RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA ISOLATED FROM SOIL

Nine strains of atypical mycobacteria and a strain of the rhodochrous taxon, originally isolated from soil samples collected on the subcoastal plains of the Northern Territory, were inoculated into cattle. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. At 4 and 10 weeks after inoculation, the cattle were tuberculin tested with bovine PPD tuberculin, avian PPD tuberculin and the appropriate homologous PPD tuberculin. Six strains induced a significant level of sensitivity to bovine PPD at the 4-week test, but only one animal gave a similar response at the 10-week test. In general, the level of sensitivity to all tuberculins declined between the 4-week and 10-week tests. At both tests the response to avian PPD was equal to, or exceeded, that to bovine PPD. The inoculation of each of the 10 strains resulted in the production of tuberculous granulomas at the subcutaneous sites and similar lesions were produced at the mesenteric lymph node site in response to 2 strains. Mycobacteria were re-isolated from 11 cattle and represented 7 strains. The significance of the soil as a reservoir of atypical mycobacteria and other organisms capable of inducing sensitivity to bovine PPD is discussed.     

THE DURATION OF THE RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA

SUMMARY Each of 4 strains of atypical mycobacteria was inoculated into 2 cattle and the responses of the cattle were studied over the following 52 weeks. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. Within 7 days palpable lesions were produced at the sites of subcutaneous inoculation in response to all the strains. After intervals varying from 3 to 26 weeks, lesions due to 3 of the strains were no longer palpable. The lesion produced in response to the fourth strain, a non-agglutinable serotype of Mycobacterium intracellulare, was still palpable at necropsy, 52 weeks post-inoculation (PI). Of the 8 cattle inoculated with mycobacteria, the latter was the only animal that had a lesion with features consistent with a mycobacterial infection and from which mycobacteria were isolated. The inoculated cattle and 4 uninoculated control cattle were turberculin tested on 8 occasions during the post-inoculation period. Bovine purified protein derivative (PPD), avian PPD and PPD tuberculins prepared from each of the atypical mycobacteria were used. In inoculated cattle, sensitivity to both avian and bovine PPD was short lived, significant levels not persisting in any animal beyond 16 weeks PI. From the results of intradermal tests on the control cattle, a 95% confidence interval for their response to any of the 6 tuberculins used, was found to be ±1.36mm. On this basis all inoculated cattle developed sensitivity to the homologous tuberculin. The animal with mycobacterial granuloma at the subcutaneous inoculation site at necropsy had never developed significant levels of sensitivity to bovine PPD, had not shown significant levels of avian sensitivity after week 16 PI nor had it shown homologous sensitivity after week 22 PI. In all animals the level of sensitivity to bovine PPD decreased between successive tests. This fact could be used to clarify the status of a reactor if non-specific bovine sensitivity was suspected. Alternatively, the comparative intradermal tuberculin test using both bovine and avian PPD may be employed.     

PATHOGENICITY FOR CATTLE OF ATYPICAL MYCOBACTERIA ISOLATED FROM FERAL PIGS AND CATTLE AND THE CORRELATION OF LESIONS WITH TUBERCULIN SENSITIVITY

Two experiments involving the inoculation of cattle with atypical mycobacteria are described. In the first experiment groups of 5 cattle were inoculated either subcutaneously or into a mesenteric lymph node with a strain of M. scrofulaceum or M. intracellulare. Four weeks and 10 weeks after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and homologous PPDs. The pathological changes observed were similar within each group of cattle inoculated with the same strain of mycobacteria. A significant interaction was demonstrated between the strain and the route of inoculation. In the second experiment 17 cattle were similarly inoculated by either of the two routes with 1 of 6 strains of M. intracellulare, a strain of M. scrofulaceum or a strain of Runyon Group IV, all of which had been isolated from feral pigs, or a strain of M. intracellulare of bovine origin. Tuberculin tests were carried out after 4 weeks and 10 weeks. Only the isolate from a bovine lymph node produced a significant level of sensitivity to bovine PPD. Cultural isolation of the mycobacteria from autopsy material was not correlated with the presence of macroscopic lesions nor with sensitivity to bovine PPD. The response to bovine PPD of cattle infected with these atypical mycobacteria decreased between 48 h and 96 h after injection of the tuberculins. As the maximum difference in the response to bovine and avian tuberculins occurs at 72 h a comparative tuberculin test should be read at this time to eliminate non-specific reactors.     

