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1.
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2.
The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge.  相似文献   

3.
日粮添加多不饱和脂肪酸对肉仔鸡抗氧化指标的影响   总被引：5，自引：0，他引：5  
本试验通过观察日粮中添加多不饱和脂肪酸(PUFA)与肉仔鸡抗氧化酶活性及过氧化产物的量的关系,研究PUFA对肉仔鸡抗氧化能力的影响.试验包括3个单因子试验,3个处理组日粮为玉米豆粕型日粮,分别添加4.5%的禽脂、玉米油和鱼油,每个处理组6个重复,每个重复10只鸡.21日龄和42日龄分别检测脾脏、肠道抗氧化酶(谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT))活性和脂质过氧化产物(丙二醛,MDA)水平.综合本试验检测的抗氧化指标(GSH-Px、SOD、CAT和MDA)可知,日粮中添加鱼油和玉米油的肉仔鸡抗氧化酶活性较对照组均有不同程度的提高,而机体脂质过氧化产物(MDA)与对照组比较无显著性差异.这表明虽然添加PUFA易使机体发生过氧化反应,但由于机体抗氧化酶系(GSH-Px、SOD、CAT)功能的增强,可以抵消PUFA过氧化的不良作用.但是肉仔鸡抗氧化酶系功能的增强可能是PUFA具有直接调控机体抗氧化酶的作用,也可能是机体过氧化产物的反馈作用导致其功能代偿性增强.添加PUFA,脾脏CAT和SOD增强主要发生在21日龄;肠道CAT和SOD活性增强则主要发生在42日龄.由此可知,日粮中添加PUFA对肉仔鸡抗氧化酶活性的影响因生长阶段不同而存在组织器官特异性.  相似文献   

4.
The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).  相似文献   

5.
Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.  相似文献   

6.
BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.  相似文献   

7.
Brucellosis is not a sustainable disease in humans. The source of human infection always resides in domestic or wild animal reservoirs. The routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. New Brucella strains or species may emerge and existing Brucella species adapt to changing social, cultural, travel and agricultural environment. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. This correlates with the fact that worldwide, the control of bovine brucellosis (due to B. abortus) has been achieved to a greater extent than the control of sheep and goat brucellosis (due to B. melitensis), these latter species being the most important domestic animals in many developing countries. The long duration and high cost of treatment of human brucellosis reduces the efficacy of the therapy. There is no human vaccine for brucellosis and the occurrence of brucellosis is directly linked to the status of animal brucellosis in a region. In this context, the Word Health Organization has defined the development of a human vaccine, besides the implementation of control and eradication programs in animals, as a high priority. The pathogenicity for humans of B. suis biovars 1, 3 and 4 is well established, whereas B. suis biovar 2 seems to be less pathogenic. Indeed, although hunters and pig farmers have repeatably experienced infectious contact with B. suis biovar 2 (found in wild boar and outdoor-rearing pigs in Europe), isolation of B. suis biovar 2 from human samples have only been seldom reported. Marine mammal brucellosis, due to two new proposed Brucella species i.e. B. cetaceae and B. pinnipediae, represents a new zoonotic threat but the pathogenicity for humans of the different Brucella species found in cetaceans and pinnipeds still has to be clearly established.  相似文献   

8.
Brucella melitensis Rev 1 organisms were salt-extracted and the cell surface proteins (BCSPs) were found to be mainly 39-42 kDa (group 2 porin proteins) in addition to 31.6, 32.5, 58.5 and 14.7 kDa proteins. DEAE-Sephadex anion-exchange column chromatography of BCSPs yielded fraction 1, which contained one major protein (39.8-42.0 kDa) and a minor protein (31.6 kDa). All these proteins were found to be immunogenic by Western blotting. Fraction 1 along with monophosphoryl lipid A and trehalose dicorynomycolate adjuvants as well as BCSPs alone induced significant (p < or = 0.05) protection in BALB/c mice. Both these immunizing agents produced almost equivalent protection to live B. melitensis Rev 1 vaccine at 15 and 30 days post challenge. Lymphocyte stimulation test as well as delayed-type hypersensitivity reaction revealed that both these preparations induced cell-mediated immune response. These preparations also induced humoral immune response as indicated by indirect ELISA. Neither of the immune responses was significantly less (p < or = 0.05) than that with live B. melitensis Rev 1 vaccine, except that their duration was short.  相似文献   

9.
为了研究山羊痒螨病对机体氧自由基及抗氧化系统的影响,分别对感染山羊和健康山羊GSH-Px、T-SOD、CAT的活力和MDA、LPO的含量进行了测定。试验结果显示,感染组山羊血清中GSH-Px活力极显著低于对照组(P<0.01),T-SOD活力极显著低于对照组(P<0.01),MDA含量显著高于对照组(P<0.05),LPO含量极显著高于对照组(P<0.01);感染组与对照组CAT活力差异不显著。试验结果表明,山羊患痒螨病后,体内氧自由基含量升高,抗氧化酶活力降低,脂质过氧化物含量上升。  相似文献   

10.
Liposomes with entrapped gentamicin were used to treat mice with infection attributable to Brucella melitensis. Liposomes bearing positive charge and formed by egg yolk lecithin, cholesterol, and stearylamine were effective in the elimination of B melitensis residing in liver and spleen. Negatively charged liposomes, formed by egg yolk lecithin, cholesterol, and dicetyl phosphate were also effective in suppression of the infection in liver, but were less so in suppression of the infection in the spleen. Free gentamicin was less effective than the encapsulated antibiotic. At 20 hours after administration of gentamicin encapsulated in liposomes, the gentamicin concentrations in liver and spleen were similar, regardless of the charge of the liposomes--neutral, positive, or negative. However, positively charged liposomes were more efficient than were other liposome types for the treatment of brucellosis caused by B melitensis.  相似文献   

