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Abstract [Objectives] This study aimed to investigate the pathogenicity， growth characteristics and drug resistance of Streptococcus suis type 2.
[Methods] Bacterial isolation and identification， biochemical experiments， determination of growth curve and correlation curve between OD600values and viable counts， drug susceptibility tests， pathogenicity analysis， and histopathological observations were carried out.
[Results] The Streptococcus strain isolated from infected pigs was identified as Streptococcus suis type 2， which was named TA01 strain. TA01 strain reached the growth peak at 6-8 h postincubation， and viable counts gradually declined after 8 h of incubation. The correlation equation between OD600values and viable counts is y=24.659x-1.076 1， R2=0.996 7. TA01 strain was sensitive to penicillin， erythromycin， florfenicol and oxacillin， and resistant to ciprofloxacin， polymyxin B and clindamycin. According to the results of pathogenicity analysis， all the mice in 3.6×109 cfu/mouse group died within 48， and these dead mice exhibited acute pyaemia septica. Based on the ReedMuench formula， it was calculated that LD50of TA01 strain was 1.137×108 cfu/mouse. Pathological examination showed obvious bluestained bacteria clusters， accompanied by neutrophil infiltration.
[Conclusions] TA01 strain was a virulent strain of Streptococcus suis type 2. Compared with Streptococcus strains which were isolated and reported in China， TA01 strain exhibited strong virulence and rapid proliferation.
Key words Streptococcus suis type 2; Isolation and identification; Growth curve; Drug sensitivity test; Pathogenicity; LD50determination
Streptococcus suis is an important zoonotic pathogen widely distributed in nature， which can not only cause diseases in pigs， but also infect humans， horses， cattle， sheep， poultry and other animals. Based on the differences in capsular antigenicity， Streptococcus suis can be divided into 35 serotypes （types 1-34 and type 1/2）[1]. Type 1， type 1/2 （strains containing both type 1 and type 2 antigens）， type 2， type 7， type 9 and type 14 are common pathogenic serotypes of Streptococcus suis. Streptococcus suis mainly causes meningitis， arthritis， endocarditis， local suppurative inflammation， and sepsis. Streptococcus suis type 2 is the most pathogenic among various streptococcal serotypes， causing the most severe economic losses to the pig industry. In the summer of 1998， Streptococcus suis type 2 infection broke out in some pig farms in Jiangsu Province， which caused serious threat to human and animal health[2]. In the summer of 2005， Streptococcus suis type 2 infection occurred in Sichuan Province， causing 38 human deaths[3]. Every year， there are many reports of human infection with Streptococcus suis in China， and most of the patients are infected through contact with infected pigs or eating infected pork. Moreover， apparent health pigs may carry a variety of serotypes of Streptococcus suis with high detection rate， which poses a potential threat to the health of people involved in pig farming and slaughtering[4-5]. Shandong Province is a large pigraising province. With the continuous expansion of breeding scale and the increase of breeding density， especially with the occurrence and prevalence of immunosuppressive diseases such as porcine circovirus disease and porcine blue ear disease[6]， the incidence rate of Streptococcus suis infection has been increasing year by year. According to the statistics of recent clinical cases in Shandong Agricultural University， Streptococcus suis infection cases account for the largest proportion of the total number of outpatient cases among bacterial diseases. Therefore， to understand the biological characteristics of epidemic strains of Streptococcus suis[7-8]and further clarify their pathogenicity is of important theoretical and practical significance to the diagnosis and control of Streptococcus suis infection.
Materials and Methods
Infected materials
There are 30 sows， 170 finisher pigs and 80 nursery pigs in a pig farm in Laiwu City. Since April， a large number of nursery pigs died successively， with 70 infection cases and 36 deaths. The infected pigs exhibited high fever， loss of appetite， lethargy and joint swelling of hind limbs， accompanied by various neurological symptoms such as swimming behavior and ataxia. Dead pigs from the pig farm were dissected. The spleens， livers， lungs and brains were collected as infected materials.
Experimental animals
Sixty 4weekold healthy Kunming mice， weighing about 20 g，were purchased from Jinan Pengyue Experimental Animal Breeding Co.， Ltd.
Reagents and instruments
EasyPure Viral DNA/RNA Kit （batch number： L41228）， purchased from Beijing Transgen Biotech Co.， Ltd.; Taq DNA polymerase and DL2000 Marker， purchased from Takara Biotechnology （Dalian） Co .， Ltd.; TSA and TSB medium， purchased from Becton， Dickinson and Company （U.S.）; fetal bovine serum （FBS）， purchased from Gibco; micro biochemical fermentation tubes， purchased from Hangzhou Tianhe Microorganism Reagent Co.， Ltd.; positive serum of different subtypes of Streptococcus suis， purchased from China Institute of Veterinary Drug Control; drug susceptibility test strips， purchased from OXOID; UV5200 spectrophotometer， purchased from BioRad.
