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The standard complement fixation (CF) test, a commercial agarose gel immunodiffusion (AGID) test (ImmuCell Corporation, Portland, Maine), and two commercial enzyme-linked immunosorbent assays (ELISAs; Allied Laboratories, Glenwood Springs, Colo. [Allied ELISA], and the CSL, Limited, [Parkville, Victoria, Australia] enzyme immunoassay [CSL ELISA]) were evaluated by using sera from the Repository for Paratuberculosis Specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of dairy cattle without diarrhea or weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. None of the cattle evaluated had been vaccinated for paratuberculosis. Among 177 M. paratuberculosis-infected cattle, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA had test sensitivities of 38.4, 26.6, 58.8, and 43.4%, respectively, and specificities of 99.0, 100.0, 95.4, and 99.0%, respectively. Only 108 of the infected cattle were confirmed by culture or by a commercial DNA probe (IDEXX Corporation, Portland, Maine) to be shedding the organism in fecal samples at the time of serological testing. Among the 108 M. paratuberculosis fecal shedders, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA were positive for 54.6, 40.7, 65.7, and 56.5% of the cows, respectively; and among the 69 cows that were nonshedders, the tests were positive for 14.5, 4.3, 47.8, and 24.6% of the cows, respectively. There was a significant difference (P < 0.05) in the rate of positive test results for all four tests between these two groups of animals. The CF test performed well when it was compared with the other serological tests if a titer of >/- 1.8 was classified as a positive test result.

To evaluate the usefulness of pulsed infrared solid-state Holmium:Yttrium-Aluminum-Garnet (Ho:YAG) laser in neurosurgery, acute responses of brain tissue to Ho:YAG irradiation and to investigate the healing processes in rats and rabbits.Animals were divided into groups according to different survival time and laser irradiation mode. Craniotomy was made and laser energy was delivered to the brain surface by two irradiation modes: 1) contact mode with the fiber in contact with the brain surface; and 2) non-contact mode with the fiber tip 5 mm above the brain surface. Gross observations were made and histological changes were studied.Acute responses were studied on rats' brain. The contact mode produced a crater lined with less than 1 mm thick thermally coagulated layer of brain tissue. The non-contact mode effectively vaporized the brain tissue even with water irrigation. Good homeostatic effect was achieved. The shock waves generated by the pulsed delivery of laser energy, however, impacted on the brain resulting in debris spreading and brain vibration. The healing processes were studied on rabbits' brain one day to six weeks after irradiation. The lesions produced by the contact mode were narrow and sharply defined. With the non-contact mode, the thickness of the coagulated layer at the bottom of the crater varied between 0.8 and 1.1 mm.Ho:YAG laser should be a clinically useful tool in neurosurgery because of its good ablation effect, shallow penetration and convenient optic fiber transmission system. Meanwhile, a continuously waved Ho:YAG laser is desired to reduce the shock wave impact.

To use an on-farm recording form to quantity the effect of specific management practices on apparent prevalence of Mycobacterium paratuberculosis in dairy cattle herds.Epidemiologic survey.26 commercial Wisconsin dairy farms.An instrument was developed on the basis of literature review and expert interviews to quantify on-farm management practices associated with increased apparent prevalence of M paratuberculosis. On-farm visits were conducted to assess how specific management practices were conducted. Apparent prevalence of M paratuberculosis infection was measured for all animals20 months old on all farms, using a commercial ELISA. Regression analysis was used to identify management variables that were significantly associated with apparent prevalence of M paratuberculosis.Regression analysis (R2 = 0.90) identified that high scores for environmental conditions, newborn calf care, grower calf care, bred heifer care, and manure handling were significantly associated with M paratuberculosis apparent prevalence in Wisconsin dairy herds.Environmental conditions, newborn calf care, grower calf management, bred heifer management, and manure handling factors may serve as a prioritized checklist for instructing owners and managers where to place emphasis in changing management practices to limit M paratuberculosis prevalence. Likewise, the factors identified as having low association with apparent prevalence may be de-emphasized in control programs, allowing dairy managers to focus time and finances on more effective components of an M paratuberculosis control program.

