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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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

Groups of l8-month-old cattle were inoculated intratracheally with 5 X 10⁵ 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-γ 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-y responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Experimental infection with Mycobacterium avium, serotype 2, 2. Oral infection with large doses of M. avium

Twelve pigs were inoculated orally with Mycobacterium avium. The doses used were 0.5, 2 or 10 mg daily for 5 days, or 10, 50 or 180 mg once (1 mg = 37 × 10⁶ viable units). Two pigs were used per dose, 1 of which was sacrificed 3 days, the other 28/31 days after the last inoculation (Table 1).Three days after inoculation, M. avium was found in the tonsils and in the intestinal mucosa of all 6 pigs, and in the mesenteric lymph nodes of 4. Viable unit counts for tonsils and intestinal mucosa were highest in pigs inoculated with 180 mg×1 and 10 mg×5. Histopathologically these pigs showed activation of the lymphoid tissue in the tonsils, Peyer patches and mesenteric lymph nodes. Twenty-eight/31 days after inoculation a spreading of the infection had taken place in all pigs, most often to the liver, less frequently to the spleen and the lungs. The kidneys and the musculature were not infected (Table 4). A correlation was apparent between the size of dose and the number of viable organisms in the tissues. Divided doses gave about 10 times higher viable counts than a single dose with the same total number of organisms (Table 5).No gross lesions were found 28/31 days after inoculation. Microscopic granulomatous lesions were found in the tonsils of 6 pigs, in the intestinal mucosa of 4 pigs, in the mandibular and mesenteric lymph nodes of 6 pigs, in the retropharyngeal lymph nodes of 3 pigs, and less frequently in the parotid and hepatic lymph nodes (Table 3).Five of 6 pigs were weakly sensitive to avian tuberculin PPD, 1000 t.u. per dose, when tested 22/25 days after inoculation; 1 of these pigs cross-reacted to human tuberculin (Table 2).     

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。     

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs: 3. Oral Infection with Small Doses of M. Avium*

Pigs were inoculated orally with Mycobacterium avium in doses ranging from 15.6 × 10² to 15.6 × 10⁶ viable units daily for 15 days (Table 1). The animals were necropsied 31–32 days after the last inoculation.Pigs given doses of 15.6 × 10⁶ and 15.6 × 10⁵ viable units showed delayed hypersensitivity to avian tuberculin 24 days after the last inoculation (Table 2). The pig inoculated with 15.6 × 10⁶ viable units showed macro- and/or microscopic lesions of the intestines and the liver, and of the mandibular, mesenteric and hepatic lymph nodes. Cultures showed growth of M. avium from the same tissues and from the spleen and the left tracheobronchial lymph node. The pig inoculated with 15.6 × 10⁵ viable units showed a pronounced granulomatous infiltration in the tonsils and the mesenteric lymph nodes. Growth of M. avium was obtained from the tonsils, the intestinal mucosa (Peyer patch) and the mandibular and mesenteric lymph nodes. Viable unit counts were high in the tonsils and in the mesenteric lymph nodes (Tables 3 and 4).Lower doses gave rise to a minimal tissue reaction and/or very low viable unit counts, and are not considered to be capable of producing a progressive tuberculosis.     

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.     

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.     

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.     

Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection

A strain of bovine herpesvirus-4 isolated from cows with mammary pustular dermatitis was used for experimental inoculation of cattle. This strain is serologically indistinguishable from the group prototype Movar 33/63 and from strain DN-599. Seronegative cattle were inoculated IV or by simultaneous intranasal, IV, intramammary (via teat channel), and intradermal inoculations. All inoculated cattle seroconverted. Clinical signs of disease or lesions were not evident, except for a dermal lesion corresponding with one intradermal inoculation site. Virus was recovered from the dermal lesion and was excreted in the milk for 17 days. Virus was recovered from esophagopharyngeal fluid at 9 and 13 days after inoculation. At different times of euthanasia (2 to 14 months after inoculation), virus was recovered from cocultures with bovine lung cells and/or explant cultures of lymph nodes, spleen, tonsils, and, in one case, kidney. In 2 animals, the virus was recovered repeatedly during 1 year from peripheral blood leukocytes by cocultivation with bovine lung cells. The number of infectious leukocytes, as determined by infectious center assay, ranged from less than 1 to 6 infectious cells/10(7) leukocytes.