Production of the vacuolation factor of Bacillus cereus isolated from vomiting-type food poisoning.

Vacuole response in HEp-2 cells was induced with culture supernatants of Bacillus cereus strains isolated from outbreaks of vomiting- and diarrheal-type food poisoning grown in rice flour and laboratory media. High vacuole response was obtained with culture supernatants of B. cereus strains isolated from vomiting-type food poisoning grown in cooked rice suspension or on a cooked rice plate, whereas no response was obtained with those of the same strains grown in brain heart infusion and trypto-soya broth media. The vacuole activity appeared only after spore formation of B. cereus. The activity was stable to proteolytic enzymes, heating, and exposing to pH 2.0 and 11.0. Of 124 strains isolated from B. cereus food poisoning that were tested, the vacuole activity was observed by 68 of 110 (61.8%) of the strains isolated from the vomiting-type food poisoning but not by all strains (14 strains) from diarrheal-type ones. Moreover, the vacuole response in the HEp-2 cells was found to be induced by 56 of 76 (73.7%) of the serotype H-1 strains isolated from vomiting-type food poisoning.     

Preliminary observations of interaction between bacteriophages and Streptococcus bovis bacteria on ruminal epithelium primoculture.

Five Streptococcus bovis strains (47/3, 59/2, 4/1, 46/2 and 44/9) isolated from calf ruminal fluid samples were examined for the adherence to cultured ruminal epithelium cells. Four strains (47/3, 59/2, 4/1 and 46/2) were able to attach to the cultured epithelial cells. However, S. bovis 47/3 strain attached to the target cells in significantly greater numbers than the other strains. Strain 44/9 did not adhere to cells of ruminal epithelium. The adherent bacteria were observed on the surface of differentiated (mainly keratinized) cells of ruminal epithelium primoculture only. The different effect of F4, F5 and F6 bacteriophages was ascertained on S. bovis bacteria adhering to rumen epithelial primoculture. A significant decrease in the number of adherent bacteria was shown after cultivation of strains 47/3 and 4/1 with F6 bacteriophage and of 47/3 strain with F4 phage. The F5 bacteriophage had no significant effect on these bacteria.     

Hydrogen Peroxide Production by Mycoplasma bovis and Mycoplasma agalactiae and Effect of In Vitro Passage on a Mycoplasma bovis Strain Producing High Levels of H2O2

Hydrogen peroxide (H2O2) production and oxygen uptake during the oxidation of NADH and L-alpha-glycerophosphate (GP) by lysed cells was determined for the type and field strains of Mycoplasma bovis and M. agalactiae. NADH oxidation by all the strains showed variable production of H2O2 ranging from 0 to 1.21 mol/mol O2 taken up. All strains were unable to oxidize GP, showing absence of GP oxidase activity. Some strains were identified that produced relatively high levels of H2O2 (> 1.0 mol/ mol O2 taken up). In vitro passage of M. bovis strain 119B96 showed reduced H2O2 production: 0.52, 0.16, and 0.07 mol/mol O2 taken up after the 50th, 100th and 200th passages, respectively. SDS-PAGE analysis showed the loss of a protein band of 32 kDa after 50 passages. These preliminary studies show that not only does H2O2 production by potentially pathogenic Mycoplasma spp. vary in the field but also that similar alterations can be induced by passage in culture. In the latter case, at least in one M. bovis strain, this alteration has been shown by SDS-PAGE to be associated with a loss of specific protein production. Further study of these phenomena is essential background for the production of more efficient vaccines for mycoplasmas.     

Isolation,Identification and Biological Characteristics Research of Streptococcus bovis from Yak

The objective of this study was to isolate and identify Streptococcus bovis from yak, and detect their hemolytic, pathogenicity and antimicrobial susceptibility. 45 fecal samples collected from the diarrheal yaks in Aba state in Northwest Sichuan province were used to isolate Streptococcus bovis with sheep blood culture media under 37℃ and 5% CO₂ condition cultured for 24 h. The isolated bacteria were identified by 16S rRNA amplification and sequencing. Total 14 Streptococcus bovis were identified, among which, 8 Streptococcus lutetiensis strains and 6 Streptococcus gallolyticus strains. 9 Streptococcus bovis strains showed α hemolytic, and 5 Streptococcus bovis showed β hemolytic. Both Streptococcus lutetiensis and Streptococcus gallolyticus caused the experimental mice mild diarrhea. The isolated 14 Streptococcus bovis were sensitive to penicillin, cefotaxime, vacomycin, acetylspiramycin, ciprofloxacin, rifampicin, teicoplanin, ampicillin and gentamicin, and highly tolerant to streptomycin, kanamycin, lincomycin, erythrocin, tetracycline and clindamycin.This study illustrated the biological characteristics of the isolated Streptococcus bovis from yak, which made the basis for the prevention and control of diseases caused by Streptococcus bovis.     

