Serological reactivity to Mycobacterium bovis protein antigens in cattle

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.     

Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis

The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD, and lipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bovis infected cattle and 223 cattle from a TB free herd. ELISA results were analyzed using receiver operating characteristic (ROC) curves in relation to culture results. The areas under the three ROC curves were 71 ± 49% SE (MPB70), 71 ± 27% SE (bovine PPD), and 56 ± 4% SE (LAM).     

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.     

新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份,用牛支原体特异性引物进行PCR检测,将检测为阳性的样本进行病原培养纯化,对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析,通过测定颜色变化单位(CCU)测定分离株生长曲线,并对分离株进行药物敏感性试验。【结果】PCR结果显示,10份鼻拭子中检测出7份牛支原体阳性样本,1份病死牛肺脏组织也检测为阳性;在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落,纯化后分离株菌落形态为典型的煎蛋状;Dienes染色可见明显的深蓝色中心脐;生化试验结果显示,分离株不水解明胶、精氨酸、七叶苷,不发酵乳糖、葡萄糖和甘露醇,不分解尿素,可还原氯化三苯基四氮唑;16S rRNA测序结果显示,分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高,均为99.9%;生长曲线测定结果显示,分离株在培...     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.     

Genomic, protein and antigenic variability of mycoplasma bovis

Restriction endonuclease analysis (REA) with three enzymes SmaI, PstI, BamHI- was used to identify 13 different genomic groups among 37 Mycoplasma bovis strains. One genomic group was comprised of 14 strains. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns for one strain chosen from each genomic group and an international reference strain PG45 were all similar. Antigenic variability in M. bovis species was investigated by immunoblotting, using serum from a calf that had been naturally infected with M. bovis and three M. bovis-specific monoclonal antibodies — mAbs N₂, I₂ and 5D7. Twenty M. Bovis field strains were tested, comprising one from each genomic group, six from the same genomic group and the reference strain. Antigenic profiles obtained with calf serum differed markedly one from the other, the heterogeneity being equally great among the strains belonging to the same genomic group as those coming from different groups. A stable antigen common to 164 out of 168 strains was detected by mAb N₂, whilst with mAbs I₂ and 5D7, two different membrane antigenic systems were demonstrated that were strikingly variable. These variations in expression occurred not only from one strain to another, but also within the same lineage of clones from a single cell.     

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Immunoblotting analysis of the reaction of wildebeest, sheep and cattle sera with the structural antigens of alcelaphine herpesvirus-1 (malignant catarrhal fever virus)

Malignant catarrhal fever (MCF) is a disease of cattle and some other ruminants caused by alcelaphine herpesvirus-1 (AHV-1), a virus of wildebeest. The disease also occurs in the absence of wildebeest and is then thought to be caused by a viral agent harboured by the sheep. The structural proteins of AHV-1 have been used as antigens for the immunoblotting analysis of sera from wildebeest, sheep and cattle infected by either AHV-1 or the "sheep-associated" form of the disease. Wildebeest sera showed a uniform response reacting strongly with six polypeptides. Sheep sera also gave positive results but individual sera reacted with varying subsets of the antigens recognized by wildebeest. These results support the earlier suggestion that sheep harbour a herpesvirus related to AHV-1. A bovine serum from a case of MCF caused by AHV-1 also reacted only with a subset of the six wildebeest-reactive polypeptides. Sera from cattle affected with the "sheep-associated" form of the disease gave reactions in only two of the eight cases tested; both positive sera reacted to a few polypeptides only.     

Tuberculosis as a zoonosis from a veterinary perspective

Tuberculosis is an important disease among many zoonoses, because both Mycobacterium tuberculosis and Mycobacterium bovis, which are the major causes of tuberculosis, are highly pathogenic, infect many animal species and thus are likely to be the source of infection in humans. In particular, monkeys are highly susceptible to these bacteria and are important spreaders. Recently, two outbreaks of M. tuberculosis occurred in four different kinds of monkeys and humans were also infected with the disease in Japan. In zoos, tuberculosis was reported not only in monkeys, but also in several different kinds of animals, including elephants. Pets such as dogs and cats are believed to be generally less susceptible to M. tuberculosis, but in this article we introduce a case of infection from man to dog by close contact. Japan is one of the few countries that have been able to control M. bovis infection. In other countries, however, cases of bovine tuberculosis and human M. bovis infection have been reported, and thus further attention is still required in the future.     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.     

Interaction of antigen presenting cells with mycobacteria

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNF and IL-10 whereas macrophages produced TNF, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.     

Genotyping of Mycobacterium bovis by geographic location within Mexico

The spacer oligonucleotide typing (spoligotyping) method was used to differentiate 62 Mycobacterium bovis isolates obtained from tissues with macroscopic lesions typical of tuberculosis in dairy cattle from different regions of Mexico. Our purpose was to see if a strain from one region was genetically different from those of other regions (with the long-term aim of doing molecular trace back of isolates obtained in the laboratory). Results from the genetic analysis indicate that M. bovis isolates cannot be grouped by geographic location due to a wide range of genetic types involved in dairy cattle infections. Isolates even from the same herd showed different spoligotypes but some isolates from different region had similar genetic patterns. Genetic typing without epidemiologic information does not seem to be a plausible method to trace back animals to source of origin to detect and eliminate sources of infection.     

