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Immunofluorescent demonstration of Campylobacter hyointestinalis and Campylobacter sputorum subsp mucosalis in swine intestines with lesions of proliferative enteritis.

An indirect fluorescent antibody technique was developed to identify Campylobacter spp in lesions of swine proliferative enteritis (SPE). Rabbit antisera to C hyointestinalis and C sputorum subsp mucosalis were produced. Bacterial smears stained by fluorescent antibody test with homologous antisera differentiated C hyointestinalis from subsp mucosalis. Ileal frozen sections from 29 pigs with histologic lesions of SPE had specific fluorescent staining of C hyointestinalis in all 29 and subsp mucosalis in 24. Bacterial structures of C hyointestinalis were seen in large numbers and were broadly distributed in intestinal luminal exudate, mucosal necrotic tissues, surface epithelium, lamina propria, and proliferative cryptal epithelium. Numerous C hyointestinalis organisms were always present in the apical cytoplasm of proliferative cryptal epithelium. Fluorescent subsp mucosalis bacteria were seen less frequently and were distributed focally in the mucosa. Numerous subsp mucosalis organisms were more common in cellular debris and in necrotic tissues of surface mucosa, and less common in the epithelial cells of proliferative crypts. Ileal sections from 13 pigs without SPE had no fluorescent staining of C hyointestinalis and subsp mucosalis.

Animals↗

Campylobacter hyointestinalis subsp. lawsonii subsp. nov., isolated from the porcine stomach, and an emended description of Campylobacter hyointestinalis.

The taxonomic relationships of seven isolates obtained from porcine stomachs (the "CHY" group), which resembled (but were distinct from) the type strain and other reference strains of Campylobacter hyointestinalis, were examined by using phenotypic and genomic methods. The phenotypic characteristics and ultrastructure of the new organisms were characteristic of Campylobacter species, although they could be distinguished from all previously described taxa. A numerical analysis of 38 phenotypic characters revealed that the new isolates formed a distinct group at a similarity level of 90.1% and could be clearly distinguished from reference strains representing 20 related taxa, principally species and subspecies belonging to the genera Campylobacter, Arcobacter, and Helicobacter. DNA-DNA hybridization studies revealed that the porcine stomach strains were genomically homogeneous (levels of relatedness, 84 to 90%), although the levels of DNA homology with type and reference strains of C. hyointestinalis were relatively high (56 to 71%). Differences in the DNA base compositions of the CHY group and C. hyointestinalis strains were also observed. Our data indicate that the new porcine isolates should be considered members of a subspecies of C. hyointestinalis, for which we propose the name Campylobacter hyointestinalis subsp. lawsonii subsp. nov. The type strain is strain CHY 5 (= LMG 14432 = NCTC 12901 = CCUG 34538). The description of C. hyointestinalis is emended accordingly, and a description of Campylobacter hyointestinalis subsp. hyointestinalis subsp. nov. is given.

Animals↗

Campylobacter hyointestinalis subsp. hyointestinalis, a common Campylobacter species in reindeer.

AIMS: To study the prevalence of Campylobacter spp. in the faecal material of reindeer, and to identify the isolates by means of a polyphasic approach. In addition, to study the genetic diversity of Camp. hyointestinalis subsp. hyointestinalis reindeer isolates by pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: The material, collected during the slaughter period in autumn 1998, comprised 399 faecal contents from the reindeer (Rangifer tarandus), a semi-domesticated, meat-producing ruminant of northern Finland. These samples came from 16 herds in the areas of eight reindeer slaughterhouses. Samples were cultured by methods suitable for isolation of fastidious Campylobacter species. Of all samples, 6% (24/399) were Campylobacter-positive. Phenotypic characteristics, SDS-PAGE protein patterns, dot blot DNA-DNA hybridization, 23S rDNA restriction fragment polymorphism analysis and PFGE identified the isolates as Camp. hyointestinalis subsp. hyointestinalis. CONCLUSIONS: Campylobacter hyointestinalis subsp. hyointestinalis was the only Campylobacter species isolated from reindeer in this study. The isolates showed high genomic diversity in PFGE with the restriction enzymes SmaI and KpnI. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE analysis is a useful subtyping method for epidemiological studies. Contaminated reindeer meat can be a source for human infections.

