A Sero-Epidemiological Study of Haemophilus Somnus Infection in Dairy Cattle

Akhtar, S., Farver, T.B. and Riemann, H.P., 1997. A sero-epidemiological study of Haemophilus somnus infection in dairy cattle. Veterinary Research Communications, 21 (4), 229-239Five repeated cross-sectional serological surveys of 790 dairy cattle in 4 dairy herds between December 1985 and February 1987 provided an opportunity to study the changes in the seroprevalence of Haemophilus somnus across the 5 surveys and with respect to some demographic and disease variables. The demographic variables included were age (heifers or cows) and farm, representing two groups of herds (two herds in each group, located in the Central and Northern Valleys of California). The serological status of cattle as either negative or positive against H. somnus, Campylobacter fetus and Leptospira hardjo were determined with enzyme linked-immunosorbent assays. Logistic regression analysis was used to compute maximum likelihood estimates of adjusted odds ratios and their respective 95% confidence intervals.The baseline risk of being H. somnus seropositive in the cattle observed at first sampling did not vary significantly during the study period after adjustment for the effects of covariates. Only at first sampling were cows about twice as likely to be H. somnus seropositive than heifers. At samplings 1 and 3, but not at 2 and 5, being in the herds of the Central Valley appeared protective. In contrast, at sampling 4 the cattle in herds in the Central Valley were about 7 times more likely to be H. somnus seropositive. C. fetus-positive cattle were about 3 times more likely to be H. somnus seropositive at sampling 1 only. The relationship between H. somnus status and L. hardjo was not significant during the study period.     

Seroprevalence and association with abortion of leptospirosis in cattle in Ontario.

Sera were collected using a systematic random sampling from 348 cattle herds in Ontario, in proportion to the cattle population in different areas. One cow in five from 296 dairy herds and one in three from 52 beef herds were sampled. The sera were analyzed for prevalence of antibodies to Leptospira interrogans serovar grippotyphosa, hardjo, icterohaemorhagiae and pomona using the microscopic agglutination test. Herd seroprevalence (one or more animals with titer greater than or equal to 80) in beef and dairy herds combined was grippotyphosa 2%, hardjo 13.8%, icterohaemorrhagiae 10.1% and pomona 25.8%; 39% of all herds showed evidence of leptospiral infection with one or more serovars; 44.2% of 52 beef herds had serological evidence of infection with serovar hardjo compared to 8.4% of 296 dairy herds (P less than 0.0001). Seroprevalence of other serovars was not significantly different between beef and dairy herds. The proportion of beef animals seropositive for hardjo and for pomona increased with age, particularly for hardjo; 26.5% of beef animals aged nine years or over were seropositive for hardjo. Dairy animals showed a significant rise of hardjo but not pomona titers with age. The seroprevalence of pomona infection was significantly higher in dairy cattle in eastern Ontario than in other regions. Thirty-four (6.1%) of 553 aborted bovine fetuses had leptospires detected by immunofluorescence techniques. Sixty-five percent of these fetuses were from submissions made between November and January. Leptospires were identified as serovar hardjo by specific immunofluorescence. There appeared, however, to be a paradoxical serological response in that eight aborting cows had antibody titers to pomona rather than hardjo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pestivirus infections in Norway. Serological investigations in cattle, sheep and pigs.

Serum samples from 1,133 dairy cows (187 herds), 3,712 ewes (103 flocks) and 1,317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28% of the cattle herds and 18% of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

Prevalence of bovine parvovirus infection in Ontario dairy cattle.

