Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility.

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.

[Bifidobacterium adolescentis suppresses the humoral immune response to an autochthonous intestinal bacterium--experiments with gnotobiotic rats]. / Bifidobacterium adolescentis supprimiert die humorale Immunantwort gegen ein autochthones Darmbakterium--Versuche an gnotobiotischen Ratten.

On the basis of association-experiments with gnotobiotic rats, we described the immunogenicity of two selected bacterial species (Bifidobacterium adolescentis and Bacteroides thetaiotaomicron). B. adolescentis is a gram-positive lactic acid producing bacterium, strains of which are claimed to have probiotic properties. B. thetaiotaomicron is a gram-negative rod, autochthonous to the human as well as to the rats' intestinal tract. Colonization of the gut was monitored by determination of bacterial cell counts in the animals' feces. In order to investigate the systemic immune reaction, the amounts of specific serum-IgG and -IgA against both bacterial species were measured in the serum. The intestinal immune reaction was examined by measuring the specific IgA in the rats' feces. Knowing about the antibody levels in gnotobiotic rats induced by monoassociation we subsequently disassociated two groups of rats in order to investigate the impact of B. adolescentis on the immune reaction against B. thetaiotaomicron. One group was disassociated simultaneously with B. adolescentis and B. thetaiotaomicron, the second group was disassociated with these bacteria in sequence. B. adolescentis was merely able to induce a mucosal immune reaction, while B. thetaiotaomicron challenged the mucosal as well as the systemic immune system. Furthermore B. adolescentis obviously suppressed the systemic and mucosal immune reaction against the autochthonous B. thetaiotaomicron.

Serum antibody responses of cats to soluble whole cell antigens and isolated fimbriae of feline Porphyromonas salivosa (macacae) and associations with periodontal disease.

The whole cell soluble antigens of two strains (NCTC 11632 and VPB 3313) of feline Porphyromonas salivosa (macacae) were analyzed by Western blotting using serum taken from 40 domestic cats with various grades of periodontal disease. Nine strongly immunogenic protein bands (66, 52, 42, 29, 27, 23, 22, 21 and 19kDa) were selected from both strains for further study. Both strains showed a significant association between overall periodontal grade and serum responses to the 66 and 21kDa bands with significant responses across both strains to all other bands except the 52kDa band. Similarly, both strains showed a significant association between the total colony forming units and serum responses to the 66 and 42kDa bands with significant responses across both strains to all other bands except the 19kDa band. When sera from 25 of these cats were tested by Western blotting against the isolated fimbriae of VPB 3313, there was a significant association between the grade of response of cats to the 42kDa fimbrial preparation and (1) the total reactivity of the mouth (the sum of the responses to all individual whole cell antigens), (2) the total colony forming units of P. salivosa (macacae) at the premolar site, and (3) to their responsiveness to the 42kDa band in the soluble whole cell antigen preparations. These findings suggest that P. salivosa (macacae) is a strong immunogen in the mouths of cats and those cats with more severe periodontal disease have a greater serum antibody reactivity to various soluble whole cell antigens, specifically including the fimbriae of this organism, than those with less severe periodontal disease. Overall, the findings suggest that this organism may be a contributor to periodontal disease in cats.

Bovine abortions associated with Bacteroides fragilis fetal infection.

Two Santa Gertrudis cattle from a herd of 105 aborted within a 24-hour period. Bacteroides fragilis was isolated from tissues of each aborted fetus. Histopathologic lesions included placentitis and bronchopneumonia in which gram-negative, rod-shaped organisms were visible. The diagnostic workup failed to reveal other causes of abortion. Anaerobes are rarely implicated in bovine abortions, and no other report was found that described abortion in cattle due to B. fragilis.

