bacteria isolation : bhi - t 80 agar : bhi - t 80 agar and 
 broth were used for isolation of e. rhusiopathiae strains from tissue 
 samples . the strains were identified and confirmed as e. rhusiopathiae by 
 polymerase chain reaction ( pcr )   after gram stain 
 and microscopic examination . all strains were isolated from cases of acute swine erysipelas 
 causing sudden death of fattening pigs and postpartum sows in 2009 ( 1 strain ) and 2013 ( 7 
 strains ) and were named after the isolation districts and time ( table 1table 1.strains used in the study and eight field strains isolated from cases of 
 septicemia causing acute death of fattening pigs and postpartum sows in eastern 
 chinastrainsdate isolatedarea isolatedgenbankaccession numberserotypecountry ofisolationserotype 1a ( fujisawa)unknownunknownab259652.11ajapang4t10unknownunknownkj6450721aunknownsy09102727/10/2009siyangkj6450801achinanz13070101/07/2013nanzhangkj6450731achinahx13070909/07/2013hexiankj6450741achinaxc13071010/07/2013xuanchengkj6450751achinash13072323/07/2013sihongkj6450761achinayc13082020/08/2013yanchengkj6450771achinayc13082828/08/2013yanchengkj6450781achinayc13111515/11/2013yanchengkj6450791achinaa ) isolates yc130820 , yc130828 and yc131115 were originated from three different 
 farms in yancheng . ) . a ) isolates yc130820 , yc130828 and yc131115 were originated from three different 
 farms in yancheng . g4t10 strain ( serotype 1a ) , a widely used commercial vaccine in 
 china , was used as a reference attenuated strain with acriflavine resistance . serotyping : serotyping was done on all the 8 field e. 
 rhusiopathiae strains as described previously with some modifications . tanja opriessnig ( veterinary diagnostic laboratory , college of 
 veterinary medicine , iowa state university , ames , iowa , u.s.a . ) . in brief , a pure culture 
 was grown at 37c for 48 hr in 30 ml of bhi broth ( binhe microorganism 
 reagent , hangzhou , china ) supplemented with 10% bovine serum ( tianhang biological 
 technology , deqing , china ) . the culture was treated with formalin solution ( final 
 concentration of 1% ) ( xilong chemical corporation , shantou , china ) , held at room temperature 
 for 24 hr , harvested by centrifugation and washed three times in 1  pbs buffer containing 
 5% formalin . washed cells were suspended in 1 ml of distilled water and 
 autoclaved at 121c for 45 min . the supernatant was collected and delivered to dr . analysis of the 432-bp hypervariable region in the spaa gene of different 
 strains : primers erko-1f ( 5-gtgaaacaccgtattttagta-3 ) and erko-2r ( 5- 
 ttcaagaagttcctgtagttt-3 ) were used to amplify the highly variable region of the 
 spaa gene . briefly , genomic 
 dna of e. rhusiopathiae strains was prepared using bacterial dna kit 
 ( omega , norcross , ga , u.s.a . ) . polymerase chain reaction ( pcr ) was performed as described 
 elsewhere   under the following conditions with 
 some modifications : primestar hs dna polymerase ( takara , tokyo , japan ) was 
 used to perform pcr ; a denaturation step at 94c for 3 min followed by 35 cycles of 
 denaturation at 94c for 45 sec ; annealing at 55c for 30 sec and extension at 72c for 30 
 sec ; and final extension of 5 min . for each strain , pcr was performed three times , and each 
 time , the pcr products were ligated with the pmd19-t simple vector 
 ( takara ) , and then , three colonies of each ligation product were sent to 
 invitrogen ( life technologies , shanghai , china ) for sequencing . pulsed - field gel electrophoresis : chromosomal dnas from the strains were 
 digested with smai , and pfge was performed 3 times according to opriessnig 
 et al .. electrophoresis was 
 carried out for 23 hr at 14c and 6 v with pulse times from an initial 1.2 sec to a final 30 
 sec . pfge patterns were detected by uv transillumination after staining by sybr gold nucleic 
 acid gel stain ( s11494 , life technologies ) . a lambda ladder pfg marker ( n0340s , neb , 
 ipswitch , ma , u.s.a . ) was used as dna size standard . acriflavine resistance test : the acriflavine resistance of all the eight 
 strains was examined by streaking strains onto agar plates containing 0.04 , 0.03 , 0.02 , 
 0.015 , 0.01 , 0.075 , 0.005 , 0.0025 , 0.00125 or 0% acriflavine ( aladdin , shanghai , china ) . the 
 acriflavine agar plates were prepared by adding 1% acriflavine ( dissolved in distilled 
