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.     

Rarity of tylosin resistance in human pathogenic bacteria

Of 3812 human cultures of Staphylococcus aureus, Streptococcus pyogenes and Campylobacter species only 1.0 per cent were resistant to tylosin, an antibiotic used extensively in animals but not in man. There was no evidence for a significant animal source of these resistant cultures, a result which provides further evidence for the rarity of the flow of resistant organisms (or their genes) from animal sources to human beings.     

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.     

奶牛临床型乳房炎的细菌分离鉴定与耐药性分析

2011年山西省多个奶牛场发生了较严重的乳房炎,对76份采集的奶样进行细菌分离鉴定并采用药敏纸片法检测主要分离菌的抗生素耐药情况。所分离细菌以革兰氏阳性菌为主,革兰氏阳性球菌和其他革兰氏阳性菌分别占60.67%和23.59%。分离出多种病原菌和机会致病菌,主要的病原菌有链球菌、金黄色葡萄球菌、大肠杆菌等,检出率为2.24%~11.24%,其中无乳链球菌的检出率最高;机会致病菌有粪链球菌、微球菌、克雷伯菌、凝固酶阴性葡萄球菌等,检出率为1.12%~11.24%,其中粪链球菌和微球菌的检出率较大,分别为11.24%和6.74%。药敏试验检测结果显示,在所选的15种药物中,主要分离菌均对丁胺卡那霉素、氟哌酸和恩诺沙星3种药物敏感;大肠杆菌、克雷伯菌、凝固酶阴性葡萄球菌对青霉素类和β-内酰胺/β-内酰胺酶抑制剂类药物产生了极强的耐药性,耐药率均为100%;乳房链球菌对该类药物也产生不同程度的耐药,耐药率为20%~100%;对链霉素产生100%耐药的细菌有大肠杆菌、克雷伯菌、无乳链球菌、乳房链球菌、停乳链球菌等细菌;部分分离菌对卡那霉素、庆大霉素、链霉素、四环素、红霉素、先锋霉素Ⅴ、复方新诺明等药物产生不同程度耐药。被检奶牛场混合感染较为严重,应进一步加强环境卫生管理,临床治疗应合理有效用药。     

用PCR技术检测牛胎儿弯曲杆菌

在国内首次用PCR技术检测牛胎儿弯曲杆菌(Campylobacterfetus),试验结果表明,灭菌生理盐水中的胎儿弯曲杆菌胎儿亚种或性病亚种均可检出;而空肠弯曲杆菌、葡萄球菌、链球菌、大肠杆菌等均为阴性。本法特异性和灵敏度很高,甚至可检出样品中的一个菌体。应用前景良好。     

The barren camel with endometritis--isolation of Trichomonas fetus and different bacteria.

This study constitutes the first reported isolation of Trichomonas fetus from the uterus of breeding camels. Twenty-four of 68 camels, which were barren for 1-4 years were found to be positive for T. fetus. One preputial washing of a camel bull which had served the herd was found to be positive for T. fetus. The uterine flora of all camels were examined. Ten different bacterial and 2 fungal species were isolated. All samples were negative for Campylobacter sp., Taylorella equigenitalis and Streptococcus zooepidemicus.     

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.     

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.     

Effect of irradiation on the viability of Toxoplasma gondii cysts in tissues of mice and pigs

Muscles from tongue, heart, and limbs of 14 pigs inoculated orally with Toxoplasma gondii oocysts were irradiated with 10, 20, 25, and 30 krad of gamma (cesium-137 and cobalt-60) irradiation. Viability of T gondii cysts was assayed by feeding porcine muscles to T gondii-free cats and/or by inoculation of sediment from acid-pepsin digested porcine muscle into mice. Cats fed 500-g samples of muscles irradiated with up to 20 krad shed T gondii oocysts. Cats fed muscles irradiated with 25 or 30 krad did not shed oocysts. Mice were inoculated with 8 isolates of T gondii, and tissue cysts in their brains irradiated with up to 40 krad were infective to mice; however, there was a 10,000-fold reduction in the viability of organisms in tissue cysts irradiated with 40 krad, compared with that in nonirradiated cysts. At 50 krad of gamma irradiation, there were no detectable infective organisms in infected mouse brains.     

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.     

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

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

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

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

Bactericidal effects of ozone at nonspermicidal concentrations.

