THAI NGUYEN UNIVERSITYTHAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN MANH CUONG INVESTIGATING SOME BIOLOGICAL CHARACTERISTICS OF STREPTOCOCCUS SUIS CAUSING PIG DISEASES IN THA
Trang 1THAI 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
Trang 2This thesis was completed at:
THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY
Scientific advisors: 1 Assoc Prof Dr To Long Thanh
2 Dr Nguyen Van Quang
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
Trang 3LIST OF PUBLICATION RELATED TO THE THESIS
1 Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang, DoHong 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.
2 Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang,Nguyen Quang Tuyen, Do Hong Anh (2019), "Isolation, serotypedetermination 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.
3 Nguyen Manh Cuong, To Long Thanh, Nguyen Van Quang,Nguyen Quang Tuyen, Do Hong Anh (2018), "Results ofexperimental 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
Trang 41 Urgency of the thesis
Pig production in Thai Nguyen province in recent years hasdeveloped rapidly quantity and improved quality, meeting thedemand of pig product consumption in the province and export-orientation, contributing to job creation and increasing income foranimal 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 facesmany difficulties, especially in the field of disease control, whichgreatly affects the efficiency of livestock production In addition toinfectious 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 ofpost-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 withrespiratory infection accompanied with coughing showed thatStreptococcus accounted for 74.0% Investigation results of Do NgocThuy 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 2 ,7 and 9 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 3 cases suffering from S.
Trang 5suis 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 foodsafety 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 urgentrequirement, making contribution to improvement of productionefficiency, 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"
2 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 Nguyenprovince
3 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 pigsraised 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 provincehas contributed to decreasing the rate of pigs suffering from
pneumonia, arthritis caused by S suis.
- Applying the treatment regimens for pneumonia, arthritis in pigsreaches high efficiency, contributing to increased income for animalproducers and sustainable development of pig husbandry in Thai
Nguyen province
Trang 6- 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
4 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 1 REVIEW OF LITERATURE
Chapter 2 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 withpneumonia and arthritis over the years in some districts and cities ofThai Nguyen province from 2015-2017
- Investigating the rate of prevalence and mortality of pigs caused bypneumonia, arthritis in pigs raised in Thai Nguyen from 2015-2017 at
different ages and seasons.
Trang 72.2.2 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 experimentalAutovaccine
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, synovialfluid, 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 withAutovaccine against pneumonia, arthritis caused by S suis
- Media and chemicals used according to the thesis procedure of thedivision 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)
Trang 8- Chemicals and bio-products used for agglutination reactions,
serology and PCR technique, IHA reaction
- Antibiotics: Cefanew-LA, Marflo-45%, Marphamox-LA and C-Namin produced by Marphavet Veterinary phar maceutical Company
Gluco-K 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 PCRand serology
- Common laboratory instruments, inoculation chamber, autoclaves,centrifuges, shaking machines, PCR technique used for investigation ofbacteria belonging to the Central Veterinary Diagnostic Center, AnimalHealth Department and Institute of Life Sciences of Thai NguyenUniversity
2.4 Methods of the thesis
2.4.1 Method of epidemiological thesis
Using methods of descriptive epidemiology and practicalepidemiology 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 theinflamed joint of sick pigs then place in cotton swab tube, stored at
40C 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 forculture 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 Centerand 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 ThiMinh Hang (2009a)
- LD50 calculation method according to Reed L.J & Muench H (1938)
Trang 9- 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)
- Experimenting treatment regimens for pigs infected by pneumonia,arthritis based on the determination of susceptibility to antibiotics of
S suis bacteria strains to select highly sensitive antibiotic drugs,
being allowed in Vietnam
- The method of manufacturing Autovaccine is tested from isolated
S suis bacteria strains according to the process of Department of
Microbiology, Institute of Veterinary Medicine
- Testing sterile, safe and effective of Autovaccine tested according
to TCVN 8684: 2011 and QCVN 01: 187-2018
- Method of antibody titre determination in pigs injected withAutovaccine by Indirect Haemaglunation (IHA) according to the OIEvaccine evaluation procedure (2018)
Chapter 3 RESULTS AND DISCUSSION 3.1 The situation of pigs affected and died of pneumonia, arthritis from 2015 to 2017 in Thai Nguyen province
3.1.1 Prevalence of pigs affected and motarlity of pigs with pneumonia, arthritis from 2015-2017
Table 3.1: Prevalence of affected pigs and mortality rates of pigs associated with pneumonia and arthritis from 2015-2017
r of
pigs
Percen
t (%) Numbe
r of pigs
Percen
t (%) Numbe
r of pigs
Percen
t (%) Numbe
r of pigs
Percentag
e (%) Numbe
r of pigs
Percen
t (%) Numbe
r of pigs
Perce nt (%))
Trang 10investigation With pneumonia, in 2017 the herds of pigs accountedfor the highest morbidity and mortality rates (16.34% and 15.44%respectively), followed by 2016 (the rates was 13.71% and 12.03%respectively) and lowest was in 2015 (the rates were 12.88% and11.29% respectively) For arthritis, the highest prevalence andmortality rates were in 2017 (13.88% and 8.18% respectively),followed by 2016 (10.53% and 6.45% respectively) and the lowestwere in 2015 (9.50% and 5.73%) Similarly, the highest number ofpigs infected and died of pneumonia and arthritis was in 2017 (6.87%and 3.66%, respectively), followed by 2016 (5.02% and 2.89%respectively) and the lowest were in 2015 (3.97% and 2.61%
respectively).