Anatomical observation
Two dead pigs were dissected， and changes in various organs were observed and recorded.
Staining， microscopic examination， separation and purification of the bacterial strain The lungs of infected pigs were collected， inoculated to TSA medium containing 5% fetal bovine serum by streak plate method， and cultured at 37 ℃ for 18 h. A single colony was selected for Gram staining， which was inoculated to TSA medium containing 5% fetal bovine serum. A 3-4 mm wellcircumscribed， transparent and colorless hemolysis zone formed around the colony， suggesting typical β hemolysis. After being incubated to the third generation， a single colony was picked， inoculated to TSB medium containing 7 ml of 5% fetal bovine serum， and cultured at 37 ℃ for 8 h with shaking at 180 r/min. The bacterial strain was isolated and purified for subsequent experiments.
PCR identification and primer design
A 100 μl aliquot of bacterial liquid was collected for PCR identification.Specific primers were designed based on the sequence of Streptococcus eftu gene published in GenBank （Table 1）. The expected size of amplified fragment is 197 bp. The total PCR reaction volume was 20 μl， containing 10 μl of Premix Taq， 1 μl of each of upstream and downstream primers， 3 μl of DNA template， and 5 μl of ddH2O. The PCR amplification was started with initial denaturation at 95 ℃ for 6 min， followed by 35 cycles of denaturation at 95 ℃ for 30 s， annealing at 56 ℃ for 30 s， and extension at 72 ℃ for 45 s; the amplification was completed by holding the reaction mixture at 72 ℃ for 7 min. After the amplification， 10 μl of PCR product was identified by 1.5% agarose gel electrophoresis.
Biochemical experiment
The isolated and purified bacterial liquid was transferred into micro biochemical fermentation tubes that contained trehalose， 5% lactose， esculin， sodium hippurate， sorbitol and mannitol respectively. The fermentation results were observed after 24 h.
Establishment of growth curve and correlation curve between OD600values and viable counts of TA01 strain
The bacterial liquid was activated at 37 ℃， inoculated into 30 ml of TSB medium at a ratio of 2%， and cultured at 37 ℃ in a constant temperature incubator with shaking at 220 r/min. At 1-24 hpostincubation， 1 ml of bacterial liquid was respectively collected and centrifuged at 5 000 r/min for 5 min to establish the growth curve with incubation time as the abscissa and logarithm of bacterial concentration[lg（cfu/ml）]as the ordinate. The measurement was repeated three times at each time point， and the results were averaged to reduce the test error.
After 5 h of incubation， 2 ml of bacterial liquid was centrifuged at 5 000 r/min for 5 min. The supernatant was discarded and the precipitate was resuspended with an equal volume of PBS. The bacterial suspension was diluted 1， 2， 3， 4， 5， 6， 7， 8， 9 and 10fold， respectively. OD600values of different dilutions of bacterial suspension were detected using a spectrophotometer at 600 nm. Drug sensitivity test
The sensitivity of the isolated strain to penicillin， erythromycin， florfenicol， ciprofloxacin， polymyxin B， clindamycin， oxacillin and rifampicin was detected according to the instructions of drug susceptibility test strips.
Pathogenicity analysis
The isolated strain was inoculated to TSA medium containing 5% fetal bovine serum by streak plate method， and cultured at 37 ℃for 18-20 h as the F1 generation. A single colony of the F2 generation was picked， inoculated to TSA medium containing 5% fetal bovine serum and cultured at 37 ℃ for 14 h. Bacterial cells were washed. The bacterial concentration was calculated according to the linear relationship between OD600values and viable counts and diluted to the required level. Sixty 4weekold healthy Kunming mice were randomly divided into 6 groups， 10 per group. Groups 1-5 were the experimental groups （3.6×1010cfu/mouse group， 3.6×109 cfu/mouse group， 3.6×108 cfu/mouse group， 3.6×107 cfu/mouse group and 3.6×106 cfu/mouse group）， and group 6 was the control group. Mice in groups 1-5 were injected intraperitoneally with 0.1 ml of 3.6×1010， 3.6×109， 3.6×108， 3.6×107， and 3.6×106 cfu/ml bacterial liquid， while mice in group 6 were injected intraperitoneally with 0.1 ml of PBS. After injection， these mice were put back to the original cages and raised under the same condition. The incidence of the mice was observed and recorded for 72 h continuously. LD50of mice was calculated by the ReedMuench formula.
The bacterial strain isolated from the lungs of dead mice was subjected to an antigenantibody agglutination test to verify the subtype of the isolate.
