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.     

The comparative performance of the single intradermal test and the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations of differing potencies

In national bovine tuberculosis (BTB) control programmes, testing is generally conducted using a single source of bovine purified protein derivative (PPD) tuberculin. Alternative tuberculin sources should be identified as part of a broad risk management strategy as problems of supply or quality cannot be discounted. This study was conducted to compare the impact of different potencies of a single bovine PPD tuberculin on the field performance of the single intradermal comparative tuberculin test (SICTT) and single intradermal test (SIT). Three trial potencies of bovine PPD tuberculin, as assayed in naturally infected bovines, namely, low (1192IU/dose), normal (6184IU/dose) and high (12,554IU/dose) were used. Three SICTTs (using) were conducted on 2102 animals. Test results were compared based on reactor-status and changes in skin-thickness at the bovine tuberculin injection site. There was a significant difference in the number of reactors detected using the high and low potency tuberculins. In the SICTT, high and low potency tuberculin detected 40% more and 50% fewer reactors, respectively, than normal potency tuberculin. Furthermore, use of the low potency tuberculin in the SICTT failed to detect 20% of 35 animals with visible lesions, and in the SIT 11% of the visible lesion animals would have been classified as negative. Tuberculin potency is critical to the performance of both the SICTT and SIT. Tuberculin of different potencies will affect reactor disclosure rates, confounding between-year or between-country comparisons. Independent checks of tuberculin potency are an important aspect of quality control in national BTB control programmes.     

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.     

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.     

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.     

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.     

Suppression of skin reactivity to bovine tuberculin in repeat tests

In cattle sensitised with killed Mycobacterium bovis an intradermal injection of either 0.3mg or 0.1mg bovine purified protein derivative tuberculin in the caudal fold suppressed skin reactivity to both bovine and avian tuberculin in comparative cervical tuberculin tests carried out 4 and 7 days later. Complete return to original sensitivity did not occur in all cattle when re-tested 60 days later but this level of sensitivity was not significantly different from that measured in initial tests. There were large individual variations in skin reaction to the sensitising dose in all treatment groups as found by others working with naturally infected cattle.     

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.     

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.     

Studies on the efficiency of absorbed bovine PPD in tuberculin and serological tests

The passive haemagglutination, enzyme-linked immunosorbent assay and indirect fluorescent antibody tests were applied to study the non-specific reactions in experimentally infected guinea pigs and tuberculin positive bovines. These cross-reactions were greatly decreased after absorption of either sera with avian PPD or bovine PPD antigen with anti-avian PPD serum. The use of both absorbed sera and antigen raised the specificity of PHA and ELISA to 100%. The use of absorbed sera rendered the IFA specific in 95%. The absorption has reduced the sensitivity of ELISA, IFA and PHA by 14, 27 and 29%, respectively.     

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.     

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.     

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.     

The experience of new European Union Member States concerning the control of bovine tuberculosis

On 1 May 2004, 10 new States joined the European Union, including Cyprus (CY), the Czech Republic (CR), Estonia (ES), Hungary (HU), Latvia (LA), Lithuania (LI), Malta (MA), Poland (PO), Slovakia (SK), and Slovenia (SN). Using OIE and published data, this paper summarises the status of bovine and human tuberculosis in animals in these countries between 1996 and 2003. National control programmes against bovine tuberculosis in cattle have been successful: the current herd incidence of this disease in cattle is currently lower than 0.2%, so all countries meet the OIE requirements for freedom from the disease. Furthermore, two countries have already been officially declared bovine tuberculosis-free EU States: the CR on 31 March 2004 (European Commission Decision No. 2004/320/EC) and SK on 4 March 2005 (Commission Decision No. 2005/179/EC). The last outbreak of bovine tuberculosis was diagnosed in cattle in CY (1928), ES (1986), LA (1989), SK (1993), CR (1995), and MA (2001). However, several issues of concern remain including the potential existence of a wildlife reservoir, the presence of Mycobacterium bovis, M. caprae, and other members of the M. tuberculosis complex (particularly M. tuberculosis or M. microti) in imported domestic or wild animals, and the potential for delayed detection of bovine tuberculosis in those States where annual tuberculin testing is no longer performed on cattle older than 24 months.     

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 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.     

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three calves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.