Relationship between serologic recognition of Escherichia coli 0111:B4 (J5) and clinical coliform mastitis in cattle

Serum IgG1 ELISA titers recognizing gram-negative core antigens (Escherichia coli [J5]) were studied at a large dairy in central California. Population mean log10 titer was 2.7357 (equivalent to 1:544) with a SE of 0.03843. Titers increased with increased lactation number (unstandardized regression coefficient = 0.06733). Changes in lactation number accounted for only 6.77% of titer variation. Titers less than 1:240 were associated with 5.33 times the risk of clinical coliform mastitis. Also, older cattle were at greater risk to develop clinical coliform mastitis. These factors apparently affect incidence in a nonlinear fashion, with greatly increased risk associated with titers less than 1:240 and with fourth or greater lactations.     

Seroprevalence of antibodies against gram-negative core antigens in rabbits, using an Escherichia coli J5 antigen-capture enzyme-linked immunosorbent assay

OBJECTIVE: To determine the seroprevalence of antibodies to gram-negative core antigens (GNCA) in specific-pathogen-free (SPF) rabbits (ie, free of Pasteurella multocida) and rabbits of undefined bacterial status (conventional). SAMPLE POPULATION: Serum samples were obtained from 7 groups of rabbits. The SPF rabbits comprised 2 adult groups and 1 immature group, whereas the 4 groups of conventional rabbits were all adults. PROCEDURE: A seroprevalence survey was conducted on rabbit sera for antibodies against GNCA, using an Escherichia coli J5 antigen-capture ELISA. RESULTS: Collective geometric mean titer (GMT) of adult rabbits was 1:6,463. The GMT of each of the 6 groups of adult rabbits was 1:956, 1:1,133, 1:4,525, 1:5,338, 1:7,669, and 1:25,600. Titers of populations differed significantly. CONCLUSION: Data analysis revealed there were anti-GNCA antibodies in rabbits. Similar to other species, the prevalence of IgM and IgG anti-GNCA antibodies increased with age. The IgG response was more marked than the IgM response. The SPF rabbits had lower IgG anti-GNCA titers than conventional rabbits, indicating possible cross-reactive epitopes between P multocida and Enterobacteriaceae. Rabbits with the highest anti-GNCA titers were those used in polyclonal antibody production, possibly stemming from endotoxin contamination of antigen or adjuvant. CLINICAL RELEVANCE: The possible cross-reactive antibodies directed at homologous wall components of Pasteurellaceae and Enterobacteriaceae could prove to be a possible heterotypic vaccination strategy for the protection of rabbits against pasteurellosis. Investigators should determine whether antigen impurity (endotoxin contamination) influences epitope focus during polyclonal antibody production and whether it affects sera variability among rabbits.     

Distribution of lipopolysaccharide core types among avian pathogenic Escherichia coli in relation to the major phylogenetic groups

Five distinct lipopolysaccharide (LPS) core types, namely R1-R4 and K12 have been identified in Escherichia coli. The aims of this study were to determine, primarily by means of PCR, the distribution of those oligosaccharide core types among avian pathogenic E. coli and their relationship to phylogenetic groups. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes encoding temperature sensitive haemagglutinin (tsh), increased serum survival (iss) and colicin V (cvaC) were determined. Of the 143 clinical isolates examined 62% possessed the R1 core, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were of K12 core type. The distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P=0.51). Three genes, tsh, iss and cvaC were found in E. coli of all four core types. The genes tsh (P<0.001) and iss (P=0.03412) were significantly associated with the R4 core oligosaccharide type. The isolates containing R4 core type LPS were mainly confined to phylogenetic group D. The widespread R1 core type showed less ability to possess virulence genes and 83% were in the phylogenetic group A. Results of this study indicated that E. coli with R1, R2, R3 and R4 were important in causing infections in chickens and further, the E. coli with R4 core type were less common among commensals, possessed more virulence genes and were related to phylogenetic groups pathogenic for poultry.     

