Characterization of Brachyspira hyodysenteriae isolates from Italy by multilocus sequence typing and multiple locus variable number tandem repeat analysis.

AIMS: To evaluate and compare the capabilities of multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) techniques to characterize Brachyspira hyodysenteriae isolates and to investigate the relationship between pleuromutilin resistance and genetic variability. METHODS AND RESULTS: MLST genotyping was performed on 180 B. hyodysenteriae isolates, and the results were evaluated considering profiles from 108 other strains previously reported in the database. In total, 37 sequence types were obtained. The MLVA approach completely characterized 172 strains and grouped the isolates into 22 different profiles. The combination of MLST and MLVA showed a slight increase in the discriminatory power, identifying 33 joint profiles. An antibiotic resistance analysis showed a reduction in the susceptibility to pleuromutilins over time, and a weak association between susceptibility to valnemulin and inclusion in clonal complex 4. CONCLUSION: MLST and MLVA are reliable methods for characterizing B. hyodysenteriae strains and they have comparable discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping of B. hyodysenteriae isolates and a database of all the genetic profiles collected during the diagnostic activities could support traditional epidemiological investigations in identifying infection sources and routes of transmission among herds, and in developing more effective control measures.

An update of Brachyspira hyodysenteriae serotyping.

Brachyspira (B.) hyodysenteriae the causative agent of swine dysentery (SD) has been divided into 9 serotypes on basis of its lipooligosaccharide (LOS). Knowledge on circulating serotypes in Europe, however, is rare. Regarding that immunity to SD is serotype specific an update of B. hyodysenteriae serotyping was undertaken. A LOS band of 10 to 25kDa was identified being appropriate for this purpose. Isolates from Germany, Spain, Denmark, USA and Japan were characterized in the immunoblot by sera raised to serotypes 1 through 7, serogroups H and I (reference strains) and to eight German strains. In total, 57 (51%) isolates responded to at least one of the antisera. Regarding German isolates (n=75) only 35 (46.7%) were identified but mainly by antisera to German strains. Positive Spanish isolates (12 of 17) yielded similar results. In contrast, positively reacting Danish isolates (9 of 12) were mainly identified by antisera to the reference strains as it was the case for recent U.S. (1 of 8) and Japanese isolates (3 of 5). Results indicate that B. hyodysenteriae has a high degree of serological heterogeneity that has probably differently developed in diverse geographical areas over time. This situation represents a challenge for vaccine development.

Understanding the molecular epidemiology and global relationships of Brachyspira hyodysenteriae from swine herds in the United States: a multi-locus sequence typing approach.

Outbreaks of mucohemorrhagic diarrhea in pigs caused by Brachyspira hyodysenteriae in the late 2000s indicated the re-emergence of Swine Dysentery (SD) in the U.S. Although the clinical disease was absent in the U.S. since the early 1990s, it continued to cause significant economic losses to other swine rearing countries worldwide. This study aims to fill the gap in knowledge pertaining to the re-emergence and epidemiology of B. hyodysenteriae in the U.S. and its global relationships using a multi-locus sequence typing (MLST) approach. Fifty-nine post re-emergent isolates originating from a variety of sources in the U.S. were characterized by MLST, analyzed for epidemiological relationships (within and between multiple sites of swine systems), and were compared with pre re-emergent isolates from the U.S. Information for an additional 272 global isolates from the MLST database was utilized for international comparisons. Thirteen nucleotide sequence types (STs) including a predominant genotype (ST93) were identified in the post re-emergent U.S. isolates; some of which showed genetic similarity to the pre re-emergent STs thereby suggesting its likely role in the re-emergence of SD. In the U.S., in general, no more than one ST was found on a site; multiple sites of a common system shared a ST; and STs found in the U.S. were distinct from those identified globally. Of the 110 STs characterized from ten countries, only two were found in more than one country. The U.S. and global populations, identified as clonal and heterogeneous based on STs, showed close relatedness based on amino acid types (AATs). One predicted founder type (AAT9) and multiple predicted subgroup founder types identified for both the U.S. and the global population indicate the potential microevolution of this pathogen. This study elucidates the strain diversity and microevolution of B. hyodysenteriae, and highlights the utility of MLST for epidemiological and surveillance studies.

