Experimental studies on the effect of BCG in protection and treatment of murine leprosy in rats
Following the original observation by Fernandez (1939) that BCG vaccination of orphan who had been proved negative to both tuberculin and lepromin, resulted in doubtlessly higher positive conversion rates than could be expected from a simple repetition of the test, BCG vaccination to prevent leprosy has been suggested for many years (Wade 1956, Fernandez 1962). Consequently, numerous clinical and field trials have been carried out in attempts to prove the effectiveness of BCG vaccination both as a stimulant for lepromin positive conversion and as a prophylactic implementation against leprosy (Azulay 1948, Flock and Destombes 1950, Convit et al. 1952, Lowe and McNulty 1953, Souza Campos 1953, Brown et al. 1968,Bechelli et al. 1968).
Although it is s1ron91y suggested that BCG vaccination is beneficial to individuals exposed to infection with leprosy, the preventive action of BCG vaccination against leprosy has not been demonstrated conclusively. Eventually, contradictory conclusions have been arrived at Congresses, Conferences and Seminars on leprosy, which have sometime advised its use (Madrid Congress 1953) while others have not (Tokyo Congress 1958).
As slated by Rees and Waters (1963), although the murine leprosy neither involves nerves nor manifests a "tuberculoid" picture, nor responds to all the same therapeutic drugs as the human disease, there are fundamental similarites in problems shill requiring elucidation which it is suggested can be better studied at this time in murine leprosy. Experimental murine lepros yhas been widely utilized as the model in leprosy research (Carpenter 1951, Lew and Carpenter1953. Terada 1944, T7da 1943, Nishimura 1956, Shimizu 1953, Hanks and Gray 1954).
There appeared many reports that explored the BCG effect on the prevention of murine leprosy in experimental animals. (Azulay 1954, Hanks and Fernandez 1956, Makamura et al.1953, Lew 1955, Muir and Henderson 1927, Ohjima et al. 1958, Hauler and Zitti 1953). However, a careful analysis of reports on the effect of BCG vaccination against murinele prosy in experimental animals indicated that the results were mostly fragmentally and lacked quantitative experimentation in their approaches. Usually, BCG vaccination resulted infavorable protective effect on murine leprosy in experimental animals (Carpenter 1951, Azulay 1954, Lew 1955, Hanks and Fernandez 1956, Nishimura et al. 1953, Ohjima et at. 1958),but some negative results have also been reported (Muir and Henderson 1927, Hauler and Zitti 1953).
In this study it was attempted 1) to provide a conclusive evidence for the prophylactic effectiveness of BCG vaccination against murine leprosy in rats and 2) to explore the possibility of vaccine treatment of fully developed murine leprosy with BCG.
Materials and Methods
Animals: Albino rats, 8 to 9 weeks old and weighing 120 to 140gm, of both sex were used. Prior to use, they were tested for tuberculin sensitivity by inoculating intracutaneously with 0.1 ml of a 5% solution of old Tuberculin in saline, and the rats that showed doubtful tuberculin reactiveness were excluded from the experiments.
Murine Leproy Bacilli: The Hawain strain of Mycobacterium lepraemurium was used. The strain has been maintained at the Department by serial passage in rat's testicle perevery 6 months, The infected (5 months old) testicles were removed and ground in mortar and pestle, and 5%(W/V) suspension of bacilli was prepared with sterile physiological saline and filtered through 6 layers of cheese gauze. For animal inoculation 0.5 ml of 5% suspension was injected intramuscularly on inner left hind thigh.
BCG: Within 24 hours before experiment freshly prepared W-G vaccine (50 mg per ml)was purchased from NIH, Korea. For animal inoculation 0. 1 ml of BCG vaccine was given intracutaneously on one side of the back. Experimental Design:
1. Prophylactic effect of BCG vaccination.
(1) Effects on the development of lepromata and involvement of Iymph nodes: 16 weeks after BU vaccination, rats of experimental group were challenged with Myco. lepraemurium. The experiment was terminated 10 weeks after challenge with M)taco. lepraemurium. To a half of rats of experimental group autoclaved murine leprosy
material was Inoculated 2 weeks after BCG vaccination to find whether or not such an injection would enhance the prophylactic effect of BCG.
(2) Effects on the involvements of viscera and lymph nodes: 2 months after BCG vaccination, rats of experimental group were challenged with Myco. lepraemurium.
Experiment was terminated 16 weeks after challenge, and weight of spleen and bacillary contents in the viscera were determined. By similar procedure, effectiveness of vaccination with heated BCG was also tested.
2. Therapeutic effect of BCG vaccination.
(1) Effects on the development of lepromata and involvements of 1)'mph nodes: Rats of experimental group were challenged with Myco. lepraemurium 3 days after BCG vaccination, and experiment was terminated 24 weeks after challenge.
(2) Effects on the involvement of viscera and lymph nodes:
a. BCG and Myco. lepraemurium were given simultaneously and experiment was terminates 24 weeks after the injections.
b. Following simultaneous inoculation with BCG and Myco. lepraemurium, 3 more additional BCG vaccines were given at 2 weeks intervals. Experiment was terminated 16 weeks after the last BCG vaccination.
3. Control experiment.
4. Effect of BCG on the prognosis of murine leprosy.
BCG vaccine was given to the rats that had developed full murine leprosy (36 weeks after inoculation of Myco. lepraemurium) and the rats were observed for another 26 weeks.
Criteria of evaluating BCG effect:
(1) Determination of bacillary involvement: Number of bacilli in film specimens from lymph nodes and visceral organs were determined and recorded arbitrarily as -, 1+, 2+, 3+, 4+ and 5+ according to the number of AF bacilli observed under microscope.
(2) Lepromata developed at the site of inoculation with Myco. lepraemurium were carefully excised and their sizes were compared by weight. Number of bacilli in the lepromata was determined with homogenate of lepromata.
(3) Lymph nodes and viscera: Lymph nodes (inguinal, axillary, and retroperitoneal) and viscera (left lung, liver, spleen, left kidney) were used and number of bacilli were deter-mined from film specimens.
1. Intracutaneous inoculation of BCG to the rats 16 weeks before injections of murine leprosy material, decreased the rate of development of lepromata and restricted the distribution of leprosy baci17i. Thus BCG conferred a degree of protection against murine leprosy.
2. Inoculation of either live or heat·killed BCG 8 weeks before injections of murine leprosy material exerted a similar protective effect against murine leprosy in rats, and there observed no significant differences in the development of lepromata and in the extent of spleen involvement between them.
3. BCG injected at approximately the same time as murine leprosy material also decreased the rate of development of lepromata and restricted the distribution of leprosy bacilli in the rats.
4. Multiple BCG vaccination (three more additional BCG injections at 2 weeks intervals following simultaneous inoculation with BCG and murine leprosy bacilli) failed to reduce the lepromata and the number of leprosy bacilli than those observed in the rats with single dose of BCG inoculation.
5. When BCG was injected into rats which had been infected with murine leprosy thirty-six weeks previously, 7 out of ten were alive after another twenty-six weeks. In a similar group of rats which did not receive BCG, only two rats were alive after twenty-six weeks.6. It appeared that the assessment of size of lepromata and number of murine leprosy bacilli in lepromata and in spleen among the viscera would be the most practical and efficient criteria for the evaluation of effect of BCG against murine leprosy in rats.