Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion

Of 38 aborted ovine fetuses from 23 sheep flocks 29 C. fetus subsp. fetus and 22 C. jejuni were isolated and examined biochemically and serologically for heat-stable antigens. Serologic examinations were carried out by passive haemagglutination test. In case of C. fetus subsp. fetus strains alkaline antigen extraction was used. Antisera to two serogroups of C. fetus and to Penner serotype reference strains 1 to 60 were produced in rabbits. Abortion was caused in 18 (78.3%) flocks by C. fetus subsp. fetus and in 5 (21.7%) flocks by C. jejuni. Six C. fetus subsp. fetus strains grew well at both 43 and 25 degrees C. With one exception all C. fetus subsp. fetus were resistant, whereas all 29 C. fetus subsp. venerealis strains were sensitive to 30 micrograms/ml cefoxitin and cefamandole. These two cephalosporins can be used to differentiate the two subspecies of C. fetus. Passive haemagglutination test using alkaline antigen extraction is a proper method for the examination of heat-stable antigens of both C. fetus subspecies. Out of 24 C. fetus subsp. fetus strains 13 belonged to serogroup A(01), and 11 to serogroup B(02). C. jejuni strains examined belonged to Penner serogroup 1 (6 strains), to serogroup 5 (4 strains) and to serogroup 8 (4 strains).     

Serogroups of Campylobacter jejuni from man and animals

A total of 186 campylobacter strains from aborted calf and sheep fetuses, from scouring dogs, rabbits and man, and from retailed poultry were isolated and examined biochemically and serologically for heat stable antigens. Immune sera were produced in rabbits against Penner reference strains from 1 to 60, and against two field isolates. Out of 186 biochemically tested strains 179 (96.2%) proved C. jejuni and only 6 (3.2%) C. coli. One strain has been identified as C. laridis. In cattle and sheep 3.2 and 21.7% respectively of all campylobacter abortions were due to C. jejuni infection. The same agent caused 12.7% of diarrhoea of dogs. The campylobacter infection rate of freshly slaughtered and dressed chicken varied between 25 and 64.3%. Out of the serologically examined 140 C. jejuni strains 118 (84.3%) could be assigned to 16 Penner serogroups and 13 (9.3%) to 2 further serogroups. Serogroups 8 (31.4%), 1 (19.3%) and 2 (12.1%) occurred most frequently. The human isolates represented the widest serotype distribution, as 32 tested strains belonged to 12 serogroups. All those serogroups which caused abortion or diarrhoea in animals or were isolated from poultry carcases were isolated also from man with diarrhoea, but some serogroups were found only in man.     

Campylobacter jejuni abortion in a heifer

Campylobacter jejuni was isolated from the lung and stomach contents of an aborted bovine fetus. The bacterial isolate was classified according to morphologic, biochemical, and thermotolerance characteristics as well as sensitivity to antibiotics and biochemical agents. Serotyping was specific for C jejuni and excluded this isolate from the more common bovine causes of campylobacteriosis, C fetus subsp venerealis or C fetus subsp fetus. This report stresses the need for careful speciation of all Campylobacter isolates from aborted fetuses, especially in conditions where routine vaccination has not reduced abortion rates in the herd. Campylobacter jejuni is much more prevalent in causing human infections than is C fetus subsp venerealis or C fetus subsp fetus. Therefore, from a public health standpoint identification is important.     

Isolation of Campylobacter Fetus Subsp. Jejuni from Faeces of Norwegian Poultry

Pooled faeces samples from 106 poultry flocks in Norway were examined. Campylobacter fetus subsp. jejuni was isolated from 10 of 100 chicken flocks and from 4 of 6 turkey flocks. Eight of the 14 isolates were classified as biotype C. jejuni, which is frequently associated with human campylobacteriosis. Five strains belonged to the biotype C. coli. One strain was resistant to nalidixic acid but differed from the biotype NARTC in its ability to hydrolyse hippurate. The results indicate that C. fetus subsp. jejuni is widespread among Norwegian poultry.     

Bulk growth procedures and a button agglutination test for Campylobacter

Bulk-cell yields were obtained from 4 Campylobacter spp incubated aerobically without the use of a special atmosphere. A button agglutination test was developed for quantitation of blood serum antibodies against C fetus subsp venerealis, C fetus subsp fetus, C jejuni, and "C hyointestinalis." The test was sensitive, and different individuals reading it usually attained the same titers. Cells of C fetus subsp venerealis, C fetus subsp fetus, and "C hyointestinalis" grown aerobically in broth made satisfactory antigens for the button test, but some cell pools of C jejuni had a tendency to autoagglutinate. Cells of C jejuni grown on blood agar had less tendency to autoagglutinate.     