Clinical aspects of experimental peritonitis in horses

Sixteen adult horses were randomly divided into 4 equal groups (GI, GII, GIII and GIV) of 4 animals and each group was injected intraperitoneally with one of the following suspension:GI (100 x 10.000.000 colony-forming units (CFU) of Escherichia coli diluted in 500ml of 0.9 percent saline); GII (100 x 10.000.000 CFU of Bacteroides fragilis in 500ml of 0.9 percent saline); GIII (100 x 10.000.000 CFU of E. coli in combination with 100 x 10.000.000 CFU of B. fragilis in 500 ml of 0.9 percent saline); GIV (500ml of 0.9 percent saline). Abdominal wall sensitivity to external pressure and tension, diarrhea, decreased intestinal sounds and increased of heart rate were the clinical signs more frequently observed in inoculated horses. Horses inoculated with pure cultures of either E. coli or B. fragilis demonstrated mild and self-limiting peritonitis, while those inoculated with the combination of both bacteria demonstrated clinical signs of higher intensity and duration.

Serological classification and virulence determination of Dichelobacter nodosus isolated from Alberta and British Columbia sheep.

Ovine footrot is a contagious disease of sheep that occurs in temperature climates. It is caused by the strict anaerobe, Dichelobacter nodosus. Benign and virulent organisms are differentiated according to serotype and protease production. This study was conducted to identify the presence of virulent serotypes of D. nodosus in sheep flocks in Alberta and British Columbia. Dichelobacter nodosus was detected in lame sheep from 11 of 15 (73%) flocks in Alberta and in 4 of 5 (80%) British Columbia flocks. It was recovered from 57 of 107 (53%) lame sheep. In Alberta, 4 distinct serotypes were isolated from the 11 positive flocks while in British Columbia a total of 6 different serotypes were isolated. One British Columbia isolate could not be classified into existing serotypes. Of the 19 field strains tested, all but 3 were defined as virulent based upon the rapid rise in protease activity in vitro which was maintained between 3 and 5 d. The knowledge of the serotype and virulence of the D. nodosus isolated from affected animals can assist in the control and prevention of ovine footrot.

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus.

OBJECTIVE: To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter Nodosus (strain A). PROCEDURE: Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded. RESULTS: The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects. CONCLUSION: Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.

Detection of bacteria in equine synovial fluid by use of the polymerase chain reaction.

Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus, and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL. Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens. Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours. Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of a 531 base-pair segment of bacterial DNA corresponding to a region of the 16S ribosomal gene. Duplicate samples of inoculated synovial fluid were prepared for microbial culture. Bacteria were detected in all samples inoculated with bacteria but not in control synovial fluid samples. Under experimental conditions there was no difference between microbial culture and PCR analyses for detection of bacteria. Experimentally, PCR was able to detect bacteria in synovial fluid within 24 hours of inoculation.

Isolation of Bacteroides ureolyticus from vaginal discharge of mares.

A total of seven Bacteroides ureolyticus strains were isolated from the cervix and the clitoral fossa of mares with vaginal discharge. No other bacteria capable of causing metritis or vaginitis were isolated from the samples. The isolated strains resembled Taylorella equigenitalis. Both species are catalase, oxidase and alkaline phosphatase positive, but, in addition to these characteristics, B. ureolyticus strains produced urease and they could not tolerate 10% O2. They also failed to be agglutinated in a hyperimmune serum raised against T. equigenitalis; however, B. ureolyticus and T. equigenitalis were agglutinated in the slide agglutination test in a serum produced against one of the B. ureolyticus isolates. Further investigations are needed to clarify the pathologic role of B. ureolyticus in genital infections of mares and other animals.

Development of gene probes of Dichelobacter nodosus for differentiating strains causing virulent, intermediate or benign ovine footrot.