Histopathological observation
Preparation of organ slices from infected pigs
The joints， brains， lymph nodes and lungs were collected from dissected pigs and fixed with 10% formalin solution to prepare routine paraffin sections for H.E. staining and microscopic examination.
Preparation of organ slices from mice
The lungs， spleens and hearts were collected from mice in experimental and control groups and fixed with 10% formalin solution to prepare routine paraffin sections for H.E. staining and microscopic examination.
Results and Analysis
Anatomical observation
Two dead pigs were dissected. The results of anatomical observation showed pulmonary congestion and hemorrhage with fibrinous exudate on the surface that adhered to the pleura; epicardial hemorrhage; joint swelling with purulent exudate on the cut surface; congestion and swelling of lymph nodes all over the body; meningeal congestion and hemorrhage. The clinical manifestations and anatomical features of infected pigs were consistent with the basic characteristics of streptococcosis， and these pigs were suspected to be infected with Streptococcus suis. The results of anatomical observation were shown in Fig. 1.
Bacterial isolation， identification， staining and microscopic observation
There were a large number of transparent， smooth， small round colonies on the TSA medium containing 5% fetal bovine serum. The colonies were picked for Gram staining and identified as by single， paired or long chainshaped Grampositive bacteria by microscopic examination （Fig. 2）. The isolated bacterial strain was named TA01 strain.
PCR identification
The isolated bacterial strain was identified by PCR using the designed specific primers. A 197 bp band was amplified （Fig. 3）， which was consistent with the expected result， indicating that the isolated strain was Streptococcus suis.
Biochemical experiment
According to the above results， the TA01 strain was initially identified as Streptococcus suis. According to the results of biological experiment， the TA01 strain could ferment 5% lactose and trehalose to produce acid and ferment esculin， but it could neither ferment mannitol and sorbitol nor hydrolyze sodium hippurate， which is in line with the biochemical characteristics of Streptococcus suis type 2.
Establishment of growth curve and correlation curve between OD600values and viable counts of TA01 strain
As shown in Fig. 4A， TA01 strain entered the logarithmic growth phase after being cultured in TSB medium for about 3 h and reached the growth peak at 6-8 h postincubation， which entered the growth decline phase after 8 h of incubation with gradually decreasing viable counts. As shown in Fig. 4B， there was a linear relationship between OD600values and viable counts of TA01 strain during growth retardation phase and logarithmic growth phase. The correlation equation is y=24.659x-1.076 1， R2=0.996 7. Where， x is OD600value and R2 is the correlation.
Drug sensitivity test
As could be seen in Table 2， the isolated strain was sensitive to penicillin， erythromycin， florfenicol and oxacillin， moderately sensitive to rifampicin， and resistant to ciprofloxacin， polymyxin B and clindamycin.
Pathogenicity analysis
Mice in experimental groups exhibited varying degrees of lethargy and rough hairs with individual deaths. Moreover， mice grew well in control group and 3.6×106 cfu/mouse group， with no death; three mice in 3.6 × 107 cfu/mouse group died within 48 h; seven mice in 3.6×108 cfu/mouse group died within 48 h; all the mice in 3.6×109 cfu/mouse group died within 48 h; all the mice in 3.6×1010cfu/mouse group died within 24 h. The results of pathogenicity analysis were shown in Fig. 5. According to the ReedMuench formula， it was calculated that LD50of the isolated strain was 1.137×108 cfu/mouse. The anatomical observation of dead mice showed acute pyaemia septica. The lungs were collected and inoculated to TSA medium containing 5% fetal bovine serum. The obtained bacterial cells were mixed with positive sera of different Streptococcus suis subtypes on the agglutination plate. The results showed that the bacterial cells were agglutinated by positive serum of Streptococcus suis type 2 but showed no aggregation after being mixed with positive sera of other subtypes. According to the results of antigenantibody agglutination test and LD50determination， the isolated strain was a virulent strain of Streptococcus suis type 2.
Histopathological observation
Histopathological observation of various organs of infected pigs
Necrotic tissues in the joint cavity were covered with bluestained bacteria clusters （Fig. 6A）. The alveolar wall exhibited congestion and hemorrhage， accompanied by infiltration and proliferation of inflammatory cells such as neutrophils and macrophages; the alveolar cavity was filled with redstained serous fluid （Fig. 6B）.
The meninx was congested with neutrophil infiltration （Fig. 6C）. Moreover， congestion and hemorrhage of lymph nodes were observed， with infiltration of a large number of neutrophils （Fig. 6D）.
Histopathological observation of various organs of mice
Mice in experimental group exhibited lung congestion with light red serous fluid exudating from the alveolar space （Fig. 7A）. There was infiltration of a large number of neutrophils in the red pulp area of spleen （Fig. 7B）. Granular degeneration of myocardial fibers was observed， with interstitial congestion and hemorrhage， and neutrophil infiltration （Fig. 7C）. However， there were no abnormalities in the lungs， spleens and myocardia of mice in control group （Fig. 7D-F）.
