Molecular and antigenic characterization of a Mycoplasma bovis strain causing an outbreak of infectious keratoconjunctivitis.

An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).     

Increasing prevalence of Mycoplasma bovis in Danish cattle

A study on the prevalence of mycoplasmas in pneumonic bovine lungs was performed on material submitted for diagnostic purposes at the Danish Veterinary Laboratory, Copenhagen. Among the 50 examined cases 43 (86.0%) were found to be infected with mycoplasmas. The predominant mycoplasmas were Ureaplasma spp. (72.0%), M. dispar (48.0%) and M. bovis (24.0%). Other mycoplasmas were M. bovirhinis (20.0%) and M. bovigenitalium (6.0%). Among the infected lungs multiple species infections were predominant (76.7%) over single species infections (23.3%) with M. dispar-Ureaplasma (25.6%), M. bovis-Ureaplasma (18.6%) and M. dispar-M. bovirhinis-Ureaplasma (11.6%) infections being the most frequently encountered combinations. There appears to be an increasing prevalence of M. bovis (24.0%) as compared to earlier reports (0.6-2.0%), thus calling for special attention upon this mycoplasma. Pulsed field gel electrophoresis (PFGE) analysis of 11 field isolates of M. bovis from 9 different farms revealed different profiles except for 2 isolates which were recovered from the same farm. Because mycoplasmas belonging to the 'M. mycoides cluster' were not encountered during this study; it appears that the Danish cattle population is still free from this group of mycoplasma in spite of their presence in some other European countries.     

Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection

Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.     

Cross-reactive proteins among eight bovine mycoplasmas detected by monoclonal antibodies

Whole cell proteins of eight bovine mycoplasmas (M. bovoculi, M. bovis, M. dispar, M. bovirhinis, M. arginini, M. verecundum, M. canadense, M. alkalescens) were separated by SDS-PAGE and transferred to nitrocellulose paper. Rabbit anti-M. bovoculi serum was found to react with immunoblots of all mycoplasma species tested. These cross-reactive proteins were in the range of 35,000-100,000 molecular weight. Monoclonal antibody MA25.5 developed against a M. bovoculi 94 kDa surface protein cross-reacted with a band of 62 kDa from M. dispar and three bands of 89, 85 and 74 kDa from M. arginini only while MA18.13 that recognized a band of 57 kDa from M. bovoculi did not react with the other species. The role of MA25.5 monoclonal antibody in inhibiting the growth of M. bovoculi, M. dispar and M. arginini was tested using the metabolic-inhibition (MI) test. Monoclonal antibody MA25.5 inhibited the growth of M. bovoculi and also inhibited M. dispar growth but at lower MI titers, while it showed no effect on the growth of M. arginini.     

牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

为获得牛支原体（Mycoplasma bovis,M.bovis）VspX蛋白单克隆抗体,将编码该蛋白的基因克隆、表达并纯化,作为免疫原,以QuickAntibody-Mouse 5W为免疫佐剂,免疫BALB/c小鼠。经3次免疫后,将小鼠脾细胞与SP2/0骨髓瘤细胞融合,经3次亚克隆筛选后,共获得5株能稳定分泌抗VspX蛋白抗体的杂交瘤细胞株,分别命名为1A8、3A3、3C12、3H9及4D11。亚型鉴定表明,3C12重链为IgG2b,其余4株为IgG1,轻链均为κ链。间接ELISA结果表明,5株细胞培养上清的抗体效价在1：1×10⁴~1：2×10⁵,腹水效价在1：1×10⁵~1：8×10⁵。选其中两株杂交瘤细胞株3H9和4D11的腹水纯化,进行亲和力测定,解离常数分别为6.3×10⁹和7.8×10⁹,属高亲和力抗体。Western blotting结果显示,5株单抗均能与牛支原体发生特异性反应,而单抗4D11与羊无乳支原体标准株PG2和丝状支原体丝状亚种标准株PG3均不反应。流式细胞术结果表明,单抗4D11与牛支原体表面的VspX的结合呈剂量依赖性。间接免疫荧光结果表明,单抗4D11可以识别黏附到胚胎牛肺细胞上的重组VspX蛋白。本试验成功制备的单克隆抗体为VspX蛋白功能的研究奠定基础。     

Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801

To obtain the monoclonal antibody (McAb) against VspX protein of Mycoplasma bovis (M. bovis),VspX gene was amplified, expressed and purified. Then, BALB/c mice were immunized subcutaneously three times with the purified recombinant VspX (rVspX) mixed with QuickAntibody-Mouse 5W adjuvant. Three days after the last injection, spleen cells were collected aseptically, and fused with SP2/0 myeloma cells in the presence of polyethylene glycol. By the clone selection, five stable hybridomas against VspX protein were obtained, separately named as 1A8, 3A3, 3C12, 3H9 and 4D11. Antibody titers in cell supernatant were from 1:1×10⁴ to 1:2×10⁵, while from 1:1×10⁵ to 1:8×10⁵ in ascites of mice by indirect ELISA. The subtypes were determined to be IgG1 and IgG2b class, and all light chains were κ chain. The affinity constant of McAb 3H9 and 4D11 were 6.3×10⁹ and 7.8×10⁹, respectively, and they belonged to high-affinity antibodies. Western blotting results showed that all of five McAbs could specifically react with M.bovis, however, McAb 4D11 could not react with Mycoplasma arginini PG2 and Mycoplasma mycoides subsp. PG3. Flow cytometry showed that McAb 4D11 reacted with surface VspX of M. bovis in a dose-dependent manner. Indirect immunofluorescence assay demonstrated that 4D11 McAb was able to detect rVspX protein binding to embryonic bovine lung cells. In the present study, McAbs against rVspX protein had been successfully prepared, which provided a basis for future researches about the function of VspX protein and the pathogenesis of M. bovis.     

Examination of cattle with respiratory diseases for Mycoplasma and bacterial bronchopneumonia agents

A total of 247 mycoplasma strains was isolated from 435 lungs, tracheobronchial secretions and nasal swabs originating from cattle with symptoms of bronchopneumonia. Mycoplasma (M.) bovis was found 89 times (36%) and was the most common mycoplasma species in the lungs. M. bovirhinis, M. bovigenitalium, M. spec. and Acholeplasma (A.) laidlawii were isolated 158 times (64%). Among these mycoplasmas M. bovirhinis was the most widespread species (114 isolations). In 55 cases (62%) M. bovis was associated with Pasteurella or Actinomyces (A.) pyogenes. The other mycoplasma species were found in 67 cases (42%) together with these bacteria. Without mycoplasmas Pasteurella and A. pyogenes occurred in 33 of the probes investigated (21%). Beside mycoplasmas Haemophilus (H.) somnus was isolated from 16 of 162 tracheobronchial secretions investigated. The results confirm earlier suppositions that in most of the cases bronchopneumonia of cattle is a multifactorial event, frequently associated with mycoplasmas--especially M. bovis.     

Infectious bovine keratoconjunctivitis: experimental induction of infection in calves with mycoplasmas and Moraxella bovis.

Eyes of 14 calves were exposed by conjunctival instillation to cultures of either Mycoplasma conjunctivae (6 calves) or Acholeplasma laidlawii (8 calves). Calves were observed for clinical signs of infectious bovine keratoconjunctivitis (IBK), and eyes were examined for the test organisms by bacteriologic cultural technique for 60 days. Acholeplasma laidlawii became established in the eyes of 5 of 8 calves; M conjunctivae became established in the eyes of 4 of 6 calves. On day 28, eyes of 9 of the 14 calves were exposed by conjunctival instillation to Moraxella bovis, and all developed IBK. Five calves exposed to Moraxenjunctivae or A laidlawii, but not to Mor bovis, did not develop IBK. Four calves not exposed to M conjunctivae or A laidlawii, but exposed to Mor bovis, developed IBK. Mycoplasmas do not have a major role in IBK, but might produce ancillary effects similar to those of infectious bovine rhinotracheitis virus, wind, ultraviolet radiation, dust, and other irritants.     

Analysis of various antigens in golden hamster testis by monoclonal antibodies.

A total of 38 hybridomas producing monoclonal antibodies (mAbs) was established by immunizing BALB/c mice with extracts of the golden hamster testis. Six mAbs stained the acrosome of developing spermatids by immunofluorescence. Two mAbs (1A11 and 4D8) reacted with spermatid components other than acrosome. The mAbs 1C9 and 4D3 recognized a 103 kilodalton (kDa) protein on immunoblots, and were reactive to spermatocytes and early spermatids, but not to late spermatids and spermatozoa. This finding suggests that the protein functions for meiosis or early spermiogenesis. Four mAbs (3G2, 2E5, 2G3, and 3F10) stained all stages of spermatogenic cells. The remaining 24 mAbs showed a positive reaction to the basement membrane of the seminiferous tubule. Two of them, 3D6 and 3E5, recognized approximately 150 kDa major proteins, indicating that the antigen is an extracellular matrix.     