An ELISA was developed to detect antibodies to the 41-kd flagellin (P41) of Borrelia burgdorferi in serum obtained from cattle. Absorption studies, immunoblot analysis, immunoelectron microscopy, and correlation of results of the P41-ELISA and the P39-ELISA as well as measurement of the antibody to P41 in calves challenge-exposed with Borrelia theileri were used to assess the specificity of the P41-ELISA. Antigens derived from Escherichia coli, Leptospira interrogans serovar hardjo, and B burgdorferi were used for absorption studies and immunoblot analysis. Antibodies to P41 of B burgdorferi cross-reacted with antigens of E coli, but were not cross-reactive with L hardjo. A value 3 SD higher than the mean of the negative-control population of cattle was defined as the minimum value (cutoff value) for a positive result by the P41-ELISA. Use of this value for classification of test results reduced the predicted rate of false-positive results attributable to E coli cross-reactivity to 1%. Immunoblot analysis revealed that test-positive serum from cattle reacted mainly with 41-, 39-, 34-, and 31-kd proteins of B burgdorferi, as well as several smaller proteins. Immunoelectron microscopy revealed that serum from cattle that was test-positive by the P41-ELISA bound to the flagellin and outer membrane of B burgdorferi. Results of absorption studies, immunoblot analysis, and immunoelectron microscopy were correlated and indicated that serum from cattle that was test-positive by P41-ELISA had stronger reactivity to B burgdorferi antigens than to antigens of E coli or L hardjo.(ABSTRACT TRUNCATED AT 250 WORDS)

A random sample of Wisconsin dairy herds, stratified by herd size, were tested for paratuberculosis by use of an absorbed ELISA procedure. The ELISA was optimized for overall accuracy by means of receiver operating characteristic curve analysis, and had a sensitivity and specificity of 50.9 and 94.9%, respectively. Herd prevalence was analyzed for correlation with responses to a management practices questionnaire completed by the herd owners. One hundred and fifty-eight herds and 4,990 cattle were tested. Of these, 50% of herds and 7.29% of cattle had positive test results. Calculation of true prevalence from the apparent prevalence indicated that 4.79% of cattle and 34% of the Wisconsin dairy herds tested had serologic evidence of paratuberculosis. Among the 54 herds classified as positive on the basis of true prevalence estimation, the mean number of test positive cattle was 20.3%. The geographic distribution of herds with positive results was not uniform. More infected herds were found in the southern and western districts of Wisconsin than in the eastern district. The west-central district had a larger number of infected herds than did other districts. By use of chi 2 analysis, the only management factor found to be significantly associated with herd prevalence was housing of calves after weaning (P = 0.03). Specifically, in herds with higher prevalence, calves were separated after weaning into calf barns and hutches rather than into pens in the cow barn more often than in herds with lower prevalence. This factor was also considered significant by use of logistic regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to successfully apply laboratory tests to the solution of clinical problems, a clinician needs to select tests that are appropriate to the suspected disease, collect and submit the necessary specimens, and recognize sources of variation that affect test results. The clinician must have confidence in the accuracy and precision of analytical methods used to produce results. An understanding of reference ranges and test performance in healthy and diseased animals is necessary for accurate interpretation and clinical decision making.

Commercial laying hens were examined microbiologically at necropsy 31 or 42 weeks after aerosol vaccination with the F strain of Mycoplasma gallisepticum (MG). Mycoplasma isolates were studied in Western blots probed with polyclonal antiserum raised in rabbits to F strain immunogen. The persistence of the vaccine strain was demonstrated by detection of a 75-kilodalton immunoreactive protein, which was present in all MG isolates and thought to be a unique marker of the F strain. Use of PCA-F to probe Western blots allowed simultaneous identification of non-MG isolates, non-F strains of MG, and the F strain of MG.