Dissemination of clonal groups of Brachyspira hyodysenteriae amongst pig farms in Spain, and their relationships to isolates from other countries.

BACKGROUND: Swine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. METHODOLOGY/PRINCIPAL FINDINGS: A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. CONCLUSIONS/SIGNIFICANCE: This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains.

Phenotypic and genetic diversity among intestinal spirochaetes (genus Brachyspira) in free-living wild mallards (Anas platyrhynchos) sampled in southern Sweden.

Brachyspira spp. are anaerobic intestinal spirochaetes that colonize vertebrates. Some species cause enteric diseases in pigs, chickens and possibly in humans, whereas others display a commensual relationship with their hosts. The aims were to investigate the prevalence among colonized free-living wild mallards (Anas platyrhynchos) of three enteropathogenic Brachyspira spp., and to describe the biodiversity of Brachyspira spp. isolates. Isolates from 150 birds were screened by PCR for 3 pathogenic Brachyspira spp., and 35 isolates from 20 mallards, 4 pigs and 1 chicken were subjected to phenotypic tests, 9 diagnostic PCRs, sequencing of the 16S rRNA and NADH oxidase (nox) genes, phylogenetic analysis and nox gene restriction enzyme analysis in silico. Of the 150 birds, 47%, 33% and 11% were positive by PCR for Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira hyodysenteriae, respectively. Thirty-one characterized isolates were provisionally identified as B. intermedia, Brachyspira alvinipulli, "Brachyspira pulli", or B. pilosicoli, whereas 4 were of indeterminate species affiliation. Many isolates were phylogenetically related to isolates from livestock. Isolates identified by PCR as B. pilosicoli displayed particularly high biodiversity. Up to five different Brachyspira genotypes were found from the same bird. Sequencing of amplicons from isolates that displayed ambiguous results as judged from PCR and phenotyping showed that several diagnostic PCRs were non-specific. Nox gene restriction enzyme analysis in silico correctly identified 2 of 34 characterized isolates. A culture technique based on filtration that produced uncontaminated spirochaete isolates was described. The results show that mallard intestines support a high degree of biodiversity among Brachyspira spp.

In vitro antimicrobial susceptibility of Brachyspira hyodysenteriae strains isolated in Japan from 1985 to 2009.

The antimicrobial susceptibilities of 72 Brachyspira hyodysenteriae isolates collected from clinical cases of swine dysentery (SD) in 11 prefectures in Japan between 1985 and 2009 were investigated by an agar dilution method using five antimicrobial agents. There is a tendency of Japanese field isolates of B. hyodysenteriae to acquire resistance to the main antimicrobials used in SD treatment such as tiamulin, valnemulin, and efrotomycin. A responsible approach for selection and use of antimicrobial agents is required for SD treatment.

Multiple-locus variable-number tandem-repeat analysis of the swine dysentery pathogen, Brachyspira hyodysenteriae.

The spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe colonic infection of pigs that has a considerable economic impact in many swine-producing countries. In spite of its importance, knowledge about the global epidemiology and population structure of B. hyodysenteriae is limited. Progress in this area has been hampered by the lack of a low-cost, portable, and discriminatory method for strain typing. The aim of the current study was to develop and test a multiple-locus variable-number tandem-repeat analysis (MLVA) method that could be used in basic veterinary diagnostic microbiology laboratories equipped with PCR technology or in more advanced laboratories with access to capillary electrophoresis. Based on eight loci, and when performed on isolates from different farms in different countries, as well as type and reference strains, the MLVA technique developed was highly discriminatory (Hunter and Gaston discriminatory index, 0.938 [95% confidence interval, 0.9175 to 0.9584]) while retaining a high phylogenetic value. Using the technique, the species was shown to be diverse (44 MLVA types from 172 isolates and strains), although isolates were stable in herds over time. The population structure appeared to be clonal. The finding of B. hyodysenteriae MLVA type 3 in piggeries in three European countries, as well as other, related, strains in different countries, suggests that spreading of the pathogen via carrier pigs is likely. MLVA overcame drawbacks associated with previous typing techniques for B. hyodysenteriae and was a powerful method for epidemiologic and population structure studies on this important pathogenic spirochete.

Characterization and epidemiological relationships of Spanish Brachyspira hyodysenteriae field isolates.

