L. A. Corner

Post mortem diagnosis of Mycobacterium bovis infection in cattle

A tentative diagnosis of bovine tuberculosis can be made following the macroscopic detection at necropsy of typical lesions. Histo-pathological examination of the lesion may increase the confidence of the diagnosis but bacteriological isolation of Mycobacterium bovis from the lesion is the only way to make a definitive diagnosis. The sensitivity of gross post mortem examination is affected by the method employed and the anatomical sites examined. Careful examination of as few as 6 pairs of lymph nodes, the lungs and the mesenteric lymph nodes can result in 95% of cattle with macroscopic lesions being identified. Although during post mortem inspection of carcases at abattoir all the principle sites where lesions are to be found were examined, this procedure was found to be insensitive for the detection of lesions. To determine the significance of cattle that give a positive reaction in diagnostic tests but do not have visible lesions (NVL), a bacteriological examination is necessary. NVLs may be due to early infection, poor necropsy technique or infection with mycobacteria other than M. bovis. M. bovis was found to survive best in frozen tissue and the tissue preservative, sodium tetraborate, was found to have adverse effects on viability. It was found desirable to use two different culture media for the primary isolation of M. bovis; agar media for rapid growth and egg media for control of contamination. Additional control of contamination was achieved without adversely affecting the viability by treating the specimen before culture with 0.075% hexadecylpyridinium chloride. The addition of CO2 to the incubation atmosphere did not enhance the recovery of M. bovis. Conventional identification of isolates of M. bovis is by biochemical tests and cultural characteristics, but methods employing monoclonal antibodies and DNA probes may be used to obtain a rapid identification.

A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1%. The most favourable results were obtained with the interferon gamma (IFN-gamma) assay which had a sensitivity of 81.8% and a specificity of 99.1%. Respective figures for the single intradermal tuberculin test were 68.1% and 96.7%. The use of MPB-70 as the antigen in the IFN-gamma assay reduced the sensitivity of this assay, without producing any useful increase in specificity. The IFN-gamma assay was also demonstrated to be a practical diagnostic test for use with large groups of cattle.

Revaccination of Neonatal Calves with Mycobacterium bovis BCG Reduces the Level of Protection against Bovine Tuberculosis Induced by a Single Vaccination

ABSTRACT Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-γ) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4+, CD8+, and WC1+ γδ T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-γ and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.

Serological reactivity to Mycobacterium bovis protein antigens in cattle

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass. The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.

Determination of the optimum concentration of decontaminants for the primary isolation of Mycobacterium bovis

The majority of tissue specimens submitted for the isolation of Mycobacterium bovis contain contaminating microorganisms and therefore require selective decontamination before bacteriological examination. The purpose of this study was to identify the preferred decontaminant amongst four commonly used reagents. The four decontaminants used in the study were 1-hexadecylpyridinium chloride, sodium hydroxide, benzalkonium chloride and oxalic acid. A comparison was made of the toxicity of the four decontaminants for M. bovis and their ability to control contamination. Used at the recommended concentrations, all reagents showed a significant degree of toxicity. The toxicity of the decontaminants for three field strains and one laboratory strain of M. bovis were similar, but a second laboratory strain, AN5, was more susceptible. It was also observed that as the concentration of each reagent decreased an abrupt change from control to lack of control of contaminating micro-organisms occurred. Hexadecylpyridinium chloride was found to be the best all-round reagent because at concentrations that effectively controlled contamination this reagent was the least toxic to M. bovis. A protocol for handling specimens based on an assessment of the risk of contamination is recommended.

Purification of a Major Mycobacterium bovis Antigen for the Diagnosis of Bovine Tuberculosis

A Mycobacterium bovis antigen has been purified from culture filtrate by chromatofocusing. This antigen is a major component of culture filtrate and cell extracts and shows a considerable degree of micro‐heterogeneity in electric charge and molecular weight. Studies with monoclonal and polyclonal antibodies raised against the purified antigen show that some of its antigenic determinants also occur in higher molecular weight species in culture filtrate and particularly in whole cell preparations. Immunoblotting and ELISA studies, using sera from M. bovis‐infected animals, showed that this antigen is one of the most immunoreactive components of M. bovis, recognized by the majority of animals with detectable antibody response to M. bovis. The specificity of the purified antigen is far superior to that of the crude culture filtrate, with very few false positive results. The purified antigen also elicits strong in vivo and in vitro cell‐mediated responses The amino acid compositions of two variants of this antigen have been determined and found to be similar to that of MPB‐70.

Analysis of ovine IL-1β production in vivo and in vitro by enzyme immunoassay and immunohistochemistry

A monoclonal antibody (mAb) specific for ovine IL-1 beta was produced and, in conjunction with a polyclonal rabbit antiserum, used to develop a sensitive enzyme immunoassay (EIA) for ovine interleukin 1 beta (IL-1 beta). The mAb neutralised the activity of recombinant ovine IL-1 beta (rOvIL-1 beta) and native OvIL-1 in an ovine thymocyte proliferation assay. However, it did not neutralise the biological activity of rOvIL-1 beta in the murine NOB1/CTLL assay. The mAb did not react with rOvIL-1 alpha, IL-2, IL-4, IL-8, tumor necrosis factor-alpha, gamma-interferon or recombinant human IL-1 beta in indirect EIA. Immunohistological staining of activated alveolar macrophages and frozen lymph node sections sections demonstrated that the mAb detected IL-1 beta secreted by ovine macrophages (CD11c-positive). The EIA was highly sensitive, detecting less than 50 pg ml-1 of rOvIL-1 beta and low levels of native IL-1 beta in supernatants from lipopolysaccharide-stimulated macrophages. The EIA did not detect heat-inactivated IL-1 beta.

An experimental ovine foot abscess model using a Fusobacterium necrophorum biotype AB

An experimental procedure is described for the production of foot abscess in sheep that mimics the natural disease. Lesions were produced by the intradermal inoculation of suspensions of Fusobacterium necrophorum biotype AB containing from 5 x 10(2) to 5 x 10(8) bacteria, into interdigital skin devitalized by freezing with liquid nitrogen. A dose of 5 x 10(5) bacteria induced the development of foot abscess in 3 of 4 and 8 of 8 inoculated feet. It was found that to produce foot abscess in devitalized tissue required between 10(3) and 10(6) fewer bacteria than were necessary to produce similar lesions in healthy tissue.

Cloning of cervine interferon-gamma cDNA by polymerase chain reaction.

As the first step in the development of a cervine IFN-gamma assay for the diagnosis of tuberculosis in deer, cervine IFN-gamma, cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-y sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-gamma protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-gamma also detects ovine but not cervine IFN-gamma. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-gamma. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-gamma as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.