Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)     

Treponema hyodysenteriae growth under various culture conditions

The influence of various culture conditions on the growth of Treponema hyodysenteriae was determined. Six different anaerobically prepared culture broths were tested for the ability to support growth of strains B78, B204 and B169. Each medium contained glucose (0.2%) and 10% (v/v, final concn.) heat-inactivated fetal calf serum. Brain-heart infusion (BHIS), heart infusion (HS) and veal infusion (VS) broths gave the highest cell yields of the spirochete with the shortest incubation times. Vigorous mixing of the cultures and the introduction of O2 (1%, final concn.) into the culture atmosphere were necessary for optimum growth. Although BHIS broth was found to be the best for routine cultivation of the 3 strains, HS broth was more suitable for investigating the physiology of growing cells, inasmuch as cell growth in this medium was limited unless a growth substrate was added. Glucose, fructose, sucrose, galactose, trehalose, N-acetyl-glucosamine, glucosamine, mannose, maltose and pyruvate were growth substrates for all 3 strains. During the growth of B204 cells in HS broth under N2:O2 (99:1), glucose and O2 were consumed and CO2, H2, acetate and butyrate were produced. In HS agar-containing medium, cells of strains B78 and B204 formed spreading colonies typical in appearance to those of other spirochetes.     

Development of an experimental model allowing discrimination between virulent and avirulent isolates of Serpulina (Treponema) hyodysenteriae.

Variation in virulence among different strains of Serpulina hyodysenteriae was studied by oral inoculation of specific pathogen free piglets and CD-1 mice. Piglets infected with serotype 2 reference strain B204 and an untypable field strain LHV-90-9-I had severe diarrhea tainted intermittently with mucus and fresh blood. The piglets inoculated with B169, B8044, B6933, and ACK300-8 reference strains representing serotypes 3, 5, 6, and 7 respectively developed moderate diarrhea. However, reference strains B234 and A-1 of serotypes 1 and 4, respectively, failed to cause any diarrhea. None of the S. hyodysenteriae strains caused diarrhea in mice. The results indicate a great variation in virulence among strains of different serotypes of S. hyodysenteriae. Mice were less susceptible to infection with S. hyodysenteriae.     

Antimicrobial susceptibility testing of Serpulina hyodysenteriae

Summary The macrobroth dilution technique was used to test the in-vitro effectiveness of 4 commonly used antimicrobial agents against 23 Australian isolates and 7 overseas strains of Serpulina hyodysenteriae. Minimum inhibitory concentrations and minimum bactericidal concentrations were determined. The growth of 90% of isolates was inhibited by dimetridazole at a concentration of 4 μg/mL, and by tiamulin at 8 μg/mL Australian isolates resistant to both antimicrobial agents were identified. Lincomycin was less effective than these antimicrobial agents, with 90% of isolates requiring a concentration of 128 μg/mL for inhibition of growth, and 54% being susceptible at 64 μg/mL. Tylosin did not prevent the growth of the majority of S hyodysenteriae isolates tested, and 90% were resistant to concentrations of 128 μg/mL. Resistant isolates came from different geographical areas. Resistance was not related to overall genetic background of the spirochaetes, and was not correlated with the presence of plasmids or the serogroup of the isolates.     

Prevalence of Treponema hyodysenteriae in healthy pigs.

The prevalence of Treponema hyodysenteriae in faecal samples from healthy pigs of various ages in different farrowing units was investigated.Samples from herds designated as Category I were processed within 2 hrs. of sampling. Samples from herds designated as Category II were transported 2 to 3 days before cultivation procedures started. T. hyodysenteriae was demonstrated in 53.7 % to 93 % of the samples collected from Category I herds. No marked difference in the frequency of positive samples from the various age groups of pigs was found. In Category II herds, the organism was demonstrated in 10 % of the samples.The degree of beta-haemolysis shown by isolated strains was grouped into 3 groups: weak, moderate and strong. Strongly betahaemolytic strains, supposedly enteropathogenic, were demonstrated in all Category I herds. Such strains were found in 4.6 % to 25 % of the positive samples in these herds. In Category II herds, 2 out of 17 positive samples harboured strongly beta-haemolytic strains of T. hyodysenteriae.The amount of growth of T. hyodysenteriae on primary plates inoculated with sample material originating from the 2 categories of herds was mostly moderate or abundant. Strongly beta-haemolytic isolates originating from Category I herds produced abundant growth on primary plates in approx. 60 % of samples harbouring such strains. In samples from Category I herds with strains producing weak or moderate beta-haemolysis sparse and moderate amount of growth of the organism was predominant.     

Swine dysentery: pathogenicity of Treponema hyodysenteriae.

When pure cultures of Treponema hyodysenteriae were orally inoculated into pigs, severe disease characteristic of swine dysentery developed. Less severe lesions were produced by oral inoculation of infective minced colon. Noninoculated pigs were used as controls. Inoculations of surgically isolated porcine colonic loops with either pure cultures or infective minced colon produced lesions only in the injected loop; the adjacent noninjected colon remained normal. Pigs and other experimental animals, including rabbits, guinea pigs, and mice, inoculated with washed cultures by various parenteral routes remained normal.     

Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly β-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.     

Parenteral immunization of pigs against infection with Treponema hyodysenteriae.