Mycobacterium fortuitum infection interference with Mycobacterium bovis diagnostics: natural infection cases and a pilot experimental infection

Mycobacterium fortuitum and at least 1 unidentified species of soil mycobacteria were isolated from lymph nodes from 4 of 5 African buffalo (Syncerus caffer) that had been culled because of positive test results using the Bovigam assay. The buffalo were part of a group of 16 free-ranging buffalo captured in the far north of the Kruger National Park (South Africa) assumed to be free of bovine tuberculosis. No Mycobacterium bovis was isolated. To investigate the possible cause of the apparent false-positive diagnosis, the Mycobacterium isolates were inoculated into 4 experimental cattle and their immune responses monitored over a 13-week period, using the gamma interferon assay. The immune reactivity was predominantly directed toward avian tuberculin purified protein derivative (PPD) and lasted for approximately 8 weeks. During that period 3 of 4 cattle yielded positive test results on 1 or 2 occasions. The immune responsiveness was boosted when the inoculations were repeated after 15 weeks, which led to 2 subsequent positive reactions in the experimental animal that did not react previously. Including an additional stimulatory antigen, sensitin prepared from M. fortuitum in the gamma interferon assay, showed that it was able to elicit a detectable gamma interferon response in all 4 experimentally inoculated cattle when applied in parallel with bovine and avian tuberculin PPD for the stimulation of blood samples. The implications of occasional cross-reactive responses in natural cases of infection with environmental mycobacteria in the diagnosis of bovine tuberculosis in African buffalo and cattle in South Africa are discussed.     

TUBERCULIN SENSITIVITY OF CATTLE INOCULATED WITH ATYPICAL MYCOBACTERIA ISOLATED FROM CATTLE, FERAL PIGS AND TROUGH WATER

Each of 12 cattle was inoculated either subcutaneously and intradermally or into a mesenteric lymph node with 1 of 8 species of live atypical mycobacteria isolated from cattle, cattle trough water and feral pigs. Seventy-eight days after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and homologous heat-concentrated syntheic medium tuberculins. They were killed 85 days after inoculation. Organisms were cultured from caseous granu-lomas at all sites in cattle inoculated with M. avium serotype 2. M. simiae was recovered from a granuloma at the subcutaneous site. Acid-fast bacilli were isolated from the mesenteric lymph node inoculated with trough water organisms. At 72 h, all the cattle had produced skin reactions of 4 mm or more to the homologous tuberculins and all except 1 produced a similar response to avian PPD. Only isolates of bovine origin sensitised cattle to bovine PPD to this degree, and these reactions were less than the corresponding response to avian PPD.     

Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 2. South-eastern England.

A tuberculin testing trial was carried out in eight counties of south-eastern England to compare the specificity for bovine tuberculosis of Weybridge human PPD with that of Rotterdam bovine PPD. The matching of these two tuberculins for potency in naturally infected cattle had already been established, the bovine PPD being approximately one-and-a-half times more potent than the human PPD per unit of weight. In 1110 cattle in 25 herds with histories of long-standing freedom from tuberculosis and in which non-specific tuberculin sensitivity was present, cross reactions were less to the bovine PPD than to the human PPD, showing that in the environment of this trial the bovine PPD was more specific than the human PPD. Induration diameter was a satisfactory alternative to skin thickening as a measure of tuberculin reactions in cattle under field conditions. Due to the steep slope of the dose-response curves of the avian PPD in the different groups of non-tuberculous cattle, the discriminating power of the comparative test, using avian and mammalian tuberculins, was less at lower doses of tuberculin. Concentrations of 1-0 mg per ml of bovine PPD and 0-5 mg per ml of avian PPD are recommended for use in a comparative tuberculin test.     

