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.     

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.     

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.     

THE INFLUENCE OF AGE ON THE SUSCEPTIBILITY OF BULLS TO CAMPYLOBACTER FETUS SUBSP VENEREALIS

Observations were carried out on 22 Hereford bulls to determine the age at which they bacame carriers of C. fetus. During preliminary investigations, 5 bulbs were repeatedly exposed to the organism by artificial or natural means from the age of 18 months. All animals became longterm carriers on reaching an age of between 40 and 70 months. Prior to this, a temporary carrier state lasting from 4 to 29 days was produced in 4 bulls. In a subsequent experiment, 17 bulls of 3 different age groups were artificially exposed to C. fetus on 5 occasions over a 2-year period. Thirteen bulls became long-term carriers, including 9 (53%) when less than 4 years old. A transient carrier state occurred in 4 bulls, 2 of which subsequently became long-term carriers. It usually lasted 1-2 weeks, although in 1 animal the organism persisted for 11 weeks. These results are discussed in relation to the findings of other workers and it is concluded that the use of young bulls to limit the spread of C. fetus in infected herds is of dubious value.     

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.     

Effect of acrosomal defects on fertility of bulls used in artificial insemination and natural breeding

Four bulls that produced spermatozoa with a high percentage of abnormal acrosomes were individually placed in pens with females for 21 days. Frozen semen from 2 of the bulls was used for artificial insemination. One of the bulls was placed in a competitive mating situation with normal bulls at pasture. First service pregnancy rates were determined by transrectal ultrasonography 28 days after bull removal from breeding pens, or after the last artificial insemination. The results of competitive mating at pasture were determined from breeding observations, the phenotypic characteristics of calves sired, and blood typing for parentage. The results of these studies suggest that bulls that produce a high percentage of spermatozoa with indented acrosomes may have normal fertility when used in artificial insemination or in single sire mating; however, their fertility may be low when breeding competitively with bulls with normal spermiograms.     

Molecular epidemiology of Campylobacter fetus subsp. fetus on bovine artificial insemination stations using pulsed field gel electrophoresis

The presence of Campylobacter fetus subspecies fetus (Cff) on bovine artificial insemination (AI)-stations can have major economical consequences. More knowledge on the epidemiology of C. fetus is needed to control Cff infections at AI-stations. We assessed the epidemiology of Cff on AI-stations and the molecular relationship between Cff strains isolated from outbreaks on AI-stations. Thirteen Cff strains (two Cff strains per outbreak and one sporadic case) isolated from bulls housed on different AI-stations were selected and compared with ten unrelated bovine and ovine Cff isolates from different geographical regions. Molecular typing by pulsed field gel electrophoresis (PFGE) with the restriction enzymes SmaI, SalI and KpnI, yielded unique profiles for most unrelated strains but indistinguishable profiles for all isolates from the same outbreak. Computer aided analysis using a composite data set of SmaI, SalI and KpnI restriction profiles revealed separate clusters for outbreak strains. Thus, PFGE profiling of Cff strains is a valuable tool to discriminate between strains derived from separate outbreaks and to identify routes of infection.     

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.     

Failure to detect bovine immunodeficiency virus contamination of stud bull spermatozoa, blood leukocytes, or semen leukocytes in samples supplied by artificial insemination centers

OBJECTIVE: To determine whether bovine immunodeficiency virus (BIV) infection could be detected in spermatozoa, blood leukocytes, or semen leukocytes from stud bulls in artificial insemination centers. ANIMALS: 30 bulls at 3 artificial insemination centers. PROCEDURE: Polymerase chain reaction testing that used 3 sets of primer pairs targeting pol and env regions of the BIV proviral genome was performed on DNA extracted from semen leukocytes, spermatozoa, and blood leukocytes from each bull. Southern blot analysis was performed to increase sensitivity of detection. Western blot analysis of plasma samples was used to detect antibodies against BIV. RESULTS: BIV provirus was not detected in DNA samples obtained from semen leukocytes, spermatozoa, or blood leukocytes, and antibodies against BIV were not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to our report of high point prevalence of BIV contamination of semen from a single artificial insemination center, bulls of the study reported here did not appear to be infected. Maximum risk of BIV infection in similar bulls was estimated at 10% with a confidence level of 95%.     