In 2017, pneumonia and arthritis in pigs in Thai Nguyen werehigher morbidity and mortality prevalences than in the previous yearsbecause at that time the price of porkers was the lowest since the past
10 years so it had a great influence on the pig industry commercialpigs that were not able to be sold led to farms and family farmssuffer from heavy losses Animal producers almost neglectedmanagement and care of animals, sanitation, prevention andtreatment of animal diseases so a lot of diseases occured
3.1.2 Morbidity and mortality rates of pigs associated with pneumonia and arthritis in different seasons
Table 3.2: Morbidity and mortality rates of pigs associated with
pneumonia and arthritis different seasons
Seasons Number of investigate
d pigs
Pneumonia Arthritis Morbidity Mortality Morbidity Mortality Number
of pigs Percen t (%) Number of pigs Percen t (%)
Numbe
r of pigs
Percen t (%)
Number
of pigs
Percen t (%)
Trang 11were 9.06% and 8.10% respectively; for arthritis 12.16% and 7.39%compared with 9.52% and 5.78% respectively;.
The results of the thesis showed that pneumonia and arthritis inpigs in Thai Nguyen province often occured throughout the year, but
in the months of the Winter-Spring seasons, it is cold, and drizzly andhumid, adversely affecting the health of animals, creating favouritecondition for diseases occur, so both the diseases occured at a higher
by piglets and the lowest was in gilts and sows (15.35, 14.11) and5.31% respectively) For arthritis, the highest prevalence was in post-weaning pigs (16.02%), followed by piglets under 1 month of age(12.02%) and the lowest was in gilts and sows (3.04%)
Table 3.3: Morbidity and mortality rates of pigs infected by
pneumonia and arthritis at different ages
of pigs
Percent (%)
Number
of pigs
Percent (%)
Number
of pigs
Percent (%)
Number
of pigs
Percent (%)
Trang 12post-in pigs with streptococcal disease, most pigs were post-infected durpost-ing theperiod from 5 to 10 weeks of age, few pigs were infected over 32weeks of age or several hours after birth
Investigation results of survival of S.suis in some areas of Thua
Thien Hue province in spring-summer season of 2015 by Bui Thi
Hien et al (2016) also showed that the prevalence of S suis infection
in healthy pigs was 11.4%
3.2 Results of some biological characteristics of isolated S suis strains 3.2.1 Isolation of S suis bacteria from pigs with pneumonia and arthritis in Thai Nguyen
Table 3.4 showed that in 403 samples from pigs withpneumonia and arthritis at different ages, there were 212 samples that
S suis isolated accounting for 52.60%; The highest prevalence was
that of post-weaning pigs> 1 - 3 months of age (58.91%) and thelowest was that of piglets less than 1 month of age (45.79%) Our
results were in agreement with those of S suis isolated from
specimens of pigs infected with PRRS in Bac Giang by TruongQuang Hai and et al (2012), which was 51.67% and by Le Van Dung
et al (2013) , which was 55.10%, but higher than finding of NguyenThi Kim Lan and others (2016, 2017) when isolating bacteria causingpneumonia in pigs in Bac Ninh province from lungs andretropharyngeal lymph nodes of pigs found that the prevalence of S.suis was 38.67% and in pigs raised in Tuyen Quang province was
13.0% As a result, isolation of S suis in pigs in Thai Nguyen
showed that this bacterium had played an important role in
pneumonia and arthritis in pigs for the past years in Thai Nguyen Table 3.4: Results of S suis isolated from pigs at different age
suffering pneumonia and arthritis
Number of positive samples (+)
Perce nt (%)
Tota l (%)
Trang 13Results of investigating some biological and chemical
characteristics of S suis bacteria isolated from pigs's specimens with
pneumonia and arthritis in Thai Nguyen province found that all
strains had typical biological and chemical characteristics of S suis
bacteria as described by scientific documents
3.2.3 Results of investigating some biological and chemical characteristic of S suis bacteria isolated by API 20 Strep kit system
Results of investigating some biological and chemical
characteristics of S suis bacteria isolated by API 20 Strep kit system,
of which 20 reactions prepared in plastic tray produced by BioMrieuxcompany, including: VP, HIP ESC, PYRA, αGAL, βGUR, βGAL,PAL, LAP, ADH, RIB, ARA, MAN, SOR, LAC, TRE, INU, RAF,
AMD, GLYG The results showed that S suis strains isolated from
pigs in Thai Nguyen had all the typical biological and chemical
characteristics of S suis bacteria.