Evaluation of the adhesive capacity of Escherichia coli isolates associated with avian cellulitis

It has been shown that Escherichia coli isolates from lesions of cellulitis belong to a limited number of clonal groups distinct from those of isolates found in the environment of these birds. In this study, different in vitro methods were used to evaluate adherence properties of E. coli isolates from cellulitis lesions and environments of high- and low-cellulitis prevalence broiler flocks. One hundred isolates were tested by hemagglutination. Adherence to frozen sections of chicken skin and binding to soluble fibronectin were examined for 40 of these 100 isolates by immunofluorescence and by immunocytofluorometry, respectively. Localization of bacterial adherence to skin tissues was confirmed by immunohistochemistry. It was demonstrated that O78:K80 isolates from cellulitis lesions adhered to skin sections to a much greater extent in deeper than in superficial tissue layers. A greater bacterial adherence following growth in TSB at 37 C was demonstrated for isolates from flocks with high prevalence of cellulitis than for isolates from flocks with low prevalence of cellulitis. MANOVA analysis results showed a significant difference between superficial and deep tissue layers only for one set of isolates from flocks with high prevalence of cellulitis. Hemagglutinating activity was variable among the O78:K80 isolates obtained from flocks with high prevalence of cellulitis. The results obtained for some O78:K80 isolates following growth in TSB suggest a role for type 1 fimbriae or F1 in adherence to skin sections. This was reinforced by the finding that adherence was inhibited by D-mannose. Poultry E. coli isolates that express F1 had no affinity for soluble fibronectin, although localization of the adherence in skin sections suggested a role for extracellular matrix components such as collagen and insoluble fibronectin.     

Mortality in swine herds endemically infected with Haemophilus pleuropneumoniae: effect of immunization with cross-reacting lipopolysaccharide core antigens of Escherichia coli

The benefit of increased immunity to cross-reacting lipopolysaccharide core antigens of gram-negative bacteria induced by vaccination with an Rc mutant of Escherichia coli 0111:B4 (strain J5) was evaluated in commercial swine herds endemically infected with Haemophilus pleuropneumoniae. Weanling pigs were vaccinated IM with E coli J5 (group 1) before the expected time of H pleuropneumoniae infection. Clinical signs, antibiotic treatment frequency, mortality, growth performance (days to market weight), and serologic responses of the pigs were monitored for approximately 5 months after vaccination. The results were compared with those of pigs vaccinated IM with a commercial H pleuropneumoniae bacterin (group 2) and with those of nonvaccinated control pigs of the same age (group 3). The treatment frequency and growth performance were similar in the 3 groups. However, vaccination with E coli J5 or with the H pleuropneumoniae bacterin lowered mortality, compared with mortality in the controls. Serum titers against E coli J5 increased after vaccination with the E coli J5 bacterin, but were not increased by vaccination with the H pleuropneumoniae. In contrast, serum titer to E coli J5 increased in all treatment groups as a result of H pleuropneumoniae infection or exposure. The protection against lethal H pleuropneumoniae infections in swine that was provided by vaccination with the E coli J5 and the H pleuropneumoniae bacterin appeared to be immunologically distinct on the basis of serologic analysis, indicating the possibility of different mechanisms of protection.     

Biotyping of clinical isolates of Escherichia coli of animal origin, using the Analytab API 20E system.

Using the Analytab (API 20E) Enterobacteriaceae system of biochemical identification, a total of 506 Escherichia coli isolates from different animal species were coded numerically or biotyped. Fifty-four different biotypes were identified, 11 accounting for 83.1% of the isolates examined. Three of these profiles accounted for 65.3% of the isolates and were found in almost all animal species. Some of the biotypes were found in only one animal species: six in cattle, five in horses, 15 in pigs, two in sheep, two in birds, one in dogs and one in a porpoise. Biotypes, as determined here, could not be related to a particular pathology and more work is needed to assess the extent and significance of this relative biotype specificity among animal species. The use of other, more sophisticated, typing systems, i.e. plasmid "fingerprinting", or restriction endonuclease analysis of chromosomal DNA, would have to be investigated.     

Effect of ammonia on the quantitative clearance of Escherichia coli from lungs, air sacs, and livers of turkeys aerosol vaccinated against Escherichia coli

Turkeys were given an aerosol vaccine to determine their ability to clear a virulent inhaled pathogenic strain of Escherichia coli, while they were being maintained in the presence of atmospheric NH3. Turkeys were exposed to 2 concentrations of NH3 (10 and 40 microliters/L of air). More E coli was found in lungs, air sacs, and livers of turkeys exposed to NH3. Turkeys not exposed to NH3 had better clearance of E coli. Vaccination against E coli improved the rate of clearance of E coli in birds not exposed to NH3.     