牦牛源牛链球菌的分离鉴定及部分生物学特性研究

本研究旨在从腹泻牦牛粪便中分离鉴定牛链球菌,并分析其溶血性、对小鼠的致病性及对抗菌药物的敏感性。将川西北阿坝州45份腹泻牦牛粪便于血平板上划线,37℃、5% CO₂培养24 h分离细菌,经16S rRNA序列扩增测序和系统发育分析鉴定出14株牛链球菌,其中8株巴黎链球菌,6株解没食子酸链球菌巴氏亚种;9株呈α溶血,5株呈β溶血。分离鉴定的牦牛源巴黎链球菌和解没食子酸链球菌巴氏亚种能引起试验小鼠的轻度腹泻;药敏试验结果显示分离菌株对青霉素、头孢噻肟、万古霉素、乙酰螺旋霉素、环丙沙星、利福平、替考拉宁、氨苄西林和庆大霉素共9种抗生素高度敏感,对链霉素、卡那霉素、林可霉素、红霉素、四环素和克林霉素耐药率较高。本研究阐明了牦牛源链球菌的部分生物学特性,为该病的防控奠定了基础。     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。     

Detection of cell detachment activity induced by Moraxella bovis

OBJECTIVE: To characterize the effect that filtrate obtained from cultures of Moraxella bovis has on cultured corneal epithelial cells and other types of cultured mammalian cells. SAMPLE POPULATION: Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis. PROCEDURE: Moraxella bovis was cultured, and bacteria were removed by filtration. The resulting bacterial culture filtrate was incubated with various types of cultured cells, and effects of the filtrate on detachment of various mammalian cell types was quantified by the use of neutral red dye. Additionally, bacterial culture filtrate was treated with protease inhibitors as well as trypsin and heat prior to incubation with cultured mammalian cells. RESULTS: Bacterial culture filtrate of M bovis caused all types of cultured cells to detach from each other and from the substrate, with the maximal effect evident 2 hours after initiating incubation. Detached cells were alive, and detachment was reversible. Serine protease inhibitors (phenylmethylsulfonylfluoride and alpha2-macroglobulin) inhibited cell detachment attributable to bacterial culture filtrate. Heating and treatment with trypsin also inhibited cell detachment. CONCLUSIONS AND CLINICAL RELEVANCE: Moraxella bovis produces a soluble factor that causes reversible detachment of cultured cells. This activity may play a role in the pathogenesis of infectious bovine keratoconjunctivitis.     

牛链球菌在瘤胃中产酸的代谢机制及调控

牛链球菌(S.bovis)是瘤胃主要的乳酸产生菌,在饲喂高精料饲粮导致瘤胃乳酸中毒进程中扮演重要角色。已有研究证实S.bovis利用碳水化合物代谢产酸主要受葡萄糖转运方式、酵解产酸途径中酶和中间代谢物调控。另外,研究也发现环境p H、增殖生长阶段及分解代谢控制蛋白(Ccp A)等对其产酸速率和模式也有显著影响。本文对近年来有关S.bovis利用饲料中碳水化合物发酵产酸代谢途径及影响因素研究加以综述,为从微生物代谢角度解析瘤胃乳酸中毒机制提供参考。     

Induction of anti-phagocytic surface properties of Staphylococcus aureus from bovine mastitis by growth in milk whey

The respiratory burst activity of bovine polymorphonuclear (PMN) cells in response to milk whey- and TSB-grown S. aureus strains isolated from bovine mastitis was studied in whole blood chemiluminescence (CL) and in a CL system with purified bovine neutrophils. In both cases milk whey-grown S. aureus strains elicited significantly less CL than homologous strains grown in TSB. Ingestion of milk whey-grown S. aureus strains by bovine neutrophils was also considerably lower than that of the corresponding homologous organisms grown in TSB. Binding of complement factor C3 to serum-opsonized milk whey-grown S. aureus strains was lower compared with TSB-grown homologous organisms. Moreover, 5 of 6 S. aureus strains grown in milk whey were significantly more resistant to in vivo clearance from the peritoneal cavity of mice compared with homologous bacteria grown in TSB. S. aureus strains grown in TSB exhibited hydrophobic surface properties, whereas homologous strains grown in milk whey were hydrophilic.     