Discussion
Streptococcus suis is a capsular grampositive bacterium， of which type 2 is the most common and most virulent serotype among the 35 serotypes. Streptococcus suis naturally infects pigs through the upper respiratory tract， especially the tonsil and nasal cavity. In this study， a bacterial strain was isolated from the lung lesions of dead pigs， which was identified as Streptococcus suis type 2 by morphological identification， PCR amplification and antigenantibody agglutination test. It was calculated that LD50of the isolated strain was 1.137×108 cfu/mouse， which was much lower compared to other type 2 strains[9-10]， indicating that TA01 strain was a virulent strain of Streptococcus suis type 2. Currently， with the prevalence of immunosuppressive diseases such as porcine reproductive and respiratory syndrome and porcine circovirus disease， the infection rate of Streptococcus suis type 2 infection has increased， however， few studies have been reported about the growth characteristics of Streptococcus suis type 2. In this study， the growth curve and OD600value of Streptococcus suis type 2 were determined by liquid culture method. The results showed that the isolated strain entered the logarithmic growth phase after being cultured in TSB medium containing 5% fetal bovine serum at 37 ℃for about 3 h， and viable counts gradually declined after 8 h of incubation.
In recent years， some scholars have investigated the drug resistance of Streptococcus isolates and found that drug resistance of Streptococcus suis type 2 has been gradually increased. Li et al.[11]determined the drug resistance of 31 strains of Streptococcus suis type 2 in Shanghai and found that all isolates were resistant to five antibiotics including amikacin， ciprofloxacin and ampicillin. Jiao et al.[12]detected the drug resistance of 19 strains of Streptococcus suis type 2 isolated from Anhui Province and reported that all these 19 strains were resistant to more than three antibiotics， and the antibiotic resistance profile included vibramycin， tetracycline， sulfafurazole and bacitracin. In this study， TA01 strain was resistant to ciprofloxacin， polymyxin B and clindamycin. Therefore， it is necessary to apply drugs scientifically and alternately during the prevention and control of streptococcosis in pig farms. Sensitive drugs can be selected through drug sensitivity test for targeted treatment.
The virulence， pathogenicity and immunity of Streptococcus suis type 2 has been a topic of great concern to researchers. So far， some studies have been reported about models of Streptococcus suis type 2 infection[13]. In this study， Kunming mice were selected to construct vaccinationchallenge models. After intraperitoneal injection of 0.1 ml of 3.6×109 cfu/ml TA01 strain， all the mice died within 48 h. Dead mice were dissected， and pyaemia septica was observed， which confirmed that the isolated strain was virulent and could be used as an experimental strain for vaccinationchallenge test or a candidate strain for vaccines.
At present， vaccine immunization and drug application are the main methods adopted in China to prevent and control Streptococcus suis type 2 infection， but the effect of vaccine immunization needs to be further improved. Therefore， it is particularly important to develop safe and effective vaccines against Streptococcus suis. In this study， drug resistance， growth curve， OD600value， LD50， pathogenicity and pathological characteristics of TA01 strain were investigated， which had certain reference significance to the prevention and control of Streptococcus suis type 2 infection and vaccine development in China. References
[1] LIU J， TIAN ZY， XIAO YC， et al. The role of porcine monocyte derived dendritic cells （MoDC） in the inflammation storm caused by Streptococcus suis serotype 2 infection[J]. PLoS One， 2016， 11（3）：e0151256.
[2] DEKKER N， BOUMA A， DAEMEN I， et al. Effect of simultaneous exposure of pigs to Streptococcus suis serotypes 2 and 9 on their colonization and transmission， and on mortality[J]. Pathogens， 2017， 6（4）：46.
[3] ZHU QY， ZHANG Q， LI KC， et al. Analysis on the virulence genes and molecular typing of Streptococcus suis type 2 strains isolated from patients[J]. China Preventive Medicine， 2016， 17（3）：202-206.（in Chinese）
[4] DUTKIEWICZ J， ZAJA CV， SROKA J， et al. Streptococcus suis： A reemerging pathogen associated with occupational exposure to pigs or pork products. Part IIpathogenesis[J]. Ann Agric Environ Med， 2018， 25（1）：186-203.
[5] KOLIOU MG， ANDREOU K， LAMNISOS D， et al. Risk factors for carriage of Streptococcus pneumoniae in children[J]. BMC Pediatr， 2018， 18（1）：144.
[6] LI Q， ZHANG YH， DU DC， et al. Characterization and functional analysis of PnuC that is involved in the oxidative stress tolerance and virulence of Streptococcus suis serotype 2[J].Vet Microbiol， 2018， 216（3）：198-206.
