibody titre was determinated in 100% of pigs; at the dilution of 1/16, this percentage was 62.8%, decreasing by 25.7%; at the dilution of 1/32: in 22.8%; At the 1/64 dilution, negative titre of agglutinating antibody was determinated With serotype antigens: At the dilution of 1/8, this percentage was 100%; at the dilution of 1/16, there were 65.7%, decreasing by 25.72%; at the dilution of 1/32, this percentage was 25.7%; At the dilution of 1/64, negative titre of agglutinating antibody was determinated Table 3.12: Results of antibody titre determination of the experimental pigs 120 days after autovaccination Antigen used for testing S suis serotype S suis serotype S suis serotype Number of pigs tested by serums (pig) Exprimental Control Exprimental Control Exprimental Control 35 35 35 1/8 Number of Percent positive (%) samples (+) 35 35 35 100 100 100 Antibody titre 1/16 1/32 Number Number of of Percent Percent positive positive (%) (%) samples samples (+) (+) 24 68.5 10 28.5 0 0 22 62.8 22.8 0 0 23 65.7 25.7 0 0 1/64 Number of Percent positive (%) samples (+) 0 0 0 0 0 0 The results of immune response in pigs immunized with Autovaccine produced from three antigens of S suis serotype 2, and showed that the rate of pigs with immune response increased gradually on 30 to 60 days after immunization with Autovaccine and was maintained until 90 days after immunization with Autovaccine, then the amount of antibodies in the blood gradually decreased Experimental Autovaccine stimulated the production of antibodies that were good for experimental pigs, producing antibodies meeting requirement of an Autovaccine 3.4 Results of experimental Autovaccine for preventing pigs raised in Thai Nguyen from pneumonia and arthritis caused by S suis 3.4.1 Results of determining the efficacy of Autovaccine for pneumonia and arthritis of pigs 26 27 Table 3.13: Results of experimental Autovaccine for preventing pigs raised in Thai Nguyen from pneumonia and arthritis caused by S suis Pneumonia Index Location (district/city) Phu Binh district PhoYen district Thai Nguyen city Total NonPhu Binh district PhoYen district autovaccinated Thai Nguyen city pigs Total Autovaccinate d pigs Number of pigs monitored (pig) 760 750 680 2.190 810 730 690 2.230 Number of pigs infected by pneumoni a (pig) Percent (%) 45 41 31 117 156 147 126 429 5.92 5.46 4.55 5.34 19.25 20.13 18.26 19.23 Arthritis Number of pigs infected Percent by (%) arthriti s (pig) 31 4.07 27 3.60 21 3.08 79 3.60 105 12.96 101 13.83 89 12.89 295 13.22 Table 3.13 showed that pigs vaccinated with Autovaccine had significantly lower rates of pneumonia and arthritis compared to unvaccinated pigs With pneumonia, Autovaccine injection pigs have 117 infected, accounting for 5.34%; while the non-infected pigs with pneumonia had the rate of 19.23% With arthritis, Autovaccine pigs had 79 infected pigs, accounting for 3.60%, while 295 non-vaccinated pigs had 13.22% Apply the formula of Halloran M.E et al (1997) we calculated the effective rate of pneumonia was 72.24% and the effectiveness of arthritis was 72.76% Thus, the use of preventive autovaccinne for pigs in Thai Nguyen is suitable in preventing and limiting the rate of pneumonia and arthritis caused by S suis 3.4.2 Results of experimental treatment regimen of pigs with pneumonia Table 3.14 showed that pigs immunized with Autovaccine had a significantly lower prevalence of pneumonia and arthritis compared to unvaccinated pigs With pneumonia, pigs that were immunized with Autovaccine, there were 117 of which affected, accounting for 5.34%; while pigs with pneumonia unvaccinated with Autovaccine 19.23% of 27 28 pigs were affected For arthritis pigs that were immunized with Autovaccine of which there were 79 pigs affected, accounting for 3.60%, while 295 unvaccinated pigs, this prevalence was 13.22% Table 3.14: Results of experimental treatment regiment of pigs with pneumonia Treatment regimen Drug Number of Administratio pigs treated n and dosage (pig) 1ml/25kg B.W/day (4mg CEFANEW-LA ceftiofur /kg (ceftiofur:10g/100ml) B.w) one injection/3 days 1ml/10kg B.W/day Gluco-K-C- Namin one injection/ day 1ml/30kg B.W/day MARFLO-45% (15mg (florfenicol:45g/100ml) florfenicol/ day one injection/3 days 1ml/10kg B.W/ day one Gluco-K-C- Namin injection day /day 1ml/10kg B.W/day (15mg MARPHAMOX- LA amoxicillin /kg (amoxicillin:15g/100ml) B.W) one injection//2 days 1ml/10kg Gluco-K-C- Namin B.W/day; one injection/ day Total Treatm Number of ent Percent time recovered pigs (%) (pig) X+m x 85 6± 0.15 79 92.94 75 7± 0.13 68 90.66 80 8± 0.16 71 87.50 217 90.41 240 Applying the formula of Halloran M.E et all (1997), we calculated the effectiveness for prevention of pneumonia was 72.24% and the effectiveness for prevention of arthritis was 72.76% Thus, the use of preventive autovaccinne for pigs in Thai Nguyen was suitable to 28 29 preventing and lowering the prevalence of pneumonia and arthritis caused by S suis Table 3.15: Results of experimental treatment regimen of pigs with arthritis Regime n Drug CEFANEW-LA (ceftiofur:10g/100ml) Gluco-K-C- Namin MARFLO-45% (florfenicol:45g/100ml) Gluco-K-C- Namin MARPHAMOX- LA (amoxicillin:15g/100ml) Administration and dosage 1ml/25kg B.W/ day (4mg ceftiofur /kg B.W) one injection /3 days 1ml/10kg B.W/day one injection/day 1ml/30kg B.W/day (15mg florfenicol/kg B.W) one injection/3 days 1ml/10kg B.W /day one injection/day 1ml/10kg B.W/day (15mg amoxicillin/kg B.W) one injection/2 days Numbe r of pigs treated (pig) Treatmen t day X + mx Number