Prevalence of Brachyspira species isolated from diarrhoeic pigs in Brazil.

Pathogenic intestinal spirochaetes of pigs include Brachyspira (formerly Serpulina) hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic spirochetosis (PCS). The purpose of this study was to assess the relative importance of Brachyspira species in diarrhoeal disease of growing pigs on farms in southern Brazil. The intensity and pattern of haemolysis, the production of indole and the hydrolysis of hippurate by reference and field porcine intestinal spirochaetes were compared with 16S-ribosomal RNA (mRNA)- and 23S-rRNA-based polymerase chain reaction assays for the identification of B hyodysenteriae and B pilosicoli. Between July and October 1998, 206 rectal swabs were taken from pigs on 17 farms with a history of diarrhoea developing within 30 days after they had been moved from nursery to growing facilities. Of 49 beta-haemolytic spirochaetes that were cultured, 29 (59.2 per cent) were grown in pure culture for phenotypic and genotypic characterisation, leaving 20 untyped. Of the 29 typed isolates, eight isolates obtained from six farms were identified as B hyodysenteriae, and 15 isolates obtained from seven other farms were identified as B pilosicoli; the remaining six isolates were identified as weakly beta-haemolytic commensal spirochaetes. There was complete agreement between the results of the phenotypic and genotypic analyses.

In vitro activity of four antimicrobial agents against North American isolates of porcine Serpulina pilosicoli.

Porcine colonic spirochetosis is a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production. The disease is caused by Serpulina pilosicoli, a newly recognized species of pathogenic intestinal spirochete. Antimicrobial therapy aimed at reducing the infection may be helpful in controlling spirochetal diarrhea. In this study, the in vitro antimicrobial susceptibilities of the reference isolate S. pilosicoli P43/6/78 from the United Kingdom and 19 field isolates obtained from pigs in Canada (n = 5) and the United States (n = 14) were determined against the antimicrobial agents carbadox, gentamicin, lincomycin, and tiamulin, all of which are commonly used for control of the related pathogenic intestinal spirochete S. hyodysenteriae. Additionally, the susceptibility or resistance of each isolate against each antimicrobial agent was estimated on the basis of available data on the in vitro antimicrobial susceptibility breakpoints of S. hyodysenteriae. Each isolate was identified on the basis of phenotypic and genotypic markers, and the minimum inhibitory concentration of each antimicrobial agent was determined by the agar-dilution method. All the isolates were susceptible to carbadox and tiamulin. The percentages of isolates susceptible, intermediate, and resistant to lincomycin were 42.1%, 42.1%, and 15.8%, respectively. Slightly less than half of the isolates (47.4%) were susceptible to gentamicin, and the remainder (52.6%) were resistant. Implementation of rational control measures to reduce infection by S. pilosicoli should improve overall health and productivity in swine herds.

Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated "Serpulina canis" sp. nov.

The spirochetes inhabiting the large intestines of humans and animals consist of a diverse group of related organisms. Intestinal spirochetosis caused by Serpulina pilosicoli is a newly recognized enteric disease of human beings and animals with potential public health significance. The purpose of this study was to determine the species identity of canine intestinal spirochetes by comparing 30 isolates obtained from dogs in Australia (n = 25) and the United States (n = 5) with reference strains representing Serpulina species and Brachyspira aalborgi, by phenotypic and genetically based typing methods. All of the canine isolates were indole negative and produced a weak beta-hemolysis when cultured anaerobically on agar medium containing blood. Four isolates were identified as S. pilosicoli by 16S rRNA-specific PCR assays, rRNA gene restriction fragment length polymorphism or ribotyping, and multilocus enzyme electrophoresis. The remaining 26 isolates formed a cluster related to porcine Serpulina innocens as determined by multilocus enzyme electrophoresis but had a unique ribotype pattern. The data suggested the existence of a novel Serpulina species, provisionally designated "Serpulina canis," colonizing the intestines of dogs.

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.

Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.

Laboratory identification and enteropathogenicity testing of Serpulina pilosicoli associated with porcine colonic spirochetosis.

