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.

Outer membrane-associated serine protease of intestinal spirochetes.

Pathogenic intestinal spirochetes cause damage to the intestinal mucosa of humans and animals by an unknown mechanism. The purpose of this study was to assess the pathogenic intestinal spirochetes Serpulina hyodysenteriae, Serpulina pilosicoli, and Brachyspira aalborgi and the non-pathogenic commensal intestinal spirochetes Serpulina innocens and Treponema succinifaciens for protease activity. A partially heat stable, subtilisin-like, serine protease was identified in the outer membrane of all spirochetes and thus may be essential for survival in the intestinal environment. The outer membrane protease may indirectly contribute to intestinal damage caused by pathogenic spirochetes during association with the mucosal surface of the host.

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.

Phenotypic and genotypic profiles of human, canine, and porcine spirochetes associated with colonic spirochetosis correlates with in vivo brush border attachment.

A group of phenotypically and genotypically distinct weakly beta-hemolytic intestinal spirochetes (WBHIS) have been associated with a diarrheal disease of humans, dogs and swine, designated colonic spirochetosis (CS). Because attachment of spirochetes to the brush border of colonic enterocytes is a consistent feature of CS, it may represent an important virulence mechanism. In this study, pure cultures of WBHIS obtained from humans, dogs, and swine with clinical signs or lesions of CS were compared with Serpulina innocens using biochemical, genotypic and an in vivo brush border attachment assay CS-associated WBHIS did not form genotypic and an in vivo brush border attachment assay CS-associated WBHIS did not form indole, but hydrolyzed hippurate. Analysis of genomic DNA using arbitrarily primed-PCR (AP-PCR) revealed that the CS-associated WBHIS had a closely related pattern which was distinctly different from that of S. innocens. For in vivo brush border attachment assays, one-day old chicks were inoculated by crop gavage with either sterile trypticase soy broth or broth containing either S. innocens or CS-associated WBHIS. On day 7 post-inoculation, the ceca of sham-inoculated control chicks and S. innocens-inoculated chicks had tall columnar enterocytes without spirochetes, and no spirochetes were isolated by culture on selective medium. Focal to segmental attachment of spirochetes to the brush border of superficial enterocytes was present in the ceca of chicks inoculated with WBHIS, and weakly beta-hemolytic spirochetes with effacement of the microvillous brush border of colonic enterocytes. Complete agreement between hippurate hydrolysis, specific- and AP-PCR assays and in vivo brush border attachment studies confirms the enteropathogencity of CS-associated WBHIS.

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.

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.

Developmental and hormonal regulation of UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferase in mouse mammary gland.

The developmental and hormonal regulation of UDP-GlcNA:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme which initiates the biosynthesis of asparagine-linked glycoproteins, was investigated in mouse mammary glands. An anti-peptide antibody raised against the carboxyl-terminal 11 amino acids of mouse GPT, immunoadsorbed GPT activity and recognized a protein of expected size (approximately 48 kDa) on Western blots. Mouse mammary glands at different stages of development were examined for GPT activity, immunoreactive protein, and GPT mRNA. All three parameters showed a similar trend, i.e. they were low in tissues from virgin and pregnant animals, increased steadily during lactation, reaching a peak around mid to late lactation, and declined thereafter in glands from post-lactating animals. At mid-lactation, the increase in GPT activity, immunoreactive protein, and GPT mRNA relative to the virgin stage was 9.5-, 3.3- and 5.4-fold respectively, on a per cell basis. These data suggest possible transcriptional and post-transcriptional modulation of GPT gene expression during development of mouse mammary gland. The results on hormonal regulation of GPT in mouse mammary explants and primary mouse mammary epithelial cells showed that all three parameters cited above were stimulated maximally by the combined presence of insulin, hydrocortisone, and prolactin, indicating that the hormonal regulation of GPT expression is also mediated at the level of RNA.

Role of sulfhydryl groups in the function of glucosidase I from mammary gland.

Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.

Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization.

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.