This research aimed to describe the genetic and phenotypic diversity of 74 Spanish Brachyspira hyodysenteriae field isolates, to establish epidemiological relationships between the isolates and to confirm the presence of tiamulin-resistant isolates in Spain. For these purposes, we performed biochemical tests in combination with diagnostic PCR analysis for the identification of Brachyspira spp. and for detection of the smpA/smpB gene. We also used antimicrobial susceptibility tests, random amplified polymorphic DNA (RAPD) and a new pulsed-field gel electrophoresis (PFGE) protocol. The combination of RAPD and PFGE allowed the study of epidemiological relationships. Both indole-negative and tiamulin-resistant isolates of B. hyodysenteriae are reported in Spain for the first time. The genetic analyses indicated a relationship between these Spanish isolates and indole-negative isolates previously obtained from Germany and Belgium.

Identification and antimicrobial susceptibility of porcine bacteria that inhibit the growth of Brachyspira hyodysenteriae in vitro.

AIMS: To identify bacilli, lactic acid bacteria and bifidobacteria that inhibit the growth of Brachyspira hyodysenteriae. METHODS AND RESULTS: A total of 80 isolates were obtained from various porcine intestinal compartments using selective conditions and grouped into 15 similarity clusters based on whole-cell protein profiles. Random amplified polymorphic DNA PCR patterns identified 24 genotypes. 16S rDNA sequencing assigned all genotypes, except eight aerobes, to established species (Bacillus subtilis, Enterococcus faecium, Lactobacillus salivarius, Lactobacillus mucosae, Lactobacillus reuteri, Lactobacillus amylovorus, Bifidobacterium thermophilum). According to their minimum inhibitory concentrations, four strains (Ent. faecium, Lact. reuteri, Lact. amylovorus, Bif. thermophilum) were susceptible to all clinically relevant antibiotics. Two lactobacilli showing multiresistance harboured the erm(B) determinant. A cross-section of eight representative strains was examined for growth suppression of two strains of Brach. hyodysenteriae, the aetiological agent of swine dysentery, and compared with intestinal strains derived from other animal sources. The Brachyspira strains were inhibited by strains of Lact. salivarius, Bif. thermophilum, Ent. faecium and B. subtilis. CONCLUSIONS: Three porcine strains of Ent. faecium, Bif. thermophilum and B. subtilis were found to be suitable as probiotic candidates because of their well-established identity, antibiotic susceptibility and antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, antagonistic activity of well-characterized porcine strains against Brach. hyodysenteriae is presented. These findings suggest that certain intestinal strains might have a potential as probiotic feed additives for prevention of swine dysentery.

Characterization of Brachyspira hyodysenteriae isolates from Korea.

This study was done to characterize diversity in 10 Brachyspira hyodysenteriae isolates in Korea. The isolates were compared with 14 well-characterized non-Korean strains of various Brachyspira species. All Korean isolates showed strong beta haemolysis and had blunt cell ends with 7-14 periplasmic flagella. They produced indole, and did not ferment fructose. They were alpha-glucosidase positive and alpha-galatosidase negative using the APIZYM kit. Using polyclonal antisera raised in rabbits against recognized serotypes, all isolates showed a strong reaction to B. hyodysenteriae antisera E, A and B. Using multilocus enzyme electrophoresis (MLEE) with 15 enzymes and 5 buffer systems, the Korean and non-Korean isolates were divided into 22 electrophoretic types (ETs) and 5 divisions (A, B, C, D and E). Division A corresponded to B. hyodysenteriae, B to B. innocens, C to B. intermedia, D to B. murdochii and E to B. pilosicoli. The 10 Korean isolates of B. hyodysenteriae were relatively diverse, being divided into 9 ETs within MLEE division A. They were all distinct from the non-Korean strains.

