Occurrence and significance of different Campylobacter types in domestic animals in Hungary

Experiences, including results of original experimental work on Campylobacter fetus, C. jejuni and C. coli induced diseases of cattle, sheep, dogs, rabbits poultry and men in Hungary are reviewed. Out of 31 cases of abortion in cows 29 (93.5%) were causes by C. fetus subsp. venerealis and only one case each (3.2%) by C. fetus subsp. fetus and C. jejuni, respectively. Out of the 29 strains of C. fetus subsp. venerealis, 26 belonged to serogroup 01 (A) and only 3 to serogroup 02 (B). Campylobacter abortions in sheep flocks were caused in 18 cases (78.3%) by C. fetus subsp. fetus and in 5 cases (21.7%) by C. jejuni. The latter strains belonged to Penner's serogroup 1 (6 strains), 5 (4 strains) and 8 (5 strains), respectively. In scouring dogs 12.7% of the cases were caused by C. jejuni. The same pathogen caused diarrhoea also in young rabbits. Isolated strains belonged to serogroup 2. In cases of Campylobacter hepatitis of laying hens, egg production has been reduced by 8 to 15% for 2 to 3 weeks. Row poultry meat represents often the source of infection for men. The 32 strains of C. jejuni isolated from faecal samples of men affected with diarrhoea belonged to 12 serogroups.     

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.     

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.     

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.     

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 strains of human and chicken origin are invasive in chickens after oral challenge

The aim of the study was to evaluate the colonizing ability and the invasive capacity of selected Campylobacter jejuni strains of importance for the epidemiology of C jejuni in Danish broiler chickens. Four C. jejuni strains were selected for experimental colonization studies in day-old and 14-day-old chickens hatched from specific pathogen free (SPF) eggs. Of the four C. jejuni strains tested, three were Penner heat-stable serotype 2, flaA type 1/1, the most common type found among broilers and human cases in Denmark. The fourth strain was Penner heat-stable serotype 19, which has been shown to be associated with the Guillain Barré Syndrome (GBS) in humans. The minimum dose for establishing colonization in the day-old chickens was approximately 2 cfu, whereas two- to threefold higher doses were required for establishing colonization in the 14-day-old chickens. Two of the C. jejuni strains were shown to be invasive in orally challenged chickens as well as in three different human epithelial cell lines.     

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.     

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.     

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.     

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.     

Intestinal carriage of Campylobacter jejuni and Salmonella by chicken flocks at slaughter.

Campylobacter jejuni were isolated in large numbers from the majority of birds sampled in colonic swabs from 28 of 60 flocks at slaughter. By contrast only small numbers of birds from 11 of the same 60 flocks yielded Salmonella enteritidis serotypes. Three C. jejuni isolates from each flock were serotyped on the basis of their heat-stable antigens, using antisera prepared against 16 serotypes common in Campylobacter diarrhea in man. The majority (72 of 83) of the chicken isolates could be serotyped.     

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.     

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies.
Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis.
Procedure The results of identification of 99 bacterial strains as determined by conventional phenotyping or by poly-merase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis ; the remaining strains were field isolates putatively identified as C fetus . In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling.
Results The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus ; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories.
Conclusion Our results indicate that misidentification of C fetus i n routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.     

Antimicrobial resistance in Campylobacter from broilers: association with production type and antimicrobial use

The isolation and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli strains from broilers arriving in French slaughterhouses, were analysed according to production types (i.e. standard, export or free-range) and antimicrobial (i.e. coccidiostats, growth promoters or therapeutic agents) administration in flocks. Prevalence was 56.6% in standard, 51.3% in export and 80.0% in free-range broilers. Three hundred and ninety-three strains were identified. Two-thirds of the strains belonged to the species C. jejuni. The others were C. coli. Antimicrobial susceptibility testing was carried out for ampicillin, nalidixic acid, enrofloxacin, tetracycline, erythromycin and gentamicin according to a dilution method. The percentages of resistant strains were, 23, 25, 17, 57, 0.3 and 0% for C. jejuni and 29, 43, 40, 70, 31 and 0% for C. coli. Statistical analysis revealed significant difference in distribution of C. jejuni and C. coli and antimicrobial resistance according to production type or antimicrobial administration.     

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.     

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.     

Serology of Campylobacter fetus fetus strains from four outbreaks of ovine abortion

As a result of a Ministry of Agriculture & Fisheries survey on ovine abortions, 76 isolates of Campylobacterfetus fetus were obtained. These isolates were from four farms in the southern Hawkes Bay, with an abortion incidence of 2.8% to 9.1%. Antisera to eight different strains of C. fetus fetus were made in rabbits. Strains were then examined using whole cell tube agglutination tests and sensitised Staphylococcal Protein A slide agglutination tests. Heat labile antigens were examined by absorbing antisera with heat-treated bacteria. Two broad serogroups were found, but within-group variation was demonstrated by cross-absorbing antisera. The isolates from one farm were all of a single broad serogroup. Both serogroups were found on the remaining three farms. Evidence for the presence of two major serogroups was also obtained by immobilisation tests and antigen analysis by gel diffusion.     

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.     

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.     

Pathology of Campylobacter jejuni abortion in sheep

Campylobacter jejuni was inoculated intravenously into pregnant ewes on gestation days 114 and 123 to reproduce ovine abortion. All ewes aborted 7-12 days post-inoculation. High numbers of C. jejuni were isolated from ewe tissues (caruncle, bile, cecal feces), fetal tissues, and placenta. C. jejuni colonies were identified in caruncles and placenta by light microscopy and immunoperoxidase techniques. Histologically, inoculated ewes had a severe purulent endometritis with vasculitis. Placentas from inoculated ewes and field cases showed necrosis and purulent inflammation; however, placentas from inoculated ewes had large numbers of bacterial colonies compared to few bacteria found in field cases. Histologically, only one fetus from the inoculated ewes showed lesions (purulent bronchopneumonia), whereas all fetuses from field cases had a distinct bronchopneumonia, and one fetus showed multifocal hepatic necrosis. These results suggest that C. jejuni (serotypes Penner 1 and Lior 2) is an important abortifacient organism for sheep.