 water ) to the sterilized basal medium ( bhi - t 80 agar ) . the isolates were cultivated in 3 
 ml bhi - t 80 broth overnight at 37c and were then streaked onto the agar 
 plates . the vaccine strain ( g4t10 ) the growth on the plates was observed after incubation at 37c for 48 
 hr . acriflavine resistance was determined by the highest concentration at which a strain 
 showed almost the same growth as on the agar that did not contain acriflavine [ 8 , 9 , 11 ] . attenuation of strain hx130709 : strain hx130709a ( derived from strain 
 hx130709 ) was attenuated by 55 passages on bhi - t 80 agar containing a gradually increasing 
 concentration ( 0.0025% to 0.03% ) of acriflavine dye . finally , the 432-bp partial 
 spaa genes of all the archived strains were amplified for sequence 
 analysis , and the virulence of each archived strain was confirmed in mice . mouse pathogenicity tests : one hundred and sixty - five female icr mice of 
 six - week - age were purchased from the laboratory animal centre of nantong university 
 ( nantong , china ) . the animals were cared for in accordance with the guide for the 
 care and use of laboratory animals ( 1996 , published by national academy press , 
 nw , washington , dc , u.s.a . ) . the animal experiments were approved by the institutional 
 animal care and ethics committee of nanjing agricultural university ( approval no . a total of 90 female icr mice of six - week - age were given subcutaneous injections in the 
 right groin with 10 colony forming units ( cfu)/ 0.1 ml of the 
 isolates or the vaccine strain . a group of 10 mice served as an untreated control . since a 
 positive correlation between the isolate s cfu and od value had been built in advance ( data 
 not shown ) , the number of bacteria injected into each mouse was almost the same . mice were 
 observed every 12 hr to detect clinical signs of the disease for the subsequent 14 days 
 . another 60 female icr mice of six - week - age were given subcutaneous injections in the right 
 groin with 10 cfu/ 0.1 ml of the isolate strain hx130709 ( f0 ) 
 and different passages of the attenuated strain hx130709a ( f5 , f10 , f15 , f20 , f25 , f30 , f35 , 
 f40 , f45 , f50 and f55 ) . antimicrobial susceptibility test : each isolate was cultured in 3 
 ml bhi - t 80 broth overnight at 37c . then , 0.1 ml of the 
 bacterial culture of each isolate was dropped onto bhi - t 80 agar , after which , antibiotic 
 susceptibility discs ( binhe microorganism reagent ) were put onto the bhi - t 80 agar . the 
 diameter of the bacteriostatic circle was measured after incubation at 37c for 24 hr . the 
 susceptibility was determined according to instructions of the clinical and laboratory 
 standards institute ( clsi ) . serotyping and acriflavine resistance : all the eight e. 
 rhusiopathiae isolates were determined to be serotype 1a ( fig . 1.genetic relationship between 8 e. rhusiopathiae field isolates and 1 
 vaccine strain and schematic representation of 8 different pfge patterns obtained 
 after restriction digestion with sma i. the classification and 
 divergence of isolates were calculated by the unweighted pair group method with 
 averages from the pfge results . at 5% divergence , 3 pfge groups ( a - c ) were present ; 
 group a was subgrouped into a1/a2 at 3.5% divergence . ) , which was finished by the laboratory of dr . tanja opriessnig ( veterinary diagnostic 
 laboratory , college of veterinary medicine , iowa state university ) . the results of the 
 acriflavine resistant test are shown in table 
 2table 2.acriflavine resistance of e. rhusiopathiae field isolates and 
 vaccine reference strainsstrainmic of acriflavine(% w / v)acriflavineresistancesy0910270.02rnz1307010.005shx1307090.0025sxc1307100.02rsh1307230.02ryc1308200.0025syc1308280.005syc1311150.0175rg4t10 ( vaccine)0.02ra ) resistant ( r ) or sensitive ( s ) to 0.01% acriflavine .. out of the total 8 isolates , 3 ( 37.5% ) showed culture growth on the bhi - t 80 
 agar containing 0.02% acriflavine , which was identical to the growth of 
 g4t10 strain ( positive control ) . genetic relationship between 8 e. rhusiopathiae field isolates and 1 
 vaccine strain and schematic representation of 8 different pfge patterns obtained 
 after restriction digestion with sma i. the classification and 
 divergence of isolates were calculated by the unweighted pair group method with 