分枝杆菌分型三重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定分枝杆菌的三重PCR方法,并比较分析其在临床检测中的可靠性。根据已发表的结核分枝杆菌、牛分枝杆菌和非洲分枝杆菌rv 3036c基因,结核分枝杆菌rv 1970f基因(RD7)和牛分枝杆菌pncA基因的序列,改造并设计合成了3对特异性扩增引物,建立了一种能对分枝杆菌样品进行初步鉴定的三重PCR方法。结果显示该方法可针对rv 3036c、rv 1970f和pncA基因分别扩增出大小为500、125和249 bp的目的片段,能特异性检测出结核分枝杆菌(500和125 bp两条带)和牛分枝杆菌(500和249 bp两条带),并可将结核分枝杆菌、牛分枝杆菌与其他分枝杆菌加以区分。本方法的检测灵敏度为50 pg/μL模板基因组DNA。对86株抗酸染色阳性菌进行三重PCR鉴定,鉴定结果与细菌16S rDNA和ITS序列测定结果一致,检测准确度为100%,优于生长特征和生化试验鉴定。     

犊牛支原体肺炎继发牛A型多杀性巴氏杆菌感染的诊治

某奶牛场犊牛相继发生肺炎和关节炎,为确诊该牛场犊牛群发病的原因并提出防控方案,本试验剖检新生犊牛并采集病料,分别开展牛支原体及其他病原菌的分离培养、PCR鉴定及药敏分析;进行牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒PCR检测;制作犊牛肺脏组织病理切片并进行观察和评估。从犊牛肺脏组织分离到牛支原体和牛A型多杀性巴氏杆菌;牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒检测均为阴性;肺脏组织病理切片可见肺泡结构破坏、出血及大量炎性细胞浸润;药敏试验结果显示,牛支原体和牛A型多杀性巴氏杆菌分别对泰乐菌素和头孢唑啉敏感,但对青霉素、庆大霉素、林可霉素和氨苄西林均呈现耐药。该犊牛群确诊为牛支原体肺炎继发牛A型多杀性巴氏杆菌感染,采用泰乐菌素联合头孢唑啉肌肉注射,配合对症治疗和规范管理,有效控制了该场犊牛疾病。     

Establishment and Application of Mycobacterium Typing Triple PCR Detection Method

This study was aimed to establish a triple PCR method to rapidly identify Mycobacterium species, and evaluate its testing reliability.Three pairs of primer that were respectively specific to rv 3036c, rv 1970f and pncA genes of Mycobacterium were designed to establish a triple PCR for preliminary identification of Mycobacterium tuberculosis(M.tuberculosis), Mycobacterium bovis(M.bovis) and other Mycobacterium spp.PCR products were the expected sizes of 500(rv3036c), 125(rv1970f) and 249 bp(pncA), and contained two DNA bands(500 and 125 bp) with M.tuberculosis DNA template, two DNA bands(500 and 249 bp) with M.bovis DNA template.No band or non-specific band appeared with Mycobacterium spp.except M.tuberculosis and M.bovis DNA templates.The sensitivity of the triple PCR was calculated to 50 pg/μL template of genomic DNA.86 acid-fast bacteria were detected by the triple PCR, 16S rDNA and ITS gene sequencing, growth test and biochemical test, and the results were consistent between triple PCR and 16S rDNA and ITS gene sequencing.The detecting accuracy of triple PCR was 100%, and higher than growth test and biochemical test.     

Vaccination of cattle against bovine schistosomosis: current status and future prospects: a review

Bovine schistosomosis, caused by Schistosoma bovis, constitutes a serious veterinary problem in many parts of the world. The vaccination approaches for the control of bovine schistosomosis include the use of irradiation-attenuated S. bovis cercarial or schistosomular vaccines, S. bovis adult worms or whole-egg antigens and defined antigen vaccine. Irradiated S. bovis cercarial or schistosomular vaccines provide partial protection against S. bovis infection. However, this type of vaccine requires live infectious cercariae or viable schistosomula for induction of protection. Unfortunately, experimental immunizations with dead schistosome antigens have been largely unsuccessful. The surge of new techniques in cellular immunology and molecular biology has made possible the development of potential candidate vaccine antigens from various species of schistosomes including S. bovis. The efficiency of these vaccines has been evaluated in experimentally infected calves. These vaccines will probably replace the irradiated S. bovis vaccines. A broad-spectrum antischistosome vaccine which can kill a variety of human and animal schistosome species is yet to be produced.     

Isolation of mycoplasmas from milk, lungs and prepuce of cattle

M. bovis was mainly isolated from 6.25% of milk samples collected from cows with clinical and sub-clinical mastitis. The main mycoplasma strain isolated from the respiratory tract of calves with symptoms of bronchopneumonia was M. bovirhinis (12.1% of samples). M. bovigenitalium was most frequently recovered from bull's prepuce (67.1% of samples).