Comparative efficacy of ten commercial Campylobacter fetus vaccines in the pregnant guinea pig: challenge with Campylobacter fetus serotype A.

Efficacy of ten commercial Campylobacter fetus vaccines was tested in pregnant guinea pigs and compared with that of an experimental vaccine prepared from the challenge-exposure strain. If the first lot of vaccine failed to protect 50% of the guinea pigs, one or two additional lots of that vaccine were purchased and retested. Three vaccines for cattle, evaluated, as the most effective of those tested, protected 62%, 72%, and 89% of the guinea pigs from abortion; the experimental vaccine protected 98%. The two vaccines for sheep protected 50% and 61% of the guinea pigs from abortion. With the other five vaccines produced for immunizing cattle, protection was from 0% to 36%, with the exception of one lot of a vaccine that protected 74%. Blood infection was found at necropsy in only 6% of the guinea pigs given vaccines that protected 50% or more from abortion, but was found in 66% of those given vaccines that protected less than 50%. Similarly, tissue infection was found at necropsy in only 18% of the guinea pigs given vaccines that protected more than 50%, but was found in 91% of those given vaccines that protected less than 50% from abortion. Oil-emulsion adjuvants appeared to enhance protection from abortion and infection. Nodules persisted at the injection site in most of the guinea pigs immunized with vaccines containing oil-emulsion adjuvants, but rarely persisted in guinea pigs given aqueous-phase adjuvant vaccines. Comparison of efficacy of the vaccines in guinea pigs with efficacy in sheep and cattle remains to be made.     

Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides

Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol-water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non-C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non-C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus.     

An examination of Campylobacter fetus subsp. fetus by restriction endonuclease analysis and serology

Restriction endonuclease analysis was used to examine 70 different strains of Campylobacter fetus subsp. fetus, which were isolated from aborted sheep foetuses. The strains could be divided into seven types based on the DNA fragment patterns obtained by electrophoresis after digestion with the enzyme BstEII. With one exception, digestion with the enzyme XhoI allowed the strains to be grouped identically to that for BstEII. Antisera were made against formalized whole cells representative of each of the seven different restriction types. Analysis of these sera by tube agglutination tests using whole cells revealed five different serogroups. Examination of the 70 strains with absorbed antisera demonstrated a complex relationship between the restriction type and the external antigens of C. fetus subsp. fetus.     

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.     

牛源胎儿弯曲菌PCR检测方法的建立

根据胎儿弯曲菌的16S rRNA基因序列设计引物,以微需氧培养的胎儿弯曲菌标准株菌体裂解物为模板,进行PCR扩增目的片段。同法检测了胎儿弯曲菌的10个参考菌株,结果均为阳性。采用异硫氰酸胍快速提取流产牛阴道分泌物中胎儿弯曲菌的DNA,然后用PCR方法检测,5份样品阳性,该试验可在24 h内完成,比病原分离法快5~7 d;而检测空肠弯曲菌、大肠杆菌、布氏杆菌、葡萄球菌、链球菌等相关病原时,均无特异性扩增产物,表明该检测方法具有快速、特异、实用性强的特点。     

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.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke' Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987.

METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939–2945) from the NZRM, representing restriction types a–g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE.

RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a–g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study.

CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke' Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke's Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987. METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939-2945) from the NZRM, representing restriction types a-g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE. RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a-g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study. CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke's Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

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).     

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.     

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.     

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.     

胎儿弯杆菌病TaqMan实时荧光定量PCR检测方法的建立

为建立胎儿弯杆菌(C.fetus)定量检测方法,本研究根据C.fetus毒力因子表面蛋白(SapA)基因序列设计引物和一条特异的TaqMan水解探针,建立了一种敏感、特异、重复性好的快速检测C.fetus的TaqMan荧光定量PCR方法.对该方法的特异性与敏感性研究,结果显示,该方法检测C.fetus结果均为阳性,而非C.fetus均为阴性;对带有SapA基因的阳性质粒的检测敏感性为10~8拷贝～10~2拷贝/μL范围内具有良好的线性关系,可敏感地检测到模板中13个拷贝的细菌DNA,其灵敏度是常规PCR方法的100倍.该方法具有简便、快速、特异性强、敏感性高等特点.该方法为C.fetus快速检测试剂盒的研制打下了良好的基础.     

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.