[7] SHI LH， ZHANG LZ， BAO M. The epidemiological investigation， prevention and control of swine streptococcosis in western Liaoning Province[J]. Heilongjiang Animal Science and Veterinary Medicine， 2007（10）：72-74.（in Chinese）
[8] WANG YT， WANG YG， WU DD， et al. The epidemiological investigation of swine streptococcosis in Heilongjiang Province during 2007-2015[J]. Heilongjiang Animal Science and Veterinary Medicine， 2016（18）：109-111.（in Chinese）
[9] WANG SH. Isolation， identification and analysis of biological characteristics of Streptococcus suis[D]. Luoyang： Henan University of Science and Technology， 2017.（in Chinese）
[10] DING YX. Drug resistance and virulence characteristics of bovinederived Streptococcus and their correlations in Inner Mongolia Area[D]. Hohhot： Inner Mongolia Agricultural University， 2015.（in Chinese）
[11] LI CY， YUE XW， HOU YX， et al. Isolation of Streptococcus suis type 2 in Shanghai area and testing of drug resistance[J]. Chinese Journal of Veterinary Parasitology， 2011， 19（6）：54-59.（in Chinese）
[12] JIAO AX， XU DW， ZHAO HW， et al. Virulence genes， hemolytic and antibiotic resistance in Streptococcus suis serotype 2 isolated from Anhui Province， China[J]. Chinese Journal of Zoonoses， 2014， 30（12）：1181-1186.（in Chinese）
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Editor： Chunmei WU Proofreader： Xinxiu ZHU
[Methods] Bacterial isolation and identification， biochemical experiments， determination of growth curve and correlation curve between OD600values and viable counts， drug susceptibility tests， pathogenicity analysis， and histopathological observations were carried out.
[Results] The Streptococcus strain isolated from infected pigs was identified as Streptococcus suis type 2， which was named TA01 strain. TA01 strain reached the growth peak at 6-8 h postincubation， and viable counts gradually declined after 8 h of incubation. The correlation equation between OD600values and viable counts is y=24.659x-1.076 1， R2=0.996 7. TA01 strain was sensitive to penicillin， erythromycin， florfenicol and oxacillin， and resistant to ciprofloxacin， polymyxin B and clindamycin. According to the results of pathogenicity analysis， all the mice in 3.6×109 cfu/mouse group died within 48， and these dead mice exhibited acute pyaemia septica. Based on the ReedMuench formula， it was calculated that LD50of TA01 strain was 1.137×108 cfu/mouse. Pathological examination showed obvious bluestained bacteria clusters， accompanied by neutrophil infiltration.
[Conclusions] TA01 strain was a virulent strain of Streptococcus suis type 2. Compared with Streptococcus strains which were isolated and reported in China， TA01 strain exhibited strong virulence and rapid proliferation.
Key words Streptococcus suis type 2; Isolation and identification; Growth curve; Drug sensitivity test; Pathogenicity; LD50determination
Streptococcus suis is an important zoonotic pathogen widely distributed in nature， which can not only cause diseases in pigs， but also infect humans， horses， cattle， sheep， poultry and other animals. Based on the differences in capsular antigenicity， Streptococcus suis can be divided into 35 serotypes （types 1-34 and type 1/2）[1]. Type 1， type 1/2 （strains containing both type 1 and type 2 antigens）， type 2， type 7， type 9 and type 14 are common pathogenic serotypes of Streptococcus suis. Streptococcus suis mainly causes meningitis， arthritis， endocarditis， local suppurative inflammation， and sepsis. Streptococcus suis type 2 is the most pathogenic among various streptococcal serotypes， causing the most severe economic losses to the pig industry. In the summer of 1998， Streptococcus suis type 2 infection broke out in some pig farms in Jiangsu Province， which caused serious threat to human and animal health[2]. In the summer of 2005， Streptococcus suis type 2 infection occurred in Sichuan Province， causing 38 human deaths[3]. Every year， there are many reports of human infection with Streptococcus suis in China， and most of the patients are infected through contact with infected pigs or eating infected pork. Moreover， apparent health pigs may carry a variety of serotypes of Streptococcus suis with high detection rate， which poses a potential threat to the health of people involved in pig farming and slaughtering[4-5]. Shandong Province is a large pigraising province. With the continuous expansion of breeding scale and the increase of breeding density， especially with the occurrence and prevalence of immunosuppressive diseases such as porcine circovirus disease and porcine blue ear disease[6]， the incidence rate of Streptococcus suis infection has been increasing year by year. According to the statistics of recent clinical cases in Shandong Agricultural University， Streptococcus suis infection cases account for the largest proportion of the total number of outpatient cases among bacterial diseases. Therefore， to understand the biological characteristics of epidemic strains of Streptococcus suis[7-8]and further clarify their pathogenicity is of important theoretical and practical significance to the diagnosis and control of Streptococcus suis infection.
