A New Model to Calibrate a Reference Standard for Bovine Tuberculin Purified Protein Derivative in the Target Species.

Klaas Frankena,Margaret Good,Anthony Duignan,Mart C M De Jong,Liesbeth Jacobs,Tonny Van Dijk

doi:10.3389/fvets.2018.00232

Abstract

Since 1986, use of a Bovine International Standard (BIS) for bovine tuberculin has been required to ensure national and international uniformity regarding the potency designation of bovine tuberculin Purified Protein Derivative (PPDb) preparations produced by multiple manufacturers. The BIS is the unique golden standard in the guinea pig potency assay, representing 100% potency, where potencies of production batches are calculated as relative potencies in comparison with the potency of the BIS which was set at 32,500 international Unit (IU) per mg. The stock supply and lifetime of the BIS is limited.The aim of this study was to develop a model to determine the potency of a newly produced in-house Reference Standard (RS) for PPDb with great accuracy in the target species (cattle) and to prove its precision and accuracy in the guinea pig potency test. First simulations were done to estimate the required number of cattle needed. Then, 30 naturally bTB infected cattle were subjected to a tuberculin skin test using multiple injections of both the RS and the BIS. Both were applied randomly in the same volume and concentration (1 dose). The potency of the RS against the BIS was directly derived from the least square means (LSMEANS) and was estimated as 1.067 (95% CI: 1.025–1.109), equal to a potency of 34,700 ± 1,400 IU/mg. In six guinea pig potency assays the RS was used to assign potencies to production batches of PPDb. Here, precision and accuracy of the RS was determined according to the parallel-line assay. Relative potencies were estimated by exponentiation of the common slope. The corresponding 95% confidence intervals were obtained according to Fieller's theorem. In sensitized guinea pigs, the relative potency of the RS against the BIS was 1.115 (95% CI: 0.871–1.432), corresponding to an absolute potency of 36,238 IU/mg (95% CI: 28,308–46,540).In conclusion: the method used to determine the potency of the RS against the BIS in naturally bTB infected cattle, resulted in a highly accurate potency estimate of the RS. The RS can be used in the guinea pig test to assign potencies to PPDb production batches with high precision and accuracy.

Highlights

Bovine tuberculosis is a zoonotic livestock infection most frequently caused by the bacterium Mycobacterium bovis which is often found to be endemic in cattle but which can infect several species of mammals and marsupials
Statistical Analysis The goal of the analysis was to determine whether production batches get comparable potency estimates when assayed against either one of both standards (RS or Bovine International Standard (BIS)), the relative potency of the Reference Standard (RS) to the BIS in guinea pigs was calculated
The suitability of the new RS as a M. bovis reference standard to assign potency to individual production batches of PPDb was assessed in the guinea pig potency test, the prescribed release test for PPD1

Summary

INTRODUCTION

Bovine tuberculosis (bTB) is a zoonotic livestock infection most frequently caused by the bacterium Mycobacterium bovis which is often found to be endemic in cattle but which can infect several species of mammals and marsupials. To control and eradicate bTB, multiple tests have been developed to detect infected cattle. The widely used tuberculin skin tests are based on the development of a delayed type hypersensitivity reaction in cattle infected by a MTBC after an intradermal injection with bovine (M. bovis) tuberculin Purified Protein Derivative (PPDb) [9, 10]. Since the development of the tuberculin skin tests in cattle, various companies worldwide commenced production of PPDb. To ensure national and international uniformity regarding the potency designation of the PPDb preparations it was essential to define a bovine tuberculin standard. A trial in natural bTB infected cattle was designed and performed to determine the potency of the RS in cattle with great precision and accuracy. The potency of the new RS was compared with the BIS in sensitized guinea pigs

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DISCUSSION