The efficacy of two media, an Edward-type medium (EPJ) and a modified SP4-type medium (SP4-PS), were compared for primary isolation of Mycoplasma gallisepticum (MG) from commercial layer chickens (n = 58) vaccinated with the live F strain of MG. Three groups of chickens that differed in the interval after vaccinal exposure to the F strain (32, 41, and 102 weeks) were studied at necropsy. Mycoplasma isolation was attempted from the trachea, sinus, and cloaca using lavage and swab techniques but was successful only from the trachea and sinus. MG was isolated from 39 (8.4%) of 463 culture attempts from 58 tracheal inocula and 58 sinus inocula. Isolation of MG was successful more frequently using EPJ medium than SP4-PS medium, and isolation occurred more often from the sinus than from the trachea. Of the 58 chickens studied, 19 (33%) were shown by culture to be infected with MG. Isolation was successful only from 32- and 41-week post-vaccination exposure groups. However, all chickens studied were serologically positive for MG antibody by rapid-plate agglutination and hemagglutination-inhibition assays.

An immunobinding assay capable of distinguishing among Mycoplasma synoviae, M. gallisepticum, M. gallopavonis, and M. meleagridis was developed. A low-protein-binding membrane filter served as the solid support, and a 96-well microsample filtration manifold was used. The assay detected 50 ng of mycoplasmal protein, or approximately 3 x 10(4) colony-forming units, from pure or mixed cultures. No cross-reactions were observed among the mycoplasma species tested. The assay was inexpensive, easy to interpret, and required approximately 3 hr to perform. The 96-well format allowed screening of numerous cultures and use of several antisera in a single assay.

Medical records from 333 cats with diabetes mellitus were studied retrospectively, using epidemiologic methods to determine the incidence of and risk factors for diabetes mellitus in this species. Abstracts were derived, using the Veterinary Medical Data Program with its 17 participating academic institutions in the United States and Canada. A reference population of 135,651 cats was derived from the same hospital population and time span (July 1980 to June 1986). The incidence of diabetes mellitus in cats was determined to be 2.45 cases/1,000 cat-years-of-risk during the 6-year study period. Breed had no detectable effect on risk for diabetes mellitus. In contrast, body weight, age, gender, and neutering had a significant (P less than or equal to 0.01) effect. Body weight of cats was categorized as being less than or greater than or equal to 6.8 kg. The higher body weight, probably indicating obesity, contributed a 2.2-fold increase in risk, even after adjustment for age and gender (adjusted odds ratio). The etiologic fraction for high body weight was 3.8%, suggesting that an estimated 3.8% of cases of diabetes mellitus was attributable to this factor alone. Over 50% of diabetic cats were greater than 10 years old, and the etiologic fraction for age greater than 7 years alone was 73.5%. Age was a significant (P less than 0.001) and the most important single risk factor for development of the disease in cats, with adjusted odds ratios of 8.3 and 14.4 for age 7 to 10 years and greater than 10 years, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Five strains of Mycoplasma gallisepticum (MG) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for the presence of carbohydrate-containing components. Staining with periodic acid-Schiff (PAS) demonstrated carbohydrate components in three of the five strains studied. The PAS-reactive bands counterstained for protein, indicating a possible glycoprotein nature. Western blot analysis using three biotinylated lectin probes demonstrated the presence of additional glycoconjugates in the blot profiles of each MG strain. The carbohydrate specificity of lectin binding was demonstrated by competition experiments using specific sugars. Differences in the number, electrophoretic mobility and the morphology of PAS and lectin reactive bands were reproducible among separate preparations of each MG strain. These findings indicate substantial phenotypic diversity among the five MG strains in their ability to produce or acquire glycoconjugates.

Twenty-one dogs with idiopathic superficial recurrent pyoderma were entered into a double-blind, placebo-controlled study to evaluate the efficacy of a commercial staphylococcal bacterin. The study spanned an 18-week period. All dogs were administered sodium oxacillin orally for the initial 6 weeks of the study. Dogs were given the bacterin or placebo SC, twice weekly at 3- or 4-day intervals, beginning at week 0 and continuing for 18 weeks. Dogs given antibiotics plus the bacterin (n = 13) had a significantly (P less than 0.05) better treatment response than those given antibiotic plus placebo.