Materials and Methods
Infected materials
There are 30 sows， 170 finisher pigs and 80 nursery pigs in a pig farm in Laiwu City. Since April， a large number of nursery pigs died successively， with 70 infection cases and 36 deaths. The infected pigs exhibited high fever， loss of appetite， lethargy and joint swelling of hind limbs， accompanied by various neurological symptoms such as swimming behavior and ataxia. Dead pigs from the pig farm were dissected. The spleens， livers， lungs and brains were collected as infected materials.
Experimental animals
Sixty 4weekold healthy Kunming mice， weighing about 20 g，were purchased from Jinan Pengyue Experimental Animal Breeding Co.， Ltd.
Reagents and instruments
EasyPure Viral DNA/RNA Kit （batch number： L41228）， purchased from Beijing Transgen Biotech Co.， Ltd.; Taq DNA polymerase and DL2000 Marker， purchased from Takara Biotechnology （Dalian） Co .， Ltd.; TSA and TSB medium， purchased from Becton， Dickinson and Company （U.S.）; fetal bovine serum （FBS）， purchased from Gibco; micro biochemical fermentation tubes， purchased from Hangzhou Tianhe Microorganism Reagent Co.， Ltd.; positive serum of different subtypes of Streptococcus suis， purchased from China Institute of Veterinary Drug Control; drug susceptibility test strips， purchased from OXOID; UV5200 spectrophotometer， purchased from BioRad.
Anatomical observation
Two dead pigs were dissected， and changes in various organs were observed and recorded.
Staining， microscopic examination， separation and purification of the bacterial strain The lungs of infected pigs were collected， inoculated to TSA medium containing 5% fetal bovine serum by streak plate method， and cultured at 37 ℃ for 18 h. A single colony was selected for Gram staining， which was inoculated to TSA medium containing 5% fetal bovine serum. A 3-4 mm wellcircumscribed， transparent and colorless hemolysis zone formed around the colony， suggesting typical β hemolysis. After being incubated to the third generation， a single colony was picked， inoculated to TSB medium containing 7 ml of 5% fetal bovine serum， and cultured at 37 ℃ for 8 h with shaking at 180 r/min. The bacterial strain was isolated and purified for subsequent experiments.
PCR identification and primer design
A 100 μl aliquot of bacterial liquid was collected for PCR identification.Specific primers were designed based on the sequence of Streptococcus eftu gene published in GenBank （Table 1）. The expected size of amplified fragment is 197 bp. The total PCR reaction volume was 20 μl， containing 10 μl of Premix Taq， 1 μl of each of upstream and downstream primers， 3 μl of DNA template， and 5 μl of ddH2O. The PCR amplification was started with initial denaturation at 95 ℃ for 6 min， followed by 35 cycles of denaturation at 95 ℃ for 30 s， annealing at 56 ℃ for 30 s， and extension at 72 ℃ for 45 s; the amplification was completed by holding the reaction mixture at 72 ℃ for 7 min. After the amplification， 10 μl of PCR product was identified by 1.5% agarose gel electrophoresis.
Biochemical experiment
The isolated and purified bacterial liquid was transferred into micro biochemical fermentation tubes that contained trehalose， 5% lactose， esculin， sodium hippurate， sorbitol and mannitol respectively. The fermentation results were observed after 24 h.
Establishment of growth curve and correlation curve between OD600values and viable counts of TA01 strain
The bacterial liquid was activated at 37 ℃， inoculated into 30 ml of TSB medium at a ratio of 2%， and cultured at 37 ℃ in a constant temperature incubator with shaking at 220 r/min. At 1-24 hpostincubation， 1 ml of bacterial liquid was respectively collected and centrifuged at 5 000 r/min for 5 min to establish the growth curve with incubation time as the abscissa and logarithm of bacterial concentration[lg（cfu/ml）]as the ordinate. The measurement was repeated three times at each time point， and the results were averaged to reduce the test error.
After 5 h of incubation， 2 ml of bacterial liquid was centrifuged at 5 000 r/min for 5 min. The supernatant was discarded and the precipitate was resuspended with an equal volume of PBS. The bacterial suspension was diluted 1， 2， 3， 4， 5， 6， 7， 8， 9 and 10fold， respectively. OD600values of different dilutions of bacterial suspension were detected using a spectrophotometer at 600 nm. Drug sensitivity test
The sensitivity of the isolated strain to penicillin， erythromycin， florfenicol， ciprofloxacin， polymyxin B， clindamycin， oxacillin and rifampicin was detected according to the instructions of drug susceptibility test strips.
