Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.

Age of cattle as related to resistance to infection with Mycobacterium paratuberculosis.

Two 1-month-old calves, four 9-month old calves, and 4 adult (5 to 11 years old) cattle were exposed to Mycobacterium paratuberculosis. At 150 days after exposure, all cattle were killed and necropsied, and at that time, tissues of the 1-month-old calves had more bacilli and lesions than did tissues of the 9-month-old calves or the adult cattle. All cattle responded to immunologic tests during the experiment.

Analysis of the effect of inapparent bovine paratuberculosis.

Inapparent paratuberculosis was studied in a Guernsey herd with a history of paratuberculosis (Johne's disease); the herd was maintained at between 900 and 950 cattle. Fecal specimens and intestinal tissue specimens from any of these that were slaughtered were examined culturally for the presence of Mycobacterium paratuberculosis. The reasons given for culling and slaughtering of cows from this herd were compared with infection status determined by culturing. Less than one-third of the culturally confirmed paratuberculous cattle were culled because of clinically apparent paratuberculosis. Mastitis was the reason for culling 3.6% of the non-infected cattle and 22.6% of the cattle with inapparent paratuberculosis. Infertility was also significantly higher in cows with inapparent paratuberculosis than in noninfected cows in the same herd. Separation of parturient dams and calves from the rest of the herd was shown to materially reduce the level of infection and incidence of clinical paratuberculosis.

Subcutaneous exposure of calves to Myobacterium paratuberculosis compared with intravenous and oral exposures.

Three groups of calves, 21 days of age, were exposed to Mycobacterium paratuberculosis by oral, intravenous, and subcutaneous routes. Fecal cultural examination and intradermal tests were made, and at 150 days after exposure, the calves were euthanatized and tissues were collected for histopathologic and bacteriologic cultural examination. Calves in the 3 groups were infected. Those exposed intravenously had significantly higher colony counts than did either of the other groups. There was no significant difference between colony counts of cultures from tissues of calves exposed orally and those of calves exposed subcutaneously.

In vitro lymphocyte transformation as a herd survey method for bovine paratuberculosis.

The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.

Intrauterine inoculation of Mycobacterium paratuberculosis into guinea pigs and cattle.

Mycobacterium paratuberculosis was inoculated intrauterinely into guinea pigs and cattle near the time of insemination to assess the effect and subsequent distribution of the organisms. Guinea pigs inoculated intrauterinely with 1 of 3 concentrations of M paratuberculosis or not inoculated were caged with male guinea pigs for 10 days. Guinea pigs given the largest dose had the lowest rate of pregnancy when examined. At necropsy, tissues were cultured to determine extent of infection. Abortion followed hypersensitivity reactions to johnin in some highly infected animals. Mycobacterium paratuberculosis was recovered in culture from the body and horns of the uterus of cows necropsied 1, 2, 3, and 7 days after inoculation and from 1 of 3 cows necropsied 2 weeks after inoculation. The organisms were not detected in extrauterine organs of any cattle or in uterine specimens taken at 3 or 4 weeks after inoculation. The evidence indicates that the small numbers of M paratuberculosis sometimes found in semen from paratuberculous bulls probably would be destroyed, rather than leading to systemic infection of the dam or to persistent hypersensitivity.

Comparison of techniques for measuring the local and systemic responses to tuberculin in cattle.

Thermography of tuberculin skin reactions in cattle sensitized with Mycobacterium avium, Mycobacterium bovis, or Mycobacterium paratuberculosis was rapid and sensitive, but was not as specifically related to the homologous sensitization as was increased skin thickness at 48 and 72 hours. Systemic temperature responses were more completely monitored by radiotelemetry of intraperitoneally located temperature transmitters than by rectal thermometry, but the temperatures indicated by the 2 methods were nearly identical. The intraperitoneal transmitters showed that the systemic responses consisted of 1, 2, or 3 temperature peaks, and in 1 animal which had previously been exposed to a heterologous tuberculin, a hypothermic response developed. The systemic temperature responses were more specifically related to homologous sensitization than were dermal responses, but were more cumbersome to measure.

Prevalence of Mycobacterium paratuberculosis in ileocecal lymph nodes of cattle culled in the United States.

During 1983 and 1984, ileocecal lymph node specimens were obtained from clinically normal cattle at 76 US Department of Agriculture-inspected cull cattle slaughterhouses in 32 states and Puerto Rico. Mycobacterium paratuberculosis was isolated from the lymph nodes of 119 of 7,540 cattle. The prevalence of bovine paratuberculosis was 1.6% overall, with 2.9% in dairy culls and 0.8% in beef culls. The prevalence for females and males did not appear to differ significantly.