The California Dairy Herd Improvement Association records of 29 California dairies which experienced clinical mycoplasmal mastitis between January 1975 and December 1977 were examined and compared to the records of selected control herds. A 15-fold greater risk of clinical mycoplasmal mastitis was found among large herds as compared to small herds. On average, herds with clinical mycoplasmal mastitis culled 5 % more cows than did control herds (33 % vs 28 %). No difference was found in average milk production. These findings compare closely with the findings of a previous report where infected herds were identified by the presence of pathogenic mycoplasma in bulk tank milk. The similarity of results support the use of frequent bacteriologic culture of bulk tank milk as a routine surveillance strategy for mycoplasmal mastitis in endemic areas. The similarity of results also supports the use of routine clinical diagnostic data in the study of the epidemiology of diseases of veterinary importance.

Third-stage and fourth-stageDirofilaria immitislarvae exhibited positive thermotaxis when placed in a thermal gradient. Negative thermotaxis was not observed. Positive thermotaxis may be important for the successful transmission and for directing third and fourth-stage larval migration toward predilection sites in the Host.

Microbiological cultural, cytologic, and immunologic observations were made on 30 calves. The eyes, nares, and bronchioalveolar region were subjected to microbiological cultural examination for mycoplasmas. Four of the examinations of 30 eyes, 15 of those of 30 nasal tissues, and 25 of those of the 30 bronchioalveolar regions from the 30 calves were positive for mycoplasmas. Mycoplasma bovis and M bovirhinis were the most prevalent species. Cytologic examinations of peripheral blood and bronchioalveolar washes did not show pathologic changes. Results of indirect hemagglutination, enzyme-linked immunosorbent assay, lymphocyte-stimulation tests on peripheral blood cells, and skin testing demonstrated only a low prevalence of immune recognition of M bovis. Infection and immune response were studied in 3 calves for 10 weeks before, and for 4 weeks after, intratracheal administration of live M bovis.

The effect of vaccination on milk production was evaluated in vaccinated and control cows experimentally challenged in two of four quarters with live Mycoplasma bovis. During the first three weeks after experimental challenge, six of eight unchallenged quarters on vaccinated cows and seven of eight unchallenged quarters on control cows became infected. Most of these quarters secreted normal milk, with negative California Mastitis Test scores and maintained normal milk production throughout most of the study (although some quarters on control cows remained infected). All challenged quarters became infected, had strong California Mastitis Test reactions, and had a drastic (greater than 85%) loss in milk production. Thereafter, four of eight challenged quarters on control cows remained infected, had mostly positive California Mastitis Test scores, produced mostly normal-appearing milk, and recovered some productive capabilities. By the end of the study no M. bovis could be recovered from challenged quarters on vaccinated cows and the milk appeared mostly normal. The California Mastitis Test scores on these quarters, however, remained elevated and milk production remained very low.

The correlation between 24-hour urine protein excretion and the protein-to-creatinine ratio (U-P/C) from random, voided urine specimens was assessed in 16 healthy Beagles (9 to 11 months old) and in 14 dogs with suspected renal proteinuria. Initially, a voided urine specimen was obtained from each dog, and the U-P/C was determined. An attempt was not made to standardize the time of collection of the voided urine. Subsequently, each dog was placed in a metabolism cage, and 24-hour urine specimens were collected for quantitative protein analysis. The Coomassie blue technique was used to measure urine protein. The correlation between the U-P/C and the 24-hour urine protein excretion (mg/kg/24 hr), evaluated by linear-regression analysis, was found to be significant (r = 0.975, P less than 0.01). These results substantiate previous findings and indicate that random, voided urine specimens may be used to compute the ratio and to accurately reflect 24-hour urinary protein loss in the dog.