of recovered pigs (pig) Percent (%) 55 ± 0.10 50 91.90 58 ± 0.17 52 89.65 55 ± 0.13 47 85.45 Based on the results of antibiotic susceptibility test, we selected antibiotics with high susceptibility to S suis isolates to formulate treatment regimens for affected pigs Results are presented in tables 3.14 and 3.15 The results in Table 3.14 showed that experimental treatment for pigs with pneumonia, antibiotics were used including: CEFANEWLA; Marflo-45% and Marphamox-LA At the same time, during the treatment, Gluco-K-C-Namin is added to increase pig resistance to diseases The results showed that all of three treatment regimens had good results, high incidence of pigs recovering from illness, 240 pigs with pneumonia, 217 of which recovered, reaching 90.41% Among treatment regimens, the highest one is regimen using CEFANEW-LA at ml/25 kg B.W for treatment of 85 pigs with pneumonia, 79 of 29 30 which recoverd from the ilness, reaching 92.94% and treatment time perod was from 6-8 days For exprimental treatment of swine arthritis, we also used the same regimens tin treating swine pneumonia (Table 3.15) The results showed that all of three treatment regimens for pigs with arthritis had good results, high incidence of pigs recovering from the ilness, in 168 pigs with arthritis were treated 149 of which recovered, reaching 88,69% The highest one is regimen with CEFANEW-LA was used at dosage of ml/25 kg B.W for treatment of 55 pigs with arthritis, 50 of which recovered from the ilness, reaching 91.90%, treatment period time was from - days From the experimental results of the above regimens, we have recommended for veterinarians and animal producers in Thai Nguyen province to apply them in treating pigs with pneumonia, arthritis In particular, it is recommended that regimen containing ceftiofur should be used for treatment of these diseases to reach high effectiveness, CONCLUSION AND SUGGESTION CONCLUSION From the results of the thesis, we have the following conclusions below: 1.1 The pig herds raised in Thai Nguyen province had high rates of infection and mortality; with average rates of pneumonia were 14.17% and 12.91% respectively; arthritis these were 11.13% and 6.85% respectively - The pigs that infected and killed by the pneumonia had the highest rates in post-weaning age (16.79 and 15.35%, respectively), the lowest rates were in the gilts and sows (8.18 and 5.31%, respectively) The rates of pigs were infected and killed by arthritis were also highest in post-weaning pigs (16.02 and 7.81% respectively) and the lowest were in gilts and sows, respectively 3.04 and 2.85% 1.2 S suis strains were isolated from samples of pigs infected by the pneumonia and arthritis in Thai Nguyen with the rate of 52.60%; The highest rates were pigs in post-weaning (58.91%) and the lowest rates were piglets under month of age (45.79%) The isolated S suis strains 30 31 were characterized by specific biological characteristics of genus and species as described scientific articles and were highly susceptible to ceftiofur (84.52%), florfenicol (81.54%) and amoxicillin (80.35%) - The isolated S suis strains belonging to the serotype (58.16%), serotype (16.33%), serotype (7.18%), serotype 29 (2.64%), serotype 21 (1, 96%) and the number of unidentified strains were 13.72%; the genes for virulence factor were arcA, mrp, sly and epf, distributed in combinations as arcA/sly, arcA/mrp, arcA/epf and arcA/mrp/sly/epf - Selecting 03 S suis strains belonging to the serotype 2, and as S.TN2-2, S.TN7-5, S.TN9-7, all of these strains had the stability in biological characteristics and high virulence after recultured generations, causing mortality of 100% for injected white mice within 12-36 hours, they have been standards which can use as the master seeds to research production of Autovaccine 1.3 All exprimental Autovaccine produced from S suis strains serotype 2, and (S.TN2-2, S.TN7-5, S.TN9-7) have standardlized about concentration, purity, safe and efficiency; immune response increases for experimental pigs, produced antibodies and immune responses which last 120 days for vaccinated pigs 1.4 The efficiency of Autovaccine to prevent the pneumonia and arthritis for the pig's herds were 72.24% and 72.76% respectively The treatment therapies for infected pig's herds by the pneumonia and arthritis in Thai Nguyen were highly effective, the rates of pig's herds recovered from the pneumonia were 87.50% to 92.94% and athritis 85.45% to 91.90% In this regard, N0 therapy method was applied using ceftiofur for treating pneumonia and arthritis for the highest efficiency, the rate of recovery from 91.90 to 92.94% respectively SUGGESTION - Continuing to thesis and complete the process of manufacturing and using Autovaccine for pneumonia, arthritis of pigs caused by S.suis bacteria - Applying experimental regimens to treat pigs with pneumonia, arthritis caused by S suis bacteria in the localities of Thai Nguyen province 31 32 32 ... Scientific advisors: Assoc Prof Dr To Long Thanh Dr Nguyen Van Quang Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the university examination committee At:... pneumonia, arthritis in pigs caused by Streptococcus suis (S suis) also occurs quite commonly leading to economic loss for farmers The disease is caused by S suis in pigs with pathological manifestations... with Streptococcus suis Derived from the practical situation of production and food safety at present, aiming at better understanding of pneumonia and arthritis in pigs associated with S suis,