The effect of culture media on bacteriocin production in various strains of bacteria]

Some staphylococcus and enterococcus strains were used to investigate the effect of culture medium on bacteriocin production. Staphylococcus cohnii SC7, Staphylococcus sp. ZTJ 151, S. saprophyticus SS 877, Enterococcus faecium EF1 and E. faecalis EFG2 were isolated from the rumen wall and contents of lambs, calves and fallow deer, Enterococcus gallinarum EG10 and E. avium EA12 were isolated from the caecum of Japanese quail. The tested bacteria belong to producers with a wide antimicrobial effectiveness spectrum, they have low to medium adherence and urease activity (Tab. III). These culture media were used to test the effect of culture medium on bacteriocin production: nutrient agar no. 2 and VL agar enriched with 2% of glucose and lactose (ZAG, ZAL, VLG, VLL), agar for isolation of faecal streptococci (SA) and the base for blood agar no. 4 and no. 2 (KA4, KA2). The strains Streptococcus bovis AO 24/85 and Staphylococcus aureus Oxford 209 P were used as indicator bacteria. Tables I and II show the results of these tests. The tested strains produced the widest inhibition zones (6 mm) with both indicators on SA medium, and this indicates massive bacteriocin production. On ZAG medium, the zones of enterococci with the AO 24/85 strain were larger size than those of staphylococci, but the zones were dim. All strains with the 209P indicator produced dim zones of the 2mm size. The larger inhibition zones (2-5mm) in comparison with staphylococci were observed in enterococci on the ZAL medium with the AO24/85 strain. The production of tested strains was balanced on VLG agar with respect to the use of both indicators.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis.

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.     

Immunisation against lactic acidosis in cattle.

The present study was designed to investigate the efficacy of control of lactic acidosis by immunisation against lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus. Ten steers were allocated to two treatment groups. One group was immunised with a vaccine containing S. bovis (strain Sb-5) and Lactobacillus (LB-27) cells, and the other was a non-immunised control group. The vaccine, using Freund's complete adjuvant for primary immunisation and Freund's incomplete adjuvant for boosters, was administered intramuscularly. After primary immunisation, boosters were given at 2 to 4 week intervals. Both anti- S. bovis and anti- Lactobacillus IgG levels in saliva increased significantly (P < 0.01) after the 1st booster which were lower (P < 0.05) than the IgG levels after the 2nd and 3rd boosters, but were not significantly different (P > 0.05) from the IgG levels prior to a grain challenge (after the 4th booster). There were positive correlations between the anti- S.bovis and anti- Lactobacillus IgG in serum and saliva. Compared with the control group, steers in the immunised group had higher (P < 0.05) feed intakes, lower (P < 0.05) rumen concentrations of lactate and lower numbers of S. bovis and Lactobacillus. Three of the control animals were withdrawn from the grain challenge due to their rumen pH persisting below 5.2, while only one animal in the immunised group was withdrawn. These results suggest that the risk of lactic acidosis can be reduced by immunisation against S. bovis and Lactobacillus.     

Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils

The cytotoxicity of Moraxella bovis whole cells and culture filtrates was studied, using 51Cr-labeled bovine and human blood neutrophils. The cytotoxicity of living M bovis was directly related to the concentration of bacteria in the neutrophil cultures, and was maximal at an approximate neutrophil to bacteria ratio of 1:10. Cytotoxicity was maximal by 30 minutes after living bacteria were added to the suspension of the 51Cr-labeled neutrophils. Expression of the cytotoxicity was dependent on the presence of Ca2+ in the media, and was independent of the presence of Mg2+. Cytotoxic activity was eliminated by inactivating M bovis in buffers containing formalin or sodium azide. Hemolytic and nonhemolytic isolates of M bovis were examined for cytotoxic activity. All 7 of the hemolytic isolates were cytotoxic for bovine neutrophils, but all 4 of the nonhemolytic isolates were devoid of cytotoxic activity. None of the 11 isolates were cytotoxic for human neutrophils. Sterile filtrates from 6-hour shaker cultures of a hemolytic M bovis isolate were cytotoxic for bovine neutrophils. Cytotoxicity of the filtrate was eliminated by heating, incubation with trypsin, or addition of EDTA to the media. Bacterial homogenates or sterile filtrates prepared from statistically incubated cultures of M bovis were not toxic for bovine neutrophils.     

