Identification of herds with cattle persistently infected with bovine viral diarrhea virus by virological evaluation of three calves

For the identification of herds with cattle persistently infected (PI) with bovine viral diarrhea virus, 1,272 animals from 20 herds were subjected to serum neutralizing (SN) test using the Nose strain and virus isolation. Eighteen PI cattle were detected from 5 herds. On the phylogenetic tree based on the nucleotide sequences of the 5' untranslated region, the isolates from the PI cattle were classified into genotypes-1a or -1b. Of 3 unvaccinated calves aged 6 to 12 months selected from each herd, the probabilities of obtaining 2 or more non-PI cattle with SN antibody titers of 64 or more (P(SN)), one or more PI cattle (P(VI)), and either of the conditions (P(Total)) were calculated using the hypergeometric probability model. P(Total) for the 5 herds with PI cattle was 1.000. P(SN) for 3 herds with many PI cattle within the selected age group was as low as 0.500 or less, and P(VI) was as high as 0.886 or more. P(SN) in the 2 other herds with few PI cattle was 1.000, and P(VI) was as low as 0.375 or less. P(Total) in 13 of 15 herds without PI cattle was 0.000, and was 0.714 or 0.774 for the 2 other herds. These results suggest that herds with PI cattle can be predicted with high accuracy when both SN test and virus isolation are performed on only 3 unvaccinated calves aged 6 to 12 months selected from a herd.     

Competitive ELISA for the detection of antibodies to Rift Valley fever virus in goats and cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.     

Serological characteristics of affected cattle during an outbreak of bovine enzootic encephalomyelitis caused by Akabane virus

During an outbreak of bovine enzootic encephalomyelitis caused by the Akabane virus (AKAV) in 2010, 210 serum samples were collected from the affected cattle, and serological investigations for the AKAV were performed using a serum neutralization test (SNT) and an enzyme-linked immunosorbent assay (ELISA). The seropositive rates for SNT and ELISA were 90.0 and 85.2 %, respectively. The titers of SNT (log₂) against the AKAV were higher than 4.0 in the highly affected cattle (80.0 %). This finding indicates that most affected cattle were infected with the AKAV and that strong immune responses against this virus were elicited in affected cattle. The strong immune response to the AKAV in cattle may provide insight into the occurrence of bovine encephalomyelitis caused by the AKAV.     

Field application of the combination of neutralizing test and virus isolation, so-called spot test, to detect herds including cattle persistently infected with bovine viral diarrhea virus

To detect herds including cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV), application of the combination of neutralizing antibody detection and virus isolation, so-called spot test, were performed on sera of 3 calves selected from each of 26 farms. Nine farms were judged as positive because 64 or more antibody titers were detected from 2 or more calves or BVDV was isolated from one or more calves. PI cattle were detected from 8 of the 9 farms. The positive judgment on one farm was obtained only when the indicator virus used on the neutralizing test was genotypically identical with the isolate from the farm. These results suggest that the spot test can be effective in detecting herds with PI cattle and that the accuracy may be influenced by the genotypes of the indicator viruses.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of infectious bovine rhinotracheitis viral antibody in cattle.

An enzyme-linked immunosorbent assay was developed to detect bovine serum antibody to infectious bovine rhinotracheitis virus. The specificity of this assay in 304 bovine sera, collected from an infectious bovine rhinotracheitis virus-free herd, was 100%; in sera from 62 cattle inoculated with an intranasal vaccine, its diagnostic sensitivity was 27.4% at one month and 100% at six months, postvaccination. In 303 bovine sera with standard serum neutralizing antibody titers of greater than or equal to 1:2 it showed 100% sensitivity; and in 463 random diagnostic samples, comparative tests indicated that enzyme-linked immunosorbent assay detected more seropositive animals (61.6%) than the standard serum neutralizing test (49.9%). The enzyme-linked immunosorbent assay method was considered to be technically superior as a routine diagnostic test for the detection of infectious bovine rhinotracheitis viral antibody in bovine sera.     

Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.     

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.     

A short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.     