3.2.4 Result of antibiotic susceptibility test of isolated S suis strains
Table 3.5: Result of antibiotic susceptibility test of
isolated S suis strains
Orde
Numbe
r of strains tested
Trang 14Table 3.5 showed that S suis strains were highly susceptible to
ceftiofur (84.52%), florfenicol (81.54%), amoxicillin (80.35%),ampicillin (72.61%) and were resistant to several antibiotics such aserythromycin (82.73%), colistin (78.57%), neomycin (72.02%) andpenicillin G (58.33%) Our results were in agreement with those ofNguyen Thi Kim Lan and others (2017), when testing the
susceptibility of S suis strains isolated in pigs in Bac Ninh province
to some antibiotics, it showed high susceptibility to ceftiofur(81.82%), amoxicillin (79.55%), florfenicol (75.00%) and ampicillin(70.45%) The above results were the basis for us to select antibiotics
for treatment of streptococcal disease associated with S suis in pigs
in Thai Nguyen to reach high effectiveness
3.2.5 Results of serotyping isolated S.suis strains
Table 3.6 showed that number of S suis serotype 2 strains was
the highest (58.17%), followed by serotype 9 (16.34%), serotype 7(7.19%), the lowest was serotype 21 and 29 (1.96 and 2.61%
respectively); S suis serotype was not determinated in the rest 21 strains,
accounting for 13.72% For serotype 2, the highest was in post-weaningpigs (64.00%), followed by piglets <1 month old (58.97%) and the
lowest was in porkers (51.21%) Results of other S suis serotypes of S.
suis isolated in Thai Nguyen were not determinated.
Table 3.6:Results of serotyping isolated S suis strains
Trang 15r of positiv e strains (+)
Percen t (%)
Numbe
r of positiv e strains (+)
Percen t (%)
Numbe
r of positiv e strains (+)
Percen t (%)
Numbe
r of positiv e strains (+)
Percen t (%)
Numbe
r of positiv e strains (+)
Percen t (%)
Piglets
( ≤ 1 month
old)
39 23 58.97 2 5.13 6 15.39 0 0 1 2.56 7 17.94 Post weaned
piglets (>1- 3
months old) 50 32 64.00 3 6.00 7 14.00 1 2.00 1 2.00 6 12.00Porkers (>3 - 6
months old) 41 21 51.21 3 7.32 9 21.96 1 2.44 1 2.44 6 14.64Gilts, sows
( ≥ 6 months
old)
23 13 56.52 2 8.69 3 13.02 1 4.34 1 4.34 2 8.69
total 153 89 58.17 11 7.19 25 16.34 3 1.96 4 2.61 21 13.72
The results of our thesis were in agreement with those of some
authors such as Cu Huu Phu (2011), identified S suis serotype 2 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 ofpigs with PRRS in Bac Giang, serotype 2 accounted for the highest rate(56.29%), followed by serotype 9 (17.03%) and the lowest was serotype
7 (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%) 5types 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 authorssuch as Do Ngoc Thuy and et al (2009) from healthy pigs and pigs withpneumonia 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 wereserotype 2, 7, 9 and 7 combinations of virulence genes Investigation of
Cu Huu Phu et al (2013) showed that serotype 2 strains carried the mostgene combinations which were scatteredly distributed in 5 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