Studies were conducted to determine prevalence and dynamics of bovine parvovirus (BPV) infection. Dairy cows from 29 randomly selected herds in southwestern Ontario were tested twice, one year apart, for the presence of hemagglutination inhibition (HI) antibodies against BPV. Fifty-one percent of 1141 cows tested had BPV-HI titers > 1:32. One year later, the seroprevalence was 83% in 1131 cows from the same farms. The herd mean seroprevalence was 49% and 86% for the year-1 and year-2 samples, respectively. Evidence of BPV infection was found in 96% (27/28) of herds in year-1 and 100% of herds in year-2. A comparison of titers from 716 cows tested twice showed evidence of frequent BPV infection. Sixty-two percent of 326 animals selected in a systematic manner from 40 Guelph area dairy farms had BPV-HI titers > 1:32. The herd mean seroprevalence was 64% Two herds had no animals with titers above the critical titer (1:32) while in one-quarter of the herds all animals exceeded the critical titer.     

Frequency of bluetongue and bovine parvovirus infection in cattle in South Carolina dairy herds

A survey of South Carolina diary cattle in 12 commercial herds was conducted, using serologic testing to determine the frequency and impact on reproduction of bluetongue (BT) and bovine parvovirus (BFV) infections. Results of the study of serum antibodies titers and dairy cattle health records indicated that the majority of the cattle surveyed was adequately protected against the major reproductive tract diseases through vaccination programs. The frequency of reproductive dysfunction was common in vaccinated herds, however. Six of the 12 herds in the survey had cattle which were BT serotest reactors, although total numbers of these reactor cattle were relatively small. Further, reproductive performance of reactor cows indicated that BT was of little consequence and was not associated with reproductive problems. All herds contained BPV serotest reactors and the latter comprised 59.7% of the total number of cows surveyed. The BPV seroreactor cows were commonly associated with the group of reproductive problem cows which experienced higher rates of embryonic mortality and more services per conception than did nonreactor cattle. The results of the survey emphasize the need for continued investigation and efforts to control BT, and especially BPV infection, in dairy cattle.     

Epidemiology and impact of Neospora caninum infection in three Queensland tropical dairy herds

This study investigated the epidemiology of Neospora caninum in three tropical dairy herds in North Queensland, Australia. All animals in the herds were bled, and the sera were tested by ELISA for N. caninum antibodies. Herd records were examined, and the number of calves carried to term and the number of abortions which occurred over the lifetime of each animal were recorded to determine the abortion rate for each animal. Pedigrees were constructed for two of the herds to investigate whether vertical transmission was occurring. The seroprevalence of N. caninum ranged from 23% to 34%. The abortion rate in seropositive animals was significantly (p < 0.001) higher than in seronegative animals in all three herds (12-20.1% cf. 3.6-7%). Overall, the probability of a calf being seropositive was 3.5 times higher when the dam was also seropositive than when the dam was seronegative. Subsequent selective breeding employed by one herd reduced the N. caninum seroprevalence from 23% to 5% over a 9-year period. This study shows that N. caninum infection is prevalent in North Queensland dairy cattle, and both post-natal infection and vertical transmission are common.     

A longitudinal study of seroprevalence and seroconversion of Neospora caninum infection in dairy cattle in northeast Thailand

A long-term study was carried out in 11 dairy herds in the Khon Kaen province of northeast Thailand between August 2001 and November 2004. The objective was to investigate seroprevalence dynamics of Neospora caninum infection in the herds and to demonstrate patterns of seroconversion in individual cattle. Each herd was visited once a year, in total four times, and sera from cattle > 3 months of age and farm dogs as well as a sample from the bulk milk were collected. All samples were analysed for presence of specific antibodies by an N. caninum iscom ELISA. The overall percentage of antibody-positive cattle was constant and varied only between 10 and 13% over the 4 years, but the variation in within-herd seroprevalence between herds was substantial. Two herds had > or = 20% seropositive animals at all samplings and consistently high bulk milk OD, whereas two herds had no seropositive animal at the last two samplings and low bulk milk OD. Five herds had a decreasing trend of within-herd seroprevalence, whereas the remaining six herds had a higher portion of test-positive individuals at the end of the study. A total of 424 individuals were sampled more than once; 344 (81%) and 32 (8%) were consistently antibody-negative and antibody-positive, respectively. The proportions of animals that changed from being seronegative to seropositive and from being seropositive to seronegative between the years were 3.9-4.6% and 19-39%, respectively. Apparent vertical and horizontal transmission rates were 58% (95% CI; 44-71%) and 5% (95% CI; 3-7%), respectively. In conclusion, the overall percentage of N. caninum antibody-positive cattle was constant over the years, but the within-herd seroprevalence varied substantially between the herds. Seroconversions were likely to occur in individual cattle although most animals had consistent serological status throughout the study.     

