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.     

RESPONSE OF CATTLE TO INOCULATION WITH ATYPICAL MYCOBACTERIA OF BOVINE ORIGIN

SUMMARY Ten strains of atypical mycobacteria originally isolated from cattle were inoculated into cattle. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. Four weeks and 10 weeks after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and the appropriate homologous PPD. Three strains produced a significant level of sensitivity to bovine PPD at the 4-week test but by the 10-week test no animal gave a significant response. The sensitivity to all tuberculins was less at the 10-week test than at the 4-week test. At both tests the response to avian PPD was equal to or exceeded that to bovine PPD. Of 4 strains originally from cattle sensitive to mammalian tuberculin only 2 produced sensitivity of bovine PPD in this experiment. Cultural isolation of mycobacteria from necropsy material was correlated neither with sensitivity to bovine PPD nor with the presence of lesions.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

猪、牛源非结核分枝杆菌的分离与鉴定

采集猪颌下淋巴结,猪肠系膜淋巴结,牛颌下淋巴结和牛肠系膜淋巴结各200份,使用改良罗氏培养基进行分离培养和传代培养,通过进行生长特性试验、生化鉴定试验和鉴别培养基生长试验对所分离出的分枝杆菌进行菌型鉴定。结果显示:猪颌下淋巴结中分离出耻垢分枝杆菌4株,鸟分枝杆菌4株,胞内分枝杆菌2株,胃分枝杆菌2株,蟾蜍分枝杆菌1株,龟分枝杆菌龟亚种杆菌1株;猪肠系膜淋巴结未分离出非结核分枝杆菌;牛肠系膜淋巴结分离出瘰疬分枝杆菌2株,加地斯分枝杆菌1株;牛肠系膜淋巴结分离出,瘰疬分枝杆菌5株,金色分枝杆菌2株,戈登分枝杆菌2株,蟾蜍分枝杆菌2株。猪、牛的感染率均为3.5%。     

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.     

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.     

牛源性分枝杆菌的犊牛变态反应试验

本文以我国自行分离的牛源性分枝杆菌回归本动物试验。将牛分枝杆菌等７种分枝杆菌注射犊牛，再以其分枝杆菌素进行变态反应，以观察其交叉反应的情况。试验结果表明：各分枝杆菌均表现出相互交错的变态反应。提示在结核、副结核的变态反应检疫过程中，应注意由其它分枝杆菌引起的非特异性干扰。     

The epizootiological significance of positive bacteriological findings on Mycobacterium tuberculosis and Mycobacterium bovis in humans

The relation between the active form of tuberculosis in persons working in agriculture and incidence of tuberculosis in cattle was analyzed in 1974 to 1978, i.e. in the period after the elimination of bovine tuberculosis in Czechoslovakia (in 1968). M. tuberculosis was isolated in 15 cases and M. bovis in four cases of persons employed by the farms on which the Regional Hygienic Station, Brno, was responsible for the microbiological diagnostics of tuberculosis. Direct contact with animals was demonstrated in eight patients; M. tuberculosis was isolated from seven of these patients and M. bovis from one. Seven cattle herds were exposed to spontaneous infection by M. tuberculosis and in one of them tuberculosis was not demonstrated during complex examination. In three herds the examination revealed only a sensitivity of cattle to mammalian tuberculin. In other three herds tuberculosis was detected by allergic tests, patho-anatomic examination and bacteriological examination. M. tuberculosis in cattle was detected in two herds. The occurrence of bovine tuberculosis caused by a cattle tender with a positive finding of M. bovis in sputum was demonstrated in one herd. Virulence for the tested cattle was found in one strain (isolated from a mesenterial lymph node of cattle) of the four strains of M. tuberculosis used for the experimental infection of 17 animals. On the other hand, in three strains of M. tuberculosis, trials with experimental infection demonstrated only allergy to mammalian tuberculin and changes at the sites of subcutaneous inoculation of mycobacteria of regressive nature; these mostly disappeared within 90 days from infection.     

Lymphocyte blastogenesis in bluetongue virus or Mycobacterium bovis-inoculated bovine fetuses

Bovine fetuses were inoculated with Mycobacterium bovis, bluetongue virus or placebo at approximately 125 days of gestation, and blastogenic responses of peripheral blood lymphocytes and lymph node cells were determined at various time intervals after inoculation. Lymphocytes from all fetuses were stimulated by phytohemagglutinin, concanavalin A, and pokeweed mitogen, and peripheral blood lymphocytes gave consistently greater stimulation indices than did prescapular lymph node cells. Bluetongue virus infection did not consistently suppress mitogen induced lymphocyte blastogenesis. Lymphocytes taken from fetuses at 20 or 50 days after Mycobacterium bovis inoculation were not stimulated by purified protein derivative (PPD), whereas lymphocytes taken from adult cattle at similar intervals after Mycobacterium bovis inoculation were stimulated by PPD. Although lymphocytes from bovine fetuses may be stimulated by mitogens, antigen specific blastogenesis to a known inducer of cellular immunity was not detected by 175 days of gestation.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.     

ISOLATION AND CHARACTERISATION OF BOVINE HERPESVIRUS MAMMILLITIS VIRUS AND ITS PATHOGENICITY FOR CATTLE

Ten viruses isolated from swabs and vesicular fluid collected from the teats of dairy cattle on 4 properties in Northern Victoria were identified as bovine herpes mammillitis (BHM) viruses by their physico-chemical and morphological properties and serological relationship to each other and a Scottish Strain of BHM virus. The viruses, isolated in bovine kidney and testicular cell cultures, produced cytopathic effects characterised by very large syncytia and eosinophilic intranuclear inculsion bodies. The intradermal inoculation of BHM virus into two cattle produced necrosis and ulceration of the skin of the teats about the area of inoculation and the development of serum neutralising antibody. After healing of the ulcers on day 37 after inoculation, the cattle were intravenously inoculated with corticosteroid for 6 days but BHM virus was not re-isolated from the teat skin or vaginal or nasal swabs.     

Isolation and identification of mycobacteria from cattle slaughtered in Pakistan.

Specimens of lung, liver and mesenteric lymph node from cows and buffaloes slaughtered in the Lahore area were cultured to investigate the type of mycobacteria involved in bovine tuberculosis. Employing the concentration method, 56 out of 530 cattle were found to be culture positive for acid-fast bacteria, 48 being Mycobacterium bovis and eight atypical mycobacteria. No M tuberculosis or M avium was isolated. Most of the isolated M bovis strains were found to be highly virulent for rabbits.     

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.