The use of Ag85 complex as antigen in ELISA for the diagnosis of bovine tuberculosis in dairy cows in Brazil

Several enzyme-linked immunosorbent assay (ELISA) methods have been investigated to evaluate their performance in the diagnosis of tuberculosis in cattle. Increased production of antibodies to the proteins of antigen 85 complex (Ag85) after experimental and natural infection in cattle shows that they are strongly immunogenic in vivo. The purpose of this study was to evaluate the use of Ag85 as an antigen in ELISA for the diagnosis of bovine tuberculosis in dairy cows in Brazil. The test groups consisted of 46 serum samples from intradermal tuberculin test (ITT)-positive animals (Group A) and 46 samples from ITT-negative animals (Group B). Group C comprised 12 samples from a tuberculosis-free herd and was the control group of the study. Samples were tested in an ELISA using Ag85 as antigen. Differences between the mean ODs of groups A and B and A and C were significant (P < 0.01), but no significant difference (P > 0.05) was observed between groups B and C. The sensitivity of the ELISA using Ag85 was 91.3% (42/46) and its specificity was 94.8% (55/58). These results were not significantly different (P > 0.05) from those observed in a previous study of an ELISA using purified protein derivative (PPD). We concluded that, although Ag85 can be used as antigen for ELISA tests in the diagnosis of bovine tuberculosis with good sensitivity and specificity rates, no significant advantages were observed in relation to the ELISA using PPD that could justify the purification and utilization of Ag85 as a single antigen in routine methods of diagnosis.     

11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24 h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应（delayed type hypersensitivity,DTH）,而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异（P>0.05）。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛（P<0.05）。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。     

A complementary diagnosis of naturally occurring tuberculosis in water buffaloes (Bubalus bubalis) in Rio de Janeiro using a MPB70-ELISA, Brazil

As tuberculosis is still a worldwide infection and buffalo breeding represents an important economic activity in various countries, the purpose of this study was to employ an enzyme-linked immunosorbent assay (ELISA) using MPB70 as a capture antigen for the diagnosis of naturally occurring tuberculosis in water buffaloes in Brazil. After the introduction of newly acquired cattle onto a tuberculosis (TB) free farm, an outbreak of TB was recorded in a mixed herd comprising water buffaloes (21) and cattle (46). The entire herd was tested by intradermal tuberculin injection (ITT) and positive animals were slaughtered and tested by culture, polymerase chain reaction (PCR), and ELISA. From the 21 buffaloes sampled, three were reactive by ITT. All the three had positive culture and ELISA, while PCR was positive in two of them. Besides that, one ITT-negative buffalo was slaughtered and presented positive results by both culture and ELISA, and was considered as anergic. Although there were only few animals, those findings demonstrate the diagnostic usefulness of an MPB70-ELISA to correctly detect Mycobacterium bovis tuberculosis in water buffaloes.     

结核分枝杆菌三种分泌蛋白在原核表达载体中的克隆、表达与鉴定

对结核分枝杆菌的三种分泌蛋白Ag85B,ESAT-6和MPT－64基因进行克隆、鉴定与表达，为结核病诊断、重组疫苗应用和免疫效应检测打下基础。以结核杆菌H37RV株基因组DNA为模板，用PCR法对基因Ag85B、ESAT－6、MPT－64进行扩增，产物与载体质粒pET22b和pGEX-4T-1构建表达Ag85B、ESAT－6、MPT－64的重组质粒，将此重组质粒先转化到大肠杆菌DH5a内抽提质粒，酶切检验，再转化入表达宿主大肠杆菌BL21（DE3）PLysS菌株内，对转化菌株以IPTG进行诱导后，破菌，离心，上清进行SDS－PAGE电泳，电泳发现转化了重组质粒的菌株有表达蛋白，所表达的蛋白质相对分子质量为30000、6000、50000。结果表明：目的基因克隆到菌株内，重组结核杆菌分泌蛋白Ag85B、ESAT－6、MPT－64的成功表达为结核病诊断、重组疫苗应用和免疫效应检测以及上述三种抗原、抗体的大规模制备打下基础。     

Evaluation of five ELISA test kits for the measurement of antibodies against Mycobacterium avium subspecies paratuberculosis in bovine serum

Five commercially available ELISA tests for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum were evaluated at the individual animal level using sera from 286 paratuberculosis-free and 110 paratuberculosis-infected dairy cattle. Sensitivity (Se) and specificity (Sp) of the tests were estimated after determination of the cut-off dtheta by TG-ROC analysis or using the cut-off values recommended by the manufacturers, respectively. When the dtheta cut-off values were applied, the five ELISA tests showed sub-optimal Se and Sp. Adopting the cut-offs recommended by the manufacturers, the Sp of four of the five ELISA increased, two tests reaching Sp > or = 99.0%. Test sensitivity clearly depended on the disease state of the animals examined. Se was significantly higher in clinically diseased than in latently infected dairy cattle. Calculation of the positive and negative predictive values indicated that, depending on the test, a considerable proportion of false positive and false negative results have to be expected. Therefore, the suitability of antibody detection for the diagnosis of paratuberculosis in individual animals is questioned.     

