Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.     

羊布鲁氏菌甲酰基转移酶的生物信息学研究

本研究旨在分析和预测羊布鲁氏菌甲酰基转移酶基因及其编码蛋白的结构与功能,用于指导其生物学功能的实验室研究。综合利用生物信息学软件(DNAMAN和Vector NTI)、数据库(prosite和PDB)和网络服务器对甲酰基转移酶进行基本性质分析、三维结构预测和功能预测,展示了甲酰基转移酶的氨基酸分布情况,二级结构中形成螺旋、折叠、卷曲的氨基酸区段及结构域;预测并综合阐释了B细胞表位的柔韧性、表面可及性和亲水性参数。生物信息学预测和分析结果可为研究在布鲁氏菌致病及机体免疫应答中甲酰基转移酶的结构与功能的关系提供丰富资料。     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Protection against Brucellosis in Goats,Five Years after Vaccination with Reduced-Dose Brucella melitensis Rev 1 Vaccine

The protection conferred by the reduced-dose Rev 1 Brucella melitensis vaccine in goats that had been immunized 5 years previously was evaluated. Sixteen goats vaccinated 5 years before with Rev 1 (1 x 10(5) cfu) and 5 non-vaccinated goats were challenged with B. melitensis 16M (4 x 10(5) cfu) using the conjunctival route. After giving birth or aborting, the goats were sacrificed and tissue samples were taken for bacteriological study. The challenge strain was recovered in 12%, of the animals from the vaccinated group, and in (80% of the control group. It is concluded, therefore that the use of reduced-dose Rev 1 protects goats vaccinated in endemic areas for at least 5 years after immunization.     

Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M

Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.     

羊种布鲁氏菌wzt基因的原核表达及间接ELISA方法的建立

为建立检测血清中布鲁氏菌抗体的间接ELISA方法,本试验采用PCR技术从羊种布鲁氏菌QY1菌株中扩增得到wzt基因片段,连接到pET-30a载体上,构建质粒pET-30a-wzt,将鉴定正确的质粒转化E.coli BL21（DE3）感受态细胞,经原核表达系统对其进行表达,表达产物用SDS-PAGE和Western blotting进行分析后,用亲和层析镍柱纯化wzt重组蛋白备用。以wzt重组蛋白为检测抗原,逐步优化条件后建立布鲁氏菌间接ELISA检测方法。结果显示,试验成功构建了pET-30a-wzt原核表达载体,并在BL21（DE3）宿主菌中表达;SDS-PAGE和Western blotting结果表明,重组蛋白约为35 ku,表达形式为上清,条带单一、无杂带,有很好的反应原性和特异性。ELISA优化试验确定了最佳包被浓度为15 μg/mL,血清最佳稀释度为1：80,酶标抗体的最佳稀释度为1：5 000;通过检测24份阴性样品确定临界值,当样品D_{450 nm}值≥ 0.30为阳性,样品D_{450 nm}值<0.30时为阴性;特异性试验表明,该方法不与小肠耶尔森菌、大肠杆菌发生交叉反应;批内及批间变异系数均<10%;用该方法对120份血清样本进行检测,并与虎红凝集试验进行相符性验证,符合率为96%。本试验建立的间接ELISA方法为布鲁氏菌病的检测提供了可靠的技术手段。     

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n = 118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA).The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.     

Comparative Evaluation of a Field-based Dot-ELISA Kit with Three Other Serological Tests for the Detection of Brucella Antibodies in Goats

A dot-ELISA (d-ELISA) test was evaluated and compared with the serum agglutination test (SAT), micro-complement fixation test (CFT) and a plate-ELISA (p-ELISA) for field use in screening herds of goats against brucellosis. During the standardization of the dot-ELISA kit on 1732 caprine serum samples, 1571 samples out of 1666 were found to be negative in d-ELISA, SAT and micro-CFT, while 59 were positive in different combinations. Of a further 66 serum samples, 34 were negative and 31 were positive in different combinations in d-ELISA, SAT, micro-CFT and p-ELISA. A total of 1584 goats belonging to different herds were then screened for brucellosis. Of the 694 serum samples screened in the first batch using d-ELISA, a positive reaction was observed in 26 cases. Further screening of these cases revealed 13 and 21 goats as positive reactors in SAT and CFT, respectively. In a second batch of 890 goats there were 109 positive reactors in d-ELISA. Among these 109 goats, 34, 40 and 80 goats were positive reactors in SAT, CFT and p-ELISA, respectively. The results of d-ELISA correlated well with those of p-ELISA. Dot-ELISA was found to be a more suitable and rapid test for screening large numbers of goats in the field.     

Seroprevalence of Toxoplasma gondii infection in Norwegian dairy goats

Background

Toxoplasma gondii is a major problem for the sheep industry as it may cause reproduction problems. The importance of T. gondii in Norwegian goat herds is uncertain, but outbreaks of toxoplasmosis in dairy goat farms have been recorded. The aim of this study was to describe the prevalence of T. gondii infection in Norwegian dairy goats by using serology.

