[Frontiers in Bioscience 3, c27-33, May 1, 1998] |
LURIE’S TUBERCLE-COUNT METHOD TO TEST TB VACCINE EFFICACY IN RABBITS
Departments of Environmental Health Sciences, Molecular Microbiology and Immunology, and Epidemiology, School of Hygiene and Public Health; and the Department of Pathology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205
Received 3/9/98 Accepted 3/14/98
3. DESCRIPTION OF THE TUBERCLE-COUNT METHOD
This method of assessing TB vaccine efficacy in rabbits was developed by Lurie in 1952 (1-3). He also used this method for determining the innate resistance of inbred rabbit families (4), as well as the virulence of the infecting strain of tubercle bacillus (5). Specifically, he allowed rabbits to inhale an aerosol of a given number of human-type tubercle bacilli, sacrificed the animals 5 weeks later, and counted the number of grossly visible tubercles in their lungs (figures 1 and 2). [The actual inhaled dose was then calculated from the weight of the rabbit and the number of bacilli cultured from the aerosol (6, also see 7)].
Figure 1. Formalin-fixed lungs of a rabbit that inhaled about 33,000 virulent human-type tubercle bacilli (strain H37Rv) 5 weeks previously. Upon dissection, these lungs contained 131 grossly visible primary tubercles, with no apparent secondary tubercles. The ratio of the number of bacilli inhaled to the number of primary tubercles produced was about 250. Effective BCG (and other vaccines for tuberculosis) should increase this ratio at least 5-fold (3). Small areas of caseous necrosis are visible in many of the tubercles. This photograph shows the ventral surfaces of the right upper, middle and azygous lobes on the left and of the entire left lung (upper and lower lobes) on the right. The right lower lobe (RLL) had been removed for culture. This RLL contained 23 grossly visible tubercles and 1.35 X 105 culturable tubercle bacilli. X 1.0
Figure 2. A tissue section of a primary lesion similar to those shown in figure 1. From left to right are (a) one of the small sites of necrosis, (b) a (surrounding) area of large epithelioid macrophages, and (x) an outside area that is densely infiltrated by smaller macrophages, lymphocytes, and plasma cells. Azure-eosin stain. X250.
The vaccine’s efficacy was then given a quantitative value. Specifically, the number of inhaled units of 1 to 3 bacilli divided by the number of grossly visible primary tubercles present at necropsy 5 weeks later provided the "ratio." This ratio is the number of inhaled bacillary units required to generate one such visible tubercle. When compared to non-vaccinated controls, rabbits that are effectively immunized need to inhale more tubercle bacilli to produce one visible tubercle, i.e., the vaccine prevented many microscopic lesions from reaching grossly visible size. The higher the ratio, the more effective was the vaccine in preventing grossly visible disease.
Bovine-type bacilli, strain Ravenel S, that are fully virulent for the rabbit produce one grossly visible primary lesion for every three bacillary units inhaled (1,6), regardless of the factors just listed. For this reason, Lurie used human-type tubercle bacilli (strain H37Rv) in which 50 to 600 bacillary units must be inhaled to produce one grossly visible primary lesion, depending on the native resistance of the rabbit (1-4,8). After BCG vaccination, Lurie’s resistant strain of rabbits needed to inhale 3000 (rather than 600) bacillary units to produce one grossly visible primary pulmonary lesion; i.e. the ratio was increased 5-fold (3). Commercially available unvaccinated New Zealand White rabbits must inhale 200 to 500 units of 1 to 3 virulent human-type tubercle bacilli (strain H37Rv) to produce one primary pulmonary tubercle that is grossly visible at 5 weeks (unpublished observations with David N. McMurray).
The beauty of this tubercle count method is that it provides a direct measure of how many tubercle bacilli must be inhaled to produce a grossly evident form of the disease. It measures the vaccine’s ability to prevent short-term and long-term pulmonary tuberculosis, because 5-week tubercles caused by human-type bacilli take months to heal in the rabbit (1). In humans, such tubercles would also take many months to heal or stabilize, and some of these lesions would progress to clinically active disease. Thus, this tubercle count method seems to be a precise way to compare the efficacy of available vaccines before they are used in clinical trials.