Characterization of Brachyspira hyodysenteriae isolates from Korea

This study was done to characterize diversity in 10 Brachyspira hyodysenteriae isolates in Korea. The isolates were compared with 14 well-characterized non-Korean strains of various Brachyspira species. All Korean isolates showed strong beta haemolysis and had blunt cell ends with 7~14 periplasmic flagella. They produced indole, and did not ferment fructose. They were alpha-glucosidase positive and alpha-galatosidase negative using the APIZYM kit. Using polyclonal antisera raised in rabbits against recognized serotypes, all isolates showed a strong reaction to B. hyodysenteriae antisera E, A and B. Using multilocus enzyme electrophoresis (MLEE) with 15 enzymes and 5 buffer systems, the Korean and non-Korean isolates were divided into 22 electrophoretic types (ETs) and 5 divisions (A, B, C, D and E). Division A corresponded to B. hyodysenteriae, B to B. innocens, C to B. intermedia, D to B. murdochii and E to B. pilosicoli. The 10 Korean isolates of B. hyodysenteriae were relatively diverse, being divided into 9 ETs within MLEE division A. They were all distinct from the non-Korean strains.

Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin.

Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe diarrhoeal disease in pigs. Few drugs are available to treat the disease, owing to both antimicrobial resistance and withdrawal of drugs authorized for use in pigs. Tiamulin is the drug of choice in many countries, but isolates with decreased susceptibility have recently been reported. The mechanism of tiamulin resistance in B. hyodysenteriae is not known and this facet is essential to understand the dissemination of the trait. To study the resistance epidemiology of B. hyodysenteriae, further characterization of a set of isolates from Germany (n = 16) and the UK (n = 6) with decreased susceptibility to tiamulin was performed. The relatedness between the isolates was studied by comparing PFGE patterns, and the in vitro susceptibility to five other antimicrobials (aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin) was also determined. For comparison of the antimicrobial-susceptibility pattern, Swedish (n = 20) and British (n = 4) tiamulin-susceptible isolates were tested. The German isolates represented several different PFGE patterns, indicating that tiamulin usage has been sufficient to select clones with decreased tiamulin susceptibility at different farms in Germany. The PFGE pattern for the six British isolates with decreased tiamulin susceptibility was identical to that of the German isolates, and they had a similar antimicrobial-susceptibility pattern, except for resistance to aivlosin, which was only found in a few German isolates. No other co-resistance with tiamulin was found.

Brachyspira hyodysenteriae and other strongly beta-haemolytic and indole-positive spirochaetes isolated from mallards (Anas platyrhynchos).

The aims of the current study were to collect intestinal spirochaetes (genus Brachyspira) from farmed and wild mallards (Anas platyrhynchos) and to identify and classify those isolates that phenotypically resembled Brachyspira hyodysenteriae, an enteric pathogen of pigs. The isolation rate of Brachyspira spp. was high from both farmed (93 %) and wild mallards (78 %). In wild mallards, it appeared that Brachyspira spp. were more likely to be found in migratory birds (multivariate analysis: RR = 1.8, 95 % CI 1.1-3.1) than in mallards sampled in a public park. Pure cultures of putative B. hyodysenteriae were obtained from 22 birds. All five isolates from farmed mallards and ten randomly selected isolates with this phenotype were used for further studies. All isolates from farmed mallards and two of the isolates from wild mallards were PCR-positive for the tlyA gene of B. hyodysenteriae. Two isolates from farmed mallards were selected for pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis. These isolates clustered with the type and reference strains of B. hyodysenteriae. 16S rDNA sequence analysis performed on 11 of the strains showed that they were all closely related to each other and to the B. hyodysenteriae-Brachyspira intermedia cluster. Three of the mallard isolates had 16S rDNA sequences that were identical to those of B. hyodysenteriae strains R1 and NIV-1 previously isolated from common rheas (Rhea americana). To conclude, the isolates from farmed mallards and two isolates from wild mallards were classified as B. hyodysenteriae based on the fact that they could not be differentiated by any of the applied methods from type, reference and field strains of B. hyodysenteriae. The remaining isolates could not be assigned irrefutably to any of the presently recognized Brachyspira species. These results point to a broader host spectrum of B. hyodysenteriae than is generally recognized, and to the presence in mallards of strongly beta-haemolytic and indole-producing spirochaetes that possess many, but not all, of the currently recognized characteristics of B. hyodysenteriae.

Detection of same sized 4.3 Kb extrachromosomal DNA elements in weakly beta-haemolytic human intestinal spirochaetes and Serpulina pilosicoli of swine origin.

