Evaluation of three competitive ELISAs and a fluorescence polarisation assay for the diagnosis of bovine brucellosis.

Bovine brucellosis is an infectious disease of worldwide public health and economic importance. The usual tests for the diagnosis of this disease include the Rose-Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT) and indirect ELISA. New tests such as competitive ELISAs (C-ELISA) and fluorescence polarisation assay (FPA) have been developed. However, C-ELISA may correspond to different protocols and a wide variation may exist in their diagnostic performance. The aim of this study was to evaluate three commercially available C-ELISA kits (C-ELISA1-3) and FPA for the diagnosis of bovine brucellosis and compare test performance with RBT, CFT, indirect ELISA and FPA. Sera submitted to EU laboratories in 2011 from 5111 adult cattle were tested. Individual test sensitivities (Se) and specificities (Sp) were estimated. Threshold assessment using the receiver operating characteristic method was also performed. The most sensitive tests were FPA (99.0%; 95% confidence interval [CI], 97.9-100%), C-ELISA1 (98.4%; 95% CI, 97.0-99.8%) and RBT (97.7%; 95% CI, 95.9-99.3%). The most specific tests were CFT (99.98%; 95% CI, 99.93-100%), SAT (99.98%; 95% CI, 99.93-100%) and RBT (99.89%; 95% CI, 99.79-99.99%). Among the new tests, none of the three C-ELISA kits studied could be recommended as a single screening test because of their low specificity, especially when used in a herd. C-ELISA3 could not be recommended as confirmatory test on individual animals to determine whether false positive serological test results had occurred.

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules.

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay. cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healthy horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16-35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.

The first International Standard anti-Brucella melitensis Serum.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis.

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.

An indirect ELISA for the detection of antibody in milk from sheep experimentally infected with Brucella melitensis biovar 3.

An indirect ELISA was evaluated for the detection of Brucella antibodies in milk (m-ELISA) from sheep experimentally infected with B. melitensis biovar 3. At the end of the second reproductive cycle (13 months post infection), the milk of 22 lactating sheep was tested using the m-ELISA. Sera from the same sheep were also tested for Brucella antibodies using the Rose Bengal test (RBT) and the complement fixation test (CFT). The first serum sampling after parturition showed 100% sensitivity in both the RBT and the CFT (confidence interval [CI] 94-100%), but in subsequent samplings the sensitivity of the RBT decreased to 73% (CI 55-85%). Similarly, the sensitivity of the CFT decreased two months after the first sampling, when respective sensitivities of 95% (CI 81-98%) and 81% (CI 61-93%) were recorded for the final two samplings. The sensitivity of the m-ELISA decreased initially (68% on the third sampling, CI 50-81%), but then increased to 95% (CI 81-98%) for the final sampling. When disease prevalence in a flock is below 5%, the estimated probability of not detecting an infected flock through m-ELISA bulk milk testing is over 25%. Under field conditions in Italy (average sheep flock size of 70), the probability that the infection will not be detected is over 25% when four (or less) infected milking sheep are present in the flock. The results show that the m-ELISA is not a reliable screening test for bulk milk samples when the prevalence of brucellosis in a sheep flock is low.

The persistence of Brucella melitensis in experimentally infected ewes through three reproductive cycles.

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.

The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis.

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).

Evaluation of an indirect ELISA for the detection of Brucella antibodies in cow's milk.

An indirect ELISA was developed by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS A&M) for the detection of Brucella antibodies in cow's milk. Specific monoclonal antibody was used against a bovine IgG(1) epitope and complies with European Commission requirements. The test accuracy was evaluated on milk samples from the regions of Abruzzo and Molise in Italy. The negative samples came from 1,250 officially brucellosis-free herds from the Molise region (Italy). The positive samples were taken from three herds in the Abruzzo and Molise regions where animals positive to the official tests were present and Brucella abortus was isolated. Test specificity was 99.8% (with a confidence interval [CI] of 99.6%-99.9%), while sensitivity was 100% (CI 91.2%-100%). The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:256 in negative milk. The probability of identifying an infected herd in the dairy cattle population. Under study was 88.6% (CI 73.9%-95.3%).

Use of the complement fixation and brucellin skin tests to identify cattle vaccinated with Brucella abortus strain RB51.