Complement fixation titers in cattle following intranasal inoculation of Hemophilus somnus.

Five bulls were inoculated intranasally with a live culture of Hemophilus somnus originally isolated from a clinical case of Hemophilus septicemia. Preinoculation and postinoculation blood samples were taken at weekly intervals for nine weeks for measuring complement fixation titers and daily postinoculation temperatures were taken for one week. Three animals had transient fever and slight lethargy was observed in two animals had a transitory rise in complement fixation titers in the second to fifth weeks postexposure while one animal which had been seronegative on preinoculation testing produced little serological response to the organism. The experiment demonstrated that the nasal instillation of young cattle using an originally pathogenic H. somnus isolate is capable of stimulating only transitory complement fixation antibody titer.     

Serological detection of multiple retroviral infections in cattle: bovine leukemia virus, bovine syncytial virus and bovine visna virus

Individual experimental animals used in our studies on bovine leukemia virus (BLV) are routinely screened for the presence of antibodies to the three bovine lymphotropic retroviruses. We utilized these screening methods to examine frozen sera from eight herds for antibodies to BLV, bovine visna virus (BVV) and bovine syncytial virus (BSV). Serum samples from 235 animals in four dairy and four beef herds were analyzed. Detection methods used included indirect fluorescent antibody tests of virus-infected cell cultures (BLV, BSV, BVV) and agar gel immunodiffusion (BLV). Sera from the BLV-infected animals in the dairy herds showed the highest single (50%, 49/97) and multiple (30%, 29/97) infections compared with 5% (7/138) and less than 1% (1/138), respectively in the beef herds. Single BVV infections were not detected in the dairy herds, but 11% (11/97) of the sera contained antibodies to BVV plus BLV or BSV. Five sera from beef cattle had antibodies only to BVV and four were obtained from one herd. Only one beef serum of the 138 tested demonstrated multiple antibodies (BLV, BVV).     

Haemophilus somnus: a comparison among three serological tests and a serological survey in beef and dairy cattle.

Serological tests for the detection of antibodies against Haemophilus somnus were carried out in herds of beef and dairy cattle using three different techniques: agglutination, complement fixation and counterimmunoelectrophoresis. The agglutination test appeared to detect more seroreactors than the complement fixation and counterimmunoelectrophoresis tests. Results of the three tests indicated that there were more positive reactors in beef cattle and dairy cattle from infected herds than in dairy cattle from clinically normal herds.     

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.     

Isolation of Haemophilus somnus from dairy cattle in kwaZulu-Natal. An emerging cause of 'dirty cow syndrome' and infertility?

Haemophilus somnus was consistently isolated from vaginal discharges of dairy cows submitted from field cases of vaginitis, cervicitis and/or metritis in the KwaZulu-Natal Midlands during the period July 1995 - December 2000 and from the East Griqualand area in November/December 2000. The purulent vaginal discharges, red granular vaginitis and cervicitis, and pain on palpation described in these cases was very similar to that reported in outbreaks of H.somnus endometritis syndrome in Australia, Europe and North America. In all the herds involved in these outbreaks, natural breeding with bulls was employed. Although there was a good cure rate in clinically-affected animals treated with tetracyclines, culling rates for chronic infertility were unacceptably high. Employment of artificial insemination in these herds improved pregnancy rates in cows that had calved previously, but many cows that had formerly been infected failed to conceive.     