High technology diagnostics: detection of enterotoxigenic Escherichia coli, using DNA probes

A relatively new technique termed colony blot hybridization appears to be a reliable replacement for the ligated porcine gut loop assay previously used to detect stable toxin-B producing Escherichia coli. The highly reproducible blot assay enables screening of large numbers of isolants for the presence of E coli stable-B toxin genes, thus avoiding variations in phenotypic expression and poor repeatability inherent in the in vivo assays. Availability of this diagnostic test can aid practitioners in identifying enterotoxigenic E coli related disease in swine and in determining the benefits of vaccination programs.     

大肠埃希菌O111∶B4内毒素对大鼠肝脏表达HSP70的影响

应用免疫组织化学技术研究了HSP70在内毒素损伤大鼠肝脏中表达的影响。将48只SD大鼠随机分为对照组(Ⅰ组)和内毒素(ET)组(Ⅱ组),每组各24只。两组大鼠分别经尾静脉注射等量无热源生理盐水和内毒素5 mg/kg体重后,分别在3、4、8、12 h采集肝脏作为检测样本,应用HE染色和免疫组织化学染色技术进行检测。结果表明,肝脏的病理变化在内毒素攻击3 h后明显增多,4 h达到峰值,而在8 h后逐渐下降;在对照组和ET组中均有HSP70表达,但ET组的表达极显著明显高于对照组(P0.01),且ET组HSP70表达量随时间的延长而下降。说明在体内内毒素可诱导HSP70在肝脏中的表达。     

大肠杆菌O111:B4内毒素对大鼠肝脏Fas蛋白表达的影响

为探讨大肠杆菌O111:B4内毒素(ET)对大鼠肝脏Fas蛋白表达的影响,本研究将48只SD大鼠随机分为试验组和对照组,试验组尾静脉注射ET,对照组尾静脉注射等体积无热源生理盐水;两组大鼠分别于注射后3h、4h、8h、12 h各迫杀6只,采集肝脏分别用于Fas蛋白表达的western blot检测和流式细胞术(FCM)检测.结果显示,试验组大鼠Fas蛋白的表达明显高于对照组(p＜0.01),并且呈上升趋势.该结果表明,ET能够有效上调大鼠肝细胞Fas蛋白的表达.     

Mutant prevention concentration of orbifloxacin: comparison between Escherichia coli,Pseudomonas aeruginosa,and Staphylococcus pseudintermedius of canine origin

Background

The mutant prevention concentration (MPC) is an important parameter to evaluate the likelihood of growth of fluoroquinolone-resistant mutants for antimicrobial-pathogen combinations. The MPCs of fluoroquinolones for different canine pathogens have not been compared. In this study, we compared for the first time orbifloxacin MPCs between susceptible strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus pseudintermedius of canine origin.

Methods

More than 10¹⁰ CFU/ml of 10 strains of each bacterial species were inoculated onto Muller-Hinton agar supplemented with different concentrations of orbifloxacin from 1× to 64× minimum inhibitory concentration (MIC) and the MPCs were recorded. MICs of original strains and of mutants arising after exposure to sub-MPC concentrations (one per original strain) were determined in the presence or absence of efflux pump inhibitors (EPIs). The effects of quinolone resistance-determining region (QRDR) mutations were also examined.

Results

MPCs were significantly higher for P. aeruginosa (16–128 μg/ml) than for E. coli (0.5–32 μg/ml). MPCs for S. pseudintermedius varied between the low-susceptible (16–128 μg/ml) and the high-susceptible strains (4–16 μg/ml) and were the most broadly distributed among the three species. Regarding resistance mechanisms, only one QRDR mutation in gyrA was found in all of the 10 mutants of E. coli and in 4 of the 10 mutants of P. aeruginosa, whereas mutations in both grlA and gyrA were found in 3 mutants and one mutation in grlA was found in 2 mutants among the 10 mutants of S. pseudintermedius. In the presence of an EPI, the MICs of P. aeruginosa mutants decreased markedly, those of E. coli mutants decreased moderately, and those of S. pseudintermedius mutants were unaffected.

