Bovine genital campylobacteriosis (vibriosis): vaccination of experimentally infected bulls

The therapeutic efficacy of a Campylobacter fetus subsp venerealis bacterin was determined in experimentally infected bulls. Ten of twelve 5-year-old Angus bulls became infected after being infused intrapreputially with C fetus subsp venerealis. Of the 10 bulls, 6 were vaccinated with 5 ml of C fetus subsp venerealis vaccine on 2 occasions 4 weeks apart. Preputial washings of the vaccinated bulls were culturally negative by the 8th week after primary vaccination. None of the 18 heifers exposed to the vaccinated bulls became infected. The 4 infected, nonvaccinated bulls remained culturally positive to C fetus (P less than 0.002), and each bull infected at least 1 heifer (P less than 0.001). Two noninfected, nonvaccinated bulls remained culturally negative and did not infect any heifer. The 4 infected, nonvaccinated bulls were then vaccinated. Two bulls remained infected 9 weeks after primary vaccination, as determined by the virgin heifer test and cultural examination of preputial washings. Serologic data from 7 sampling periods were different (P less than 0.001) for vaccinated vs nonvaccinated bulls at 4 (against K antigen) or 6 (against O antigen) weeks after primary vaccination. Vaccination was effective in eliminating the infection in most of the infected bulls, but cannot be recommended as the sole measure of control in infected herds.     

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.     

PASSIVE TRANSMISSION OF CAMPYLOBACTER FETUS BY IMMUNISED BULLS

Two groups of 24 heifers were mated with 3 immunised and 3 susceptible bulls respectively. Six heifers in each group were infected artificially with Campylobacter fetus sub-sp. fetus biotype venerealis. Among susceptible heifers mated with immunised bulls, 13/18 became pregnant and 5/18 yielded evidence of infection with C. fetus. Among susceptible heifers mated with susceptible bulls, 7/18 became pregnant and 10/18 yielded evidence of infection. C. fetus was isolated on one occasion from an immunised bull, but the immunised bulls failed to develop carrier status and one was shown to be refractory to artificial challenge. Susceptible bulls developed carrier status during the breeding period. It is suggested that passive transmission of C. fetus by immunised bulls can occur under conditions of intensive sexual activity.     

A DUAL VACCINE FOR THE IMMUNISATION OF CATTLE AGAINST VIBRIOSIS

Single subcutaneous injections of a mineral oil adjuvant vaccine containing 20 mg dry weight of Campylobacter fetus subsp fetus biotype venerealis cells and 20 mg dry weight of C. fetus subsp fetus biotype intermedius cells per 5 mil dose protected 2- and 3-year-old heifers and 3- and 4-year-old cows against genital infection with either organism.     

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.     

Vaccination studies for the control of campylobacteriosis in Jamaican cattle

Following the first diagnosis of campylobacteriosis in Jamaican cattle a field study was undertaken to determine the pathogenicity of Campylobacter fetus subspecies venerealis Jam (Jamaican strain) and to evaluate the effectiveness of vaccination in controlling the disease. A total of 46 nonpregnant yearling heifers and four two-year-old bulls were used in two separate experiments. The results showed that C fetus subspecies venerealis Jam readily colonised the reproductive tract of susceptible heifers and persisted in some animals (68 per cent of unvaccinated and 33 per cent of vaccinated animals) for the duration of the experiment. Pregnancy was confirmed in 13 of 18 (72 per cent) culture-negative heifers but in only eight of 28 (29 per cent) of the heifers with two or more positive cultures. Vaccination appeared to be curative because 44 per cent of vaccinated heifers were cleared of infection whereas 85 per cent of unvaccinated, inoculated heifers remained infected for at least 17 weeks. Vaccination improved the fertility level of the infected heifers threefold. Infection was not established in vaccinated bulls used for breeding infected heifers.     

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.     

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.     

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.     

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.     

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.     

Prevalence of genital campylobacteriosis and trichomonosis in crossbred breeding bulls kept on zero-grazed smallholder dairy farms in the Tanga region of Tanzania

A survey to demonstrate the presence or absence of genital campylobacteriosis and trichomonosis in cross-bred breeding bulls kept under smallholding dairy farms in the Tanga region of Tanzania was carried out during the period of January-June 1996. Sheath washings, swabs and preputial scrapings were collected from 58 randomly selected bulls. Campylobacter fetus subsp. venerealis was demonstrated in 3/58 (5.1%) and Tritrichomonas foetus in 0/58 (0%) of all bulls tested. Bull-level variables of level of taurine genes (62.5% taurine genes, F2; 75% taurine genes, F3) and age were not significantly associated with campylobacteriosis (P > 0.05). The result of the study identifies Campylobacterfetus subsp. venerelias as the agent of enzootic infertility in smallholder herds and suggests that may be a significant problem.     

Campylobacter fetus in artificial insemination unit and slaughterhouse bulls in Ontario.

Preputial fluid samples were collected from 90 bulls in two Ontario artificial insemination units using a penial glove swab technique previously developed by one of us for use in donor bulls. No Campylobacter fetus organisms were identified from the prepuce or from samples of semen collected at the same time from these bulls. The distal genitalia of 200 bulls were collected at a slaughter house. One isolation of a Campylobacter fetus subspecies venerealis was obtained on a culture from the fornix area of the prepuce of one of these bulls.     

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.     

Antigenic and restriction enzyme analysis of isolates of Campylobacter fetus subsp venerealis recovered from persistently infected cattle

Thirty-two isolates of Campylobacter fetus subsp venerealis were obtained from 1 bull and 4 heifers with experimentally induced infection. When whole-cell antigens of isolates were cross titrated with antisera to the infecting strain, isolates from 3 heifers had limited antigenic variation, whereas whole-cell antigens of isolates from 2 cattle (the bull and a heifer) differed serologically from those of the infecting strain. Changes were detected specifically in 6 heat-labile antigens. Of the 6 heat-labile factors evaluated, all were initially present on the infecting parent strain, but not on early isolates obtained from 4 of the 5 cattle. Restriction enzyme analysis revealed minor variation in the DNA fingerprints of isolates obtained from individual cattle, thus implying stability of the Campylobacter genome once persistent infection is established. Isolates with identical restriction enzyme patterns expressed different heat-labile antigens. Correlation could not be found between the DNA electrophoretic pattern and the expression of heat-labile antigens.     

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.     

Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium.

One hundred and five bulls from an artificial insemination unit were tested for the presence of Campylobacter fetus subspecies venerealis. The method involved the inoculation of preputial samples into a new transport enrichment medium prior to culture and immunofluorescence tests. Seventeen bulls (16%) were found to be either positive or suspected carriers of C. fetus at one or more sampling times. The average age of these 17 bulls was about two years greater than the average age of all the bulls in the unit. A combined treatment of vaccination and dihydrostreptomycin sulfate injection suppressed or eliminated the organism from carrier bulls. The use of transport enrichment medium has increased our capability and effectiveness to monitor the presence of C. fetus in artificial insemination bulls.     

BOVINE VIBRIOSIS VACCINATION

An adjuvant vaccine was prepared from an Australian isolate of Campy-lobacter fetus subsp fetus biotype intermedius and injected into 23 virgin Guernsey heifers. Ten nonvaccinated animals served as controls. When challenged by the intravaginal route with a culture of either the homologous strain or biotype venerealis, weekly swabs from the anterior vagina continued to yield either biotype in 8 of 10 nonvaccinates at 6 weeks as compared with 3 of 23 vaccinates. Serology in vaccinated heifers and rabbits showed that the vaccine produced high titres of antibody against both homologous and heterologous strains.     

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.     

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.