Animals↗

Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates.

Campylobacter hyointestinalis was isolated from five members of the same family who had previously consumed raw milk. Pulsed-field gel electrophoresis of genomic DNAs from the five strains, after digestion with restriction endonuclease SalI, revealed that three strains had identical genome patterns and therefore appeared to be related, whereas the other two had completely different genome patterns and appeared to be unrelated. We report here for the first time the isolation of C. hyointestinalis from family members who had consumed raw milk. Our study also demonstrates the usefulness of pulsed-field gel electrophoresis for epidemiologic studies of this unusual campylobacter.

Bacteriological Techniques↗

"Campylobacter hyointestinalis" sp. nov.: a new species of Campylobacter found in the intestines of pigs and other animals.

The name "Campylobacter hyointestinalis" sp. nov. is proposed for a Campylobacter species that was isolated from the intestines of pigs with proliferative enteritis. "C. hyointestinalis" is also found in the feces of cattle and has been isolated from the intestine of a hamster. "C. hyointestinalis" is distinguished from previously described catalase-positive Campylobacter species by colony morphology, ability to produce H2S in triple sugar iron agar, ability to grow anaerobically in 0.1% trimethylamine N-oxide hydrochloride, resistance to nalidixic acid, susceptibility to cephalothin and metronidazole, and hydrogenase activity. Sixteen "C. hyointestinalis" strains were highly related (greater than or equal to 76%) by DNA-DNA hybridization (hydroxyapatite method, 50 and 65 degrees C). Other Campylobacter species were less than or equal to 30% related to "C. hyointestinalis." The type strain of "C. hyointestinalis" is designated 80-4577-4 (= ATCC 35217), and its DNA has a guanine-plus-cytosine content of 36 mol%.

Animals↗

Isolation of "Campylobacter hyointestinalis" from a human.

We isolated "Campylobacter hyointestinalis" from the rectal culture of a homosexual man with proctitis. Phenotypic characterization of the isolate was confirmed by DNA hybridization by using the taxonomic spot blot. "C. hyointestinalis" was previously isolated only from animals but should be considered in the identification of Campylobacter species isolated from humans.

Adult↗

Effects of subtherapeutic administration of antimicrobial agents to beef cattle on the prevalence of antimicrobial resistance in Campylobacter jejuni and Campylobacter hyointestinalis.

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (> or =94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.

Animals↗

Species-specific cloned DNA probes for the identification of Campylobacter hyointestinalis.

Conventional microbiological methods for isolating and identifying Campylobacter species are laborious, tedious, and subjective. Because of the increasing importance of Campylobacter species in human and animal diseases and the recent emergence of many new species and atypical strains, we are developing chromosomal DNA probes for rapid and simple identification of Campylobacter species, especially those of veterinary importance. We report the cloning and characterization of chromosomal DNA fragments from Campylobacter hyointestinalis, an organism isolated from pigs with proliferative enteritis. To obtain C. hyointestinalis-specific probes, chromosomal DNA fragments from C. hyointestinalis were cloned into plasmid vector pGEM-3Z. Recombinant plasmids were screened for C. hyointestinalis-specific inserts by DNA hybridization, using chromosomal DNA from either C. hyointestinalis or C. fetus which had been 32P labeled. Recombinants which hybridized to C. hyointestinalis, but not C. fetus, DNA were 32P labeled and screened further for sensitivity and specificity. Three probes were identified that were species specific and capable of detecting 10(4) C. hyointestinalis organisms by bacterial spot blotting in 48 h. We anticipate that these probes will be useful for routine species identification and for epidemiological studies.

Animals↗

Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications.

Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0%, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96.4 to 100%. Sequence similarities between strains representing the two different subspecies ranged from 95.7 to 99.0%. An intervening sequence was identified in certain of the C. hyointestinalis subsp. lawsonii strains. C. hyointestinalis strains occupied two distinct branches in a phylogenetic analysis of the genus Campylobacter, emphasizing the need for multiple strain analysis when using 16S rRNA gene sequence comparisons for taxonomic investigations.

Animals↗

Campylobacter hyointestinalis associated with human gastrointestinal disease in the United States.