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.     

Mycoplasma bovis-free breeding of cattle]

On a cattle farm latently infected by M. bovis, field studies aiming at the formation of a mycoplasma free herd, were carried out with a group of newborn female calves. These calves were strictly separated from their dams and any other cattle immediately after parturition. Intensive investigations for mycoplasmas were made over 30 months (mycoplasma isolation from nasal swabs, antibody detection by means of indirect hemagglutination test and ELISA technique). M. bovis could never be isolated from the samples. Also, there were no antibodies to M. bovis. In some animals antibody titers to M. bovis occurred after having contact with cattle infected with M. bovis. The results demonstrate a practicable way to establish cattle herds free from M. bovis infection.     

Analysis of cross-reactivity of five new chicken monoclonal antibodies which recognize the apical complex of Eimeria using confocal laser immunofluorescence assay

For Apicomplexa (members) the host cell invasion is realized with the help of the organelles located at the apical tip of parasites. In this research paper the characterization of five chicken monoclonal antibodies (mabs) produced against Eimeria acervulina sporozoites is described. All mabs reacted with molecules belonging to the apical complex of chicken Eimeria sporozoites. On immunofluorescence assay (IFA) one mab, 8E-1, recognized an apical tip molecule present on all chicken Eimeria sporozoites, two mabs (8D-2 and HE-4) recognized an antigen present on the apical tip of the same two Eimeria species (E. acervulina and E. brunetti), another mab (5D-11) recognized an antigen present on the apical tip of other two species (E. acervulina and E. maxima) while one mab (8C-3) identified antigens present on the sporozoites and sporocysts wall of only E. acervulina. Besides the apical tip antigens, two mabs (HE-4 and 8D-2) recognized some proteins located in the anterior half of the sporozoites. Collectively, these mabs proved that the apical complex of chicken Eimeria sporozoites share one or more antigens that are expected to play a role in host cell recognition and invasion.     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

Analysis of the antigen-specific IFN-gamma producing T-cell subsets in cattle experimentally infected with Mycobacterium bovis

Three 10 months old cattle were infected by the intratracheal route with 10(6)cfu of a field strain of Mycobacterium bovis. Blood samples were regularly collected for in vitro IFN-gamma production after antigenic stimulation. Peripheral blood cells of infected animals produced IFN-gamma in response to crude M. bovis antigens (live and heat-inactivated BCG and protein-purified derivative (PPD)) 3-4 weeks after infection. The ratio of the response to bovine PPD versus avian PPD indicated a specific sensitisation for M. bovis antigens. Three months post-infection (PI), animals were culled and M. bovis was cultured from tubercle lesions. At different time points, the frequency of specific M. bovis IFN-gamma producing CD4+, CD8+ and WC1+ T-cells in the peripheral blood was examined by flow cytometry. Two colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules showed that both CD4+ and CD8+, but not WC1+, T-cells produced IFN-gamma following stimulation with PPD, live or killed BCG.In two animals analysed, the relative percentage of circulating IFN-gamma producing CD8+ cells decreased between week 5 and week 9 PI. The same evolution was not observed for IFN-gamma secreting CD4+ cells. Magnetic positive selection of T-cells from infected animals showed that CD4+ T-cells produced specific IFN-gamma only in the presence of antigen presenting cells (APCs). Positively selected CD8+ T-cells secreted IFN-gamma only in the presence of recombinant human IL-2 and APCs. In vitro depletion of the CD4+ T-cells, but not the depletion of CD8+ or WC1+ T-cells, resulted in abrogation of the specific IFN-gamma production showing the key role of this cell population for the specific IFN-gamma production.     

Detection of mycoplasmas in experimentally-infected bull sperm

Bull sperm pellets experimentally infected with various concentrations of Mycoplasma (M.) bovis, M. bovigenitalium, and M. californicum were stored several months at -196 degrees C and were subsequently tested for mycoplasmas, using different methods and culturing media. M. bovis was reliably identified by laboratory diagnosis up to a concentration of 10(2) cfu/pellet. M. bovigenitalium and M. californicum were found to make higher demands on the quality of culturing substrates. Recommended are 1-h broth incubation and 5-d broth breeding.     