11.
The authors studied the persistence of infection in 46 ewes experimentally infected with Brucella melitensis biovar 3 and monitored through three subsequent reproductive cycles. The entire experimental period lasted for 151 weeks. Infection of ewes and elimination of Brucella in milk, or its presence in vaginal discharges, persisted throughout the duration of the trial, as demonstrated by recurrent elimination of Brucella in milk and vaginal discharges. Brucella melitensis was recovered from the tissues of one ewe killed at the end of the trial. The strain was recovered from vaginal swabs and milk following parturition in the third reproductive cycle from an ewe that had aborted in the first cycle but was not pregnant in the second cycle. From a public health point of view, the periodical recovery of Brucella from the milk during the entire trial period illustrated that brucellosis in sheep remains a continuous occupational risk and a significant public health problem for consumers of fresh milk and milk products. That risk may persist for at least 3 years following the initial infection of the flock. Lamb antibody titres became negative in all lambs within 5 months after birth. This suggested that serological tests on lambs may have no practical diagnostic significance if performed during the first 5 months of life. Nevertheless, the birth of three infected lambs suggested that the phenomenon of latent carrier state may represent another way for B. melitensis to persist in a flock.  相似文献   

12.
An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.  相似文献   

13.
This study was designed to investigate the effects Nigella sativa L. (NS) and Urtica dioica L. (UD) on lipid peroxidation, antioxidant enzyme systems and some liver enzymes in carbon tetrachloride (CCl4)-treated rats. A total of 60 healthy male Sprague-Dawley rats were utilized in this study. The rats were randomly allotted into one of four experimental groups: A (CCl4-only treated), B (CCl4 + UD treated), C (CCl4 + NS treated) and D (CCl4 + UD + NS treated), each containing 15 animals. All groups received CCl4 [0.8 ml/kg of body weight, subcutaneously, twice a week for 90 days starting day 1]. In addition, B, C and D groups also received daily intraperitoneal injections of 0.2 ml/kg NS or/and 2 ml/kg UD oils for 45 days starting day 46. Group A, on the other hand, received only 2 ml/kg normal saline solution for 45 days starting day 46. Blood samples for the biochemical analysis were taken by cardiac puncture from five randomly chosen rats in each treatment group at beginning, at 45th and at 90th day of the experiment. The CCl4 treatment for 45 days increased the lipid peroxidation and liver enzymes, and also decreased the antioxidant enzyme levels. NS or UD treatments (alone or combination) for 45 days starting day 46 decreased the elevated lipid peroxidation and liver enzyme levels and also increased the reduced antioxidant enzyme levels. Live weights of the rats decreased in group A, and increased in groups B, C and D. It is concluded that NS and UD decrease the lipid peroxidation and liver enzymes, and increase the antioxidant defence system activity in the CCl4-treated rats.  相似文献   

14.
To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.  相似文献   

15.
Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.  相似文献   

16.
An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.  相似文献   

17.
Milk and blood samples from 704 lactating ewes were examined for the diagnosis of Brucella melitensis infection by milk-ELISA, serum-ELISA, RBPT, SAT and culture of milk. Of these ewes, 209 were from brucellosis free sheep flock, 443 from brucellosis infected sheep flock and 52 were from private sheep flocks of which status for brucellosis was not known. All the 209 ewes belonging to uninfected sheep flock were found negative in all the tests and of the remaining 495 ewes 105 were positive in serum-ELISA, 103 in milk-ELISA, 92 in RBPT, 85 in SAT, and B. melitensis biovar-1 was isolated from the milk of 29 ewes. Of the 105 serum-ELISA positive ewes, 99 were positive and 6 were negative in milk-ELISA, whereas of the 103 milk-ELISA positive ewes, 4 were negative in serum-ELISA. All together, 99 ewes were positive and 386 were negative in both the assays while 10 ewes yielded variable results. The specificity of milk-ELISA in brucellosis free flock was 100% and sensitivity and positive predictive value were 96.11% and 94.28%, respectively, in infected flocks. The Brucella antibody levels in milk and serum samples as determined by milk-ELISA and serum-ELISA were correlated significantly. The milk-ELISA for brucellosis appears to be an attractive alternative of serum-ELISA particularly in the lactating ewes.  相似文献   

18.
In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (beta-coefficient of regression analysis).  相似文献   

19.
Brucella melitensis biotype 1 was isolated in pure culture from the lungs, liver, spleen, kidney, stomach contents, abomasum and brain of an aborted caprine (Boer goat) foetus in the district of Cullinan near Pretoria. The 18 does and 1 ram in the flock of Boer goates were examined serologically by means of the complement fixation (CF) test, using Brucella abortus antigen. Six weeks later they were examined again, using B. abortus as well as B. melitensis biotype 1 antigens. No significant differences were found between the 2 CF tests using B. abortus antigen, or between the results obtained by using the B. abortus and B. melitensis antigens. Twelve goats, showing CF antibody titres, were slaughtered and examined bacteriologically. No relationship was found between the serological and bacteriological results.  相似文献   

20.
A brucella phage of the Izatnagar series, designated Iz1, was lytic for all Brucella species that are normally smooth, although the efficiency of plating varied between biovars and species. The phage was also lytic for rough strains of B melitensis and B suis and, to a lesser extent, B ovis. It displayed negligible lytic activity towards B canis and rough B abortus cultures. In its morphological and serological properties and its stability to inactivating agents, the Iz1 phage resembled other brucella phages.  相似文献   

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