Evaluation of three Brucella soluble antigens used in an indirect Elisa to discriminate S19 vaccinated from naturally infected cattle

An O-polysaccharide (O-chain) and a hot-water extracted polysaccharide (PS), both obtained from Brucella abortus 1119-3, and a B. melitensis 16M native hapten (NH) were evaluated by indirect enzyme linked immunosorbent assay (ELISA) on three groups of cattle sera. The sera tested were: (a) 75 sera from cows naturally infected with B. abortus; (b) 130 sera from non-infected and non-vaccinated cattle; and (c) 61 sera from non-infected heifers recently vaccinated with B. abortus Strain 19 (S19). Sensitivity (Se), specificity (Sp) and the capability to discriminate vaccinated cattle (ADV) were determined. Using PS antigen, Se was 100% and the Sp was 97.7%, while the highest Sp was obtained by using the O-chain (99.2% ). For the NH antigen, Se was 94.7% and the Sp was 90.0%. The ADV of the three antigens was approximately 85%. Statistical analysis showed significant differences between O-chain/PS and O-chain/NH antigens. The agreement among antigens determined by kappa coefficient was 0.899 for O-chain/PS, 0.845 for O-chain/NH and 0.795 for PS/NH.     

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.     

Estimation of sensitivity and specificity of pregnancy diagnosis using transrectal ultrasonography and ELISA for pregnancy-associated glycoprotein in dairy cows using a Bayesian latent class model

AIMS: To determine the sensitivity (Se) and specificity (Sp) of pregnancy diagnosis using transrectal ultrasonography and an ELISA for pregnancy-associated glycoprotein (PAG) in milk, in lactating dairy cows in seasonally calving herds approximately 85–100 days after the start of the herd’s breeding period.

METHODS: Paired results were used from pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk carried out approximately 85 and 100 days after the start of the breeding period, respectively, from 879 cows from four herds in Victoria, Australia. A Bayesian latent class model was used to estimate the proportion of cows pregnant, the Se and Sp of each test, and covariances between test results in pregnant and non-pregnant cows. Prior probability estimates were defined using beta distributions for the expected proportion of cows pregnant, Se and Sp for each test, and covariances between tests. Markov Chain Monte Carlo iterations identified posterior distributions for each of the unknown variables. Posterior distributions for each parameter were described using medians and 95% probability (i.e. credible) intervals (PrI). The posterior median estimates for Se and Sp for each test were used to estimate positive predictive and negative predictive values across a range of pregnancy proportions.

RESULTS: The estimate for proportion pregnant was 0.524 (95% PrI?=?0.485–0.562). For pregnancy diagnosis using transrectal ultrasonography, Se and Sp were 0.939 (95% PrI?=?0.890–0.974) and 0.943 (95% PrI?=?0.885–0.984), respectively; for ELISA, Se and Sp were 0.963 (95% PrI?=?0.919–0.990) and 0.870 (95% PrI?=?0.806–0.931), respectively. The estimated covariance between test results was 0.033 (95% PrI?=?0.008–0.046) and 0.035 (95% PrI?=?0.018–0.078) for pregnant and non-pregnant cows, respectively. Pregnancy diagnosis results using transrectal ultrasonography had a higher positive predictive value but lower negative predictive value than results from the ELISA across the range of pregnancy proportions assessed.

CONCLUSIONS AND CLINICAL RELEVANCE: Pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk had similar Se but differed in predictive values. Pregnancy diagnosis in seasonally calving herds around 85–100 days after the start of the breeding period using the ELISA is expected to result in a higher negative predictive value but lower positive predictive value than pregnancy diagnosis using transrectal ultrasonography. Thus, with the ELISA, a higher proportion of the cows with negative results will be non-pregnant, relative to results from transrectal ultrasonography, but a lower proportion of cows with positive results will be pregnant.     