Pathogenicity analysis
The isolated strain was inoculated to TSA medium containing 5% fetal bovine serum by streak plate method， and cultured at 37 ℃for 18-20 h as the F1 generation. A single colony of the F2 generation was picked， inoculated to TSA medium containing 5% fetal bovine serum and cultured at 37 ℃ for 14 h. Bacterial cells were washed. The bacterial concentration was calculated according to the linear relationship between OD600values and viable counts and diluted to the required level. Sixty 4weekold healthy Kunming mice were randomly divided into 6 groups， 10 per group. Groups 1-5 were the experimental groups （3.6×1010cfu/mouse group， 3.6×109 cfu/mouse group， 3.6×108 cfu/mouse group， 3.6×107 cfu/mouse group and 3.6×106 cfu/mouse group）， and group 6 was the control group. Mice in groups 1-5 were injected intraperitoneally with 0.1 ml of 3.6×1010， 3.6×109， 3.6×108， 3.6×107， and 3.6×106 cfu/ml bacterial liquid， while mice in group 6 were injected intraperitoneally with 0.1 ml of PBS. After injection， these mice were put back to the original cages and raised under the same condition. The incidence of the mice was observed and recorded for 72 h continuously. LD50of mice was calculated by the ReedMuench formula.
The bacterial strain isolated from the lungs of dead mice was subjected to an antigenantibody agglutination test to verify the subtype of the isolate.
Histopathological observation
Preparation of organ slices from infected pigs
The joints， brains， lymph nodes and lungs were collected from dissected pigs and fixed with 10% formalin solution to prepare routine paraffin sections for H.E. staining and microscopic examination.
Preparation of organ slices from mice
The lungs， spleens and hearts were collected from mice in experimental and control groups and fixed with 10% formalin solution to prepare routine paraffin sections for H.E. staining and microscopic examination.
Results and Analysis
Anatomical observation
Two dead pigs were dissected. The results of anatomical observation showed pulmonary congestion and hemorrhage with fibrinous exudate on the surface that adhered to the pleura; epicardial hemorrhage; joint swelling with purulent exudate on the cut surface; congestion and swelling of lymph nodes all over the body; meningeal congestion and hemorrhage. The clinical manifestations and anatomical features of infected pigs were consistent with the basic characteristics of streptococcosis， and these pigs were suspected to be infected with Streptococcus suis. The results of anatomical observation were shown in Fig. 1.
Bacterial isolation， identification， staining and microscopic observation
There were a large number of transparent， smooth， small round colonies on the TSA medium containing 5% fetal bovine serum. The colonies were picked for Gram staining and identified as by single， paired or long chainshaped Grampositive bacteria by microscopic examination （Fig. 2）. The isolated bacterial strain was named TA01 strain.
PCR identification
The isolated bacterial strain was identified by PCR using the designed specific primers. A 197 bp band was amplified （Fig. 3）， which was consistent with the expected result， indicating that the isolated strain was Streptococcus suis.
Biochemical experiment
According to the above results， the TA01 strain was initially identified as Streptococcus suis. According to the results of biological experiment， the TA01 strain could ferment 5% lactose and trehalose to produce acid and ferment esculin， but it could neither ferment mannitol and sorbitol nor hydrolyze sodium hippurate， which is in line with the biochemical characteristics of Streptococcus suis type 2.
Establishment of growth curve and correlation curve between OD600values and viable counts of TA01 strain
As shown in Fig. 4A， TA01 strain entered the logarithmic growth phase after being cultured in TSB medium for about 3 h and reached the growth peak at 6-8 h postincubation， which entered the growth decline phase after 8 h of incubation with gradually decreasing viable counts. As shown in Fig. 4B， there was a linear relationship between OD600values and viable counts of TA01 strain during growth retardation phase and logarithmic growth phase. The correlation equation is y=24.659x-1.076 1， R2=0.996 7. Where， x is OD600value and R2 is the correlation.
Drug sensitivity test
As could be seen in Table 2， the isolated strain was sensitive to penicillin， erythromycin， florfenicol and oxacillin， moderately sensitive to rifampicin， and resistant to ciprofloxacin， polymyxin B and clindamycin.
Pathogenicity analysis
Mice in experimental groups exhibited varying degrees of lethargy and rough hairs with individual deaths. Moreover， mice grew well in control group and 3.6×106 cfu/mouse group， with no death; three mice in 3.6 × 107 cfu/mouse group died within 48 h; seven mice in 3.6×108 cfu/mouse group died within 48 h; all the mice in 3.6×109 cfu/mouse group died within 48 h; all the mice in 3.6×1010cfu/mouse group died within 24 h. The results of pathogenicity analysis were shown in Fig. 5. According to the ReedMuench formula， it was calculated that LD50of the isolated strain was 1.137×108 cfu/mouse. The anatomical observation of dead mice showed acute pyaemia septica. The lungs were collected and inoculated to TSA medium containing 5% fetal bovine serum. The obtained bacterial cells were mixed with positive sera of different Streptococcus suis subtypes on the agglutination plate. The results showed that the bacterial cells were agglutinated by positive serum of Streptococcus suis type 2 but showed no aggregation after being mixed with positive sera of other subtypes. According to the results of antigenantibody agglutination test and LD50determination， the isolated strain was a virulent strain of Streptococcus suis type 2.
