Tubercle bacilli of the pathogenic human strain H37Rv had previously been shown to multiply, after ingestion by cultured mouse peritoneal macrophages, within phagosomes that tended to remain unfused with secondary lysosomes. Means were sought therefore for promoting experimentally a modification of the host response so as to attain a high level of phagolysosome formation, enabling tests to be made of any effects on the course and outcome of the intracellular infection. This was achieved by exposing viable bacilli to specific rabbit antiserum before their ingestion. Quantitative assessments, using electron microscopy, now showed that a majority of the phagosomes containing intact bacilli had fused with ferritin-labeled lysosomes, and frequently the fusion was massive. Bacterial viability studies established that the serum pretreatment was not itsel bactericidal. In the course of progressive infections with strain H37Rv, monitored by counts both of viable bacterial units and of intracellular acid-fast organisms, no appreciable difference was found between the intracellular growth rates of control and antiserum-treated bacilli. Concurrent electron microscopy showed that bacilli could remain intact and multiply both in phaagolysosomes and in unfused phagosomes, ruling out the possibility of selective growth of antiserum-pretreated bacilli within the minority of phagosomes that remained unfused. It was concluded that "turning on" phagosome-lysosome fusion in normal macrophages did not influence the outcome of infection with virulent M. tuberculosis; lysosome contents manifestly failed to exercise an antibacterial effect on this organism. Nevertheless, the possibility remains that the lysosomes of specific immune macrophages have antituberculous potentiality. In that case the experimental "turning on or off" of fusion could be a decisive factor in the outcome of a virulent challenge. Should it not be, the antibacterial capabilities of immune cells would need to be ascribed to factors other than lysosomal attack, the latter being essentially for disposal of the dead organisms.

https://rupress.org/jem/article-pdf/142/1/1/1267442/1.pdf

Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival.

The authors argue that plague should be taken much more seriously by the international health community.

/pdf/plague-past-present-and-future-4yvkf2520n.pdf

Plague: past, present, and future.

A. Macrophage-Dependent Resistance in the Absence of Specific Immune Responses.... 1223 B. Augmentation of Macrophage Numbers by Local Proliferation and Influx of Monocytes from the Blood 1223 C. Granuloma Formation 1224 D. Macrophage Activation 1224 E. The Genetic Basis of Mononuclear Phagocyte-Dependent Resistance ......... 1226 F. Role of Polymorphonuclear Leukocytes .... 1226

The Role of Cell-Mediated Immunity in Bacterial Infections

Brucellosis vaccines: past, present and future.

(1973). Bacterial Assimilation of iron. CRC Critical Reviews in Microbiology: Vol. 2, No. 3, pp. 273-331.

Bacterial Assimilation of iron

The preservation of Brucella melitensis was of initerest because Elberg and Faunce (1957) had shown that a nondependent mutant derived from a streptomycindepenident strain of B. melitensis was immunogeniic wheni administered as a living vaccine. Huttoni et al. (1951) and, more recenitly, Alexander and van Drimmelen (1956), showed that lyophilization was practical for preserving the viability of the Brutcella abortus (straini 19) vaccinie. Although lyophilizationi is a geinerally accepted method for maintaining viability of bacterial cultures (Harris, 1954), the conditions for the optimal preservation of bacteria by lyophilization are not identical for all species or strains of given species. This report describes the results of limited studies on the lyophilization and storage of a live vaccine strain of B. melitensis.

Lyophilization of Brucella melitensis.

In experiments designed for study of bacteremia and serologic response of Macaca cynomolgus philippinensis to vaccination with a soluble antigen (fraction I) of Brucella melitensis and the live Rev. I vaccine strain, fraction I and Rev. I were administered both four weeks apart and simultaneously. One of three monkeys given the vaccines four weeks apart and two of three monkeys vaccinated with fraction I and Rev. I in a single injection developed transient bacteremia, as shown by blood cultures. Both methods of vaccination induced significant titers of antibody to B. melitensis, and vaccinated monkeys were protected against subsequent challenge with a virulent strain of B. melitensis.

Priming of Macaca cynomolgus philippinensis with purified antigen of Brucella melitensis before injection of Rev. I vaccine.

Immune mechanisms active against Brucella were studied under conditions of oxygen deficiency. B. melitensis grew in rabbit serum-Tyrode medium flooded with N2 and CO2 gas mixtures. Immune sera from rabbits injected with B. melitensis strain Rev I possessed growth-inhibitory activity that operated in anaerobic environments against Rev I and virulent strain 6015. When mixed with macrophages, immune sera mediated even greater inhibition of bacterial growth and slowed the spread of infection throughout the tissue culture. Although under anaerobic conditions the rate of phagocytosis was reduced, the macrophages in immune serum killed significant percentages of Brucella, suggesting that an antibacterial mechanism had been activated. Sonic extracts of macrophages prepared and tested under anaerobic conditions depressed the growth rate of strain Rev I. The extracts, however, exhibited no immediate killing capacity when tested in Tyrode solution. A factor from serum was required for depression of the growth rate.

Serum-mediated Immune Cellular Responses to Brucella melitensis IV. Infection of Macrophages Under Anaerobic Conditions

In earlier reports of immune responses to Brucella, macrophages were studied for their growth-inhibiting action mainly in the absence of added antibody [1-3]; the addition of antibody was found to exert no effect on the capacities of immune cells to inhibit growth of Brucella [2, 3]. On the other hand, in rabbits immunized with the Rev I vaccine strain of Brucella melitensis, immune serum enhanced the in-vitro functions of macrophages [4-7]. More than one function of the macrophage is involved; each in turn may be activated or enhanced by more than one immunoglobulin, as well as by nonspecific factors of serum [8]. When infection of normal macrophages is initiated by Brucella of the smooth variety, many phagocytes degenerate. A heat-stable material that reduced degeneration of macrophages accumulated in the sera of animals vaccinated with the Rev I strain. It produced its maximal effects when combined with macrophages from immunized animals. A similar factor was

Serum-mediated immune cellular responses to Brucella melitensis. VI. The sequence of cellular and serologic changes after immunization of the rabbit with the Rev I vaccine strain.

SummaryInflammatory-induced peritoneal histiocytes of rabbit, guinea pig and monkey, both normal and immunized, respond to the cytotoxic action of parasitizing brucella within 24 hours. Normal cells undergo about 30% loss whereas immune cells resist completely the cytotoxic action for at least 96 hours and retard intracellular multiplication for at least 144 hours. Cells of rabbits, guinea pigs, and monkeys react alike in these respects. The cellular immunity produced by the Rev I strain of B. melitensis was also operative against virulent B. suis.

Sanford S. Elberg

Papers

Lyophilization of Brucella melitensis.

Priming of Macaca cynomolgus philippinensis with purified antigen of Brucella melitensis before injection of Rev. I vaccine.

Serum-mediated Immune Cellular Responses to Brucella melitensis IV. Infection of Macrophages Under Anaerobic Conditions

Serum-mediated immune cellular responses to Brucella melitensis. VI. The sequence of cellular and serologic changes after immunization of the rabbit with the Rev I vaccine strain.

Response of normal and immune histiocytes of various animal species to infection by brucella melitensis.