Estimation of sensitivity and specificity of five serological tests for the diagnosis of porcine brucellosis

While serological tests are essential in surveillance and control programs of animal diseases, to date none of the common serological tests approved in the EU (complement fixation test or Rose-Bengal test) has been shown to be reliable in routine individual diagnosis of porcine brucellosis, and some more recent tests like ELISA have not been fully evaluated yet. In the absence of a gold standard, this study allowed the estimation of sensitivities and specificities of these tests with a Bayesian approach using Markov Chain Monte Carlo algorithms. The pig population that was tested included 6422 animals from Metropolitan France. Serum samples were collected from a large population of pigs, representative of European swine population and tested with five brucellosis serological tests: Rose-Bengal test (RBT), fluorescence polarization assay (FPA), indirect ELISA (I-ELISA) and two competitive ELISAs (C-ELISA). The sensitivity and the specificity of each test were estimated. When doubtful results were excluded, the most sensitive and specific test was C-ELISA(2) (Se C-ELISA(2)=0.964, [0.907; 0.994], 95% credibility interval (CrI); Sp C-ELISA(2)=0.996, [0.982; 1.0], 95% CrI). When doubtful results were considered as negative, C-ELISA(2) was still the most sensitive and specific test (Se C-ELISA(2)=0.960, [0.896; 0.994], 95% CrI and Sp C-ELISA(2)=0.994, [0.977; 0.999], 95% CrI). The same conclusions were reached when doubtful results were considered as positive (Se C-ELISA(2)=0.963, [0.904, 0.994], 95% CrI and Sp C-ELISA(2)=0.996, [0.986; 1.0], 95% CrI).     

Evaluation of three serological tests for brucellosis in naturally infected cattle using latent class analysis

Serological methods are traditionally used in diagnosis of brucellosis. However, the comparative performance of these tests and their accuracy under the local environment in Zambia has not been assessed. Thus, the objective of our study was to evaluate the diagnostic performance of three serological tests for brucellosis; Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and Fluorescence Polarisation Assay (FPA) in naturally infected cattle in Zambia without an appropriate reference test to classify animals into truly infected and non-infected. Serological test results from a study to determine sero-prevalence were used to compare the performance of RBT, c-ELISA and FPA in diagnosing brucellosis in traditional cattle. Since none of the tests can be seen as a perfect reference test or gold standard, their performance in a population of naturally infected cattle was evaluated using latent class analysis which allows the sensitivity (Se) and specificity (Sp) to be estimated in the absence of a gold standard. The highest Se was achieved by the c-ELISA (97%; Credible Posterior Interval (CPI)=93-100%) and the highest Sp by the FPA (93%; CPI=85-99%), conversely these tests also had the lowest Sp and Se, respectively, with the RBT performing well in both the Se (93%; CPI=84-98%) and Sp (81%; CPI=61-97).     

Diagnostic performance measures of ELISA and quantitative PCR tests for porcine circovirus type 2 exposure using Bayesian latent class analysis

Porcine circovirus 2 (PCV2) is believed to be a necessary but not sufficient underlying cause of porcine circovirus associated disease (PCVAD) in swine (Opriessnig et al., 2007). Since the potential threat of PCVAD is dependent on the prevalence of PCV2 in swine populations, accurate diagnostic tests are important for epidemiologic surveillance. Therefore, we evaluated the diagnostic sensitivity (Se) and specificity (Sp) of a new indirect ELISA and two quantitative PCR tests for PCV2 in a series of latent class models that used Bayesian estimation procedures. A total of 4140 samples from finisher pigs were tested for evidence of PCV2 by the ELISA and a TaqMan (TM) quantitative PCR, 995 by the ELISA and a SYBR Green (SG) dye-binding PCR, 998 by both PCRs and 993 by all three tests. Overall, the median (95% probability interval) ELISA Se and Sp was 0.85 (0.83-0.87) and 0.74 (0.68-0.79), respectively, when all three tests were analyzed together at an ELISA absorbance (optical density or OD) cutoff of ≥0.3. The TM PCR Se and Sp was 0.86 (0.84-0.88) and 0.94 (0.87-0.97), respectively, and the SG PCR Se and Sp was 0.83 (0.81-0.85) and 0.98 (0.94-1.00), respectively when all three tests were analyzed together at an ELISA OD cutoff of ≥0.3. Sensitivity analysis revealed that Sp estimates in general had less stability than Se estimates, but the SG PCR(Sp) was the most stable. Limited conditional dependence between the two PCR tests was detected. We conclude that the ELISA had the highest diagnostic Se at an absorbance cutoff of ≥0.3, while the SG PCR had the highest diagnostic Sp. The prevalence levels for exposure to PCV2 in finishing swine populations across all analyses ranged from 58 to 100%.     

Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach

Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies.     

Meta-analysis of field studies on bovine tuberculosis skin tests in United States cattle herds

Our objective was to summarize information on the diagnostic accuracy, in terms of test sensitivity (Se) and specificity (Sp), for bovine tuberculosis (bTb) tuberculin skin tests as currently used in the United States. Meta-analyses including Se and Sp estimates from field studies of bTb tuberculin tests conducted in North American cattle were conducted to provide a distribution of estimates and central tendency for Se and Sp of the caudal fold tuberculin (CFT) and serial interpretation of the CFT and comparative cervical tuberculin (CFT-CCT) tests. In total, 12 estimates for CFT and CFT-CCT test Se and Sp were identified from seven publications matching inclusion criteria. Estimates for CFT test Se ranged from 80.4% to 93.0% and CFT test Sp from 89.2% to 95.2%. Estimates for CFT-CCT test Se ranged from 74.4% to 88.4% and CFT-CCT test Sp ranged from 97.3% to 98.6%. These distributions of test Se and Sp are intended to provide a more realistic representation for U.S. bTb skin tests than previously reported. Estimation and discussion of herd-level CFT and CFT-CCT test parameters is also included. These results should be considered at the herd and individual animal level when evaluating results from tuberculin skin test results in North American cattle herds.     

Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using M. bovis BCG Tokyo culture filtrate as antigen and anti-MPB70 4C3/17 monoclonal antibody was developed for use in multiple animal species. An analysis of the C-ELISA data for cattle and bison serum panels revealed specificities of 68% to 85% and sensitivities of 85% to 89%. Receiver operator characteristics (ROC) of this data revealed areas of 81% to 92% for C-ELISA and demonstrated that C-ELISA as well as the indirect ELISA protocols, MPB70-ELISA and LAM-ELISA, discriminate M. bovis infected animals from non-infected animals for these particular panels. The kappa statistic values for agreement beyond chance between C-ELISA and MPB70-ELISA were determined after ELISA cutoffs were adjusted to minimize false positives. There were poor to excellent agreements between C-ELISA and MPB70-ELISA in all species tested (Bovidae, Cervidae, and Camelidae) that were consistently higher than the kappa statistic between C-ELISA and LAM-ELISA. The humoral response to one antigen and little or no response to the other in many animals argued for a parallel interpretation of C-ELISA and LAM-ELISA to increase sensitivity.     

Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Monoclonal antibodies (mAb) were produced by fusing Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c and ND4 mice that were immunized with killed Leptospira interrogans serovar pomona whole cells. Thirty hybridomas which produced antibodies (of the IgG1, IgG2a, IgG2b, or IgG3 isotype) that bound to epitopes on the serovar pomona whole cell antigen were identified by an indirect enzyme-linked immunosorbent assay (ELISA). Twenty-eight of these 30 mAbs cross-reacted in the indirect ELISA with at least one whole cell antigen prepared from 12 other pathogenic Leptospira serovars, and/or with whole cell antigen from the non-pathogenic Leptospira biflexa serovar patoc. The two serovar pomona-specific mAbs, which were designated M897 and M898, were obtained from the ND4 mouse and were both of the IgG1 isotype. In competitive ELISAs, M897 and M898 were inhibited from binding to the pomona antigen by bovine sera with anti-serovar pomona microscopic agglutination test (MAT) titres ranging from 100 to 6400. No significant inhibition was observed with pomona MAT-negative sera or with sera from animals experimentally infected with serovars canicola, copenhageni, grippotyphosa, hardjo type hardjobovis or sejroe. The epitopes recognized by M897 and M898 were both highly susceptible to sodium meta-periodate oxidation, indicating a carbohydrate composition. Neither of these mAbs reacted in immunoblots with the separated components of the serovar pomona whole cell antigen.     

Old and new diagnostic approaches for Q fever diagnosis: correlation among serological (CFT, ELISA) and molecular analyses

The objective of this study was to evaluate the performance of the complement fixation test (CFT) with respect to ELISA for the serological diagnosis of Q fever and to assess the role of serology as a tool for the identification of the shedder status. During 2009-2010, sera from 9635 bovines and 3872 small ruminants (3057 goats and 815 sheep) were collected and analyzed with CFT and ELISA. In addition, 2256 bovine, 139 caprine and 72 ovine samples (individual and bulk tank milk samples, fetuses, vaginal swabs and placentae) were analyzed with a real-time PCR kit. The relative sensitivity (Se) and specificity (Sp) of CFT with respect to ELISA were Se 26.56% and Sp 99.71% for cattle and Se 9.96% and Sp 99.94% for small ruminants. To evaluate the correlation between serum-positive status and shedder status, the ELISA, CFT and real-time PCR results were compared. Due to the sampling method and the data storage system, the analysis of individual associations between the serological and molecular tests was possible only for some of the bovine samples. From a statistical point of view, no agreement was observed between the serological and molecular results obtained for fetus and vaginal swab samples. Slightly better agreement was observed between the serological and molecular results obtained for the individual milk samples and between the serological (at least one positive in the examined group) and molecular results for the bulk tank milk (BTM) samples. The CFT results exhibited a better correlation with the shedder status than did the ELISA results.     

Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

Background

Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs.

Methods

Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations.

Results

The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs.

Conclusion

Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow.     

Evaluation of cellular and serological diagnostic tests for the detection of Mycobacterium bovis-infected goats

We have evaluated the comparative intradermal skin (CID) test, the interferon gamma (IFN-γ) assay, two ELISAs with bovine PPD antigen, the standard and the anamnestic using sera obtained, respectively, at the time of the tuberculin injection and 15 days later, for the diagnosis of caprine tuberculosis (TB). The sensitivity and specificity results were high for the CID test (83.7%, 100%), the IFN-γ assay (83.7%, 96%) and the anamnestic ELISA (88.6%, 95.8%). In contrast, they were comparatively low for the standard ELISA (54.9%, 88%). However, test results with the standard ELISA were positive in a group of goats with cavitating TB (100%). A combination of the CID test and the IFN-γ assay offered the highest sensitivity, 95.8%, and also high specificity, 96%. In spite of this, the evidence that the serological tests were most sensitive for the detection of goats with severe lesions (100% positivity) suggested that a combination of CID test and anamnestic ELISA may be most useful as part of an eradication campaign against caprine TB.     

An efficient peptide-based ELISA for differentiating fowl adenovirus 4–infected chickens from vaccinated chickens

Fowl adenovirus serotype 4 (FAdV4), the causative agent of hepatitis-hydropericardium syndrome (HPS), has caused major economic losses to the poultry industry worldwide. Although inactivated vaccines have been deployed widely against FAdV4, a DIVA (differentiating infected from vaccinated animals) test specific for FAdV4 has not been available. We synthesized an immunogenic peptide, corresponding to regions 66–88 aa of the 22K nonstructural protein of FAdV4, and used the peptide as coating antigen to develop an indirect ELISA for a DIVA test specific to FAdV4. Specificity analysis showed that the ELISA only reacted with sera against FAdV4, and not with sera against other pathogens tested. Moreover, the ELISA could effectively differentiate FAdV4–infected chickens from vaccinated chickens. In a test of sera from experimentally infected chickens, the ELISA had 95% and 85% concordance with an indirect immunofluorescence assay (indirect IFA) and a commercial ELISA, respectively, and the concordance was 80.5% between the ELISA and the indirect IFA in detecting clinical infection samples. Our peptide-based ELISA provides an efficient DIVA test for FAdV4 in clinical samples.     

Use of recombinant proteins in antibody tests for bovine tuberculosis

Tuberculosis (TB) in cattle remains a major zoonotic and economic problem in many countries. Since the standard diagnostic assay, the intradermal test (IDT) with bovine PPD tuberculin, has less than optimal accuracy in all situations, other diagnostic methods such as serological assays have been investigated. Because of fundamental concerns for the low sensitivity and specificity of previous ELISA protocols, a profiling ELISA with nine purified, recombinant proteins of TB complex mycobacteria, was employed on samples from four groups of cattle: (a) naturally Mycobacterium avium-exposed and experimentally Mycobacterium bovis-infected, (b) officially-certified TB-free herds, (c) exposed to M. bovis in two field TB outbreaks and scored as bovine reactors in the gamma-IFN assay for bovine TB, (d) paratuberculosis (para TB)-infected. The described ELISA proved to be highly specific. In fact, the antibody (Ab) response could be consistently detected in 3 out of 3 endotracheally-infected calves and in 1 out of 3 contact-infected calves. There was also a very low prevalence of low-titered, non-specific Ab responses in paraTB-infected animals. As for the animals exposed to field TB outbreaks, 16 out of 28 gamma-IFN positive cattle were also Ab-positive; importantly, 7 out of 12 gamma-IFN positive, IDT-negative cattle showed Ab responses to TB proteins. In general, the profile of the Ab response varied among animals; the reaction to single recombinant antigens was sometimes transient and fluctuating, whereas the panel of antigens on the whole was indeed more effective in Ab detection.