Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

Contamination of red-meat carcasses by Campylobacter fetus subsp. jejuni.

Campylobacter fetus subsp. jejuni was commonly present in the feces of unweaned calves (2 to 3 weeks old) and from two of four groups of sheep. One new season lamb (12 to 16 weeks old) carried the organism, but the bacteria were not isolated from cattle. With unweaned calves, the fractions of animals infected and carcasses contaminated were similar. Contamination of carcasses usually involved low densities of C. fetus subsp. jejuni (ca. 1 to 10/cm2), which were isolated from flank but not rump areas. The organism was recovered less frequently from chilled carcasses and deboned veal. Small numbers of C. fetus subsp. jejuni could be recovered from equipment during the processing of unweaned calves but not after routine cleaning.     

Genome map of Campylobacter fetus subsp. fetus ATCC 27374

Abstract A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by Sal I, Sma I and Not I. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4–14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA , rpoB , and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes ( flaAB ) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis , respectively, were used to identify the locations of flaAB , the gyrA and the gyrB genes on the ATCC 27374 chromosome.     

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 (≥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.     

Localization of hydrogenase and nitrate reductase in Campylobacter sputorum subsp. bubulus

Campylobacter sputorum subsp. bubulus contained hydrogenase activity after growth with lactate and nitrate and after growth with hydrogen and nitrate. After growth with hydrogen and nitrate a molar growth yield (g dry cells/mol hydrogen) of 5.6 was measured. Hydrogenase and nitrate reductase were membrane-bound enzymes. In cells with high hydrogenase activity the H⁺/O, H⁺/NO _inf2 ^sup- and H⁺/NO _inf3 ^sup- values with hydrogen as the electron donor were 3.74, 2.61 and 4.36 respectively. In cells with low hydrogenase activity these values were 2.33,-0.86 and 1.31 respectively. These values and the stoichiometry of respiration-driven proton translocation (H⁺/2e=2) led to the conclusion that hydrogenase is located at the periplasmic side of the cytoplasmic membrane. In cells with low lactate dehydrogenase activity or low hydrogenase activity the reduction of nitrate to nitrite could be separated from the reduction of nitrite to ammonia. Positive H⁺/NO _inf3 ^sup- values (between 0.9 and 1.7) with lactate or hydrogen as the electron donor were measured in these cells whereas H⁺/NO _inf2 ^sup- values were negative. From this result it was concluded that nitrate reductase is located at the cytoplasmic face of the cytoplasmic membrane. The results explain the previous observation that molar growth yields with nitrate were somewhat higher than those with nitrite.     

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.     

Structural studies of peptidoglycans in Campylobacter species.

Peptidoglycans (PG) from Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus were composed of muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1.1:1:1.7:1.1:09. Thirty percent of the amino groups of diaminopimelic acid were involved in cross-linkages between peptides. During cultivation, C. coli and C. jejuni changed from a spiral to a coccoid form. In C. coli, we could isolate PG only from the spiral forms in yields of 0.8-1.2% by dry weight. C. fetus did not change to a coccoid form, and always contained PG. Thus, it is possible that the morphological transformation from the spirals to the coccoid forms of C. coli and C. jejuni is accompanied by, and probably due to, the degradation of PG.     

Improved growth medium for Campylobacter species.

Campylobacter species were grown in a base containing proteose peptone no. 3, yeast extract, K2HPO4, (NH4)2SO4, NA2SO3, soluble starch, and agar. Concentrations and sources of organic nitrogen and growth factors were critical, and the optimal pH range was 7.0 to 7.5. Cultures tolerated 0.7% NaCl in addition to the salt present in the organic constituents and were sensitive to surface-active agents at concentrations recommended for enrichment of other gram-negative bacteria. Cultures were maintained on the proposed medium for 1 year with transfer every 2 weeks.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway.

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.     

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.     