Campylobacter hyointestinalis was isolated from stool specimens of four persons, all of whom were experiencing nonbloody, watery diarrhea. The youngest (8 months of age) and the oldest (79 years of age) individuals were females, and the other two were homosexual men. C. hyointestinalis was the only clinically significant pathogen isolated from stool specimens for three of the individuals. In case 3 (involving a 37-year-old homosexual man), Entamoeba histolytica and Shigella sonnei were also present in the stool. The identification of all C. hyointestinalis strains was made biochemically and confirmed by DNA hybridization. This study documented the isolation of C. hyointestinalis from four patients with diarrhea, and our findings suggest that the clinical significance of Campylobacter species must be expanded to include C. hyointestinalis as a potential cause of human gastrointestinal disease.

Adult↗

Susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to antimicrobial agents and characterization of quinolone-resistant strains.

OBJECTIVES: To study the susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to several antimicrobial agents and to investigate the mechanisms of nalidixic acid and ciprofloxacin resistance. METHODS: The disc diffusion method was employed to study the susceptibility of 49 C. hyointestinalis subsp. hyointestinalis strains of reindeer and bovine origin to 12 different antimicrobial agents. In addition, the MICs of nalidixic acid and ciprofloxacin were determined. The nucleotide sequence of a 270 bp fragment of the gyrA gene was determined in ciprofloxacin-susceptible and -resistant strains. The effect of a multidrug efflux pump inhibitor Phe-Arg-beta-naphthylamide (PA beta N) on the MICs of ciprofloxacin and nalidixic acid was also studied. RESULTS: The only decreased susceptibility for antimicrobial agents of this study was observed for sulphonamide compound and streptomycin (24% and 32% of the strains, respectively), and this phenomenon was observed exclusively in the bovine strains. In sequence studies, a Thr-86-->Ile change was found in strains with MICs of ciprofloxacin of > or = 64 mg/L, but this mutation was absent in strains with lower resistance levels. The use of PA beta N did not affect the MIC of ciprofloxacin but decreased the MIC of nalidixic acid 2-4-fold. CONCLUSIONS: The Finnish C. hyointestinalis subsp. hyointestinalis strains are susceptible to a majority of the antimicrobials of veterinary importance. The mechanism of ciprofloxacin resistance at lower levels (< or = 32 mg/L) is not associated with a specific mutation in the quinolone resistance-determining region of the gyrA gene. Finally, there are distinct differences in the mechanisms of ciprofloxacin resistance compared with nalidixic acid resistance within the studied species.

Animals↗

Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.

Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA.

Animals↗

Characterization of the replication region of the small cryptic plasmid of Campylobacter hyointestinalis.

The complete nucleotide sequence of a 2.5-kb cryptic plasmid from Campylobacter hyointestinalis was determined. Only one open reading frame (ORF), encoding a polypeptide of M(r) 39,667, designated RepA, could be identified within the sequence. This was confirmed by minicell analysis. Analysis of the region upstream from the ORF showed an A+T-rich region followed by four 19-bp direct repeats. Together, these features are characteristic of other replication origins (ori(s)). The promoter sequence of the repA gene was identified by primer extension analysis and both the putative -10 and -35 regions were found to lie within two potential hairpin-loop structures. RepA showed marked amino acid sequence homology to a replication-initiation protein from the Neisseria gonorrhoeae plasmid, pFA3, and with other replication-initiation proteins over two conserved motifs. A putative partitioning (par) locus was identified upstream from the ori and consisted of a perfect 9-bp inverted repeat and six putative DNA gyrase-binding sites. A putative mobilization origin (oriT) region was identified. This featured a 19-bp imperfect inverted repeat adjacent to a sequence of 12 bp which showed strong homology to the consensus sequence of the 'nick regions' in a variety of oriTs of other plasmids.

Amino Acid Sequence↗

The isolation of Campylobacter hyointestinalis from a pig in South Africa.

The isolation of a strain of Campylobacter hyointestinalis from a piglet is described. The animal originated from a farm where another animal showed signs of intestinal adenomatosis. The animal from which the isolation was made had diarrhoea, and an enteropathogenic Escherichia coli was also isolated. No pathological changes indicative of intestinal adenomatosis were detected in this animal.