Investigation of bovine venereal campyloacteriosis in beef cow herds in New Zealand

AIMS: To determine regional prevalences of beef cow herds in New Zealand positive for Campylobacter fetus subsp venerealis antibodies in samples of vaginal mucus tested using an immunoglobulin (Ig) A enzyme-linked immunosorbent assay (ELISA), and to examine the suitability of the IgA ELISA for detecting infection with C. fetus subsp venerealis under field conditions in New Zealand. METHODS: Vaginal mucus samples (n=1,230) collected from beef cow herds (n=125) throughout New Zealand (approximately 10 samples/herd) were tested for antibodies to C. fetus subsp venerealis using an IgA ELISA. Test results were compared between herds classified as having low, medium and high fertility based on pregnancy test results interpreted in relation to the duration of the mating period used. In addition, a small number of samples were collected from dairy cows that were mated using artificial insemination (AI) and had no contact with breeding bulls. The influence of putative risk factors for the spread of venereal disease and the effect of sample quality on the status of herds according to test results was assessed using multivariate logistical regression. Preputial washings from 54 bulls from nine herds classified as low fertility in which mucus samples from > or =3 cows were IgA antibody-positive were cultured for the presence of Campylobacter spp, and isolates of C. fetus subspecies were characterised using a polymerase chain reaction (PCR) test. RESULTS: One or more mucus samples was positive to the IgA ELISA in 70% of all herds tested. The prevalence of IgA antibody- positive individuals was >20% in most regions of New Zealand and did not differ significantly for cows from herds classified as high, medium or low fertility (28%, 26% and 23%, respectively; p=0.39). No relationship was found between mucus antibody status and age of breeding group, herd size, herd fertility, number of herds that female replacements or breeding bulls were sourced from, whether a serving ability test (SAT) was used to assess bulls, or the quality of samples submitted to the laboratory. Campylobacter fetus subsp venerealis was not cultured from any of the 54 bulls sampled. Four other species of Campylobacter and related organisms were cultured, viz Arcobacter cryaerophilus, Campylobacter jejuni, Campylobacter fetus subsp fetus and Helicobacter cinaedi. CONCLUSIONS: The specificity of the IgA ELISA as a diagnostic test for C. fetus subsp venerealis was found to be unsatisfactory under New Zealand conditions. It is possible that an immunological response by cows to Campylobacter species other than C. fetus subsp venerealis caused cross-reactivity in the IgA ELISA. The results do not support the hypothesis that C. fetus subsp venerealis is widespread in New Zealand.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.     

Pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus from sheep abortions in the Hawke's Bay region of New Zealand

AIM: To type Campylobacter isolates from sheep abortions from the Hawke's Bay region of New Zealand. METHODS: Campylobacter isolates were collected from aborted sheep fetuses from commercial farms in the Hawke's Bay region. Information on the Campylobacter vaccination status of flocks in the study was collected. Isolates were identified to species level using standard phenotypic tests, then typed using pulsed-field gel electrophoresis (PFGE). RESULTS: Eighty-one C. fetus subsp. fetus isolates were cultured from aborted sheep fetuses from 25 farms and four C. jejuni isolates were cultured from fetuses from three farms. The C. fetus subsp. fetus isolates were classified into six PFGE groups. A single pulsed-field type predominated amongst isolates from 19 of the 25 farms. The C. jejuni isolates comprised two types. CONCLUSIONS: A range of C. fetus subsp. fetus PFGE types was identified, and one type, B1, was found most frequently. Campylobacter fetus subsp. fetus was only isolated from samples from sheep that had not been vaccinated with C. fetus subsp. fetus vaccine that season.     

Adaption of ELISA for the detection of Campylobacter antibodies and its application in seroepidemiological studies in sheep and cattle herds

ELISA was adapted for the study of antigenic relations among important Campylobacters and for the presence of anti-campylo-bacter antibodies in 394 sheep and 265 cattle. Rabbit anti-C. jejuni, C. coli, G. fetus subsp. fetus and C. laridis heat-stable antigen sera were evaluated against 29 Campylobacter strains and 6 other bacteria. Anti-C. jejuni and G. coli reacted strongly with homologous antigens and weakly with C. fetus subsp. fetus, C. laridis and C. fecalis antigens. C. fetus subsp. fetus serum reacted mainly with its homologous antigen. C. laridis serum showed closer reactivity to C. jejuni than to C. fetus subsp. fetus, C. coli and C. fecalis. Insignificant cross-reactions were observed with Y. enterocolitica, S. dublin and E. aerogenes heat-stable antigens, Ewes vaccinated with C. fetus subsp. fetus bacterin showed higher ELISA titers against C. fetus subsp. fetus antigens than non-vaccinated ewes or rams. Twenty-five percent of the vaccinated animals showed titers as low as 95 % of the non-vaccinated animals. In cattle the lowest antibody titers against C. fetus subsp. fetus, C. jejuni, C. coli and C. laridis antigens were exhibited by the precolostrum sera followed by the postcolostrum and adult sera. These studies demonstrated the applicability of the ELISA test in seroepidemiological investigations concerning the distribution and significance of Campylobacter antibodies in food animal sera.     