Onchocerca dewittei japonica n. subsp., a common parasite from wild boar in Kyushu Island, Japan

We describe Onchocerca dewittei japonica n. subsp. from the Japanese wild boar, Sus scrofa leucomystax, in Oita, Kyushu Island, where all seven animals examined were found to be infected. This study began with efforts to identify the causative species in a recent case of zoonotic onchocerciasis. Compared with Onchocerca dewittei dewittei from Sus scrofa jubatus in Malaysia, which was reexamined here, our new subspecies has much greater space between the ridges on the females. In addition, its microfilariae (from uteri) are shorter (192-210 microns compared with 228-247 microns), and only the posterior third of the microfilarial body is coiled, instead of the entire body. The Onchocerca species parasitic in suids (these two subspecies and O. ramachandrini from the warthog in the Ethiopian region) form a group sharing several characters. Among the most unusual characters are the body swellings (a specialized apparatus for mating, known in only a few other genera). In addition, longitudinal cuticular crests were found on males of both subspecies from wild boar and on females of O. ramachandrini.     

Description of Desulfotomaculum nigrificans subsp. salinus as a new species, Desulfotomaculum salinum sp. nov

This study focused on the physiological, chemotaxonomic, and genotypic characteristics of two thermophilic spore-forming sulfate-reducing bacterial strains, 435T and 781, of which the former has previously been assigned to the subspecies Desulfotomaculum nigrificans subsp. salinus. Both strains reduced sulfate with the resulting production of H2S on media supplemented with H2 + CO2, formate, lactate, pyruvate, malate, fumarate, succinate, methanol, ethanol, propanol, butanol, butyrate, valerate, or palmitate. Lactate oxidation resulted in acetate accumulation; butyrate was oxidized completely, with acetate as an intermediate product. Growth on acetate was slow and weak. Sulfate, sulfite, thiosulfate, and elemental sulfur, but not nitrate, served as electron acceptors for growth with lactate. The bacteria performed dismutation of thiosulfate to sulfate and hydrogen sulfide. In the absence of sulfate, pyruvate but not lactate was fermented. Cytochromes of b and c types were present. The temperature and pH optima for both strains were 60-65 degrees C and pH 7.0. Bacteria grew at 0 to 4.5-6.0% NaCl in the medium, with the optimum being at 0.5-1.0%. Phylogenetic analysis based on a comparison of incomplete 16S rRNA sequences revealed that both strains belonged to the C cluster of the genus Desulfotomaculum, exhibiting 95.5-98.3% homology with the previously described species. The level of DNA-DNA hybridization of strains 435T and 781 with each other was 97%, while that with closely related species D. kuznetsovii 17T was 51-52%. Based on the phenotypic and genotypic properties of strains 435T and 781, it is suggested that they be assigned to a new species: Desulfotomaculum salinum sp. nov., comb. nov. (type strain 435T = VKM B 1492T).     

Survival and growth of Campylobacter fetus subsp. jejuni on meat and in cooked foods.

Twelve strains of Campylobacter fetus subsp. jejuni isolated from humans and animals grew at temperatures ranging from 34 to 45 degrees C and pH minima between 5.7 and 5.9. Only one strain grew at pH 5.8 with lactic acid present at a concentration similar to that in meat. All strains had decimal reduction times of less than 1 min at 60 degrees C. Further examination of a typical strain showed that it grew at 37 degrees C on high-pH meat but not at 37 degrees C on normal-pH meat. Bacterial numbers on both high (6.4)-pH and normal (5.8)-pH inoculated meat declined at a similar rate when the meat was stored at 25 degrees C. At -1 degree C, the rate of die-off was somewhat slower on normal-pH meat but was very much slower on high-pH meat. The initial fall in bacterial numbers that occurred when meat was frozen was also greater for normal-pH meat than for high-pH meat. The organism exhibited a long lag phase (1 to 2 days) when grown in cooked-meat medium at 37 degrees C and died in meat pies stored at 37 or 43 degrees C. Evaluation of the risk of Campylobacter contamination of red-meat carcasses to human health must take into account the limited potential of the organism to grow or even survive on fresh meats and in warm prepared foods.     

Abnormal testes in reindeer, Rangifer tarandus.

In a sample of reindeer from South Georgia, 4 males were abnormal in that 1 had bilateral cryptorchid testis, 2 each had 1 cryptorchid testis and 1 had a vestigial testis. The antlers of the cryptorchid males were small, but the antler cycle itself was hardly affected.     

Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen.

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.