Four cows were vaccinated with Mycoplasma bovis five times at two week intervals: three times subcutaneously in Freund's complete adjuvant, and two times with M. bovis alone in two of four quarters by intramammary infusion. The effect of vaccination on the immune response was evaluated in the serum and whey of the four vaccinated and control (placebo) cows experimentally challenged in two of four quarters with live M. bovis. Vaccination resulted in markedly increased M. bovis-specific, serum IgM, IgG and IgG2, but not IgA, reactivity. Challenge exposure with live M. bovis by intramammary infusion resulted in high specific serum IgM, IgG1 and IgG2 reactivity and a noticeable IgA response in both vaccinated and control cows. Whey from quarters on vaccinated cows had elevated, specific IgG1 reactivity at the time of challenge but no other differences were observed. Challenge exposure with live M. Bovis resulted in high antibody levels of all isotypes in quarters which were challenged, but highly elevated reactivities in unchallenged quarters occurred only with IgG1 and IgG2. These results indicate that vaccination elevated M. bovis-specific IgG1 but not other immunoglobulin reactivity in quarters on vaccinated cows, and that live organisms are necessary to elicit a local, specific IgA response.

Enzyme-linked immunosorbent assay, using monoclonal antibodies, was used to detect Mycoplasma bovis in milk samples from a dairy experiencing an epizootic of mastitis. This method was specific (100%) for M bovis. Broth enrichment increased the sensitivity from 65% to 86%, compared with standard culture methods.

The enzyme-linked immunosorbent assay (ELISA) was optimized for detection of Mycoplasma bovis-specific IgG in bovine serum. The test is rapid, reproducible, convenient, and sensitive. With this assay, the serum from naturally infected and immunized calves demonstrated the presence of antibodies early in infection and rapid increase in titers during the infection. Cross-reactivity of bovine serum with mycoplasma antigens of bovine, caprine, avian, and environmental sources was tested with this assay system. Cross-reaction was measurable in all instances, with the strongest reaction measured between M bovis and M agalactiae.

Polyclonal antisera (PCA) to three Mycoplasma gallisepticum (MG) strains (F, S6, and A5969) produced in rabbits were used in enzyme-linked immunosorbent inhibition assay (ELISIA) and Western blot analysis to examine antigenic variability among these strains of MG. In ELISIA, inhibiting antigen of the same strain used for immunization always led to the greatest percentage inhibition of PCA reactivity. Western blot analysis of antigens of four MG strains demonstrated both common and restricted patterns of immune recognition among the three PCAs.

Most bovine mastitis due to mycoplasmas is initiated by passage of mycoplasmas through the teat canal into the teat and gland cisterns. Within a few days, mycoplasma numbers increase to as much as 10(6) or 10(8), and the cows react with a strong inflammatory response. Alveolar epithelium undergoes degenerative changes and exudate replaces milk secretion. The interstitium between alveoli is invaded with lymphocytes, macrophages, plasma cells and fibroblasts. The extent and duration of these changes vary greatly. In milder cases, they may be reversed within days or weeks with a return to normal or reduced milk production. Often, destruction and atrophy of alveoli are complete with extensive fibrosis throughout the udder. Milk ducts may undergo invasive and obliterative fibrosis. Cell-mediated responses are suppressed, while hypersensitivity is suspected of enhancing the adverse responses. Immunity in cows that recover is variable and of limited duration.

An enzyme-linked immunosorbent assay (ELISA) was adapted to detect Mycoplasma californicum-specific antibodies in bovine serum. Cross-reactive antibody was found in the M californicum-positive reference serum when assayed against each of 7 solid-phase antigens of heterologous mycoplasma species. Cross-reactivity was further demonstrated by inhibition of ELISA reactivity to M californicum solid-phase antigen by incubation of sera with antigen suspensions of each heterologous species. Incubation of test sera with a cross-reacting antigen mixture containing equal proportions of the 7 cross-reactive mycoplasmas was used to minimize cross-reactivity in the M californicum-specific ELISA. Specificity of antibody reactivity to M californicum, as measured by ELISA, was determined by enzyme-linked immunosorbance inhibition, in which sera were incubated with M californicum antigen suspensions before determining ELISA reactivity to M californicum solid-phase antigen. Seropositive and suspect sera (n = 55) were obtained from 3 dairies that had bacteriologically verified epizootics of M californicum mastitis. The percentage of inhibition demonstrated in enzyme-linked immunosorbance inhibition was determined for each serum. Inhibition percentages below the 15th percentile (61% inhibition) of this distribution were classified as nonspecific.

