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Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%-100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals. [ABSTRACT FROM AUTHOR]

The virus neutralization test (VNT) is a functional immunoassay which detects the presence and quantity of neutralizing antibodies. It is a highly sensitive and specific test. As with most neutralization assays, the EHDV VNT does not react with all virus-targeting antibodies, but specifically with those antibodies that bind to VP2, the outermost capsid structural protein of the virus. The interaction between VP2 and neutralizing antibodies can block EHDV cell binding, neutralizing its infectivity. The detection and quantification of neutralizing antibodies are indicative of how protected an animal is against reinfection. The EHD VNT can therefore be a useful tool to monitor the efficacy of a vaccination campaign. VP2 is also the main determinant of EHDV serotype specificity, and so EHDV-neutralizing antibodies which target VP2 are also serotype-specific. Throughdetecting and quantifying neutralizing antibodies, the VNT can discriminate the EHDV serotype responsible for an infection and provides insights into the time of infection. It is considered the gold standard test for identifying and quantifying antibodies against EHDV serotypes present in test serum samples. The assay is performed in vitro and is based on inhibition of virus infectivity in the presence of neutralizing antibodies. A neutralizing antibody titer is determined through the presence or absence of cytopathic effect in a cell monolayer. The VNT is a relatively inexpensive assay using standard laboratory equipment; however, to perform the assay, cell cultures, significant time, intensive labor, and technical skill are required., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

According to European Union (EU) regulations, the serological tests for the eradication of bovine and ovine brucellosis are the Rose Bengal Test, Complement Fixation Test, and i-ELISA. These methods, also recommended by the World Organization for Animal Health (OIE) for international trades, have limitations related to the use of suspensions of smooth Brucellae or LPS extracts. Limitations include false-positive serological reactions to brucellosis, which in turn impedes accurate diagnosis in some herds. False positive reactions should be considered carefully during the final stages of an eradication programme and for surveillance purposes in brucellosis-free areas. In this study, we produced specific sera through the experimental infection of sheep with Y. enterocolitica O:9 and E. coli O157:H7. These are the most important cross-reactive bacteria with Brucella. We then evaluated the antibody response of groups of sheep that had been immunised towards homologous antigens and official antigens for brucellosis, in order to identify a differential diagnostic protocol to distinguish cross-reaction in Brucella-infected animals.

A serological survey was carried out in the northern prefectures of Hokkaido, Iwate and Aomori in Japan, for the presence of antibodies against Toxoplasma gondii, Chlamydiapsittaci var. ovis, Mycobacterium paratuberculosis, Coxiella burnetii, Brucella spp., Leptospirosis and Orf virus (ORFV). Out of 267 samples tested, highest overall prevalence (28.78%) was found for T. gondii. The 12.59% of tested sheep were positive for C. psittaci var. ovis. A total of 8.67% were found to be seropositive for C. burnetii. Levels of these infections were found in all three prefectures. Seroconversion to ORFV was detected in Hokkaido and Iwate Prefectures (2.57%). Animals were positive only for L. ballum (1.50%), in Hokkaido and Aomori Prefectures. No animals tested positive for Brucella spp. and M. paratuberculosis.

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis Linnaeus, 1758). The test accuracy was evaluated on milk samples from the Campania Region in Italy. A total of 100 negative samples were collected from 10 officially brucellosis-free herds in Salerno Province, while 30 positive samples were collected from 3 herds in Caserta Province, where animals gave positive results to the official tests and it was here that Brucella abortus biovar 1 had been isolated. Test sensitivity was 100%, with a confidence interval (CI) of 90.8%-100%, while specificity was 98% (CI 93%-99.4%) on individual milk samples. To simulate bulk milk samples from herds infected at various levels of infection, dilutions from 1:10 to 1:100 of positive milk samples in negative milk were also used. The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:50 in negative milk. Considering the average national water buffalo herd size, the probability of identifying infection in a water buffalo herd by bulk milk testing is 50% (CI 33.1%-66.9%) in the worst case scenario of a single infected animal contributing to the bulk milk.

Approximately 250,000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150,000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

The results of an enzyme-linked immunosorbent assay (ELISA) implemented for the detection of gamma interferon (gamma interferon) production in cattle vaccinated with Brucella abortus strain RB51 are presented. A purified protein fraction derived from RB51 (RB51 brucellin) has been used as antigenic stimulus for whole blood. The test was evaluated for 300 days in ten heifers vaccinated at calfhood with 10 x 10(9) colony-forming units of RB51 and in five control heifers. All animals came from officially brucellosis-free herds. Vaccinated animals started to give positive results from day 17 post vaccination (pv) until day 239 pv. All vaccinated animals gave a positive reaction at least once (with a stimulation index exceeding 2.5). Nevertheless, if sampling on day 20 pv is excluded (90% of vaccinated animals gave positive results), the sensitivity of the test varies from 20% to 70%, with a 40% average. A stimulation index over 2.5 was also recorded in three control animals. The results suggest that the gamma-interferon test is not suitable for the detection of cattle vaccinated with RB51, either at the individual or at the herd level.

The official methods specified in the national bovine and ovine/caprine brucellosis eradication plan are the Rose Bengal and complement fixation tests. In the current phase of the eradication plan, it is often difficult to interpret the results obtained with the official tests. Consequently, additional tests that offer greater sensitivity and specificity are thus required. For this reason, two methods, the indirect chemiluminescence enzyme-linked immunosorbent assay (i-ELISA CL) and the competitive chemiluminescence ELISA (c-ELISA CL) that use a chemiluminescent substrate to determine anti-Brucella antibodies in bovine and ovine serum were validated. The methods are based on the detection of anti-Brucella antibodies in serum by catalysis of a chemiluminescent enzyme substrate (luminol/H2O2/enhancer system) by peroxidase conjugated to secondary anti IgG antibodies in i-ELISA CL and to monoclonal anti-lipopolysaccharide (LPS) antibodies in c-ELISA CL. From the results obtained, a cut-off of 60% for bovine serum and 37.5% for ovine serum, expressed as positivity rate (PR), were established using these cut-off values, for the i-ELISA CL, 100% sensitivity and specificity was obtained for bovine serum and 100% sensitivity and 99.8% specificity for ovine serum. Cut-off values of 30% for bovine serum and 40% for ovine serum, expressed as inhibition rate, were selected for c-ELISA CL, which ensured 100% sensitivity and specificity in both cases.

A total of 7 cows, 10 sheep and 10 goats were vaccinated subcutaneously with 5 ml of a recombinant vaccine consisting of synthetic virions containing the four principal proteins (VP2, VP3, VP5 and VP7) of bluetongue virus serotype 2 (BTV-2). The same number of animals and species were vaccinated with 2.5 ml (the normal vaccination dose) and 2 cows, 2 sheep and 2 goats were inoculated with a placebo and the adjuvant added to the vaccine. Animals vaccinated with the normal dose received a booster 14 days after the first injection and 8 sheep a third vaccination 4 months after the second inoculation. One month after the third vaccination, the 8 sheep and another 4 that had never come into contact with the virus were challenged with 1 ml of 10(5.8) TCID(50) of a BTV-2 Italian field isolate. All animals showed competitive enzyme-linked immunosorbent assay (c-ELISA) antibodies starting 14 days following the first vaccination. Conversely, no animal demonstrated neutralising antibodies to BTV-2 after vaccination. Fever (>40 degrees C) was observed in 6 vaccinated animals and 2 controls between 8 and 13 days post challenge. The virus was isolated from all animals from the 7th day post challenge. There was no significant difference in the blood chemical parameters tested and no significant interaction was found in the trial group.