Comparison of the specificity of human and bovine tuberculin PPF for testing cattle. 3. National trial in Great Britain.

A field trial on a country-wide basis was undertaken to compare the specificity for bovine tuberculosis of single and comparative tuberculin tests in cattle using either Weybridge human or Weybridge bovine PPD. The tests were made on 10,305 cattle in 179 herds distributed throughout all regions of England, Scotland and Wales. Results showed that a comparative tuberculin test using avian PPD with either human or bovine PPD had a much higher efficiency than a single injection of mammalian tuberculin in the neck of cattle, and confirmed that a comparative test is still essential in the British environment. Weybridge bovine PPD gave significantly better discrimination between tuberculous and non-tuberculous cattle than Weybridge human PPD when used together with avian PPD in a comparative tuberculin test. The diameter of induration gave an absolute measure of the extent of oedema, if present, and induration diameter used in conjunction with skin thickening increased the sensitivity and specificity of the test. Rules of interpretation were developed and are presented for an intradermal comparative tuberculin test in cattle using Weybridge avian and bovine PPDs.     

Diagnosis of Mycobacterium bovis infection in calves sensitized by mycobacteria of the avium/intracellulare group

Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.     

COMPARISON OF THE EFFICIENCY OF TWO DOSES OF BOVINE PPD TUBERCULIN IN SINGLE CAUDAL FOLD TESTS ON AUSTRALIAN CATTLE

SUMMARY: The sensitivity and specificity of 0.2 mg and 0.4 mg doses of bovine PPD tuberculin were compared in Northern Territory beef cattle from tuberculous herds and herds with a prevalence of tuberculosis of less than 0.1%. Reactions were interpreted subjectively by observation and palpation, and were also measured to the nearest mm with calipers at 24 h, 48 h, 72 h and 96 h after injection of tuberculin. All cattle were examined post mortem for the presence of macroscopic and microscopic tuberculous lesions. The apparent specificity of caudal fold tests with 0.2 mg and 0.4 mg doses was determined in cattle in Victoria from tuberculosis-free dairy and beef herds. Victorian cattle reacting to the caudal fold tests were subjected to a comparative intradermal test with 0.1 mg bovine PPD and 2,500 IU avian PPD not less than 42 days later.
Tests with the 0.2 mg dose achieved the highest level of sensitivity of 95.6% at 48 h, 72 h and 96 h, while in tests with 0.4 mg the maximum reached was 94.7% at 72 h. The specificity of tests in Northern Territory cattle ranged from 85.0% to 88.3% with the 0.2 mg dose and from 80.6% to 82.3% with the 0.4 mg dose. The highest specificity was achieved with both doses at 96 h. The apparent specificity of 0.2 and 0.4 mg doses of bovine PPD in tuberculosis-free herds in Victoria was high, a false-positive reactor rate of only 0.6% occurring with caudal fold tests. All false-positive reactions were shown to be non-specific or due to previous experimental sensitisation.     

PATHOLOGY AND TUBERCULIN SENSITIVITY IN CATTLE INOCULATED WITH MYCOBACTERIUM AVIUM COMPLEX SEROTYPES 6, 14 AND 18

SUMMARY Three strains of Mycobacterium avium complex organisms, serotypes 6, 14 and 18 isolated from typical tuberculous lesions in cattle were examined for pathogenicity and ability to sensitise cattle to avian and bovine tuberculin. Each strain caused tuberculoid granulomas at the site of subcutaneous inoculation but no lesions elsewhere. Sensitisation to bovine tuberculin was detected in the caudal fold test in 11 of 18 inoculated animals 8 weeks after injection. In a simultaneous comparative cervical test, reactions to avian tuberculin were much larger than reactions to bovine tuberculin in all inoculated animals.     

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation H-Group, which differed from any known mycobacterial species. Mycobacteria of this H-group had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. H-Group mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing H-Group mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.     