Seven Dichelobacter nodosus genomic DNA clones including six specific for virulent and one for benign strains were identified. A collection of 96 footrot isolates, which in turn comprised 27 virulent isolates showing elastase activity at 7 days, 25 high intermediate isolates with elastase activity at 14 days, 24 low intermediate isolates with elastase activity at 21-28 days and 20 benign isolates with no elastase activity at up to 28 days, were used to assess these clones. Of the six virulent specific clones, five (pV238-83, pV405-239, pV470-65, pV470-145 and pV470-178) reacted with 27 virulent isolates, and 12 of 25 high intermediate isolates, but none of 24 low intermediate isolates and 20 benign isolates in dot blot hybridization. The other virulent-specific clone (pV470-13) recognized all 27 virulent and 25 intermediate isolates, and 22 of 24 low intermediate isolates and three of 20 benign isolates in dot blot hybridization. By contrast, the benign-specific clone (pB645-335) detected all 20 benign isolates and 24 low intermediate isolates, and also 13 of 25 high intermediate isolates, but none of 27 virulent isolates in dot blot hybridization. Southern hybridization analysis indicated that whereas clones pV238-83, pV405-239 and pV470-178 bound a Sau3A band of 0.5 kb, clones pV470-65 and pV470-145 recognized two Sau3A bands of 0.7 and 0.5 kb in virulent strains of serogroups A to I. However, clone pV470-13 detected a Sau3A band pattern in virulent strains different from those recognized by the other five virulent specific-clones. Besides showing a distinct Sau3A band pattern in intermediate strains, pV470-13 also reacted with three benign strains that showed binding with it in dot blot hybridization. The benign-specific clone pV645-335 detected a Sau3A band of 0.5 kb in both intermediate and benign strains of serogroups A to I. Thus the combination of pV470-13 and pB645-335, or any other virulent-specific clone, would clearly differentiate among D. nodosus strains causing virulent, intermediate or benign footrot.

Serogrouping of Bacteroides nodosus isolates from 62 sources in the United States.

Bacteroides nodosus isolates from 62 sources in the United States were obtained from sheep with infectious foot diseases. Serotypic analysis of these isolates revealed 21 serotypes (designated I-XXI). These serotypes were compared with British and Australian/New Zealand B nodosus strains by use of reciprocal tube agglutination tests. These tests, as well as the cross-matching tube agglutination tests of the US serotypes, resulted in arranging the US serotypes into 11 serogroups, and comparing these serogroups with their Australian/New Zealand serogroup and British serotype counterparts. Three US serogroups and 1 additional British serotype had little or no relationship to any of the Australian/New Zealand serogroups A-H (the vaccine strains). One or more of these unrelated serogroups were found in 29% of the sources studied. The most frequently found US serotype was serotype XV at 29%. The most frequently found US serogroups were the serogroups analogous to serogroup B (43.5%) and serogroup H (37%); the other serogroups were found in 22.6% or less of the sources studied. Evaluation of 3 sources revealed that multiple serotypes in a single flock are common, multiple serotypes from a single lesion are possible, B nodosus isolates obtained from goats (unlike those from cattle) appear identical to the isolates obtained from sheep, and disease can appear in vaccinated animals, even in a flock that appears to be harboring only a single serogroup-B serotype (the serogroup for which there are 3 strains in the current vaccine).

Detection of Dichelobacter nodosus using species-specific oligonucleotides as PCR primers.

Dichelobacter nodosus is an essential causative agent of ovine footrot, a disease of major economic significance. Four oligonucleotides complementary to variable regions of the 16S rRNA of D. nodosus were identified, synthesized and tested for their specificity and sensitivity as probes for the detection of D. nodosus. In hybridization reactions using total RNA as the target nucleic acid, three probes were found to be both sensitive and species-specific. When these probes were used as primers in PCR reactions, on both purified D. nodosus DNA and whole cells, the sensitivity of detection was increased by several orders of magnitude. Using PCR, it was possible to detect the presence of D. nodosus by direct examination of lesion material from footrot infected sheep.

Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov.

