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.     

Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.
Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests.
Animals A herd of 87 goats of Guadarrama breed.
Procedure Serial testing and segregation of animals.
Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing.
Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avian^B reactors) should be considered test positive for M bovis . By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.     

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.     

Serological and cutaneous testing of bovine tuberculosis with the A60 antigen complex from Mycobacterium bovis, strain Calmette-Guérin

A60, a major thermostable macromolecular antigen complex of Mycobacterium bouis strain Calmette-Guérin (BCG), is immunodominant in tuberculosis and able to elicit both humoral and cellular immune reactions in infected hosts. A60-based ELISA and cutaneous tests have been used, in conjunction with the PPD-based skin reaction, in a control group of healthy animals, and in a herd including tuberculous animals. Cutaneous testing with A60 yielded results comparable with those with PPD: both were negative with control cattle and positive with infected animals. Moreover, comparative cutaneous testing with avian tuberculin yielded similar results with PPD and A60. When animals from the infected herd were tested with both avian and bovine sensitins, 54% of cattle were diagnosed as fully positive, 26% suspect, and 20% negative. Serological analysis with the A60-ELISA of part of the infected herd yielded 74% positive, 21 % suspect and 5% negative results. Thus, positivity was 74% for Serological analysis and 54% for cutaneous testing, whereas positive plus suspect results were 95% for serological analysis and 80% for cutaneous testing. It can be concluded that A60 can be used in place of PPD for cutaneous testing in cattle, and that the diagnostic value of the A60-ELISA is superior to that of the PPD-cutaneous test.     

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.     

Evaluation of an ELISA-PPD for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major health problem in Brazil. The intradermal tuberculin test is the standard test for its detection, but it can lack both sensitivity and specificity. The purpose of this study was to evaluate a bovine enzyme-linked immunosorbent assay- (ELISA - PPD) under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested with the intradermal tuberculin test (ITT). Two hundred and seven cows gave a positive reaction, which represents 12.7 per cent of the cattle studied. The sensitivity and specificity rates to ITT were 87.7 per cent and 95.2 per cent, respectively. From the 1632 animals 15 per cent of each herd (220 in total) were selected to be tested by the ELISA. Differences between mean optical density (OD) of the control group, ITT -positive and ITT -negative groups were all significant (P<0.01). The sensitivity rates to ELISA - PPD were 86.7 per cent, while specificity was 90.6 per cent. The use of ELISA - PPD is suggested for situations where the investigation of the whole herd is more important than the individual testing of each cow. In addition, the ELISA - PPD can also be helpful when a collective diagnosis is desired to elucidate clinical suspicions of disease, or in the first steps of a control program, for identification of foci.     

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.     

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.     

Evaluation of relationship between plasma fibrinogen concentration and tuberculin testing in red deer

After intradermal injection of bovine purified derivative (PPD), increases in plasma fibrinogen concentration and plasma viscosity developed in red deer (Cervus elaphus) with a history of tuberculosis caused by Mycobacterium bovis. Serum haptoglobin concentrations were also found to increase under similar circumstances. The increases were reproducible and did not appear to be related to mustering, stress, or the handling associated with injection of PPD. A significant (P less than 0.05) direct relationship was found between the increase in plasma fibrinogen concentration and various markers of bovine tuberculosis infection, such as stimulation of lymphocyte transformation in response to bovine PPD and the diameter of intradermal tuberculin skin test reactions. A stronger correlation (P less than 0.01) was found with the volume of intradermal tuberculin skin test reactivity, and the strongest correlation (P less than 0.001) was with the presence of circulating antibovine PPD antibody.     

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.     

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.     

Effect of an inactivated paratuberculosis vaccine on the intradermal testing of goats for tuberculosis

The effect of an inactivated paratuberculosis vaccine on the diagnosis of tuberculosis (TB) in goats was investigated in a herd with a history of clinical paratuberculosis but which was free of TB. Cohorts of animals in 2006, 2008 and 2009, were vaccinated once at 1 month of age, and 50% of the 2006 cohort served as unvaccinated controls. The goats were aged 8 months, 20 months and 3.5 years old at the time of the survey. All animals were assessed using a single intradermal injection of bovine tuberculin purified protein derivative (PPD) (SID test), or using both bovine and avian PPD (CID test). An interferon (IFN)-γ assay using both bovine and avian PPD was carried out on the 2006 cohort and was interpreted according to three different 'cut-off' points. No unvaccinated (control) animals tested positive to any of the assays, confirming that the herd was TB-free. The SID test had a low specificity in vaccinated animals at 8 and 20 months of age, whereas the CID test demonstrated 100% specificity in animals ≥20 months-old. The specificity of IFN-γ assay was less than maximal for vaccinated animals 3.5 years old as small numbers of false positives were detected, although this depended on the chosen cut-off point. The study findings demonstrate that the use of an inactivated paratuberculosis vaccine in goats <1 month-old in a TB-free herd does not result in false positives to a CID test for TB when performed in animals ≥20 months-old.     

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.     

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.     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

PPD的批次与注射方式对PPD皮内变态反应检疫的影响

为了探究在牛结核病检疫中出现的使用不同批次的PPD和皮下注射PPD等情况是否会对检疫结果的判定造成干扰。本试验首次通过在豚鼠背部采用皮下注射法和皮内交替注射同一厂家不同批次的结核菌素提纯蛋白衍化物(Purified protein derivative of tuberculin,PPD)的方法,参照《结核菌素提纯蛋白衍化物(PPD)制造及检定规程》于每次注射后24h、48h和72h观察皮试处是否产生迟发型变态反应。试验结果为皮内组(不同批次PPD皮内注射)和皮下组(同一批次PPD皮下注射)经3次注射后的皮试结果与标准参照组的皮试结果无差异,且在每次皮试后的24h、48h和72h皮试处均未出现局部红斑硬结。本试验证明了PPD皮内变态反应检疫结果判定不受PPD皮下接种及PPD的不同批次的干扰。     

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.     

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.