 averages from the pfge results . at 5% divergence , 3 pfge groups ( a - c ) were present ; 
 group a was subgrouped into a1/a2 at 3.5% divergence . sequence analysis of the 432-bp hypervariable region in the spaa gene : 
 according to nagai et al . , 
 different nucleotide substitutions in the 432-bp hypervariable region on the 
 spaa gene of the eight e. rhusiopathiae field strains 
 and the japanese official challenge strain fujisawa comparing with the vaccine strain 
 g4t10 are shown in table 
 3table 3.substitutions in nucleotide and amino acid in a 432-bp hypervariable 
 region on the spaa gene of 8 e. rhusiopathiae field strains compared 
 with the corresponding sequence of the vaccine strain 
 g4t10substitutionpattern / strainsubstitutions ( position [ nucleotide ] , amino 
 acid  ) no . of nucleotidesubstitutionsspaa type584 ( a)590 ( t)609 ( t)769 ( a)885 ( a)d > ai > ti > mi > lnspattern 1nz130701c1ayc130820c1asy091027c1apattern 2hx130709g1bsh130723g1byc131115g1bpattern 3yc130828cg2cpattern4xc130710cg2dreference strainfujisawac1a ) original amino acid > substituted amino acid ; ns = no amino acid mutation ; 
 g = glycine ; d = aspartic acid ; n = asparagine ; a = alanine ; i = isoleucine ; t = threonine ; 
 m = methionine ; l= leucine ; e = glutamic acid . b ) japanese official challenge strain .. according to the different substitutions , spaa can be divided 
 into four spaa - types . no nucleotide substitutions in the 432-bp 
 hypervariable region on the spaa gene of all the archived attenuated 
 strains were found comparing with the parental strain hx130709 ( table 4table 4.nucleotide substitutions in a 432-bp hypervariable region on the spaa gene of 11 
 e. rhusiopathiae strains during the attenuation compared with the 
 corresponding sequence of the original strain hx130709concentration ofacriflavine ( % w / v)number ofpassagebacteria collectedfor spaa sequencingno . of nucleotidesubstitutionsspaa type0.0025f1-f40.00375f5-f7f50b0.005f8-f10f100b0.0125f11-f120.02f13-f27f15 , f20 , f250b0.0225f28-f38f30 , f350b0.025f39-f45f40 , f450b0.0275f46-f480.03f49-f55f50 , f550breference strainhx130709f0b ) . a ) original amino acid > substituted amino acid ; ns = no amino acid mutation ; 
 g = glycine ; d = aspartic acid ; n = asparagine ; a = alanine ; i = isoleucine ; t = threonine ; 
 m = methionine ; l= leucine ; e = glutamic acid . pfge and data analysis : eight distinct pfge patterns with 22 to 30 dna 
 fragment bands were produced from the genomic dna of the 8 isolate strains and 1 vaccine 
 strain g4t10 with smai digestion ( fig . 2.pfge patterns produced from 8 erysipelas field isolates and 1 vaccine strain digested 
 with sma i. lanes : 1 , g4t10 ; 2 , nz130701 ; 3 , 
 hx130709 ; 4 , xc130710 ; 5 , sh130723 ; 6 , yc130820 ; 7 , yc130828 ; 8 , sy091027 ; 9 , 
 yc131115 ; m , lambda ladder pfg marker ( n0340s , neb ) . ) . genetic relationships 
 among the isolates were compared , and the dendogram analysis was done ( fig . 3 pfge groups ( a - c ) were 
 present at 5% divergence . at 3.5% divergence , most of the 
 isolates ( 6/9 ) were within group a including the vaccine strain g4t10 . resistance to acriflavine and spaa - types showed no specific patterns and 
 were randomly distributed . data analysis of the homogeneity of the pfge patterns showed that 
 these 9 strains shared over 92.1% identity with each other . pfge patterns produced from 8 erysipelas field isolates and 1 vaccine strain digested 
 with sma i. lanes : 1 , g4t10 ; 2 , nz130701 ; 3 , 
 hx130709 ; 4 , xc130710 ; 5 , sh130723 ; 6 , yc130820 ; 7 , yc130828 ; 8 , sy091027 ; 9 , 
 yc131115 ; m , lambda ladder pfg marker ( n0340s , neb ) . the pfge patterns produced from the genomic dna of the 12 archived strains during the 
 attenuation with smai digestion are shown in supplemental fig . the pfge 
 patterns of these 12 strains were very similar with each other . a few changes of patterns 
 occurred between strains f10 and f15 , but strains f15-f55 almost shared the same pfge 
 pattern . based on the dendogram analysis , 2 pfge groups ( a - b ) were present at 5.8% 
 divergence ( fig . 3fig . 3.genetic relationship among the 12 archived e. rhusiopathiae strains 
 during the attenuation and their pathogenicity towards mice . the classification and 
 divergence of the strains were calculated by the unweighted pair group method with 
 averages from the pfge results ( supplemental fig . 