Findings

Goat serum originally collected as part of two nationwide surveillance and control programmes between 2002 and 2008 were examined for T. gondii antibodies by using direct agglutination test. In total, 55 of 73 herds (75%) had one or more serologically positive animals, while 377 of 2188 (17%) of the individual samples tested positive for T. gondii antibodies.

Conclusions

This is the first prevalence study of T. gondii infection in Norwegian goats. The results show that Norwegian goat herds are commonly exposed to T. gondii. Nevertheless, the majority of goat herds have a low prevalence of antibody positive animals, which make them vulnerable to infections with T. gondii during the gestation period.     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other α-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P<0.05 or P<0.01) from those of vaccinated ewes that neither contracted brucellosis nor excreted the pathogen. In general, all the tests detect the infection in the non-vaccinated ewes with substantial effectiveness. It can be concluded that the high levels of circulating antibodies and of antigen-specific gamma-IFN are related to active Brucella infection. Similarly, the state of protection against the disease, but not necessarily against infection, due to a previous immunization with the Rev. 1 vaccination, appears to be responsible for a low level of detectable immune response. Nevertheless, the design of the study limits conclusions to pregnant ewes and cannot be extrapolated to non-pregnant ewes or rams. Likewise, the study provides no information on animals which are carriers of B. melitensis.     

Comparison of milk-ELISA and serum-ELISA for the diagnosis of Brucella melitensis infection in sheep

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.     

Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches

The diagnostic accuracies of the modified agglutination test (MAT) and indirect ELISA test for the detection of serum antibodies against Toxoplasma gondii in sheep were evaluated through Bayesian approaches on two populations of sheep created from three different groups of animals (T. gondii-aborted ewes, colostrums-deprived newborn lambs, and ewe-lambs and adult ewes with unknown T. gondii infection status). Tests showed a high degree of agreement (kappa statistic = 0.93; 95% confidence interval = 0.87, 0.98) and a significant specificity (Sp) correlation (gamma(Sp) = 0.26; 95% credibility interval = 0.017, 0.61). When prior information was used for all unknown parameters the posterior medians for the sensitivity (Se) and Sp of the MAT and ELISA were, respectively, 92.6% (95% credibility interval = 85.2, 96.9), 95.5% (89.9, 98.7), 90.5% (83.4, 95.6), and 97.8% (94.2, 99.5). These estimates remained similar when uninformative priors were included. The Se estimates of the MAT and ELISA were higher than those obtained on pigs in other study using the same approach (Se = 80.6% and Sp = 89.5% for the MAT, and Se = 71.5% and Sp = 85.5% for the ELISA [Georgiadis, M.P., Wesley, O.J., Gardner, I.A., Singh, R., 2003. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl. Stat. 52, 63-78]. This finding supported the believe that test performances may vary when applied on different animal species. Thus, if these tests are planned to be used on animal species other than sheep or pigs, their diagnostic accuracy should be re-assessed to prevent biased inferences from their results.     

Persistent immune responses in late infection and after treatment in experimental Schistosoma bovis infections in goats

This study explored host immune responses and their possible relationship to the anti-fecundity phenomenon in Schistosoma bovis-infected goats. The design comprised a primary infection with or without treatment at week (wk) 13, and with or without challenge at wk 36. Necropsy was performed at 36 or 52 wk. Serum levels of anti-egg IgG, and anti-worm IgG and IgM, were measured by ELISA. In chronic infection, anti-worm antibodies stayed high, reflecting persisting worm burdens, whereas anti-egg IgG remained high despite minimized egg excretion. After treatment, anti-worm IgM and anti-egg IgG were minimized, but anti-worm IgG remained above the values of the uninfected controls. Histopathology showed lowered numbers of perioval granulomas in chronic infection and resolution of liver fibrosis with time, but intestinal lymphoplasmacytic perivasculitis and hepatic eosinophilic infiltrates were maintained at wk 52. Significant splenic plasmacytosis persisted after treatment. The results indicated that persistent immune responses, in chronically infected and in treated goats, may explain sustained worm fecundity depression at challenge infection.     

Immunogenicity of Major Cell Surface Protein(s) of Brucella melitensis Rev 1

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.     

Toxicity in goats caused by oleander (Nerium oleander)

Cases of poisoning by oleander (Nerium oleander) were observed in several species, except in goats. This study aimed to evaluate the pathological effects of oleander in goats. The experimental design used three goats per group: the control group, which did not receive oleander and the experimental group, which received leaves of oleander (50 mg/kg/day) for six consecutive days. On the seventh day, goats received 110 mg/kg of oleander leaves four times at one-hourly interval. A last dose of 330 mg/kg of oleander leaves was given subsequently. After the last dose was administered, clinical signs such as apathy, colic, vocalizations, hyperpnea, polyuria, and moderate rumen distention were observed. Electrocardiogram revealed second-degree atrioventricular block. Death occurred on an average at 92 min after the last dosing. Microscopic evaluation revealed renal necrosis at convoluted and collector tubules and slight myocardial degeneration was observed by unequal staining of cardiomyocytes. Data suggest that goats appear to respond to oleander poisoning in a manner similar to other species.     