精子生育相关性抗原对奶牛妊娠率影响研究

为研究精子生育相关性抗原对奶牛妊娠率的影响,测试了16头种公牛精子的生育相关性抗原,进行人工授精,比较奶牛的妊娠情况.结果显示:测试的16头种公牛精子生育相关性抗原,其中13头呈阳性,3头阴性,阴性率为18.75%.在用FAA阳性精液进行人工授精的520头奶牛中,有433头奶牛怀孕,妊娠率为83.26%.而FAA阴性授精的80头奶牛中,只有52头奶牛怀孕,妊娠率为65.0%(P<0.01).因此,奶牛人工授精FAA阳性精子妊娠率比FAA阴性精子高18个百分点,大大提高了奶牛的妊娠率.     

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.     

Isolation of Coxiella burnetii from bull semen

Previous observations on the dissemination of Coxiella burnetii between laboratory animals strongly support the hypothesis of venereal transmission. Serum and semen samples, from 57 bulls used for artificial insemination, were assayed for specific C. burnetii phase 11 antibodies and the presence of the organism respectively. Viable C. burnetii were detected in the semen of seropositive bulls. These findings indicate the possibility of sexual transmission of C. burnetii between cattle and further our knowledge of the epidemiology of the organism. The procedures used for investigations into the source of infection and route of transmission should be modified to take these findings into account.     

Comparison of two sampling tools for diagnosis of Tritrichomonas foetus in bulls and clinical interpretation of culture results.

OBJECTIVE: To compare sensitivity for diagnosing Tritrichomonas foetus infection in bulls using 2 sampling tools and to calculate negative predictive values for infection. DESIGN: Randomized clinical trial. ANIMALS: 30 Bos taurus bulls naturally or experimentally infected with T foetus. PROCEDURE: Preputial scrapings were obtained once/wk for 6 weeks using an artificial insemination pipette and a metal brush; which tool was used first for each bull was randomly determined. Samples were collected first from the left side of the prepuce and then from the right side and placed in commercially available transport media chi 2 Values and confidence limits were adjusted for effect of clustering of results by bull. RESULTS: Significant differences in sensitivity of results were not found between samples collected using the brush or pipette. Using the pipette, sensitivity was estimated to be 91.6% (95% confidence interval, 84.3 to 95.7%); negative predictive values ranged from 41 to 99% for prevalence of infection of 90 to 5%, respectively. Sensitivity was 88.8% for first sample obtained and 96.1% for second sample obtained. CONCLUSIONS AND CLINICAL RELEVANCE: Collection of preputial scrapings with an artificial insemination pipette or a metal brush and use of a commercially available culture system can provide a sensitive diagnostic test for T foetus infection in bulls. Calculated negative predictive values indicated that 1 or 2 tests would suffice in most clinical situations. For bulls from herds in which T foetus is endemic, 2 to 4 tests/bull may be required to ensure that each bull is not infected.     

Search for Neospora caninum DNA in bull semen using PCR

Neospora caninum represents one of the most frequent abortifaciant organisms worldwide. The parasite is diaplacentally transmitted from the pregnant cow to the fetus, where it normally leads to the delivery of a healthy, however persistently infected calf. Abortion thus is a relative rare event. The transmission of bovine neosporosis occurs in more than 90% of the cases vertically due to the endogenous reactivation of a persistently infected mother. Exogenous infections are therefore responsible for less than 10% of the cases.The question arises about which infection sources may be relevant in this context. In Switzerland, the role of dogs as definitive hosts has been shown to be of low significance in that respect. Recently, discussion focused on the potential of infectious bull semen following natural or artificial insemination. Thus, a few years ago a report documented the detectability of N. caninum-DNA in the semen of naturally infected bulls by nested-PCR. As a consequence, we decided to gain own experience by investigating 5 separate semen specimens per animal, originating from 20 N. caninum-seropositive bulls used for artificial insemination in Switzerland. All probes turned out to be negative by nested PCR. Based upon our laboratory experiences, the potential bull semen-associated Neospora-problem seems not to affect the Swiss bull population, thus there is no evidence to include further respective means of control.     

Tarsal lameness of dairy bulls housed at two artificial insemination centers: 24 cases (1975-1987).