Animals↗

Campylobacter hyointestinalis (new species) isolated from swine with lesions of proliferative ileitis.

Intestines from 48 swine with enteric disease were examined by bacteriologic cultural technique for the presence of various Campylobacter species. Histopathologic techniques were used to determine whether the submitted specimens had lesions of either swine proliferative ileitis or other enteric diseases. Three species of Campylobacter were identified as Campylobacter jejuni/coli, Campylobacter sputorum ss mucosalis, and Campylobacter hyointestinalis (proposed new species) on the basis of biochemical characteristics and response to various inhibitory substances. The C hyointestinalis was isolated from 18 of 27 (67%) swine with proliferative ileitis and from only 1 of 21 (5%) swine with other enteric diseases. The C sputorum ss mucosalis was obtained from 16 of 27 (59%) swine with proliferative ileitis and from 2 of 21 (10%) swine with other enteric disease. The C jejuni/coli was isolated from 2 of 27 (7%) swine with proliferative ileitis and from 8 of 21 (38%) swine with other enteric disease. The new organism, C hyointestinalis, was catalase-positive, hydrogen sulfide positive in triple sugar iron agar, glycine tolerant, intolerant to 3.0% sodium chloride, able to grow at 25 C, sensitive to cephalothin, and resistant to nalidixic acid. On the basis of these characteristics, C hyointestinalis was differentiated from other campylobacters isolated from swine and from other sources.

Animals↗

Transmission of Campylobacter hyointestinalis from a pig to a human.

We report on a case of human gastroenteritis caused by the pathogen Campylobacter hyointestinalis. Recurrent watery diarrhea and intermittent vomiting were the most significant symptoms of the previously healthy patient. Whole-cell protein electrophoresis and 16S rRNA gene sequencing were used to identify this Campylobacter species. Investigation of the patient's surroundings led to the recovery of a second C. hyointestinalis strain originating from porcine feces. Subsequent typing of the human and the porcine isolates by pulsed-field gel electrophoresis revealed similar macrorestriction profiles, indicating transmission of this pathogen.

Aged↗

Outer membrane components of Campylobacter hyointestinalis.

Outer membranes were isolated, by sodium lauryl sulphate extraction, from the American type strain, five Australian, and four English isolates of Campylobacter hyointestinalis. On SDS-PAGE examination, the protein profiles of seven strains (including the type strain) were similar, and were dominated by two major proteins of 47 and 50 kDa. Three other isolates had unique major protein profiles. The largest of these proteins was heat-modifiable in these isolates, and in the type strain. The flagellin of three isolates screened was of similar M(r) to that of Campylobacter jejuni/Campylobacter coli. The lipopolysaccharides of C. hyointestinalis isolates were heterogeneous in structure; 5/10 isolates synthesised material of M(r) value greater than that of the low M(r) C. jejuni/C. coli lipopolysaccharide. By gel excision and re-electrophoresis, it was shown that the higher M(r) materials of one isolate were not artifactual aggregates of lower M(r) species.

Animals↗

Isolation of a restriction-less mutant and development of a shuttle vector for the genetic analysis of Campylobacter hyointestinalis.

A cosmid shuttle cloning vector, pCHI15, was constructed which could be mobilized from Escherichia coli K-12 to a putative restriction-less mutant of Campylobacter hyointestinalis, C. fetus subsp. fetus, and C. fetus subsp. venerealis at a frequency of 10(-4) transconjugants per donor. A previously described C. coli shuttle vector, pILL550, could not be mobilized into the C. hyointestinalis restriction-less mutant, implying that the C. coli replicon was not functional in a C. hyointestinalis host. The type strains of C. jejuni, C. coli, C. fetus subsp. fetus, and C. hyointestinalis were analysed for their ability to be transformed by plasmid DNA which had been modified by other Campylobacter species. Each Campylobacter species was found to be most efficiently transformed by plasmid DNA that had been previously passaged in the same species. pCHI15 could be mobilized from C. coli into C. fetus subsp. fetus and the putative restriction-less mutant of C. hyointestinalis at a frequency of 3.0 x 10(-4) and 2.5 x 10(-3) transconjugants per donor, respectively.

Campylobacter↗