The International Reference Preparations of Clostridium welchii (C. perfringens) beta and epsilon toxoids.

The Central Veterinary Laboratory, Weybridge, England was requested by the WHO Expert Committee on Biological Standardization to obtain suitable materials for international standards for Clostridium welchii (C. perfringens) beta and epsilon toxoids and to arrange collaborative assays. Preparations were obtained and dispensed as freeze-dried toxoids in ampoules. The toxoids were assayed by nine laboratories in eight countries. On the basis of the results obtained, the materials have been established as the International Reference Preparations of Clostridium welchii (C. perfringens) Beta and Epsilon Toxoids.

Practical application of bovine tuberculin PPD in testing cattle in Great Britain.

A national tuberculin testing trial was carried out in 8151 cattle in 132 herds due for normal herd testing and distributed throughout the 11 animal health administrative regions of England, Scotland and Wales. The numbers selected from each region were in approximately equal proportions to the total cattle populations of the regions and comprised a representative sample of the national herd. The standard intradermal comparative tuberculin test using Weybridge avian and human purified protein derivatives (PPD) was carried out on one side of the neck, and Rotterdam bovine PPD (1 X 0 mg per ml) was injected on the opposite side in the usual 'mammalian' site. Of the cattle tested, 9 X 4 per cent were classified as positive reactors to a single intradermal injection of the bovine tuberculin applying the official EEC interpretation for tuberculin tests. A single intradermal injection of human PPD would have resulted in a significantly higher proportion (10 X 4 per cent) of cattle being designated for removal. The rules of interpretation developed by Lesslie and Hebert (1975) for a comparative tuberculin test using avian and bovine PPDs, when applied to this sample, classified 0 X 80 per cent as inconclusive reactors compared with 0 X 58 per cent for the avian/human comparative test. The avian/bovine test also classified as positive reactors 0 X 12 per cent of the cattle tested, whilst the avian/human test classified as positive 0 X 05 per cent of cattle in routine herd tests during the three-year period 1972 to 1974.

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

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

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

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

The relationship between length of incubation, bacterial growth and tuberculin yield of a strain of Mycobacterium tuberculosis.

The relationship was studied between length of incubation in a liquid synthetic medium, amount of bacterial growth, pH of the culture filtrate and tuberculin yield of a single strain, DT, of Mycobacterium tuberculosis. No sinple relationship was found between the total crop of bacteria and the tuberculoprotein content of the culture filtrate. On the other hand, protein release and the pH of the culture filtrate were closely related. In each of two trials the bacterial crop increased rapidly during the early period of incubation, although maximum growth was not reached until 9 and 13 weeks respectively. The pH of the culture filtrate reached its maximum at a value of more than 9-0 after 5 weeks incubation and then fell until the 10th week; a second rise occurred until the 13th week when the pH dropped again and levelled off slightly on the alkaline side of neutrality. The rate of increase in tuberculin yield was most rapid during the first 7 weeks of incubation. After this, the rate of increase slowed down but the yield was still rising after 18 weeks. It is concluded that for the large-scale production of mammalian tuberculin by the method used in these trials, it is profitable to incubate production cultures for 14 weeks or longer.