Six intravenous injections of formalin-inactivated Treponema hyodysenteriae were given to 8 specific-pathogen-free pigs at 6-day intervals. The 8 vaccinated and 8 control pigs were challenged intragastrically with pure cultures of T hyodysenteriae 7 and 8 days after the last intravenous injection. Clinical signs of swine dysentery were observed in all 8 control pigs, but was observed in only 1 of the immunized pigs. Three control pigs died. These findings suggest that parenteral immunization with T hyodysenteriae provided a marked degree of protection against subsequent intragastric challenge exposure with the homologous isolate of T hyodysenteriae.     

Minimal inhibitory concentrations of five antimicrobials against Treponema hyodysenteriae and Treponema innocens

The minimal inhibitory concentrations of carbadox, dimetridazole, lincomycin, ronidazole, and tiamulin against isolates of Treponema hyodysenteriae and Treponema innocens were determined by an agar-dilution method. The results obtained indicated that tiamulin was the most effective antimicrobial in vitro against T. hyodysenteriae, followed by carbadox. Dimetridazole, lincomycin, and ronidazole had poor efficacy in vitro against the T. hyodysenteriae isolates. Isolates of T. innocens were more sensitive to the various antimicrobials. Carbadox and tiamulin were the most effective in vitro, followed by ronidazole, dimetridazole, and lincomycin.     

A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy

Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.     

Identification of Treponema hyodysenteriae and Treponema innocens using two four-hour identification systems

Two 4-hour identification systems, the RapID-ANAII (Innovative Diagnostic Systems) and the ANI card (Vitek Systems), were used to identify isolates of Treponema hyodysenteriae and T. innocens. Twenty-one isolates of T. innocens and 53 isolates of T. hyodysenteriae were tested with both systems. With the ANI system, alpha-galactosidase was the only test that differentiated the two species. With the RapID-ANAII system, alpha-galactosidase and indole tests allowed differentiation of the two species. Treponema hyodysenteriae was alpha-galactosidase negative and indole positive, whereas T. innocens produces the opposite reactions. Three isolates were alpha-galactosidase negative and indole negative. These isolates represent a group intermediate between the two officially described species. The two species of swine Treponema can be identified by commercial identification kits and a third group of isolates intermediate between the two species was identified.     

Typing of Treponema hyodysenteriae by restriction endonuclease analysis

Restriction endonuclease analysis (REA) was used to type eight well-characterised strains of Treponema hyodysenteriae originating from the U.K., Canada and the U.S.A., and 16 isolates from cases of swine dysentery in Western Australia (W.A.). Several of the W.A. isolates were also serotyped by the method of Baum and Joens (1979), and the two typing techniques were compared. REA typing was more discriminatory than serotyping, being able to distinguish strains within serotypes. The new technique was neither more difficult nor more time-consuming to perform than serotyping. Within the 16 W.A. isolates, three different REA patterns were identified, with common patterns found on different farms. The eight overseas strains had seven different REA patterns, all of which could be distinguished from the patterns of the W.A. isolates.     

Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens

A total of 2450 samples of feces, intestinal contents and colon mucosal scrapings were bacteriologically examined. A total of 53 strains of Treponema sp. were isolated, and 45 strains of Bacteroides sp., 30 strains of E. coli, 30 strains of Micrococcus sp. and 10 strains of Streptococcus D isolates were randomly selected. Growth promoting studies showed statistically significant stimulation of Treponema sp. growth by yeast extract, chicken egg yolk and rumen fluid. Different growth inhibitors were also tested. For selective medium the following inhibitors were selected: spectinomycin, colistin, vancomycin, brilliant green. Optimal concentrations of these inhibitors in the medium were determined. Finally TSA medium supplemented with 0.05% yeast extract, 5% bovine blood, 0.01% DTT, 400 micrograms spectinomycin, and 250 micrograms/ml vancomycin, appeared to be optimal selective medium for intestinal Treponema sp. isolation. Quantitative studies showed that the number of Treponema C.F.U. on Songers et al. medium with spectinomycin and on spectinomycin-vancomycin medium, did not differ significantly. The number of overgrowing bacteria was statistically significantly lower on spectinomycin-vancomycin medium, than Songers et al. selective medium with spectinomycin. The TSA supplemented with blood, yeast extract 50 micrograms/ml of colistin and 1 microgram/ml of brilliant green was less selective than spectinomycin-vancomycin medium and inhibited some strains of Treponema sp. In the case of spectinomycin-vancomycin resistant of overgrowing bacteria, colistin-brilliant green medium may be suitable for isolation of Treponema sp.     

Ultrastructural characterization of colonic lesions in pigs inoculated with Treponema hyodysenteriae.

Twelve pigs were inoculated orally with pure cultures of Treponema hyodysenteriae. Pigs were necropsied at different time intervals postinoculation; colonic specimens were collected and prepared for light and electron microscopy. The earliest colonic lesion detected by electron microscopy consisted of superficial vascular congestion and dilatation, edema of the lamina propria and intercellular separation of the epithelial cells at the crypt shoulders. This lesion progressed to epithelial cell necrosis and extrusion into the lumen and extravasation of red cells. Large numbers of spirochetes were present and free, between, over and under necrotic epithelial cells whether in place or partially extruded. Spirochetal penetration of colonic enterocytes and intracytoplasmic multiplication were confirmed in this study. The spirochetes were found to invade the epithelial cells only from their lateral borders. The relationship between T. hyodysenteriae and the colonic anaerobes was not determined.