A new species, Porphyromonas circumdentaria, is proposed for pigmented, asaccharolytic strains that were isolated from the gingival margins or mouth-associated diseases of cats. This bacterium is an obligately anaerobic, gram-negative, brown- or black-pigmented, asaccharolytic, nonmotile, nonsporing, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 40 to 42 mol%. It produces major amounts of acetic, butyric, and isovaleric acids and minor amounts of propionic, isobutyric, and phenylacetic acids as end products of metabolism in cooked meat medium. Glutamate and malate dehydrogenases are present, while 6-phosphogluconate and glucose-6-phosphate dehydrogenases are absent. The major cellular fatty acid is 13-methyltetradecanoic acid (iso-C15:0 acid). P. circumdentaria strains are catalase positive and produce ammonia, and colonies fluoresce under short-wavelength UV light. These strains do not hemagglutinate erythrocytes, exhibit trypsinlike activity, or produce chymotrypsin or alpha-fucosidase. They are heavily piliated and produce a capsule. The type strain is strain VPB 3329 (= NCTC 12469). Bacteroides salivosus (D. N. Love, J. L. Johnson, R. F. Jones, and A. Calverley, Int. J. Syst. Bacteriol. 37:307-309, 1987) is an obligately anaerobic, gram-negative, pigmented, asaccharolytic, nonmotile, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 42 to 44 mol%. This organism produces major amounts of acetic, butyric, and phenylacetic acids and minor amounts of isobutyric and isovaleric acids as end products of metabolism in cooked meat medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.

Summer mastitis in heifers: studies on the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus and Bacteroidaceae in clinically healthy cattle in Denmark.

With the aim of investigating the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus, Bacteroides melaninogenicus ss. levii and Fusobacterium necrophorum, and thus the potential for development of summer mastitis, clinically healthy Danish Holstein-Friesian heifers due to calve in the autumn were sampled from the teat tip, the conjunctiva and the oral cavity at 2-6 week intervals from 1979 to 1981. The overall isolation rates of F. necrophorum, P. indolicus and B. melaninogenicus ss. levii, in order of significance, were significantly higher during the pasture period whereas no differences in isolation rates of A. pyogenes between housed and pastured animals were detected. F. necrophorum was recovered almost exclusively from the oral cavity, P. indolicus and A. pyogenes occurred most frequently in samples from the teat skin, whereas isolates of B. melaninogenicus ss. levii were evenly distributed between conjunctiva and teat tip samples. A distinct seasonal pattern of the isolation rates of summer mastitis pathogens was recorded, which corresponded closely to the seasonal activity of symbovine insects, in particular the headfly Hydrotaea irritans (Fallén). However, the high proportion of clinically healthy bacterial carriers as compared with the incidence of clinical disease strongly suggests that as yet unknown contributing or triggering factors, apart from the mere presence of the relevant bacterial species, are required for the establishment and development of clinical summer mastitis.

Enterovirulence of enterotoxigenic Bacteroides fragilis in gnotobiotic pigs.

A porcine isolate of enterotoxigenic Bacteroides fragilis colonized the intestinal tract and caused watery, nonhemorrhagic diarrhea when given orally to 12, 1- to 2-day-old gnotobiotic pigs. Diarrhea occurred 2 to 3 days post-inoculation and continued throughout the 4 to 6 day post-inoculation period. Diarrheic pigs became mildly anorexic and dehydrated. They developed intestinal lesions characterized by swelling, vacuolation, and exfoliation of enterocytes, and crypt hyperplasia throughout the large intestine and, to a lesser extent, in the distal small intestine. Bacterial adherence to, or invasion of, the intestinal mucosa was not detected. A porcine isolate of nonenterotoxigenic B. fragilis was administered orally to six control pigs. The isolate colonized the intestinal tract, but the pigs did not develop clinical disease or intestinal lesions. The pathogenetic mechanism of the disease may involve mediation by a soluble enterotoxin (or toxins) elaborated by B. fragilis.