A study was conducted to assess the use of ozone (O3) to control pathogens or contaminants of concern to animal breeders and regulatory officials. In separate experiments, samples of fresh bovine semen and Pseudomonas aeruginosa, Escherichia coli, or Campylobacter fetus subsp. venerealis were diluted with antibiotic-free milk (10(6) sperm and 10(6) organisms/mL of diluted semen), exposed in the previous day to a constantly monitored level of 5, 10, 15, or 20 micrograms/mL of O3 for 3-5 min. After 10 min at 30 degrees C, sperm motility was assessed and the samples cooled to 5 degrees C. Two and 18 h after the beginning of cooling, aliquots of each semen sample were evaluated for motility and cultured for organisms. Reductions were observed in P. aeruginosa and E. coli colony counts of 2 logs, and in C. fetus of 5 logs, after exposure for 2 h to O3 at a concentration of 5 micrograms/mL that had a moderate effect on sperm motility (reduction of 20%). Fewer than 100 colonies, i.e., a 4 logs reduction of all bacteria, were counted after dilution with ozonized-treated milk at 20 micrograms/mL of O3. However, this concentration of O3 reduced sperm motility by 50% 10 min after dilution. The results of these experiments indicate that a concentration and exposure time to O3 can be selected to reduce P. aeruginosa, E. coli, and C. fetus in contaminated bull semen diluted with milk while having only minimal effects on sperm motility.     

Diagnosis of vibriosis in bulls at breeding stations through lavages of the artificial vagina]

The objective of the study was to test the suitability of lavages of artificial vaginas which are used for collecting ejaculate for diagnosing the Campylobacter fetus subsp. fetus germs. According to the results of the investigation this method may be considered equal to the method of lavages of the prepuce. However, it is important to point out the advantages: it eliminates the danger of an accident, it enables quality lavage in the laboratory. To caputure the Campylobacter fetus the filtration through diaphragm filters is used together with the culture medium Fluid Thioglycollate medium (Difco) with an additive of agar, 10% ram blood and antibiotics (bacitracin, novobiocin). The catch rate in the prepuce lavages is 26.9%, in lavages of the artificial vagina 26.1%. The insignificant difference between the errors of the methods, i.e. the number of negative findings by employing one method and positive results with the other method, is 24.4% in lavages of the artificial vagina and 22.0% in lavages of the prepuce.     

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.     

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.     

Comparative studies on competitive exclusion of three isolates of Campylobacter fetus subsp. jejuni in chickens by native gut microflora

Resistance of young chicks to Campylobacter fetus subsp. jejuni was substantially increased by early exposure to native gut microflora. Protection was demonstrated against two human isolates and a chicken isolate of C. fetus subsp. jejuni. Significant protection against the chicken isolate was observed throughout a 91-day test period. Infection reached 100% (25/25) in the untreated group at 56 days of age and only 4% (1/25) in the group treated with native gut microflora. Campylobacter fetus subsp. jejuni was isolated from the ceca and less frequently from the gall bladder and liver of chicks that actively shed the bacteria. Cultures of feces from chicks reared on wood-shavings litter were often negative, suggesting that culturing litter as an indicator of infection has limited value.     

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.     

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.     

广东省1株猪2型链球菌的分离鉴定及部分生物学特性研究

为确诊引起广东省某规模化猪场保育仔猪发生死亡的病因,本试验采集病猪肝脏、脾脏、心脏、脑、关节液和淋巴结等病料进行细菌分离,并对分离菌株进行纯化培养、菌落形态观察、革兰氏染色、形态学观察、生化试验,用血清型特异性引物进行PCR扩增,并进行毒力基因检测、药敏试验、生长曲线测定及动物试验。结果显示,分离菌株在成牛血清琼脂平板上呈圆形、灰白色、表面光滑、边缘整齐的菌落,镜检为革兰氏阳性球菌,呈链状排列。该菌株的生化试验结果符合链球菌的特征,PCR扩增结果显示猪链球菌保守基因片段（gdh和gapdh）和猪2型链球菌特异的cps2J基因片段均为阳性,表明该分离菌株为猪2型链球菌。毒力基因检测结果表明,该菌株毒力因子基因型为gdh⁺/gapdh⁺/epf⁺/mrp⁺/sly⁺/orf2⁺/fbps⁺。分离菌株用营养肉汤培养6 h达到对数生长期,培养10 h的菌液D_{600 nm}值为0.310。药敏试验结果表明,该菌株对青霉素、阿莫西林、阿莫西林-克拉维酸钾等药物敏感,但对强力霉素耐药。动物试验结果表明,感染分离株的BALB/c小鼠未出现死亡且无明显临床症状,毒力较低。本研究为该猪场链球菌病的防控提供了重要参考信息。