Studies on the Mechanism of Host Resistance in Experimental Leptospirosis Icterohemorrhagica
Authors: Stavitsky Abram B. 
Published: January 2023

Summary and Discussion
From the studies of many pathologists, bacteriologists, and immunologists a fairly complete picture of a well-developed host resistance, whether native or acquired, to infection has been developed (21). First, the infective agent is localized at the site of entrance into the body; second, the progressive multiplication of the organisms is inhibited, and most or all of them that penetrate into the tissues are destroyed, and, finally, any noxious substances that the bacteria liberate either before or after their dissolution are rendered less injurious or completely harmless. Using this picture as a basis, it is of interest to ascertain how well the resistance to leptospirosis is developed in the species of animals studied.
Of the peripheral barriers only uninjured skin and nasal mucous membrane prevented invasion of the body by the spirochetes. When injected into the dermis the organisms were not localized and produced a very mild inflammatory reaction at the site of injection during the first 24 hours after injection, as indicated by histological examinations of the tissue and by the free diffusion to the tributary lymphatics of trypan blue inoculated into the area. Further evidence of the invasiveness of the leptospira is the production of serious systemic effects and relatively mild local effects upon intradermal injection. When injected into the peritoneal cavity, the eye (20) and meninges (14), (20) the organisms produced a progressively more intense inflammatory reaction and correspondingly more localization of the spirochetes in that order. However, it is not known which is primary, rapid escape of the organisms from the injection site before causing much local cellular injury or the non-irritant quality of the leptospiras per se. It is possibly significant that the organisms escaped from a peritoneal cavity which was artifically inflamed so that intraperitoneally-injected streptococci, horse serum and trypan blue were unable to spread through the regional lymphatics. Studies to demonstrate the formation of “Spreading factor” (22) and fibrinolysins by leptospiras were unsuccessful (20), suggesting that it is their rapid motility which enables them to bore through fibrinous networks and other localizing barriers erected by the host. Perhaps, a very closely-knit coagulum and quite complete lymphatic blockade are required to restrain their spread.
Whole blood, serum and cell-free inflammatory fluid of normal animals were withour demonstrable effect on the spirochetes in vitro, suggesting that in the naturally-resistant or susceptible host the organisms are not destroyed in vivo by these fluids per se. Therefore, if the number of spirochetes is reduced in the blood or in an inflammatory area, this reduction is probably effected by some other mechanism. Nor were the leptospiras cleared from the blood of guinea pigs, mice and rats during the first 48 hours after intracardial injection and there was no appreciable fluctuation in the number of organisms in the blood during this time. In other experiments organisms were cultured from the blood up to 120 hours after injection (20). However, owing to the technical difficulty involved in making quantitative counts of leptospiras in any medium, determinations were not possible of the actual numbers of organisms in the peripheral circulation. There was no evidence of phagocytosis of spirochetes by the reticulo-endothelial cells in the liver, spleen, adrenals and bone marrow. Observations of the inter-reaction of leptospiras and reticulo-endothelial cells in tissue culture might shed some light on the failure of these cells to clear the circulation of the spirochetes.
In studying phagocytosis of leptospiras it was realized that it would be very difficult to recognize a leptospira in a phagocytic cell using the dark-field technic. However, one might expect to see the actual engulfing of organisms by these cells in the course of numerous dark-field examinations if the process occurs at all frequently. It was therefore considered significant that in numerous dark-field examinations of wet preparations from the inflamed anterior chamber of the eye (20), peritoneal exudate and blood as well as of in vitro preparations containing serum, normal and immune phagocytes and varying numbers of leptospiras not a single instance of phagocytosis of the organisms was observed. In fact, when the organisms and phagocytes were observed for over an hour on a warm stage there was no apparent attraction between them, the two types of cells coming together infrequently and then only accidentally. Nor was phagocytosis observed in smears and sections from infected brain (20), meninges (20), liver, spleen, adrenals, bone marrow and kidneys. Whether phagocytosis of fragmented spirochetes occurs could not be determined as it was not possible to distinguish between coccoid spirochetal bodies (2) and normal intracellular granules in any type of preparation. However, even if it does occur, phagocytosis is not necessarily a primary element in anti-leptospiral resistance.
It is not known why phagocytosis of leptospiras does not occur readily, if at all. Teale (23) has emphasized the importance to the phagocytic process of sedimentation of bacteria in slowly circulating blood and the formation of fibrin meshworks with subsequent enmeshment of the organisms in these. It may be significant, therefore, that leptospiras do not sediment readily as indicated by the observation that 5 hours' centrifugation at 3000 r.p.m. was required to clump them down completely from a heavy culture. It was also shown that the organisms could wriggle through rather dense networks of fibrin without being trapped (20). Other factors of possible importance are the large surface area, irregularly shaped surface and the positive charge (24) of these organisms compared to the negative charge of most bacteria. Attempts to demonstrate leukocidin production by leptospiras were negative (20). Phagocytosis of the spirochetes conceivably may occur in vitro or in vivo under special conditions. For instance, a very high titer of antibodies may be required to alter the surface charge of leptospiras so that phagocytes may spread on and eventually engulf them. However, in experiments in which hyperimmune serum was injected into infected guinea pigs no evidence of phagocytosis was observed although the organisms were agglutinated and lysed by the antibodies.