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation “H-Group”), which differed from any known mycobacterial species. Mycobacteria of this “H-Group” had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. “H-Group” mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing “H-Group” mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.     

Comparison of the specificty of human and bovine tuberculin PPD for testing cattle. 1--Republic of Ireland.

A tuberculin testing trial in cattle was carried out in the Republic of Ireland to compare the specificity for bovine tuberculosis of a human purified protein derivative (PPD) tuberculin (Weybridge) with that of a bovine PPD (Rotterdam), and to determine whether discrimination between specific and non-specific reactions to mammalian tuberculin is better with doses of tuberculins smaller than those traditonally used for testing cattle. Tests were carried out in 510 cattle, 395 of which were shown by post mortem examination to be tuberculous and 115 non-tuberculous. Three dilutions at five-fold intervals of both mammalian tuberculins were used together with two dose levels of avian tuberculin PPD (Weybridge), and all reactions were measured both by increase in skin fold thickness and by diameter of induration. In the environment of this trial, the bovine PPD was shown to be more specific for bovine tuberculosis than the human PPD, and particularly in differentiating from "skin tuberculosis". There was no indication of greater specificity at lower doses of tuberculin. Measurement of induration diameter proved a satisfactory alternative method of reading tuberculin reactions in cattle under field conditions.     

The comparative performance of the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations from different manufacturers

Ireland currently obtains its avian and bovine tuberculin purified protein derivatives (PPDs) from a single source. Because problems of supply or quality cannot be discounted, it is prudent that Ireland identify alternative supplier(s) as part of a broad risk management strategy. Therefore, the aim of this study was to compare the performance of a number of different tuberculin combinations (that is, pairings of bovine and avian PPD; with different manufacturers) in the single intradermal comparative tuberculin test (SICTT), as currently performed in Ireland. The study was randomised, controlled and double-blinded. A total of 2172 cattle were used in the study. Each animal was tested using two SICTTs, the first based on the tuberculin combination in current use, and the second using one of six trial tuberculin combinations. Analyses were conducted to compare both reactor-status and skin increase. For each control/trial tuberculin combination, there was good agreement between the control and trial reactor-status. Differences in skin increases were mainly confined to animals categorised as either negative or severe inconclusive. However, the measured differences were minor, and unlikely to have a significant impact on the actual test outcome, either for individual animals or for herds. In conclusion, while further studies determining sensitivity and specificity in Ireland would have to be done in the event of a change in tuberculin PPD there should be minimal disruption of the national programme if alternative tuberculin PPDs meeting WHO, OIE and EU regulations were used. In this study, the precision of the guinea pig bio-assay to assess tuberculin potency was low and therefore Ireland should maintain its practice of periodically assessing potency in naturally infected cattle, even though this is not currently required under WHO, OIE or EU Regulations.     

THE USE OF BOVINE PPD TUBERCULIN IN THE SINGLE CAUDAL FOLD TEST TO DETECT TUBERCULOSIS IN BEEF CATTLE

SUMMARY The efficiency of 2 different doses of bovine PPD tuberculin was compared using the caudal fold test for the detection of tuberculosis in beef cattle. Two matched groups of 98 cattle were selected on the basis of their reactivity to HCSM tuberculin. Cattle in each group were tested with a single 0.1 ml dose of bovine PPD tuberculin containing either 0.1 mg or 0.2 mg bovine PPD respectively. Two further groups of 100 young stock from a herd with an incidence of tuberculosis of less than 0.1% were selected as controls. Tests were interpreted subjectively by palpation and observation and objectively by caliper measurement at 48, 72 and 96 h. All cattle were examined post mortem for the presence of visible lesions.     

