Isolation of Treponema hyodysenteriae from sources other than swine

Fecal samples were collected from animals and environments on 3 swine farms and cultured for Treponema hyodysenteriae. Each farm was a farrow-to-finish operation and, at the time of sampling, swine dysentery was enzootic among 8- to 22-week-old pigs. Pathogenic T hyodysenteriae was isolated from pigs on all 3 farms. On farm A, nonpathogenic T hyodysenteriae was isolated from a sample of lagoon water. On farm B, pathogenic T hyodysenteriae was isolated from a waste-holding pit. On farm C, a dog was observed to be eating feces of pigs that had swine dysentery. The dog was diarrheic and a fecal sample yielded a pathogenic isolant of T hyodysenteriae. Further isolation attempts were unsuccessful after the dog was removed from the infected premises. Isolation of pathogenic and nonpathogenic organisms from waste-holding systems emphasizes the need for cultural techniques in detecting pathogenic 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.     

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.     

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.     

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 and electrophoretic analysis of Treponema hyodysenteriae axial filaments

Axial filaments of Treponema hyodysenteriae were purified and examined by transmission electron microscopy. The size and fine structure of the filaments were similar to those of Treponema zuelzerae and Treponema phagedenis biotype reiterii. Filaments were dissociated by heat and 2-mercaptoethanol and examined by polyacrylamide-gel electrophoresis. Six protein bands were evident, with approximate molecular weights of 39,000, 29,000, 27,000, 22,000, 21,000, and 18,500 daltons. All the proteins reacted with T hyodysenteriae and T innocens rabbit antisera, using the immunoblot technique.     

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.     

Growth of Serpulina (Treponema) hyodysenteriae under iron-restricted conditions.

Reference strains of Serpulina hyodysenteriae expressed at least three iron-regulated proteins with apparent molecular masses of > 200, 134, and 109 kDa when grown under iron-restricted conditions. Cells of S. hyodysenteriae grown under these conditions also showed increased outer membrane bleb formation when examined by electron microscopy after negative staining. S. hyodysenteriae did not use the 2 most common types of siderophore, namely catechol and hydroxamate. Western blotting with serum from a pig experimentally infected with S. hyodysenteriae B204 indicated that the 109-kDa major iron-regulated protein was expressed in vivo and was conserved among all strains tested.     

The growth of Treponema hyodysenteriae and other porcine intestinal spirochaetes in a liquid medium.

A new simple method for the preparation of a liquid medium containing rabbit serum for the propagation of Treponema hyodysenteriae and other porcine intestinal spirochaetes is described. The medium, when dispensed in shallow layers and sealed under 10 per cent CO2 in nitrogen, had a redox potential not greater than -125mV and an initial pH of about 6.9 when buffered with bicarbonate. Growth of T hyodysenteriae developed more rapidly and viable counts reached higher levels at 42 degrees C than at 37 degrees C. Viable counts increased at least 10,000-fold after two to five days' incubation, depending on the temperature. Growth could be initiated from small inocula that failed to produce colonies on blood agar. Using a 1 per cent inoculum, the medium supported the growth of two strains of T hyodysenteriae through 10 serial passages.     

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.     

Effect of levamisole on the clinical and immunologic responses to oral vaccine of Treponema hyodysenteriae

Conventionally raised crossbred Hampshire pigs were vaccinated orally with attenuated Treponema hyodysenteriae in combination with an anthelmintic, levamisole or dichlorvos. Pigs in group I (n = 9) were treated with levamisole and vaccinated with attenuated T hyodysenteriae and those in group II (n = 9) were treated with levamisole and permitted to commingle (contact exposure) with group I. Pigs in group III (n = 9) were vaccinated in a similar manner and were treated with dichlorvos. Pigs in group IV (n = 9) were treated with dichlorvos and permitted to commingle with group III. Control pigs (group V; n = 9) were not given any anthelmintic, nor were they vaccinated; they were housed separately. During the 8-week interval between vaccination and challenge inoculation, 4 total days and 8 total days of diarrhea were observed in pigs in groups I and II, respectively. Likewise, 5 total days and 10 total days of diarrhea were seen in groups III and IV, respectively. In all groups, the pigs tended to shed the organism in their feces after they were vaccinated or challenge inoculated, as determined by a fluorescent antibody technique (FAT) and culture procedure (CP). Overall mean shedding patterns of 5.5% and 24.5% identified by CP and FAT, respectively, were seen in the 2 levamisole-treated groups (I and II). In contrast, mean shedding patterns of 4% and 18% of the isolation attempts were detected by CP and FAT, respectively, in the 2 dichlorvos-treated groups. Diarrhea and shedding of T hyodysenteriae in the controls (group V) did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic studies on the interaction between normal mice peritoneal phagocytes and Treponema hyodysenteriae, Treponema innocens and Bacteroides vulgatus.

One hundred and twenty female mice (CF1 strain) were divided into three groups of 40. The first group was injected intraperitoneally with broth cultures of Treponema hyodysenteriae. The second group was injected with a combination of T. hyodysenteriae and Bacteroides vulgatus. The third group was injected with Treponema innocens. Peritoneal wash from four mice of each group was collected at eight time intervals postinjection, then prepared for and examined by light and electron microscopy. Peritoneal wash from one mouse at each time interval was prepared for microbiological examination. Treponema hyodysenteriae produced peritoneal macrophage aggregation, transient neutrophilia and macrophage cytolysis. Cytolysis was characterized by rarefaction of the cytoplasm, vesiculation of the endoplasmic reticulum, mild swelling of the mitochondria and disruption of the nuclear and ctyoplasmic membranes. The combination of T. hyodysenteriae and B. vulgatus produced macrophage aggregation and marked neutrophil necrosis. Peritoneal macrophages phagocytized more T. hyodysenteriae than B. vulgatus during early postinjection intervals. Treponema innocens failed to produce cytotoxicity of peritoneal macrophages but did produce macrophage aggregation and transient neutrophilia. Treponema hyodysenteriae and T. innocens did not multiply in the mice peritoneal cavity and were reisolated up to 16 hours postinjection. Bacteroides vulgatus was reisolated up to 24 hours postinjection.     

Multilocus enzyme electrophoresis for identification and typing of Treponema hyodysenteriae and related spirochaetes

An assessment was made of multilocus enzyme electrophoresis as a means of identifying and typing spirochaetes isolated from pigs. Using five enzyme systems, 36 isolates from Australia, the U.K. and the U.S.A. were divided into 12 electrophoretic types or multilocus genotypes, comprising four major, genetically distinct groups. All 26 isolates of Treponema hyodysenteriae fell into one group, members of which showed relatively little genetic diversity. Ten isolates of non-pathogenic spirochaetes fell into three genetically different groups. Although the technique was capable of typing organisms within the groups, it was not always as discriminatory as DNA-restriction endonuclease analysis. Examination of additional enzyme loci should increase the sensitivity of the method for typing and for overall assessment of genetic relationships between spirochaetes.