Conclusions

MPCs of orbifloxacin vary between bacterial species of canine pathogens, possibly due to the diversity of the main fluoroquinolone resistance mechanism among these species. Therefore, the type of bacterial species should be taken into consideration when using fluoroquinolone drugs such as orbifloxacin in canines.     

Endotoxin-induced changes in the hemostatic system in neonatal calves: the effect of antiserum to a mutant Escherichia coli (J-5)

Changes in the hemostatic system were studied in 22 neonatal calves given a small dosage of Escherichia coli endotoxin (0.5 microgram/kg) by slow (5-hour) IV infusion. The effect of pretreatment with an antiserum to mutant of E coli O111:B4 (J-5) was evaluated. The platelet count, plasma fibrinogen concentration, prothrombin time, and activated partial thromboplastin time changed significantly from base line during and after endotoxin infusions in all calves. The mean platelet count was significantly decreased from 1 through 24 hours after endotoxin infusion was started. Mean plasma fibrinogen was decreased 2 through 12 hours after endotoxin infusion was started. The mean prothrombin time and activated partial thromboplastin time were significantly greater than base line at 3 to 6 hours and 3 to 12 hours, respectively, after endotoxin infusion was started. Serum concentration of fibrinolytic degradation products remained less than 10 micrograms/ml. Bovine J-5 antiserum did not prevent the endotoxin-induced changes in the hemostatic system of these neonatal calves.     

Characterization of the origin and body of the normal equine rear suspensory ligament using ultrasonography, magnetic resonance imaging, and histology

The suspensory ligament is difficult to image accurately, partly because it contains ligamentous fibers, as well as noncollagenous adipose and muscle tissue in the normal horse. Our hypothesis was that magnetic resonance (MR) imaging would be more accurate than ultrasonography in identifying the size of the suspensory ligament and the presence and size of noncollagenous tissues within the ligament. Eleven horses were used for ultrasonographic and MR imaging and histologic evaluation of the rear suspensory ligament. The origin and body of the normal suspensory ligament had a heterogenous appearance on MR images with two separate islands of mixed signal intensity evident throughout its otherwise hypointense cross-sectional area. Histologically, there were isolated islands of muscle, adipose, loose connective tissue and dense collagenous partitions, organized in two separate bundles that extended through the full length of the suspensory ligament origin and body to the level of its bifurcation. Comparison of MR images with corresponding histologic sections confirmed that islands of heterogenous signal intensity in normal suspensory ligaments correlated well with these bundles. Using ultrasonography, it was impossible to distinguish these islands from surrounding dense collagenous tissue consistently. MR imaging determined the cross-sectional area of the suspensory ligament more accurately than ultrasonography. Based upon these results, MR imaging is superior to ultrasonography for assessment of the suspensory ligament. The appearance associated with normal ligament anatomy needs to be understood before MR signal variation can be considered as indicative of disease in the suspensory ligament.     

Evaluation of a glutamine-containing oral rehydration solution for the treatment of calf diarrhoea using an Escherichia coli model

A high-calorie oral rehydration solution (ORS) with glutamine (n=11) was more effective in correcting plasma, extracellular fluid and blood volume than solutions without (one WHO-type solution, n=6, and two high-glucose but glutamine-free solutions, n=7, n=12). It was the only solution to improve plasma volume significantly within 48 h and sustain the improvement throughout treatment; similarly, it was the only solution to correct packed-cell volume within 48 h and sustain the benefit to the end of treatment. At the end of treatment, the glutamine-treated calves were the only ones to avoid a significant weight loss compared with their pre-diarrhoeic values. The crucial difference between this solution and those used with glutamine previously is that it gave significant nutritional support whereas WHO type solutions did not. It also had more favourable effects on hyponatraemia and metabolic acidosis than a standard ORS. Use of a high-calorie ORS for 4 days (rather than 2 days of 50:50 admixture with milk replacer) brought additional beneficial effects on blood glucose and body weight.     

Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes

Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test. K88ac always gave negative HA results with porcine red cells. However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera. K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.     