Campylobacter as the cause of diarrhea in calves]

The modified Preston medium allows the isolation of C. jejuni, C. coli and C. fetus subsp. fetus from intestinal samples of calves at an incubation temperature of 37 degrees C. In the first series of investigation, Campylobacter excretion in calves (n = 7) was followed up to the age of 4 months. In the first 4 days of life, these bacteria could not be detected in any of the animals. Thereafter first C. coli we found in all calves. In 4 animals, only strains of this species were isolated during the whole investigation period. In 3 animals C. fetus subsp. fetus could be detected repeatedly, however C. coli and sometimes C. jejuni were found, too. In the second series of investigation, isolation of Campylobacter from different parts of the gastrointestinal tract or organs was successful in 19 out of 25 diarrhoeal, moribund calves. 16 out of 19 positive animals harboured large amounts of these gramnegative bacteria in the distal jejunum and ileum. In 10 animals out of these 16, the germ colonized also the proximal jejunum and abomasum. From 6 calves, C. fetus subsp. fetus was isolated, and C. jejuni from 7 calves. C. coli was relatively rare. From the lymph nodes of the proximal and distal jejunum, Campylobacter (exclusively C. jejuni) were isolated from 5 animals. Due to the Campylobacter presence in the small intestine of diarrhoeal calves, a contribution of this bacteria within the pathogenesis of calf diarrhoea is possible. Final evaluation of their pathogenesis importance is only positive by means of virulence tests.     

Detection of Campylobacter antibodies in sheep sera by a Dot-ELISA using acid extracts from c. fetus ssp. fetus and c. jejuni strains and comparison with a complement fixation test

In this study, a dot-enzyme-linked immunosorbent assay (Dot-ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall-bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot-ELISA and CFT. Twenty-two sera showed anti-complementary activity were not suitable for CFT. Of the 22 sera showing anti-complementary activity, two sera were found to be positive in Dot-ELISA. Eighty-eight (67.2%) of the remaining 131 sera were negative by both Dot-ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot-ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot-immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot-ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose.     

IMMUNISATION OF CATTLE AGAINST VIBRIOSIS WITH VACCINES PREPARED FROM CAMPYLOBACTER FETUS SUBSP FETUS

Subcutaneous administration of vaccines prepared from cells of Campylobacter fetus subsp fetus strain A28 to heifers gave substantial protection against infertility due to C. fetus subsp venerealis strain B6. Strains A28 and B6 had different heat-stable antigens and conformed respectively to serotype B and serotype A of Berg et al (1971). The results suggested that the protective antigens were heat-labile antigens common to both strains. Although vaccines prepared from serotype B strains of C. fetus subsp fetus could be used to immunise cattle against vibriosis, the results did not suggest that their use in preference to those prepared from C. fetus subsp venerealis would offer any advantages.     

Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7-10 DNA fragments ranging in size from 40 to 373kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.     

Campylobacter fetus subsp. jejuni in poultry reared under different management systems in Nigeria

Cloacal swabs from 487 live birds in 36 flocks and 70 poultry carcasses were cultured for Campylobacter fetus subsp. jejuni. It was isolated from 12.3% of the birds in 19 flocks. Chickens, turkeys, and guinea fowl differed from one another in isolation rates of the organism. Management system affected its occurrence, and only 7.1% of eviscerated carcasses yielded it. It was concluded that bird species, management system, and immersing slaughtered poultry in boiling water before dressing affect recovery of C. fetus subsp. jejuni from live birds and carcasses.     

Antimicrobial susceptibility testing of German Campylobacter fetus subsp. venerealis isolates by agar disk diffusion method

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis and is transmitted by asymptomatic carrier bulls via contaminated semen during artificial insemination. The aim of the present study was to determine the in vitro susceptibility of Campylobacter fetus subsp. venerealis isolated from bovine specimens in the years from 2000 to 2009 in Germany to antibiotics generally used in semen treatment. The susceptibilities of 50 strains to spectinomycin (10 microg), gentamicin (10 microg), streptomycin (25 microg), penicillin (10 microg), lincomycin (10 microg), ciprofloxacin (5 microg), erythromycin (30 microg) and tetracycline (30 microg) were determined using a disk diffusion susceptibility test. All strains were susceptible to gentamicin. A considerably reduced susceptibility to one or more antimicrobial agents was detected in seven out of 50 isolates (14%) with the most frequent reduction in susceptibility to lincomycin and spectinomycin. Furthermore, strains with reduced susceptibility to more than one antimicrobial agent were always associated with reduced susceptibility to lincomycin. It is recommended to determine the antimicrobial susceptibility of Campylobacter fetus subsp. venerealis isolates in order to evaluate the efficacy of the generally used antibiotic treatment of bull semen and to detect possible resistances.     