The effect of tuberculin testing on the development of cell-mediated immune responses during Mycobacterium bovis infection

Protection against tuberculosis (TB) is associated with Th1-type cell-mediated immunity (CMI). Whilst the intradermal injection of partially purified derivatives of tuberculin (PPD) represents the classic test assessing the delayed type hypersensitivity (DTH) response used in both humans and cattle for diagnosing TB, it has been suggested that the test may modulate host CMI responses. To investigate the kinetics of the development of the DTH response and its subsequent effect on CMI responses, groups of 6-month old calves were inoculated intranasally with 8 x 10(4) cfu of Mycobacterium bovis, subjected to the comparative intradermal tuberculin test (TT) using bovine and avian PPD (PPD-B, PPD-A) at various time intervals post-infection, and immune responses compared. These included DTH, lymphocyte proliferation, IgG production, and synthesis of the cytokines: IFNgamma, IL-10, IL-4, IL-6, and IL-13. All animals were subjected to post-mortem examination. The kinetics of the development of the DTH response assessed in the TT was such that infected cattle could be identified as early as 3 weeks post-infection, which correlated with the detection of an antigen-specific IFNgamma response. Transient increases in plasma-derived IFNgamma as a result of TT during an established TB infection were more pronounced when blood was stimulated with PPD-A compared with PPD-B stimulation. This has the potential to mask diagnosis of infection as a result of the stronger avian-bias if the IFNgamma test is used the week following TT. Disease pathology was not affected by TT. A transient failure to a second TT was observed in 1 of 30 animals and the time (post-infection) at which the TT is administered may be of significance. In serum, IgG responses to PPD-B, which were undetectable prior to TT, were elevated after TT and were most pronounced in cattle that were TT at 6 weeks post-infection. Other cytokines were also affected by the TT; IL-4 mRNA levels increased and IL-6 mRNA levels decreased, whilst PPD-B specific IL-10 protein synthesis was enhanced. These observations may offer the potential for further diagnostic assays that could complement the TT and IFNgamma test.     

COMPARISON OF THE EFFICIENCY OF 4 DOSES OF BOVINE PPD TUBERCULIN IN CATTLE AND GUINEA PIGS: RECOMMENDATIONS FOR THE MOST SUITABLE DOSE FOR USE IN THE SINGLE CAUDAL FOLD TEST IN CATTLE

The potencies of 4 batches of Australian bovine PPD tuberculin relative to the standard British bovine PPD preparation 291 were determined from guinea pig assays.
Using the assayed potencies, it was concluded that doses of 0.08, 0.17, 0.24 and 0.47 mg of bovine PPD were used in single caudal fold tests in 2 trials of Northern Territory cattle. The sensitivity, specificity and test efficiency of the Australian preparations were determined.
At 72 h after injection, the time recommended to read the test, the 0.08 mg dose gave the lowest sensitivity (79.2%) which was significantly different from that of the other 3 doses. This dose also gave the highest specificity (88.9%). However, the 0.24 mg dose gave a specificity of 85.0% and a sensitivity of 95.6% resulting in the highest test efficiency (87.9%).
Change in caudal fold thickness, subjectively assessed reaction and the amount of induration were measured in individual cattle. For tuberculous cattle, regression analysis showed significant relationships (p < 0.01) between the change in caudal fold thickness, subjectively assessed reaction and log concentration of each preparation. However, there was no significant difference between the mean responses observed with the 2 highest doses. Predicted responses for untested doses were determined from the regression equations.
When the results of the trials were considered in relation to factors such as batch variation, minimisation of injection error, likely levels of desensitisation, cost and international implications, it was considered that a dose of 0.3 mg bovine PPD relative to the British standard 291 would fulfil optimum requirements for use in all parts of Australia.     

Specific in vitro lymphocyte response of chickens injected with killed Mycobacterium tuberculosis.

An in vitro microassay for lymphocyte transformation, using 3H-thymidine incorporated into avian peripheral blood leukocytes, is described. The transformation responses of 1 X 10(6) leukocytes stimulated by the nonspecific mitogen, phytohemagglutinin-M (PHA-M), were equivalent with 68- and 92-hour incubation durations. The PHA-M in concentration of 50 micronl/ml produced greater stimulation than did that of 25 or 100 micronl/ml. The transformation response to PHA-M was significantly affected by the lot of bovine fetal serum used to supplement the RPMI 1640 culture medium. The specific antigen, purified protein derivative (PPD) of tuberculin, stimulated significant transformation in cultures of 1 X 10(6) leukocytes from Mycobacterium tuberculosis-sensitized fowl. The PPD at concentration of 50 microng/ml was superior to 100 or 150 microng/ml, and 68-hour incubation was significantly better than 92-hour incubation. The magnitude of the in vitro transformation response to PPD was greater at 6 weeks after sensitization than at 2 or 4 weeks, and it was not directly related to the magnitude of the in vivo wattle test response in sensitized chickens.     