Characterization of monoclonal antibodies directed against the bovine herpesvirus-1 glycoprotein E and use for the differentiation between vaccinated and infected animals

A panel of seven monoclonal antibodies (MAbs) directed against the bovine herpesvirus-1 (BHV-1) glycoprotein E (gE) was obtained. For that purpose, mice were either tolerized to BHV-1 gE-negative virus and then immunized with wild type BHV-1 or immunized with plasmid DNA expressing the gE and gI glycoproteins. The MAbs were characterized by their reactivity with the gE protein or the gE/gI complex and by competition experiments. Results showed that the MAbs were directed against three antigenic domains, two located on the gE glycoprotein and one on the gE/gI complex. Blocking experiments were performed with sera from experimentally vaccinated and infected cattle. A competition was observed between gE-positive bovine sera and six of the seven MAbs. The bovine sera thus recognized two of the three antigenic sites. Field sera were then tested in blocking enzyme-linked immunosorbent assay using one horseradish peroxidase-conjugated MAb. A specificity of 98.2% and a sensitivity of 98.2% compared to the commercially available test were observed.     

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).     

The sustainability, feasibility and desirability of breeding livestock for disease resistance

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak.     

Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses

Following isolation of a virus (CSIRO19) from insects in Australia and its identification as bluetongue virus serotype 20 (BTV20), a nationwide survey of antibodies in cattle and sheep sera was undertaken. Initial studies using the serum neutralization (SN) test showed that the distribution of BTV20 antibodies in cattle was confined to the northern part of Australia. Group-reactive antibody tests (agar gel diffusion precipitin, AGDP, and complement-fixation, CF) showed group-reactive cattle sera south of the BTV20 zone (northern Australia), and southwards from Queensland to New South Wales. Very few group-reactive sheep sera (45 out of 16213) were found and these were of doubtful epidemiological significance. Some of these BTV group-reactive, BTV20-negative, sera were tested in SN tests against BTV1 to 17 and Ibaraki (IBA) virus. The results indicated that BTV1, or a closely related orbivirus, was active in cattle in Queensland, northern Western Australia, and New South Wales, and that antibody to BTV15 was present in some of the cattle sera in northern Western Australia and the Northern Territory. Antibody to IBA virus was present in some cattle sera in Queensland, northern Western Australia and New South Wales. SN antibody titres ?60 were also found to a number of other BTV serotypes in cattle sera in northern Western Australia and Queensland (principally, BTV2 and BTV7). Low level reactions were commonly observed against these and a number of other BTV serotypes, often in the same serum samples. Further, 22% of the group-reactive cattle sera did not react with any of the viruses in the SN tests. Such results were difficult to interpret in terms of known Australian BTV or BTV-related isolates.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States.     

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.     

A competition enzyme immunoassay for brucellosis diagnosis

Two monoclonal antibodies (MAbs) conjugated with horseradish peroxidase were used independently in a competitive enzyme immunoassay (cEIA) to detect Brucella specific antibodies in 1120 sera from Brucella-free cattle, 61 from cattle known to be infected with B. abortus, and 207 sera from vaccinated calves. The results were compared to those obtained in the complement fixation test (CFT). The cEIA with both MAbs proved to be more sensitive than the CFT because no false-negative results were obtained. In addition, discrimination between sera from infected and vaccinated animals was more evident.     

Serosurveillance for Japanese encephalitis, Akabane, and Aino viruses for Thoroughbred horses in Korea

Recent global warming trends may have a significant impact on vector-borne viral diseases, possibly affecting vector population dynamics and disease transmission. This study measured levels of hemagglutination-inhibition (HI) antibodies against Japanese encephalitis virus (JEV) and neutralizing antibodies against Akabane virus (AKAV) and Aino virus (AINV) for Thoroughbred horses in Korea. Blood samples were collected from 989 racehorses in several provinces, between October 2005 and March 2007. Sera were tested using either an HI assay or a virus neutralization test. Approximately half (49.7%; 492/989) of the horses tested were antibody-positive for JEV. The HI titer against JEV was significantly correlated with racehorse age (p < 0.05). Horses with an HI antibody titer of 1:160 or higher accounted for 3.9% of the animals tested, indicating that vectors transmitting arthropod-borne viruses bit relatively few horses. In contrast, 3.8% (19/497) and 19.5% (97/497) of horse sera collected in March 2007 were positive against AKAV and AINV, respectively. The presence of antibodies against AKAV and AINV may indicate the multiplication of AKAV and AINV in these horses.     

A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants.

A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleo-protein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.     

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.     

Evidence of Schmallenberg virus circulation in ruminants in Greece

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.