Agarose gel electrophoresis of total DNA from Italian strains of weakly beta-haemolytic human intestinal spirochaetes (w beta HIS) and porcine Serpulina pilosicoli reference strain P43/6/78 showed an extrachromosomal band having the same size and migrating at 4.3 Kb. The same results were observed after agarose gel electrophoresis of DNA obtained from the supernatant fluids of the spirochaetal cultures analysed. Swine Serpulina hyodysenteriae reference strain P18A was comparatively analysed and a 6.5 Kb extrachromosomal DNA element was found, as expected. Furthermore, S. hyodysenteriae reference strain P18A differed from all the other spirochaetes tested and had a higher number of flagella (8-12) at each cell end and was strongly beta-haemolytic. To the best of our knowledge, this is the first report on the detection of a band of extrachromosomal DNA having the same size in w beta HIS and S. pilosicoli from swine origin.

[Investigations on Brachyspira--diagnostic and therapeutic strategies in swine dysentery]. / Untersuchungen zur Brachyspira--Diagnostik und Behandlungsstrategie bei der Schweinedysenterie.

The infectious agent of swine dysentery, Brachyspira (Br.) hyodysenteriae, seems to be widespread in German pig herds. Due to different reasons the eradication is increasingly difficult. Not only the success of therapeutic procedures but also the possibilities of diagnostics are unsatisfactory. Although only the bacteriological investigation of faeces or intestinal probes by culture techniques allows the typing of Brachyspira strains and the testing of drug resistance, however, the rate of false negative results is relatively high. In comparison with the cultural method an easy, prompt and cheap immunofluorescent test (IFT) resulted in a good sensitivity (90%). The higher rate of negative results by culture techniques can not be attributed to a lower specificity of the IFT, but to an insufficient transport of samples to the laboratory. The IFT therefore has to be considered as a valuable supplement to the cultural diagnostic of Br. hyodysenteriae. It is absolutely necessary to establish strategies in eradication of swine dysentery which result in pig breeding herds free of Br. hyodysenteriae. Only weaner pigs which are reliable free of this germ guarantee a fattening period sufficiently free of swine dysentery. The principles of different measures in effective eradication are described.

Analysis of Serpulina hyodysenteriae strain variation and its molecular epidemiology using pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was applied as a molecular typing tool for the spirochaete Serpulina hyodysenteriae, the agent of swine dysentery. Analysis of a collection of 40 mainly Australian isolates, previously characterized by other methods, divided these into 23 PFGE types. This confirmed that there are many strains of the spirochaete in Australia. PFGE was more discriminatory for strain typing than both multilocus enzyme electrophoresis and serotyping. It had similar discriminatory power to restriction endonuclease analysis, but the results of PFGE were easier to interpret. When applied to 29 isolates collected from 4 farms over periods of up to 8 years, 2 PFGE patterns were found on 3 farms, and a single pattern on the other. In each case a new strain had apparently emerged as a variant of an original parent strain. PFGE was found to be a powerful technique for investigating the molecular epidemiology of swine dysentery outbreaks.

[Diagnosis of swine dysentery and spirochaetal diarrhea. 2. Effort of macrorestriction analysis for differentiation of intestinal Serpulina relative to determination by culture-biochemical markers following species classification]. / Zur Diagnostik von Schweinedysenterie und Spirochätendiarrhoe.

Genotypic differentiation by means of macrorestriction fragment profile analysis using Mlul restriction enzyme was carried out differentiating 41 Serpulina field strains from swine (38), dog (2) and a rat as well as ten type and reference strains into 40 electrophoretic types. A dendrogram was created using the average linkage between groups method. At a level of 50% similarity the patterns could be divided into six groups that roughly corresponded to the results yielded by cultural and biochemical methods formerly (FELTRUP et al. 1999). Five of these clusters corresponded to the five known porcine Serpulina species, one cluster contained the S. pilosicoli isolates from dog and rat included in this study. Interestingly all nine investigated indole negative, strongly haemolytic isolates were clustered together in one group with the S. hyodysenteriae strains, so that incidence of indole negative variants of S. hyodysenteriae was confirmed. Because of being grouped together with two S. intermedia isolates, the suitability of B 256 as S. innocens type strain is--in accord to investigations carried out by PETTERSSON et al (1996)--called in question.