This study characterized the immune responses in four vaccinated and four control cows in response to vaccination and experimental intramammary inoculation with Mycoplasma bovis. Specific antibody responses occurred in serum and milk in response to vaccination and experimental infection. Lymphocytes from peripheral blood, but not from the mammary gland of vaccinated cows had increased responsiveness to mitogens. No lymphocytes tested were responsive to M. bovis antigen. Both vaccination and experimental infection resulted in skin test reactivity. These results imply that vaccination results in immune responses which may alter the course of experimental M. bovis mastitis, but may contribute to cellular inflammation.

The effect of vaccination on mycoplasmal infection and the cellular inflammatory response was evaluated in 4 vaccinated and 4 control cows experimentally challenged in 2 of 4 quarters with live Mycoplasma bovis. In unchallenged quarters during the first three weeks after experimental challenge exposure, 6 of 8 quarters on control cows, and 7 of 8 quarters on vaccinated cows became infected with low numbers (10(2)-10(4) cfu/ml) of M bovis. During the same period all challenge-infused quarters on both control and vaccinated animals became infected with high numbers (10(9) cfu/ml) of M bovis. Thereafter, all quarters on vaccinated cows became culture-negative for M bovis, while 2 of 8 unchallenged quarters, and 4 of 8 challenged quarters on 3 of 4 control cows remained infected. A cellular inflammatory response as measured by the California Mastitis Test accompanied the experimental infection in proportion to the infection level except in challenged quarters on vaccinated cows after the first three weeks post challenge in which the cellular inflammatory response remained high despite the advent of negative M bovis culture results. This study indicates that the course of experimental M bovis mastitis can be affected by vaccination, and that vaccination results in an adverse cellular inflammatory response in challenged quarters.

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.

The DNA alkaline elution technique provides a sensitive assay for the effects of DNA-damaging drugs in mammalian cells. We have adapted this method to permit measurements of effects on DNA in solid tumors. Human colon carcinoma xenografts in nude mice were treated with a single i.p. injection of 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, and the effects on the DNA were followed for 19 hr. Drug doses in the pharmacological range produced significant reductions in DNA alkaline elution rates in assays in which X-ray was used to introduce a standard frequency of single-strand breaks. These changes in alkaline elution rate were attribute to the production of both DNA interstrand and DNA-protein cross-links, which were distinguished from each other on the basis of the extent to which the effect on elution could be reversed by proteinase K. Crosslinking increased for about 8 hr after treatment with little change thereafter up to 19 hr. A drug-resistant tumor line exhibited substantially less cross-linking than did a drug-sensitive line at all time points examined.

ELISA for use in epidemiologic field studies of bovine mastitis, were developed to measure serum antibody to Mycoplasma bovis and M. californicum. Varying levels of serological cross-reactivity to seven heterologous bovine mycoplasmal species were demonstrated in each assay. Cross-reactivity was minimized by preincubation of cattle sera with suspensions of heterologous mycoplasma antigens, prior to measuring serum antibody to solid-phase antigen. Heterologous absorption improved the immunological specificity of the assays while avoiding the need to prepare species-unique antigens. Serum antibody was measured at one serum dilution. Test results were expressed as a ratio of the reactivity of a positive and a negative reference serum. A negative reference population (n = 127) was assembled. The percentile distribution of ELISA reactivity of these 127 sera were used to establish the classification criteria for each assay. The statistical methods used, while easily applied, were found to be sensitive to outlying values in the reference population. The resulting classification criteria provided controlled or known probabilities of false-positive misclassification in the two ELISA test systems. Sera from cattle with defined exposure histories were tested and classified according to these criteria.