Histopathological observation
Histopathological observation of various organs of infected pigs
Necrotic tissues in the joint cavity were covered with bluestained bacteria clusters （Fig. 6A）. The alveolar wall exhibited congestion and hemorrhage， accompanied by infiltration and proliferation of inflammatory cells such as neutrophils and macrophages; the alveolar cavity was filled with redstained serous fluid （Fig. 6B）.
The meninx was congested with neutrophil infiltration （Fig. 6C）. Moreover， congestion and hemorrhage of lymph nodes were observed， with infiltration of a large number of neutrophils （Fig. 6D）.
Histopathological observation of various organs of mice
Mice in experimental group exhibited lung congestion with light red serous fluid exudating from the alveolar space （Fig. 7A）. There was infiltration of a large number of neutrophils in the red pulp area of spleen （Fig. 7B）. Granular degeneration of myocardial fibers was observed， with interstitial congestion and hemorrhage， and neutrophil infiltration （Fig. 7C）. However， there were no abnormalities in the lungs， spleens and myocardia of mice in control group （Fig. 7D-F）.
Discussion
Streptococcus suis is a capsular grampositive bacterium， of which type 2 is the most common and most virulent serotype among the 35 serotypes. Streptococcus suis naturally infects pigs through the upper respiratory tract， especially the tonsil and nasal cavity. In this study， a bacterial strain was isolated from the lung lesions of dead pigs， which was identified as Streptococcus suis type 2 by morphological identification， PCR amplification and antigenantibody agglutination test. It was calculated that LD50of the isolated strain was 1.137×108 cfu/mouse， which was much lower compared to other type 2 strains[9-10]， indicating that TA01 strain was a virulent strain of Streptococcus suis type 2. Currently， with the prevalence of immunosuppressive diseases such as porcine reproductive and respiratory syndrome and porcine circovirus disease， the infection rate of Streptococcus suis type 2 infection has increased， however， few studies have been reported about the growth characteristics of Streptococcus suis type 2. In this study， the growth curve and OD600value of Streptococcus suis type 2 were determined by liquid culture method. The results showed that the isolated strain entered the logarithmic growth phase after being cultured in TSB medium containing 5% fetal bovine serum at 37 ℃for about 3 h， and viable counts gradually declined after 8 h of incubation.
In recent years， some scholars have investigated the drug resistance of Streptococcus isolates and found that drug resistance of Streptococcus suis type 2 has been gradually increased. Li et al.[11]determined the drug resistance of 31 strains of Streptococcus suis type 2 in Shanghai and found that all isolates were resistant to five antibiotics including amikacin， ciprofloxacin and ampicillin. Jiao et al.[12]detected the drug resistance of 19 strains of Streptococcus suis type 2 isolated from Anhui Province and reported that all these 19 strains were resistant to more than three antibiotics， and the antibiotic resistance profile included vibramycin， tetracycline， sulfafurazole and bacitracin. In this study， TA01 strain was resistant to ciprofloxacin， polymyxin B and clindamycin. Therefore， it is necessary to apply drugs scientifically and alternately during the prevention and control of streptococcosis in pig farms. Sensitive drugs can be selected through drug sensitivity test for targeted treatment.
The virulence， pathogenicity and immunity of Streptococcus suis type 2 has been a topic of great concern to researchers. So far， some studies have been reported about models of Streptococcus suis type 2 infection[13]. In this study， Kunming mice were selected to construct vaccinationchallenge models. After intraperitoneal injection of 0.1 ml of 3.6×109 cfu/ml TA01 strain， all the mice died within 48 h. Dead mice were dissected， and pyaemia septica was observed， which confirmed that the isolated strain was virulent and could be used as an experimental strain for vaccinationchallenge test or a candidate strain for vaccines.
At present， vaccine immunization and drug application are the main methods adopted in China to prevent and control Streptococcus suis type 2 infection， but the effect of vaccine immunization needs to be further improved. Therefore， it is particularly important to develop safe and effective vaccines against Streptococcus suis. In this study， drug resistance， growth curve， OD600value， LD50， pathogenicity and pathological characteristics of TA01 strain were investigated， which had certain reference significance to the prevention and control of Streptococcus suis type 2 infection and vaccine development in China. References
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