 1 ) . most of the strains 
 ( 9/12 ) were within group a in which these 9 strains shared over 99.5% identity with 
 each other . each mouse in different groups was injected with 0.1 ml 
 pbs and 10 cfu strains f0-f55 , respectively . mice were observed daily to 
 detect clinical signs of the disease for the subsequent 14 days . only 3 groups 
 survived for the end of the experiment after receiving subcutaneous injections of 
 10 cfu of strains f50 and f55 and pbs , while other groups injected with 
 10 cfu of strains f0-f45 died within 5 days . ) . most of the strains ( 9/12 ) belonged to group a in which these 9 strains shared over 
 99.5% identity with each other . genetic relationship among the 12 archived e. rhusiopathiae strains 
 during the attenuation and their pathogenicity towards mice . the classification and 
 divergence of the strains were calculated by the unweighted pair group method with 
 averages from the pfge results ( supplemental fig . 
 1 ) . most of the strains 
 ( 9/12 ) were within group a in which these 9 strains shared over 99.5% identity with 
 each other . each mouse in different groups was injected with 0.1 ml 
 pbs and 10 cfu strains f0-f55 , respectively . mice were observed daily to 
 detect clinical signs of the disease for the subsequent 14 days . only 3 groups 
 survived for the end of the experiment after receiving subcutaneous injections of 
 10 cfu of strains f50 and f55 and pbs , while other groups injected with 
 10 cfu of strains f0-f45 died within 5 days . pathogenicity towards mice : the results of the pathogenicity towards mice 
 are shown in figs . 3 and 4fig . each mouse of the untreated control group received a subcutaneous injection of 0.1 
 ml pbs in the right groin and was observed every 12 hr as others . 
 almost every mouse injected with 10 cfu e. rhusiopathiae 
 field strains died within 72 hr , while all mice injected with 10 vaccine 
 strain g4t10 and pbs survived for the end of the experiment .. regardless of the acriflavine resistance , pfge patterns or 
 spaa types , all the 8 isolates and the early passages of the attenuated 
 strains ( f5 , f10 , f15 , f20 , f25 , f30 , f35 , f40 and f45 ) showed high levels of virulence in 
 mice . all mice died within 5 days after receiving subcutaneous injections of 10 
 cfu of the strains , while all those injected with the attenuated strains 
 ( g4t10 , f50 and f55 ) and pbs survived for the end of the 
 experiment . each mouse of the untreated control group received a subcutaneous injection of 0.1 
 ml pbs in the right groin and was observed every 12 hr as others . 
 almost every mouse injected with 10 cfu e. rhusiopathiae 
 field strains died within 72 hr , while all mice injected with 10 vaccine 
 strain g4t10 and pbs survived for the end of the experiment . antimicrobial susceptibility : diameters of the bacteriostatic circles of 
 the 8 isolates , vaccine strain g4t10 and the attenuated strain 
 hx130709a ( f55 ) against the 15 antimicrobial agents are shown in table 5table 5.antimicrobial susceptibility testing results of 8 e. 