基于结核菌素与CFP10／ESAT6的牛IFN-γ检测法在牛结核病诊断中的比较研究

本研究通过比较三种刺激物（牛结核菌素、禽结核菌素和牛型结核菌特异性抗原CFP10／ESAT6对结核菌素皮内变态反应阳性牛的IFN-γ刺激反应,探讨IFN-γ检测法在我国牛结核病诊断中的应用前景。无菌采集22头结核菌素皮内变态反应阳性牛的血液．肝素抗凝。每1mL全血与1mL RPMI1640完全培养基混合均匀,并加入0.1mL（20μg）PHA（阳性对照孔）、0．1mL（20μg PHA）CFP10／ESAT6融合蛋白、0．1mL（2000U）＆结核菌素（PPD／B）、0．1mL（2500u）禽结核菌素（PPD／A）或等量PBS（无刺激阴性对照）,37℃培养过夜。次日用夹心ELISA法检测各刺激组0．1mL培养上清的IFN-γ,以OD630表示IFN-γ浓度。结果,特异性抗原CFP10／ESAT6刺激组与牛结核菌素（PPD／B）刺激组的IFN-γ反应具有良好的相关性．相关系数为0．84。但CFP10／ESAT6刺激组IFN-γ浓度与牛和禽PPD的比较反应（以两刺激组IFN-γ浓度差值表示）间无相关性,相关系数为-0．11。分析禽PPD组的IFN-γ反应,发现实验牛中有少数牛对禽PPD有反应。以OD630=0．17为阳性反应切割值,牛PPD检出阳性牛21头,CFP10／ESAT6检出20头,牛和禽PPD比较反应（以OD值差值表示）检出14头,禽PPD阳性反应3头。扣除禽PPD阳性反应牛后,牛和禽PPD比较反应与牛PPD刺激组的相关系数增至0．54。结果表明,牛PPD的IFN-γ释放反应检测灵敏度最高。当出现牛型结核菌与环境分枝杆菌混合感染时．应用牛和禽PPD比较反应检测牛结核的准确度低,混合感染牛被误判为结核阴性牛。而基于CFP10／ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性与敏感性。     

Biologic characteristics of mycobacterium strains isolated from cattle from herds with clinical paratuberculosis

In the period from 1983 to 1986, bacteriological examination for paratuberculosis was performed in 263 samples of lymph nodes, intestinal mucous membrane and excrements of cattle, kept on a farm where clinical paratuberculosis occurred. Seventy-nine strains of mycobacteria were isolated during the culturing. On selective agar medium with mycobactin as the growth stimulator, 71 strains were isolated which had failed to grow on the conventional mycobacterium-culturing media. In the subculture, the dependence of mycobacteria on the mycobactin declined and the number of mycobacterium strains growing in the subculture on conventional mycobacting-free media doubled. Two thirds of the mycobacteria which did not depend on mycobactin during growth exhibited the same antigenic properties as Mycobacterium avium 1, 2, 3, 8 during serotypification. Ability to induce sensibility to PPD avian tuberculin or paratuberculin was demonstrated during the bioassays of mycobactin. Almost a half of the strains inducing animals' sensitivity to the above-mentioned allergens were found to be virulent to pullets that had tuberculosis in their parenchymatous organs. Of the laboratory animals, the highest virulence of the mycobactin-dependent mycobacterium strains was demonstrated in mice subjected to intravenous infection, accompanied by hyperplasia of the spleen, with reisolation of the mycobacterium culture within six eight weeks after infection.