Prevalence of serum antibodies to Mycobacterium avium subsp. paratuberculosis in cattle in Galicia (northwest Spain)

Prior to establishing a control and prevention program for Johne's disease in cattle in Galicia (northwest Spain), a survey was conducted to estimate the prevalence of the disease. For this survey, 61,069 animals of at least 1-year of age from 2735 randomly selected herds were bled and samples analyzed with a commercial ELISA. The estimated true individual-level prevalences – assuming the manufacturer's reported test sensitivity of 48.5% and specificity of 98.9% – were 3.02% in dairy cattle, 1.03% in beef cattle and 2.83% in animals from farms with both dairy and beef cattle. True herd prevalences (with herds declared positive if one or more animals tested positive) were 10.69% for dairy herds, 0% for beef herds and 2.71% for mixed herds. When herds were declared positive if at least two animals tested positive, true herd prevalences were 14.75% for dairy herds, 1.47% for beef herds and 12.01% for mixed herds. Assuming a higher specificity of 99.4%, true individual-level prevalences increased to 4.03% in dairy herds, 2.07% in beef herds and 3.84% in mixed herds. Herd prevalences were 27.77%/18.79%, 2.78%/2.40% and 5.70%/12.24% (using the one/two-animal cut-offs) in dairy, beef and mixed herds, respectively. In conclusion, these results seem to indicate that a small percentage of cows and a rather high percentage of dairy herds in this region are MAP-seropositive.     

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.     

Bluetongue infections in Louisiana cattle

Bluetongue virus (BTV) serotype 17 was isolated from cattle with clinical signs of bluetongue disease during 1978 and 1979 epizootics. Bovine sera from 6 herds located in an epizootic region were examined in 1979 for antibodies, using an immunodiffusion (ID) test. Of 300 sera, 164 (54.7%) were seropositive. Sera from statewide surveys of Louisiana cattle in July to August 1980 and December 1980 to January 1981 were tested for BTV antibodies, using the ID test. Fifty-eight of 70 herds (82.9%) and 164 of 597 (27.5%) individual cattle tested in July to August 1980 were seropositive. Fifty-four of 63 (85.7%) herds and 170 of 600 (28.3%) individual cattle tested in December 1980 to January 1981 were seropositive. Significant differences (P less than 0.01) were found in the seropositive rates between the various geographic regions of the state during each survey. Adult breeding-age cattle in 3 sentinel herds were tested for BTV antibodies beginning in 1976 and continuing through January 1981. During this interval, the seropositive rate in 2 of 3 herds was increased. Also, individual cattle in all 3 of these herds converted from seronegative to seropositive, indicating exposure during a particular interval for each herd. The age distribution of seropositive cattle in a dairy indicated that 2-year-old cattle had a seropositive rate comparable with that of older animals in the herd, suggesting that the 2-year-old animals had been exposed to a BTV before they entered the breeding herd.     

Prevalence and geographic distribution of bovine viral diarrhoea (BVD) infection in Finland 1993-1997

Bulk milk samples from every herd supplying milk to dairies in Finland were examined for the presence of antibodies to BVD virus (BVDV) annually during 1993-1997. The highest prevalence, 0.99% in 1994, declined to 0.37% in 1996; however, this favourable trend appeared to discontinue in 1997, where the prevalence remained at 0.41%. In 1993, sera of all individual animals from bulk milk antibody-positive herds were examined for the presence of these antibodies. Since 1994, only sera of animals from herds with a bulk milk absorbance reading greater than 0.250 in the EIA test were examined individually. Three geographic foci of BVDV antibody-positive dairy herds were resolved in 1994, one in the north-western, another in the eastern and a diffuse third in the southern part of Finland. A distinct limiting of the spread was apparent in 1997. Beef cattle were also studied during 1993-1997; in 1993 breeding units, in 1994 mainly beef suckler herds and in 1995-1997 serum samples of beef animals at slaughter were examined for the presence of antibodies to BVDV. The prevalence of seropositive herds in 1993 and 1994 was 30.2% and 3.2%, respectively, while the prevalence among slaughter animals ranged 0.8-1.6%. Seronegative animals in herds with > 50% of seropositive animals were examined for the presence of BVD-virus. A total of 40 dairy herds and two beef herds with viraemic (persistently infected, PI) animals was encountered during 1993-1997. A comprehensive control programme and a more specific, cooperatively funded eradication programme for dairy cattle were launched in 1994. These programmes most probably contributed to the decline in prevalence during 1994-1996.     