 rhusiopathiae field strains , the vaccine strain g4t10 
 and the attenuated strain hx130709aantibioticalsusceptibility discs  diameter of bacteriostatic circle 
 ( mm )  no . of resistantstrains ( % ) g4t10nzxcshycycycsyhxhx130701130710130723130820130828131115091027130709130709apc - g40/s42/s42/s40/s38/s39/s42/s38/s40/s54/samo40/s39/s42/s40/s39/s40/s42/s40/s43/s55/samp36/s38/s36/s34/s38/s36/s40/s34/s36/s46/scez32/s35/s35/s34/s34/s32/s36/s33/s34/s41/sctx37/s36/s36/s42/s34/s34/s38/s33/s37/s47/ssm17/s12/i6.5/r6.5/r14/i14/i6.5/r12/i6.5/r23/s4 ( 40)km6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r6.5/r10 ( 100)tc26/s30/s9/r10/r27/s27/s12/r26/s14/r39/s4 ( 40)doxy25/i30/s11/r14/r25/i26/i18/r27/i15/r36/s4 ( 40)em32/s35/s33/s34/s24/s32/s30/s35/s39/s44/sroxm30/s31/s31/s30/s22/i30/s29/s27/s30/s42/slcm36/s33s11.5/r14/r6.5/r32/s9/r30/s13/r42/s5 ( 50)sxz6.5/r6.5/r10/r8/r6.5/r6.5/r6.5/r6.5/r6.5/r10/r10 ( 100)erfx36/s6.5/r16/i11/r17/i19/i12/r34/s15/i10/r4 ( 40)cip30/s6.5/r20/i18/i18.5/i18.5/i17/i36/s18/i16/i1 ( 10)a ) penicillin ( pc - g ) , amoxicillin ( amo ) , ampicillin ( amp ) , cefazolin ( cez ) , 
 cefotaxime ( ctx ) , streptomycin ( sm ) , kanamycin ( km ) , tetracycline ( tc ) , doxycycline 
 ( doxy ) , erythromycin ( em ) , roxithromycin ( roxm ) , lincomycin ( lcm ) , sulfisoxazole 
 ( sxz ) , enrofloxacin ( erfx ) and ciprofloxacin ( cip ) . s = susceptible ; i = intermediate ; 
 r = resistance .. all the strains were highly sensitive to pc - g , amp , cez , ctx , em , roxm and 
 amo . half of the isolates ( hx130709 , xc130710 , sh 130723 and yc131115 ) were resistant to sm , 
 tc , doxy and lcm , and the other isolates ( nz130701 , yc130820 , yc130828 and sy091027 ) were 
 moderately resistant to these antibiotics . a ) penicillin ( pc - g ) , amoxicillin ( amo ) , ampicillin ( amp ) , cefazolin ( cez ) , 
 cefotaxime ( ctx ) , streptomycin ( sm ) , kanamycin ( km ) , tetracycline ( tc ) , doxycycline 
 ( doxy ) , erythromycin ( em ) , roxithromycin ( roxm ) , lincomycin ( lcm ) , sulfisoxazole 
 ( sxz ) , enrofloxacin ( erfx ) and ciprofloxacin ( cip ) . compared with strain hx130709 , the attenuated strain hx130709a ( f55 ) was much more 
 sensitive to these antibiotics , except for km , sxz , erfx and cip . swine erysipelas used to be widely spread twenty years ago in china , which was later well 
 controlled due to the wide use of antibiotics . in the present study , all the eight 
 e. rhusiopathiae isolates were from cases of septicemia and were 
 determined to be serotype 1a . the acriflavine resistance has been used as one of the tools for discriminating the vaccine 
 strain from field isolates because the live vaccine strain could grow in media containing at 
 least 0.02% acriflavine , while the field strains are usually sensitive to acriflavine . some 
 acriflavine - resistant e. rhusiopathiae strains have been isolated from 
 slaughter pigs affected by chronic arthritis [ 8 , 9 , 14 ] . it s worth 
 mentioning that among the eight e. rhusiopathiae strains isolated from 
 septicemia cases in this study , 3 ( 37.5% ) virulent isolates ( sy091027 , xc130710 and 
 sh130723 ) showed culture growth on the bhi - t 80 agar containing 0.02% acriflavine as the 
 vaccine strain g4t10 did . proved that 
 spaa plays an important role in enhancing the virulence of e. rhusiopathiae 
 strains . a method for discrimination between a japanese vaccine strain and the field strains 
 was developed based on the nucleotide sequencing of a 432-bp hypervariable region in the 
 spaa gene . in this study , spaa gene analysis showed that no mutations occurred among different 
 passages of strain hx130709a comparing with the parental strain hx130709 , which further 
 confirmed that the virulence of serotype 1a e. rhusiopathiae strains 
 isolated from acute swine erysipelas outbreaks in eastern china is unrelated with some point 
 mutations in the hypervariable region of the spaa gene . prevalence of met-203 type spaa variant in e. 