Glucose phosphate isomerase isoenzyme patterns in bovine lymphoblastoid cell lines infected with Theileria annulata and T parva, with an improved enzyme visualisation method using meldola blue

Five strains of Theileria annulata from three geographically different areas and one strain of T parva were grown in bovine lymphoblastoid cell lines. Lysates of the parasitised cells were examined by thin layer starch gel electrophoresis for multiple forms of the enzyme glucose phosphate isomerase. A reported difference between the glucose phosphate isomerase isoenzyme patterns of T annulata and T parva was confirmed. Cultures of one strain of T annulata grown in cells derived from four different cattle showed similar host and parasite isoenzyme patterns. Two strains of T annulata and one strain of T parva grown in lymphoid cells derived from the same animal showed identical host cell isoenzyme patterns whereas the isoenzyme pattern associated with each parasite was different. Strains of T annulata from different geographical areas showed major differences in their isoenzyme patterns, but no differences were detected between strains from the same geographical area. Meldola blue was found to be superior to phenazine methosulphate as an intermediate electron acceptor in the visualisation of enzyme activity.     

一株牛支原体的分离鉴定及致病性初步研究

对采集到的疑似牛支原体肺炎肺组织病料进行病原的分离,并对分离株进行形态学、生化和分子生物学鉴定,结果显示成功分离获得1株牛支原体,命名为NM001.该分离株的菌落形态呈典型的"荷包蛋状",不能发酵葡萄糖,不能水解精氨酸,不分解尿素.PCR能够扩增出牛支原体特异的P48基因条带,16S rRNA基因序列与Ningxia-...     

Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum

An in vitro assay was developed using calf corneal cells to assess the importance of fimbriae in the colonisation of the bovine ocular surface by Moraxella bovis, and the role of fimbrial antibodies in the bovine immune response and resistance to infectious bovine keratoconjunctivitis (IBK). Fimbriae promoted adherence of M. bovis to calf corneal cells in culture; 15 fimbriate isolates, representative of 6 fimbrial serogroups of M. bovis, adhered to the cells whereas 4 non-fimbriate isolates failed to do so. Fimbrial antibodies in hyperimmune rabbit serum inhibited attachment of all fimbriate strains of the homologous fimbrial serogroup but not those of 5 heterologous serogroups. The relevance of these results to the use of a polyvalent fimbrial vaccine in the control of IBK is discussed.     

Use of sodium deoxycholate to extract cell wall components of virulent Mycobacterium bovis

Mycobacterium bovis ATCC No. 19210 was grown on Middlebrook 7H-10 medium with pyruvate. Cells were harvested, and extracts were prepared, using 2% sodium deoxycholate (DOC) and 0.003M EDTA (in 0.1M Tris-HCl, 0.15M NaCl with 0.02% sodium azide [pH 8.4]). Phenylmethylsulfonyl fluoride was added, and the cells were extracted for 48 hours at 4 C. Cells were removed by centrifugation at 10,000 X g for 30 minutes. The supernatant was filter sterilized and separated into 2 fractions (peak A and peak B) by size-exclusion chromatography. The nonfractionated DOC extract and DOC peak A elicited delayed-type hypersensitivity responses at each of the protein concentrations tested (0.5, 1.5, and 4.5 micrograms) in M bovis-sensitized guinea pigs; responses were not detected, using DOC peak B. Significant differences were detected for each of the antigens when enzyme-linked immunosorbent assay values were compared, using sera from cattle before and 10 months after they were exposed to M bovis (P less than 0.01).     

Effect of nutritional conditions on changes in leukocyte populations in Japanese black calves

To clarify the effect of nutritive conditions on changes in immune cells in Japanese Black (JB) calves during the growth period, leukocyte populations were analyzed in ten healthy JB calves managed in one herd. The calves were divided into two groups: five calves in Group 1 were given insufficient nutrition, and the other five calves in Group 2 received adequate nutrition. The levels of serum total cholesterol and glucose were significantly lower in Group 1 than in Group 2 at 1 month. The numbers of CD3+, CD4+ and CD8+ cells tended to be lower in Group 1 than in Group 2 at months 1 and 2, and the difference in CD4+ was significant at month 2. The number of MHC class-II(+high) cells was significantly lower in Group 1 than in Group 2 at months 1 and 2. These results suggest that adequate nutrition might stimulate an increase in immune cells in calves during the growth period.