肠出血性大肠杆菌O157:H7紧密素单克隆抗体的制备及鉴定

以基因重组技术构建工程菌株表达肠出血性大肠杆菌O157:H7紧密素的融合蛋白。用纯化的紧密素蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,对杂交瘤细胞及时筛选,阳性孔经4次有限稀释法克隆,成功获得2株能稳定传代并分泌抗紧密素单克隆抗体(McAb)的杂交瘤细胞株。2株单抗分别制备腹水,ELISA检测效价分别为5.2×104,2.5×104。Western blot检测表明,2株单抗与融合蛋白发生特异性反应;ELISA检测表明,2株单抗可特异性检出大肠杆菌O157:H7。本研究为建立大肠杆菌O157检测方法提供了物质基础。     

Effects of fish oil supplementation on the performance and the immunological, adrenal, and somatotropic responses of weaned pigs after an Escherichia coli lipopolysaccharide challenge

Seventy-two crossbred pigs (7.58 +/- 0.30 kg BW) weaned at 28 +/- 3 d of age were used to investigate the effects of fish oil supplementation on pig performance and on immunological, adrenal, and somatotropic responses following an Escherichia coli lipopolysaccharide (LPS) challenge in a 2 x 2 factorial design. The main factors consisted of diet (7% corn oil [CO] or 7% fish oil [FO]) and immunological challenge (LPS or saline). On d 14 and 21, pigs were injected intraperitoneally with either 200 microg/kg BW of LPS or an equivalent amount of sterile saline. Blood samples were collected 3 h after injection for analysis of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), cortisol, growth hormone (GH), and insulin-like growth factor (IGF)-I. On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was determined. Lipopolysaccharide challenge decreased ADG (487 vs. 586 g; P < 0.05) and ADFI (as-fed, 776 vs. 920 g; P < 0.05) from d 14 to 21 and ADG (587 vs. 652 g; P < 0.10) from d 21 to 28. Fish oil improved ADG (554 vs. 520 g; P < 0.10) and ADFI (891 vs. 805 g; P < 0.10) from d 14 to 21. On d 14, LPS challenge x diet interactions were observed for IL-1beta (P < 0.10), PGE2 (P < 0.001), and cortisol (P < 0.05) such that these measurements responded to the LPS challenge to a lesser extent (IL-1beta: 93 vs. 114 pg/mL, P < 0.05; PGE2: 536 vs. 1,285 pg/mL, P < 0.001; cortisol: 143 vs. 206 ng/mL, P < 0.05) in pigs receiving the FO diet than in pigs fed the CO diet. In contrast, among LPS-treated pigs, pigs fed the FO diet had higher IGF-I (155 vs. 101 ng/mL; P < 0.10) than those fed the CO diet. On d 21 among LPS-treated pigs, pigs fed FO had lower IL-1beta (70 vs. 84 pg/mL; P < 0.10) and cortisol (153 vs. 205 ng/mL; P < 0.05) than those fed CO. Pigs fed FO had lower PGE2 (331 vs. 444 pg/mL; P < 0.05) and higher IGF-I (202 vs. 171 ng/mL; P < 0.10) compared with those fed CO. Lipopolysaccharide challenge decreased GH (0.27 vs. 0.33 ng/mL; P < 0.05) on d 14, whereas it had no effect on GH on d 21. During both LPS challenge periods, the challenge increased PBLP when these cells were incubated with 8 (1.46 vs. 1.32; P < 0.10) or 16 microg/mL (1.46 vs. 1.30; P < 0.05) of concanavalin A. Fish oil had no effect on PBLP. These results suggest that FO alters the release of proinflammatory cytokines, which might lead to improved pig performance during an immunological challenge.     

P10B抗菌肽和硫酸小檗碱对肉鸡源大肠杆菌的体外联合抗菌作用研究

为了解P10B抗菌肽与硫酸小檗碱联合抗菌作用,进行P10B抗菌肽与硫酸小檗碱联用的体外抑菌试验。试验菌株为20株肉鸡源大肠杆菌菌株。采用微量肉汤稀释法测定P10B与硫酸小檗碱对大肠杆菌的最低抑菌浓度;棋盘稀释法测定两个药的分级抑菌浓度指数,判断联合抗菌效果。结果表明,在联合药敏试验中,两药呈协同作用的占10%,呈相加作用的占85%,呈无关作用的占5%。平均分级抑菌浓度指数为0.825,两种药联和用药效应主要表现为累加作用。