[The diagnosis of swine dysentery and spirochaete diarrhea. 1. Cultural-biochemical differentiation of intestinal Serpulina in routine diagnosis]. / Zur Diagnostik von Schweinedysenterie und Spirochätendiarrhoe. 1. Mitteilung: Kulturell-biochemische Differenzierung intestinaler Serpulinen in der Routinediagnostik.

Frequent incidence of Serpulina strains showing all cultural and biochemical characteristics of Serpulina (S.) hyodysenteriae except of being indole negative, and alpha-galactosidase positive isolates showing strong haemolysis on Columbia agar with 5% sheep blood and trypticase soy agar with 5% ox blood, respectively, was the cause to evaluate common biochemical and cultural methods in Serpulina routine diagnostics. To this purpose ten type and reference strains as well as 47 field strains were examined for their ability to produce indole, haemolysis, hippurate cleavage, alpha-galactosidase, alpha- and beta-glucosidase activity. Two four-hour identification-systems were used, RapID ANA II and Rosco diagnostic tablets. The ability to produce indole was determined by different methods. All investigations were carried out at least two times. For the investigation of haemolytic patterns trypticase soy agar with 10% ox blood proved to be most effective. Results received using this agar could always be confirmed by the ring phenomenon. Determining the ability to produce indole by adding p-dimethylaminocinnamaldehyde to bacterial growth collected on a cotton swab was confirmed to be more sensitive than other methods. Both four-hour-systems were shown to be useful in Serpulina diagnostics, though in the RapID ANA II only four of 18 available reactions could be used and the hippurate cleavage reaction has to be carried out additionally. Using cultural and biochemical methods, it was possible to assign the type and reference strains to the correct species, as well as 46 of 47 field isolates could be identified including all five known intestinal Serpulina species from swine. 27 strains were determined as S. hyodysenteriae, nine of these isolates atypically being indole negative. In contrast one canine S. pilosicoli strain was atypical showing indole production. Therefore incidence of indole negative variants of S. hyodysenteriae as well as indole positive S. pilosicoli isolates must be taken into consideration.

[Serotyping of strains of Serpulina hyosenteriae isolated form swine with porcine dysentery symptoms in the province of Buenos Aires]. / Serotipificación de cepas de Serpulina hyodysenteriae aisladas de cerdos con cuadros de disentería porcina en la Provincia de Buenos Aires.

Seventeen Serpulina hyodysenteriae strains isolated from faeces, rectal swabs and intestinal contents of pigs with Swine Dysentery, from farms located in Buenos Aires province were serotyped. Samples on selective media (trypticase soy agar added by 5% ovine blood, 400 mg/l spectinomicin, 30 mg/l colistin, 30 mg/l vancomycin) were streaked and incubated under anaerobic atmosphere for 72 h at 42 degrees C. Suspected S. hyodysenteriae growth were identified by strong beta-hemolytic zone, without colonies, and the spirillar morphology, using the Victoria Blue 4-R stain were criteria following by S. hyodysenteriae preliminar identification. The following antigens were made by phenolic extraction from a concentrated inocula washed twice in PBS pH 7: whole-cell (WC), boiled cell (BC) and lipopolysaccharide (LPS). Two serological test were: coagglutination and immunodiffusion, using polyclonal rabbit antisera against the 9 serotypes of S. hyodysenteriae and S. innocens, using WC and BC like antigens for the first test and BC and LPS for the second. The Dot-ELISA Test was performed using BC and LPS antigens and monoclonal antibodies (AbM) against serotypes 1, 2, 3, 8, 9 of S. hyodysenteriae, AbM species-specific and AbM against S. innocens. All isolated S. hyodysenteriae strains belonged to serotype 8. Like in other countries occurred, it would exit a high regional prevalence of S. hyodysenteriae serotype, being the serotype 8 in Argentine.

Presence of 22- and 17-kDa proteins reacting with sera in mice experimentally infected with Brachyspira (Serpulina) hyodysenteriae.

The antibodies to B. (S.)hyodysenteriae in experimentally infected mice were detected by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). The reactions in MAT were serotype specific while those in ELISA were common to both strains. A further investigation with immunoblotting technique demonstrated that 22- and 17-kDa proteins reacted strongly with the sera. The proteins in ATCC 27164 strain strongly reacted with the serum from ATCC 31212 strain-infected mouse and vice versa. These proteins were sensitive to proteinase K.