Pathogenic intestinal spirochetes of swine include Serpulina hyodysenteriae, a strongly beta-hemolytic spirochete that causes swine dysentery, and S. pilosicoli, a weakly beta-hemolytic intestinal spirochete (WBHIS) that causes porcine colonic spirochetosis. Because of the existence of nonpathogenic WBHIS in the normal swine colon, it is important to develop laboratory procedures for accurate identification of S. pilosicoli. The purpose of the present study was to assess hippurate hydrolysis and polymerase chain reaction (PCR) amplification of specific 16S ribosomal RNA (rRNA) sequences for identification of porcine S. pilosicoli. Additionally, the enteropathogenicity of 8 field isolates of porcine S. pilosicoli was determined by challenge exposure of 1-day-old chicks and sequential histologic examination of the cecal mucosa. The field isolates of porcine S. pilosicoli hydrolyzed hippurate and yielded S. pilosicoli-specific products by PCR amplification of 16S rRNA sequences. Although all of the field isolates of porcine S. pilosicoli attached to the cecal epithelium of challenge-exposed chicks by day 21 postinoculation, some isolates had locally invasive phenotypes. We concluded that identification of porcine S. pilosicoli could be made on the basis of results of hippurate hydrolysis and 16S rRNA PCR amplification. Challenge inoculation of 1-day-old chicks followed by histologic examination of the cecal mucosa demonstrated the enteropathogenicity of porcine S. pilosicoli.

Restriction fragment length polymorphism of the periplasmic flagellar flaA1 gene of Serpulina species.

Forty-one reference and field isolates of intestinal spirochetes representing Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Brachyspira aalborgi, and nonclassified weakly beta-hemolytic intestinal spirochetes were compared by restriction fragment length polymorphism (RFLP) of the periplasmic flagellar (PF) flaA1 gene. Six genetically distinct groups (I through VI), each with a unique RFLP fingerprint pattern, were identified by Southern blotting analysis of EcoRV chromosomal DNA digests with a PCR-amplified digoxigenin-labeled 1-kb fragment of the S. hyodysenteriae isolate B78 PF flaA1 gene. The RFLP fingerprint patterns corresponded to known DNA homology differences between Serpulina species and to provisionally designated species described previously by using phenotypic and genotypic classification schemes. RFLP fingerprinting of the PF flaA1 gene provides a relatively simple genotypic method for identification of intestinal spirochetes without the use of radioisotopes.

Cecal spirochetosis associated with Serpulina pilosicoli in captive juvenile ring-necked pheasants.

Spirochetes similar to those described in the ceca of broilers with diarrhea and in laying hens with decreased egg production and growth were identified in the ceca of captive-raised juvenile ring-necked pheasants (Phasianus colchicus). The birds were submitted for diagnostic investigation of an illness characterized by a seromucoid ocular discharge, sneezing, swollen infraorbital sinuses, and weight loss. In addition to cecal spirochetosis, the birds had mild enteric coccidiosis, trichomoniasis, and nematodiasis (Heterakis spp.); esophageal capillariasis; and respiratory mycoplasmosis. Weakly beta-hemolytic spirochetes isolated from the ceca of one pheasant were identified as Serpulina pilosicoli with the use of a 16S rRNA sequence-specific polymerase chain reaction amplification assay. Diffuse cecal enterocyte attachment was reproduced in a 1-day-old chick challenged with the pheasant S. pilosicoli isolate. Immunohistochemical staining of sections of ceca from the pheasant and challenged chick with a Serpulina spp. flagellar antigen-specific monoclonal antibody confirmed spirochetal attachment to cecal enterocytes. The etiologic significance of the spirochete infection is unknown because respiratory signs and multiple gastroenteric pathogens dominated the clinicopathologic manifestations and an intestinal disorder was not a clinical complaint.

Light microscopic and ultrastructural changes in the ceca of chicks inoculated with human and canine Serpulina pilosicoli.

Light microscopic and ultrastructural changes were observed in chicks challenged with North American Serpulina pilosicoli, a weakly beta-hemolytic intestinal spirochete (WBHIS) associated with human and canine intestinal spirochetosis. Chicks in control groups received trypticase soy broth or canine Serpulina innocens. The birds were necropsied at weekly intervals, and the ceca were processed for bacteriologic and pathologic examinations. No WBHIS were isolated from the ceca of chicks in the control groups, but WBHIS with genotypes similar to the parent isolates were isolated from the ceca of chicks inoculated with human and canine S. pilosicoli. Gross examination revealed no significant changes in the ceca of chicks at any time post-inoculation. Light microscopic examination revealed no spirochetal attachment in the ceca of chicks in control groups. In contrast, focal to diffuse thickening of the brush border of the surface epithelium along with dilation of the crypt lumina and mild focal lamina propria heterophil infiltration were present in the ceca of chicks inoculated with human and canine S. pilosicoli. Scanning electron microscopic examination revealed focal to confluent spirochetal attachment mainly in the furrow region at the periphery of the crypt units. Transmission electron microscopic examination revealed spirochetes attached to the brush border of the cecal epithelium, causing effacement of the microvilli and disruption of the terminal web microfilaments. The cecal epithelium of chicks inoculated with the canine S. pilosicoli also had caplike elevations of the apical membrane at the point of attachment of the spirochetes together with large numbers of vesicles in the cytoplasm immediately beneath the terminal web and evidence of spirochetal invasion beyond the mucosal barrier. The changes observed suggested that the mechanism of attachment of human and canine S. pilosicoli to the cecal epithelium of chicks was analogous to but different from that described previously for other attaching and effacing gastroenteric bacterical pathogens of human beings and animals.