In the European Union, RB51 vaccine can be used only under strictly controlled conditions for the immunisation of cattle at risk of infection with Brucella abortus. A test is therefore necessary to distinguish vaccinated from unvaccinated animals. The complement fixation test with RB51 antigen (RB51-CFT), dot-blot and gamma-interferon used to identify vaccinated animals have been described, but sensitivity of the tests has been poor and positivity transient after calfhood vaccination. To avail of a rapid and accurate diagnostic tool, the authors produced, controlled and evaluated an experimental brucellin prepared from strain RB51 (RB51 brucellin). The potency of this brucellin was evaluated in guinea-pigs sensitised with RB51 and compared with a commercially available brucellin. Both allergens produced similar biological activity in guinea-pigs. The RB51 brucellin skin test was performed in 10 cattle 414 days after calfhood vaccination with RB51 when they were negative to the RB51-CFT. The skin test revealed 60% sensitivity (with a confidence interval of 95%, CI 30.8%-83.3%) and 100% specificity (CI 60.7%-100%). These findings limit the use of the skin test only for screening to detect RB51 vaccinated herds, not individual animals. Nevertheless, following intradermal inoculation of RB51 brucellin, a transient antibody increase to the RB51-CFT was observed, from day 9 to day 20 post inoculation with RB51 brucellin. This transient antibody increase, when evaluated in parallel with the RB51 brucellin skin test results, enables detection of individual vaccinated animals (sensitivity 100%; CI 76.2%-100%).

Serological surveillance of bluetongue virus in cattle, sheep and goats in Albania.

The recent spread of the bluetongue (BT) virus (BTV) in the Mediterranean Basin encouraged numerous countries to undertake entomological and serological surveillance programmes to identify affected areas and control the infection. Hitherto, no data on the presence and diffusion of BTV in Albania were available. Between October and November 2002 serum samples from 857 cattle and 870 sheep and goats were collected by the Albanian Veterinary Services in 15 districts, some bordering Yugoslavia, Macedonia and Greece, and others along the Adriatic coast. At the Albanian Veterinary Research Institute the samples were tested for the presence of BTV antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA) (bluetongue antibody test kit, IZS A&M, Teramo); in Italy, the virus neutralisation (VN) test was used to confirm the ELISA results and determine the serotype of BTV circulating. Overall seroprevalence was 18.9% in cattle and 4.4% in sheep and goats; seropositive animals occurred in all districts surveyed. The highest prevalence of BT was observed in the Tirana District, with 61% of the cattle and 20% of the sheep and goat populations BT-positive. The VN test confirmed the c-ELISA results revealing the presence of antibodies against BTV serotype 9.

Bluetongue laboratory diagnosis: a ring test to evaluate serological results using a competitive ELISA kit.

The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.

Serological response in cattle and sheep following infection or vaccination with bluetongue virus.

Data from various experimental and field studies were compiled and analysed to evaluate the serological response in sheep and cattle against different bluetongue (BT) virus (BTV) vaccine combinations (Onderstepoort Biological Products, South Africa); the accuracy of diagnostic procedures commonly used for detecting BTV antibodies was also assessed. Using the competitive enzyme-linked immunosorbent assay (c-ELISA) (IZSA&M, Teramo, Italy) and the virus neutralisation (VN) test, antibody responses were evaluated under the following vaccination regimes: monovalent modified-live vaccine against BTV-2 in cattle and sheep, monovalent modified-live vaccine against BTV-9 in sheep, and bivalent modified-live vaccine against BTV-2 and BTV-9 in cattle and sheep. The data were compared to serological results observed in cattle and sheep infected with Italian field strains of BTV-2 or BTV-9. The c-ELISA consistently detected antibodies earlier than the VN test in both livestock species and against all BTV serotypes. The highest and most rapid antibody responses were observed in sheep infected in the field. In cattle and in sheep, high VN titres were detected using monovalent vaccines, while bivalent vaccines initiated lower antibody titres that developed more slowly.

Kinetics of the antibody response in ewes experimentally infected with Brucella melitensis biovar 3.

The authors evaluated the kinetics of antibody response in 46 ewes coming from officially brucellosis free flocks that were experimentally infected with Brucella melitensis biovar 3, and monitored through three subsequent reproductive cycles. In this study, results of Rose Bengal test (RBT) and complement fixation test (CFT) were considered. Test results of 2nd and 3rd reproductive cycle show a peak in the antibody production at parturition, followed by a drop in the following months. The peak at parturition is significantly lower in the 3rd reproductive cycle compared to the 2nd. The drop in antibody production observed after parturition of the 3rd reproductive cycle is significantly higher than that observed after parturition of the 2nd reproductive cycle. Nevertheless, the infection can still be revealed at flock level after three years post infection.

Sensitivity and specificity of serological and bacteriological tests for contagious bovine pleuropneumonia.

In 1990 an outbreak of contagious bovine pleuropneumonia (CBPP) occurred in Italy. Subsequent surveillance for CBPP was based on random sampling in bovine herds, serological controls on all animals moved from the herd of origin and controls on slaughtered animals. Official tests employed were the complement fixation test (CFT) and bacteriological isolation and typing. A total of 33,856 serum samples collected from herds in CBPP-free regions were used to define CFT specificity, while samples from 595 animals from infected herds were employed to define the sensitivity. Ninety-nine animals from three infected herds were used to estimate the sensitivity of the isolation technique. Results showed the specificity of CFT (threshold +1:10) to be 98% and sensitivity to be 63.79%. The sensitivity of the test did not change significantly, regardless of whether the lesions were caused by acute or chronic infection. The sensitivity of the isolation technique was 54.1%.