Pulsed-field gel electrophoresis of Campylobacter jejuni sheep abortion isolates

Campylobacter species are a significant cause of sheep abortion in most sheep-raising countries. In New Zealand, Campylobacter fetus subsp. fetus is the leading cause of diagnosed sheep abortion and the species C. jejuni and C. coli have also been implicated. To date, strain typing information of C. jejuni sheep abortion isolates is limited. The objective of the present study was to genotype C. jejuni isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the 2000 breeding season, using pulsed-field gel electrophoresis (PFGE). In this study, C. jejuni isolates were cultured from approximately 10% of farms from which Campylobacter species were isolated from sheep abortions in the year 2000. This equated to 25 C. jejuni isolates from 21 farms. These isolates were obtained from the veterinary diagnostic laboratories and strain typed using the molecular typing technique PFGE. Ten distinct PFGE types were identified amongst the isolates. No particular PFGE type was found most frequently amongst these C. jejuni sheep abortion isolates. However, indistinguishable or similar C. jejuni PFGE types were identified from different aborted foetuses from the same flock, consistent with the role of C. jejuni as an infectious cause of abortion in sheep. These strain types were similar or indistinguishable from C. jejuni sheep abortion isolates obtained in 1999 in a smaller study (Mannering, S.A., Marchant, R.M., Middelberg, A., Perkins, N.R., West, D.M., Fenwick, S.G., 2003. Pulsed-field gel electrophoresis typing of C. fetus subsp. fetus from sheep abortions in the Hawke's Bay region of New Zealand. NZ Vet. J. 51, 33-37).     

Campylobacter as a venereal disease in cattle

Since the introduction of AI, venereal diseases caused by Tritrichomonas fetus and Campylobacter fetus subsp. venerealis haved been eradicated in The Netherlands. Campylobacter fetus subsp. fetus can cause sporadic abortion and early embryonic death. When natural breeding is practised, venereal diseases must be included in the list of differential diagnoses of fertility problems. A case study of a Campylobacter fetus subsp. fetus infection is described.     

The prevalence of bovine venereal campylobacteriosis in cattle herds in the Lake Chad basin of Nigeria

The prevalence of bovine venereal campylobacteriosis (BVC) was investigated in the Lake Chad basin of Nigeria. Preputial washings and cervico-vaginal mucus samples were obtained from 270 cattle presenting a history of abortion and lowered fertility, kept in traditional and institutional farms. All the samples investigated were cultured using standard bacteriological technique. Campylobacter fetus was isolated from six bulls and four cows. In all cattle sampled, the isolation rates were 2.2% for C. fetus subsp. venerealis and 1.5% for C. fetus subsp. fetus; the herd and within-herd prevalence rates for C. fetus were 22.2% and 3.4%, respectively, while the overall active infectivity rate was 3.7%. BVC probably contributes to lowered fertility and abortions found in cattle in the Lake Chad basin of Nigeria, associated more with C. fetus subsp. venerealis than C. fetus subsp. fetus.     

Comparison of surface antigens of some Campylobacter fetus subsp. fetus strains of ovine origin by polyacrylamide gel electrophoresis and immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were used to identify and to compare the surface antigens of eight C. fetus subsp. fetus strains. Seven strains (one of serogroup A and six of serogroup B) were isolated from aborted ovine fetuses, while one strain (serogroup A) originated from an aborted calf fetus. Saline extracts at 56 degrees C and 100 degrees C were used as antigens. Antisera were produced in rabbits. In saline extracts (56 degrees C) of the strains at least 19 fractions were identified by SDS-PAGE, with molecular masses ranging from approx. 4,800 to 205,000. The major bands appeared at 205,000, 66,000, 31,500, 25,000, 21,000 and 17,500. Despite the fact that the strains were cultured from 4 different sheep flocks and belonged to serogroup A or B, the SDS-PAGE profiles of the strains were very similar. When boiled (100 degrees C) extracts were used, a band migrating at 32,500 in sheep strains and a band at 97,500 in the calf isolate were missing. Most of the bands obtained by SDS-PAGE could be identified also by the immunoblot procedure. A or B type specificity of the ovine isolates was due to an LPS fraction, migrating at approx. 21,000, while the other LPS fractions appearing under this region although reacted with antisera did not influence the type specificity. Using alkaline extracts (pH 12) in SDS-PAGE, LPS fractions gave more pronounced profiles. In two of our C. fetus subsp. fetus isolates, plasmids with a molecular mass of 31,500 were identified.