Of all factors studied only the specific lytic antibodies affected the leptospiras in vitro and in vivo. It seems reasonable, therefore, that the only factor which was not considered on a comparative basis, namely, antibody production, may be an important one in explaining the age and species resistance in leptospirosis. As Baumgartner (25) and others (26) have shown, mature individuals of most species of laboratory animals are better producers of antibodies than young individuals. Higuchi (27) states that the rat is a strong antibody producer against leptospiras but it was not possible to determine from the German summary of his paper in Japanese whether he has experimental proof for the statement. Therefore, it is desirable to study antibody production against leptospiras and the effect of immunization on the spread and multiplication of the organisms in various species. It is of course possible, however, that another as yet undetermined defense mechanism is involved in anti-leptospiral resistance as the rôle of fever temperature in native resistance of the rabbit to pneumococcus type III (28).
There is evidence that leptospiras do not damage the tissues of the mouse and rat as much as those of the guinea pig (20), although the organisms enter and spread in the bodies of these hosts practically as readily as in the guinea pig (14). However, these facts coupled with the finding of hemorrhagic pulmonary lesions in a high percentage of mice and rats inoculated with the spirochete indicate that the resistance of these species to this infection is a relative rather than absolute one. The pathogenesis of this infection in the mouse and rat will be discussed in extenso in a future publication.
It is apparent from the observations made in this study that the resistance of the guinea pig to leptospirosis is poorly developed when compared to the ideal picture of a well-developed host resistance (19). Antibody production with lytic properties appears to be the defensive mechanism of last resort which fortunately is apparently very effective in destroying the organisms. The importance of antibodies in leptospirosis is further indicated by Larson (29) who found that human convalescent and rabbit hyperimmune sera prevented the death of susceptible young mice infected with leptospiras, even if the sera were given as late as the 4th day after infection. From observations made in previous (2) and the present studies it appears that these antibodies have a direct antispirochetal action although some workers consider it to be an antitoxic action (30). Attempts by the author to demonstrate toxin production by these organisms have, however, been unsuccessful (14).
If leptospiras are not readily phagocytosed in the body, it may justifiably be asked how the all-important lytic antibodies are formed. Of course, the answer to this question at the present state of our knowledge can be only speculative. However, it has been shown in unpublished studies by the author that guinea pigs may be successfully protected against leptospirosis by immunization with spirochete-free filtrates of leptospiral cultures. In addition, recent work by Ehrich and Harris (31) suggests that phagocytic cells in the body may not elaborate antibodies themselves but rather phagocytose complex antigens or whole bacteria and break them down into simpler, absorbable or adsorbable form. These soluble antigens are then presumably absorbed by lymphocytes which elaborate the antibodies. It seems plausible, therefore, that anti-leptospiral bodies may formed by absorption of soluble antigens without phagocytosis of the whole organisms. Of course, this hypothesis remains to be proved.
Finally, it is desirable in view of the findings in this study to consider studies on immunity in other spirochetoses. In a recent review of the literature on avian spirochetosis including some original experiments on the mechanism of immunity in that disease Knowles et. al. (32) conclude that this immunity is basically humoral in nature. The important rôle of spirocheticidal antibodies in infections with relapsing fever spirochetes was first recognized by Gabritschewsky in 1896 (33) and later put on a firm experimental foundation by the classical experiments of Novy (34). Although later studies have shown that the nature of immunity in this disease with its frequent relapses is by no means completely understood Kritchewski et. al. (35) on the basis of some of the most recent experimental evidence conclude that the immunity in relapsing fever, as well as in avian spirochetosis and syphilis is primarily due to destruction of the spirochetes by specific spirochetolytic antododies with phagocytosis of the organisms being only an accidental or secondary phenomenon. Chesney and his co-workers (36) have presented independent evidence for the humoral nature of host resistance in syphilis. In fact, Chesney states (37) that he has never seen a single instance of phagocytosis of Treponema pallidum either in human syphilis or in experimental syphilis in the rabbit. Therefore, it would seem that a common mechanism underlies antispirochetal immunity, at least in the species studied. This mechanism is primarily humoral in nature unlike that in antibacterial immunity which usually depends on the inter-reaction between opsonizing antibodies (bacteriotropins) and phagocytic cells in destroying the organisms. Studies by Taliaferro (38) on trypanocidal antibodies against Trypanosoma lewisi and duttoni suggest that the humoral mechanism is of importance in anti-trypanosome resistance, thus providing another case where the spirochetes and trypanosomes behave biologically similarly.