 rhusiopathiae isolates is increasing in japan . interestingly , four of the eight isolates in our study belonged to met-203 
 type , and the other four field isolates belonged to ala-195 type ( supplemental table 1 ) . a 
 further study should be carried out to investigate the prevalence of met-203 type 
 spaa variant in e. rhusiopathiae isolates in china . based on the dendogram analysis , the nine strains shared over 92.1% identity with each 
 other , showing no correlations with spaa - type and resistance to acriflavine 
 ( fig . results of the drug susceptibility test showed that all the strains were highly sensitive 
 to a variety of antibiotics , such as pc - g , amp , cez , ctx , em , roxm and amo , and also 
 resistant to km and sxz , which well explained why swine erysipelas was easy to control with 
 the use of antibiotics . however , nearly half of the isolates showed resistance to lcm , sm , 
 tc , doxy , sxz and erfx , because these antibiotics were widely added in the pig feeds in 
 china . with the emergence of new types of e. rhusiopathiae strains which are 
 resistant to the widely used antibiotics , vaccines seem to be a better choice in controlling 
 swine erysipelas however , attenuated vaccine strains carry the risk of regaining their 
 virulence thus , posing a hazard to susceptible swine , and the inactivated vaccines are 
 rarely used because of the higher vaccination cost . so , it is extremely urgent to develop more effective and safer subunit or 
 gene engineering vaccines with virulence factor . however , the attenuation mechanism of 
 e. rhusiopathiae strains remains unknown . though spaa 
 gene analysis showed that no mutations occurred during the attenuation , the mutations 
 probably occurred in different important points of genome . so , all the archived strains ( f5 , 
 f10 , f15 , f20 , f25 , f30 , f35 , f40 , f45 , f50 and f55 ) and the original strain hx130709 ( f0 ) 
 were analyzed using pfge to detect whether any genome changes occurred during the 
 attenuation . the pfge patterns of the above 12 strains were very similar with each other 
 ( supplemental fig . 1 ) . a few changes of the pfge patterns occurred between strains f10 and 
 f15 , but strains f15-f55 shared over 99.5% identity with each other ( fig . surprisingly , though strains f15-f55 shared almost the same 
 pfge patterns , only strains f50 and f55 were avirulent ( fig . 3 ) . this indicates the attenuation occurred between f46 and f50 and the 
 mutations of smai restriction enzyme cutting site in the genome of the 
 stains did not lead to any change of the virulence . it s possible that the mutations in the 
 genome which directly lead to virulence loss can not be detected by pfge with 
 smai digestion . in this case , comparing the entire genome of wild strain 
 and the attenuated strain may be a good way to understand the attenuation mechanism . but , 
 there is also another possibility that the strain virulence was decreased during the 
 attenuation mainly by phenotype changes instead of genotype changes . in this study , we conclude that the acute swine erysipelas outbreaks in eastern china were 
 caused by serotype 1a e. rhusiopathiae strains with different biochemical 
 characteristics , and the virulence of serotype 1a e. rhusiopathiae strains 
 is unrelated with some point mutations in hypervariable region of the spaa 
 gene . meanwhile , the attenuated strain hx130709a was obtained from a highly virulent 
 clinical strain hx130709 after 55 passages on agar plates containing acriflavine . also , its 
 virulence had been confirmed in mice , which lays the foundation for a further study of 
 virulence factor of e. rhusiopathiae with proteomics and transcriptomic 
 technologies .
recently , a series of acute swine erysipelas outbreaks occurred in eastern china . 
 eight 
 strains isolated from cases of septicemia were determined as serotype 1a , and 4 of the 
 isolates were resistant to acriflavine . 
 one isolate strain named hx130709 was attenuated 
 on agar media containing acriflavine dye . 
 the 432-bp hypervariable region in 
 spaa gene of the field and attenuated strains were amplified and 
 sequenced . 
 it was further compared with the vaccine strain g4t10 , 
 and thus , the eight field strains can be divided into four spaa - types . 
 the partial spaa gene analysis also showed that no point mutations 
 occurred among different archived passages of hx130709 during the attenuation . 
 results of 
 pulsed - field gel electrophoresis showed that eight distinct patterns with 22 to 30 dna 
 fragment bands were produced from field strains , and twelve distinct patterns with 23 to 
 27 dna fragment bands were produced from different passages of the attenuated strains . 
 mouse pathogenicity test showed that the mortality of the mice infected with 
 104 cfu field strains was 100% and the attenuation of strain hx130709 
 occurred between 46 and 50 passages . 
 all the field and attenuated strains were highly 
 sensitive to -lactam antibiotics , tetracyclines and macrolides . 
 so , we can make 
 conclusions that the acute swine erysipelas outbreaks in eastern china were caused by 
 serotype 1a e. rhusiopathiae strains with different biochemical 
 characteristics , and the virulence of serotype 1a e. rhusiopathiae 
 strains is unrelated with some point mutations in 432-bp hypervariable region of the 
 spaa gene .