糖化酶单抗的研制及其抗原识别位点的分析

为建立掺糖造假蜂蜜中残留糖化酶的检测方法,用从黑曲霉中提取的糖化酶作为免疫原,免疫BALB/c小鼠,利用杂交瘤技术获得了12株稳定分泌针对糖化酶抗体的杂交瘤细胞株。单克隆抗体亚型鉴定结果显示,10株为IgG1,2株为IgG2b,轻链均为κ轻链。Western blotting分析结果表明,12株抗体均可特异性结合糖化酶。其中6株单抗(McAb-2H4F9、6H9D8、8F2F11、8F2E9、1A8G6、1C4D5)细胞株采用体内诱生法制备的腹水效价均1∶1×10⁴以上。采用抗体叠加试验对这6株抗糖化酶单抗的抗原识别位点进行检测,反应增殖结果表明,6株单抗分别针对4类不同抗原位点,McAb-6H9D8和McAb-8F2F11针对第Ⅰ种抗原决定簇;McAb-1A8G6和McAb-1C4D5针对第Ⅱ种抗原决定簇;McAb-8F2E9针对第Ⅲ种抗原决定簇;McAb-2H4F9针对第Ⅳ种抗原决定簇。制备的抗体针对不同的抗原表位,为双抗夹心ELISA方法的建立提供前提。     

Comparison of media used for the primary isolation of Mycobacteriurm bovis by veterinary and medical diagnostic laboratories

Veterinary and medical laboratories engaged in the cultural diagnosis of bovine or human tuberculosis were requested to supply samples of the media that they routinely use for the primary isolation of M. bovis. Fourteen laboratories supplied 7 basic media types; these were Lowenstein-Jensen, Stonebrink's, modified Middlebrook 7H11 agar, tuberculosis bovine blood agar, egg yolk agar, Gerloff's egg and Herrold's egg yolk. Two strains of M. bovis were used to test the media, strain AN5, a glycerol-tolerant laboratory strain and M86/90 a glycerol-sensitive wildtype strain. AN5 grew well on all media with the exception of Herrold's and strain M86/90 did not grow on media containing glycerol and grew poorly on Herrold's medium. It is recommended that Lowenstein-Jensen with pyruvate (but without glycerol), Stonebrink's, modified Middlebrook 7H11 and tuberculosis bovine blood agar should be considered the media of choice for the primary isolation of M. bovis. Egg yolk agar also proved adequate for this purpose in the trial. This medium may be suitable for routine use but to date experience with its use is limited.     

Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).     

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M. bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.     

Assessment of Bdellovibrio bacteriovorus 109J killing of Moraxella bovis in an in vitro model of infectious bovine keratoconjunctivitis

The objective of this study was to determine the potential of Bdellovibrio bacteriovorus 109J as an alternative non-chemotherapeutic treatment of infectious bovine keratoconjunctivitis (IBK). To accomplish this, various parameters of B. bacteriovorus predation of Moraxella bovis were determined in vitro. Initial passage of B. bacteriovorus using M. bovis as prey required 10 d for active cultures to develop compared with 2 d for culture on normal Escherichia coli prey; however by the 5th passage, time to active predatory morphology was reduced to 2 d. This high passage B. bacteriovorus culture [1 × 10(10) plaque forming units (PFU)/mL] killed 76% of M. bovis [1 × 10(7) colony forming units (CFU)/mL] present in suspension broth in a 4 h assay. The minimal level of M. bovis supporting B. bacteriovorus predation was 1 × 10(4) CFU/mL. To assess the ability of B. bacteriovorus to kill M. bovis on an epithelial surface mimicking IBK, an in vitro assay with Madin-Darby bovine kidney (MDBK) cells inoculated with 4 × 10(7) CFU/mL M. bovis was used. Treatment with a B. bacteriovorus suspension (1.6 × 10(11) PFU/mL) decreased adherence of M. bovis to MDBK cells by 6-fold at 12 h of treatment, as well as decreased the number of unattached M. bovis cells by 1.4-fold. This study demonstrates that B. bacteriovorus has potential as an effective biological control of M. bovis at levels likely present in IBK-infected corneal epithelia and ocular secretions.