Natural changes in the spread of bovine viral diarrhoea virus (BVDV) among Estonian cattle

The results of a survey conducted during 1993-2000 to study the spread of bovine viral diarrhoeal virus (BVDV) among Estonian cattle are presented. The BVDV infection status of a representative random sample of cattle herds housing 20 or more dairy cows was established to estimate the prevalence of herds with active BVDV infection [potentially having persistently infected (PI) cattle--suspect PI herds]. The herds investigated comprised approximately 70% of all Estonian dairy cows. The BVDV infection status was established in 315-350 herds (making the sampling fraction about 20%) during three sampling periods: 1993-95, 1997-98, 1999-2000. BVDV antibodies were detected in herd bulk milk samples and/or sera from young stock by a liquid-phase-blocking enzyme-linked immunosorbent assay developed in the Danish Veterinary Institute for Virus Research. The results of the survey demonstrate the reduction in the prevalence of herds with active BVDV infection in the studied fraction of the Estonian cattle population. During the first sampling period (1993-95) a prevalence of 46% (+/- 5%) for suspect PI herds was observed, during the second sampling period this prevalence was 16% (+/- 3%) and in the third period it was 18% (+/- 3%). As there is no control programme for BVDV in Estonia, the observed changes reflect the natural course of the infection in the study population. A possible cause for these changes is the decreased trade in breeding animals as a result of the economic difficulties present in cattle farming during the study period. The farming practices (most large herds are managed as closed herds) and the low density of cattle farms have obviously facilitated the self-clearance of herds from the BVDV infection, diminishing the new introduction of infection into the herds.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.     

The relationship between the occurrence of undifferentiated bovine respiratory disease and titer changes to Haemophilus somnus and Mannheimia haemolytica at 3 Ontario feedlots.

The association between exposure to Haemophilus somnus and Mannheimia haemolytica (formerly Pasteurella haemolytica) and the risk of undifferentiated bovine respiratory disease (UBRD) was investigated using serological evidence of exposure coupled with a factorial design vaccine field trial. Measures of previous exposure (titer at arrival) and current exposure (titer increase in the study period) to these agents were used. The vaccine field trial involved systematic allocation of animals into groups that received either a M. haemolytica vaccine, an H. somnus vaccine, a combined M. haemolytica and H. somnus vaccine, and an unvaccinated control group. Serum was collected from the 852 animals enrolled to determine titers to H. somnus, M. haemolytica, bovine coronavirus and bovine viral diarrhea virus. Vaccination with H. somnus in combination with M. haemolytica and with M. haemolytica alone reduced the risk of UBRD. The odds ratio for vaccination with H. somnus alone and UBRD risk suggested some sparing effect, but the 95% confidence limits included unity. There was no association between serological evidence of concurrent exposure to M. haemolytica and UBRD occurrence. There was an association between titer change to H. somnus and UBRD risk. However, the association changed with time of BRD treatment; animals diagnosed and treated for UBRD on or after day 10 showed little evidence of exposure to H. somnus, despite evidence of natural H. somnus exposure in the unvaccinated group. The association between titer change to H. somnus and UBRD occurrence seen in this study may be a consequence of prolonged exposure to antibiotics, rather than a causal association.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.