Intestinal spirochetosis and giardiasis in a beagle pup with diarrhea.

This report describes the light microscopic and ultrastructural changes in a 3-month-old dog with naturally acquired intestinal spirochetosis and giardiasis. It was concluded that the pathogenetic characteristics of weakly beta-hemolytic spirochetes associated with colitis in this pup were similar to those associated with human and porcine spirochetal diarrhea.

Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis.

Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.

Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper.

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.

Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times more sensitive than conventional culture of dysenteric feces on selective medium. There was complete agreement between the results of PCR assays and anaerobic culture on selective agar medium with diagnostic specimen (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysenteriae by PCR amplification of DNA has great potential for rapid identification of S. hyodysenteriae in diagnostic specimens.

Identification and partial characterization of a group of weakly beta-hemolytic intestinal spirochetes of swine distinct from Serpulina innocens isolate B256.

Comparative analyses of a group of 16 weakly beta-hemolytic spirochetes isolated from feces and mucosal scrapings of intestines of swine in the midwestern United States, and eastern Canada revealed the existence of a phenotypically and genotypically related group of 7 isolates. Although isolates in this group differed from all known reference isolates of intestinal spirochetes of swine, partial similarity was detected with S. joneseae isolate 16, a newly identified weakly beta-hemolytic intestinal spirochete of human beings. In addition to producing weak beta-hemolysis on blood agar plates, S. innocens isolates B256 and 4/71, S. joneseae isolate 16, and the 16 field isolates lacked the characteristic ring phenomenon described for Serpulina hyodysenteriae, an enteropathogenic spirochete of swine. All but one of the field isolates of weakly beta-hemolytic intestinal spirochetes gave negative results for indole production. The same isolates yielded variable results for alpha-galactosidase production. By transmission electron microscopic examination of negatively-stained cross-sections of spirochetes, the isolates segregated into groups containing either 4 to 7 or 9 to 16 profiles of axial filaments per cell cross-section. Analyses of genomic DNA of selected isolates using whole-genome cross-hybridization revealed a single genetic type consisting of 7 field isolates of weakly beta-hemolytic intestinal spirochetes. The 7 field isolates were distinct from the reference isolates S. innocens isolates B256 and 4/71, S. hyodysenteriae isolates B78 and B204, and Treponema succinifaciens isolate 6091 based on the number of axial filaments per cell cross-section and lack of cross-hybridization signal. S. joneseae isolate 16, had the same number of axial filaments per cell cross-section and produced a weak hybridization signal with a representative isolate of the 7 weakly beta-hemolytic field isolates from swine. This report suggests the existence of a widely distributed group of closely related weakly beta-hemolytic intestinal spirochetes of swine with genotypic characteristics distinct from S. innocens isolate B256.

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)

Morphologic analysis of enteric lesions in conventional and streptomycin-treated inbred C3H/HeN mice infected with Serpulina (Treponema) hyodysenteriae.

Oral administration of streptomycin is known to enhance the susceptibility of mice to enteric pathogens by altering the indigenous flora. We examined the effect of oral streptomycin treatment on the susceptibility of inbred C3H/HeN mice to infection with Serpulina (Treponema) hyodysenteriae. A total of 56 mice were randomly divided into four groups (A-D) of 14 each. From days 0 to 7, mice in groups A and B received streptomycin in their drinking water and mice in groups C and D served as controls. On day 7, mice in groups A and C were inoculated intragastrically with S. hyodysenteriae serotype 4, strain A1, and groups B and D served as uninoculated controls and received sterile trypticase soy broth. Clinical signs were monitored daily and body weights were recorded weekly. Mice were euthanized and necropsied for bacteriologic and histopathologic examinations on day 7 (2/group) and on days 14, 21, 28, and 35 (3/group) of the experiment. Soft fecal pellets were noticed in infected groups (A and C), but no significant differences in body weights were observed between groups (P greater than 0.05). Macroscopic changes were noted only in infected groups (A and C) beginning on day 21 of the experiment and consisted of catarrhal typhlitis, cecal emptiness, and atrophy. Histologically, the cecum and colon of mice in groups A and C had goblet cell hyperplasia, which preceded crypt epithelial cell hyperplasia, inflammatory cell infiltrates, and focal necrosis of mucosal epithelium. S. hyodysenteriae was reisolated from 10 of 12 mice in each infected group (A and C) from day 14 (7th day postinoculation) through day 35 (28th day postinoculation).(ABSTRACT TRUNCATED AT 250 WORDS)