Effects of gestation and lactation on vitamin D receptor amounts in goats and sheep

During gestation and lactation, an increased demand for calcium (Ca) due to the development of fetal skeleton and excretion via milk is observed. The higher need for Ca is met by an augmented mobilisation of Ca from bones and by an increased absorption from the intestines. The main influence on this physiological process of active absorption has Vitamin D, acting through Vitamin D receptors (VDR) located in the mucosal wall of the intestines, thus increasing Ca absorption. As a consequence of inadequate Ca absorption, metabolic diseases like milk fever can develop. In this study immunohistochemical procedures were applied to colon mucosa biopsies of pregnant and lactating goats and sheep, to study the effect of late gestation, parturition and lactation on VDR amount. Colon mucosa biopsies were collected 2 weeks before parturition, 1 and 4 weeks post partum (pp), 2, 3, 4, and 5 months pp from 11 dairy goats and 11 sheep. Immunohistochemistry was performed employing a biotinylated monoclonal rat anti-VDR antibody and streptavidin peroxidase techniques. Nuclei and cytoplasm of enterocytes stained positively for VDRs. Strongest immunoreactions were observed in intermediate and superficial glandular cells. The biopsy samples taken during early lactation revealed a lower immunoreaction for VDR compared with samples taken during later stages of lactation. In conclusion, immunochemistry and biopsy technology are useful tools to assess changes in VDR expression in relation to varying demands for Ca in the process of a reproductive cycle. These results show that in dairy goats and sheep, an influence of gestation and lactation on VDR is obvious.     

Preslaughter diet management in sheep and goats: effects on physiological responses and microbial loads on skin and carcass

Sixteen crossbred buck goats (Kiko x Spanish; BW = 32.8 kg) and wether sheep (Dorset x Suffolk; BW = 39.9 kg) were used to determine the effect of preslaughter diet and feed deprivation time (FDT) on physiological responses and microbial loads on skin and carcasses. Experimental animals were fed either a concentrate (CD) or a hay diet (HD) for 4 d and then deprived of feed for either 12-h or 24-h before slaughter. Blood samples were collected for plasma cortisol and blood metabolite analyses. Longisimus muscle (LM) pH was measured. Skin and carcass swabs were obtained to assess microbial loads. Plasma creatine kinase activity (863.9 and 571.7 ± 95.21 IU) and non-esterified fatty acid concentrations (1,056.1 and 589.8 ± 105.01 mEq/L) were different (P < 0.05) between sheep and goats. Species and diet treatments had significant effects on the ultimate pH of LM. Pre-holding total coliform (TCC) and aerobic plate counts (APC) of skin were significantly different between species. Goats had lower (P < 0.05) TCC (2.1 vs. 3.0 log₁₀ CFU/cm²) and APC (8.2 vs. 8.5 log₁₀ CFU/cm²) counts in the skin compared to sheep. Preslaughter skin E. coli counts and TCC were different (P < 0.05) between species. Goats had lower (P < 0.05) counts of E. coli (2.2 vs. 2.9 log₁₀ CFU/cm²) and TCC (2.3 vs. 3.0 log₁₀ CFU/cm²) in the skin compared with those in sheep. Diet, species, and FDT had no effect (P > 0.05) on E. coli and TCC in carcass swab samples. The APC of carcass swab samples were only affected (P < 0.05) by the FDT. The results indicated that preslaughter dietary management had no significant changes on hormone and blood metabolite concentrations and sheep might be more prone for fecal contamination than goats in the holding pens at abattoir.     

Evaluation of recombinant fructose-1,6-bisphosphate aldolase ELISA test for the diagnosis of Schistosoma japonicum in water buffaloes

Fructose-1,6-bisphosphate aldolase (FBPA) is an ubiquitous enzyme essential for glycolysis, gluconeogenesis and the Calvin cycle. It has been demonstrated to induce immune responses and to be useful in the immunodiagnosis of malaria. In this study, FBPA was cloned from the adult worms of Schistosoma japonicum and tested as an antigen for the diagnosis of S. japonicum infection in water buffaloes. Enzyme-linked immunosorbent assay (ELISA) was performed on the sera from 32 infected water buffaloes and 20 negative controls using the recombinant FBPA protein or soluble worm antigen preparation (SWAP) as an antigen. The OD cut-off values were determined to be 0.57 with 100% specificity and 100% sensitivity for the FBPA ELISA and 1.13 with 93.8% specificity and 95.0% sensitivity for the SWAP ELISA. These findings indicate that the recombinant FBPA of S. japonicum should be an useful diagnostic tool for the detection of antibodies against S. japonicum.