Quantitative evaluation of a transport-enrichment medium for Campylobacter fetus

The enrichment feature of a selective serum-based transport medium for Campylobacter fetus was quantitatively examined. Preputial samples from artificial insemination bulls were spiked with known numbers of C fetus strains and inoculated into transport-enrichment medium (TEM). The survival and multiplication of these strains in TEM under different incubation periods and temperatures were assessed by plate counts. Mean enrichment values of 3.72 log and 4.42 log were observed after incubation at 37 degrees C for two and four days, respectively. There was no significant difference in the enrichment values between the C fetus subspecies venerealis strains and a C fetus subspecies fetus strain. Incubation of inoculated TEM vials at room temperature for up to two days neither improved the growth of C fetus nor affected its subsequent enrichment when the vials were reincubated at 37 degrees C. Comparison of the survival of C fetus with and without the use of TEM under simulated transport conditions demonstrated the superiority of TEM.     

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.     

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.     

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.     

The application of a transport and enrichment medium to the diagnosis of Campylobacter fetus infections in bulls

The use of a transport and enrichment medium (TEM) in the diagnosis of Campylobacter fetus infections in bulls is described. The medium significantly improved the diagnosis rate in samples which, because of the length of time between collection and receipt at the laboratory, were unsuitable for processing by direct culture. The TEM was able to support the viability, and subsequent multiplication, of C. fetus in some samples for up to 7 days before the TEM was incubated. The submission of paired samples of TEM, one containing unfiltered preputial washing (PW) and the other containing PW filtered through a 0.60 micron cellulose acetate filter, significantly increased the accuracy of diagnosis.     

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.     

A comparative study of the growth of Campylobacter fetus strains in liquid media

The growth of C. fetus fetus, C. fetus venerealis and C. fetus venerealis biotype intermedius was examined in 10 liquid media. From the data obtained, a 10% inoculum size and an oxygen level of 6% seemed imperative for consistent growth, especially for the C. fetus venerealis strain. A lowered redox potential obtained by the addition of 0, 1% cysteine-HC1 to the media was stimulatory. The medium which yielded the best growth was the one described by Dennis & Jones (1959). The fastidious C. fetus venerealis strain yielded maximum values of 0,5% packed cell volumes after 48 h cultivation in a microaerophilic atmosphere on this medium. The other strains yielded higher values.     

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.     

The development of a transport and enrichment medium for Campylobacter fetus

A transport and enrichment medium was developed for Campylobacter fetus. From inocula of between 10 and 35 organisms the medium was able to support the multiplication of 19 of 21 strains of C. fetus if the medium was incubated immediately after inoculation; when incubation was delayed for 3 days after inoculation, only seven of the 21 strains multiplied. From inocula of 100-350 organisms all 21 strains multiplied following immediate incubation, and 20 of 21 when incubation was delayed for 3 days. From inocula of about 10(4) organisms all strains multiplied following immediate or delayed incubation. The medium restricted the growth of Proteus vulgaris and Pseudomonas aeruginosa.     

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.     

Some Characteristics of Campyloracter Fetus Sursp. Jejuni Isolated from Pigs,Rirds and Man By

One hundred and two strains of Campylobacter fetus subsp. jejuni isolated from pigs, various avian species and man were subjected to biochemical characterization including hydrolysis of hippurate, tolerance of 3.5 % saline, H₂S production in TSI medium, growth on TTC agar and antibiotic sensitivity. On this basis the porcine strains could be classified into 4 groups, while the avian and human strains could each be divided into 2 groups. The avian and human strains had the same biochemical characteristics as 2 of the porcine groups.     

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.     

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.     

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).     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.     

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.     

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.     

Comparison of three sampling methods for the diagnosis of genital vibriosis in the bull

Three different methods of collecting preputial material for bacteriological examination were compared using 3 bulls infected with Campylobacter fetus subsp. fetus. The first method utilised a specially designed instrument to scrape the preputial and penile mucosa, int he second method plastic pipettes were used to aspirate material and the third method involved washing the preputial cavity with sterile peptone water. Bacteriological examination of the samples showed conclusively that scraping was the method of choice because more C. fetus positive samples were identified and there was less interference from contaminating organisms.     

Bacterial infertility in camels (Camelus dromedarius): isolation of Campylobacter fetus

This study constitutes the first reported isolation of C. fetus subsp. venerealis from cervical swabs in breeding camels. 9 of 30 camels which were barren for a long period were found to be positive for C. fetus. The methods of isolation are described. Additionally, cervical flora of 30 camels were examined. 9 different bacterial species were isolated of which Staphylococcus aureus was most frequently found. All samples were negative for Trichomonas sp. and Streptococcus zooepidemicus.     

A simple technique for mass cultivation of Campylobacter fetus.

Studies using 86 media for maximum growth of Campylobacter fetus for antigen production showed that a diphasic medium (solid base with liquid overlay) was most suitable. The solid base was double strength cystine heart agar. The liquid overlay was thioglycollate medium of Brewer (135-C) without agar. This medium yielded maximum growth of C. fetus in six days with good motility, less clumping and less filament formation than all other media tried.