Degenerative joint disease of the tarsi was diagnosed in 20 of 24 Holstein bulls with tarsal lameness at 2 artificial insemination centers from 1975 to 1987. Each of the 2 centers housed about 100 bulls/yr. Of the 24 bulls with tarsal lameness, 22 were from the artificial insemination center designated as A, and 2 were from the center designated as B. Examination of the housing and management procedures revealed that center A had concrete floors with cuboidal-shaped yards, whereas center B had deep sand flooring, with long, narrow yards. The only other difference between the 2 centers was that center A used 1- and 2-year-old bulls as teasers for older, heavier bulls to mount, whereas center B used bulls that were at least 6 years old to withstand the stress placed on their hind limbs by the weight of bulls undergoing semen collection. Radiographic lesions of tarsi of bulls from both centers ranged from distention of the tibiotarsal joint pouch to hypertrophic degenerative osteoarthritis of the distal, intertarsal, and tarsometatarsal joints. It was concluded that the concrete flooring and the semen collecting practices were responsible for the high prevalence of tarsal lameness and degenerative joint disease of the tarsi in bulls housed at center A.     

娟姗公牛引入牧区生理机能变化与种用效果分析

为全面掌握娟姗种公牛在高寒牧区的适应性和生理机能变化情况,将6月龄娟姗种公牛引入牧区后观测生理机能变化情况,待体成熟后与牦牛开展自然交配。对牦牛自然交配和人工授精方式下的繁殖性能进行对比分析,结果表明：自然交配方式下母牦牛受胎率、产犊成活率、繁殖率比采用人工授精技术的母牦牛相应指标分别高19.26%、25.71%和23.12%,流产率和初生犊牛死亡率则比采用人工授精技术的母牦牛分别低8.54和5.64个百分点。同时对两种杂交技术下牦牛所产的后代娟犏雌牛进行犊牛生产性能对比分析,结果表明：自然交配母牦牛所产的后代娟犏雌牛犊比人工授精的母牦牛所产娟犏雌牛犊的初生重、12月龄体重、初胎初乳量、初胎年均泌乳量分别高4.92 kg、21.86 kg、0.36 kg、197.89 kg,两组数据经差异显著性检验,自然交配组的娟犏雌牛犊初生重、12月龄体重、初胎年均泌乳量均极显著的高于人工授精组的相应指标（P＜0.01),初胎初乳量差异显著（P＜0.05)。     

Three types of acrosomal aberrations of bull spermatozoa and their relation to fertility

The effect of acrosomal aberrations of the spermatozoa of Finnish Ayrshire bulls on the corrected non-return rate within 60 days of the first 500 inseminations was studied. The material consisted of sperm samples examined by the artificial insemination societies. All samples had been accepted for use in artificial insemination. One Giemsa-stained slide was studied for each of the 95 bulls concerned. Samples showing distinct acrosomal defects were studied by electron microscopy. Three different types of acrosomal aberration were found. One was obviously associated with subfertility in all 6 bulls in which it was detected.     

Scrotal circumference measurements on young Holstein bulls

Scrotal circumference was measured on all young Holstein bulls entering an artificial insemination center over a 57-month period. A total of 3,008 measurements were taken on 723 bulls between the ages of 5 and 18 months. Mean scrotal circumference, standard deviation, and 10th and 25th percentile statistics were calculated for each one-month interval between 5 and 18 months of age. Scrotal circumference increased as a logarithmic function of age and was best described by a quadratic regression equation. This information provides the veterinary practitioner with indices for normal scrotal circumference measurements for young Holstein bulls, and an estimate of the scrotal circumference growth curve.     

奶牛人工授精受胎率影响因素和提高措施

奶牛人工授精是以人工方法,采用机械手段采集优质种公牛的精液,再经过机械手段将特定处理后的精液输入母牛生殖道的一种生产技术方式。人工授精操作能极大减少种公牛的养殖数量,节约养殖成本,提高优质种公牛的利用效率,加快品种改良进程,促进育种工作的开展。但由于奶牛人工受精是一个侵入性的操作,一旦操作不合理或者各个细节把握不完善,会造成繁殖母牛出现严重的繁殖障碍,降低母牛的受胎率,不利于提高品种改良和扩大养殖效益。该文主要论述奶牛人工受精受胎率的影响因素,提出相应的改进措施。     

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.