STUDIES ON THE MECHANISM OF IMMUNITY IN TYPHUS FEVER : II. ALLERGIC AND TOXIC REACTIONS PRODUCED WITH RICKETTSIA PROWAZEKI

The intradermal inoculation of Mexican typhus virus into immune guinea pigs produces a local reaction which is similar in its appearance to the lesion observed in the skin of normal animals submitted to the same treatment. The reaction in the immune animal appears earlier and fades sooner than the lesion in the normal guinea pig. The inoculation of heat-killed or formalin-killed Rickettsiae produces no significant reactions at the site of the intradermal injection in typhus immune guinea pigs. The virus, inoculated intradermally, has been recovered from the local lesion 72 hours after the injection into typhus immune guinea pigs. Normal guinea pigs and persons without a history of typhus fever present a congestion and some swelling of the skin at the site of the intradermal injection of formalinized Mexican Rickettsiae. The reaction appears 24 hours after the inoculation and fades within 48 hours. Heating the formalinized Rickettsia suspensions at 70°C. for 30 minutes renders them inactive in normal men and guinea pigs. From the experiments reported in this paper it seems that the reactions observed in typhus immune guinea pigs submitted to a second inoculation of typhus virus, belong to the group of reactions presented by tuberculous animals (Koch''s phenomenon) and the accelerated takes shown by immune persons submitted to revaccination with vaccinia virus. A heat labile substance has been demonstrated in the formalinized Rickettsia bodies, which produces a reaction in the skin of normal men and guinea pigs.     

TYPHUS FEVER : IV. FURTHER OBSERVATIONS ON THE BEHAVIOR OF RICKETTSIA PROWAZEKI IN TISSUE CULTURES

In tissue cultures grown at 32°C., typhus Rickettsiae increase rapidly within the cytoplasm of infected cells up to about the 14th day. At this time practically every cell is infected and the majority of cells are distended with organisms. This condition remains constant as long as successful cultures of the cells can be maintained (up to 52 days). Loss in virulence does not take place during this period in vitro. The number of Rickettsia-filled cells found in sections and the incubation period of the infection resulting from inoculation of cultures from each age group are definitely correlated. The behavior of typhus Rickettsiae in dividing cells is described and methods of spread of the infection other than by mitosis of cells are discussed. Normal tissues do not become infected in vitroto any considerable extent in spite of prolonged proximity to heavily infected cultures of scrotal sac exudate. Complete anaerobiosis and alterations in pH do not alter the intracellular location of the organism in tissue cultures. The organisms are not seen within nuclei of infected cells. They remain intact and infective for several weeks in cells which are kept alive but not multiplying. They disappear in less than 1 week, however, when the cells undergo degeneration.     

TYPHUS FEVER : V. THE EFFECT OF TEMPERATURE ON THE MULTIPLICATION OF RICKETTSIA PROWAZEKI IN TISSUE CULTURE

The temperature at which tissue cultures infected with typhus Rickettsiae are incubated has been shown to exert a marked influence on the intracellular multiplication of Rickettsia prowazeki. At 41°C. the organisms were not found in the cultures histologically on and after the 2nd day in vitro, and the cultures were non-virulent on and after the 3rd day in vitro, in spite of good preservation and growth of the cells. At 37.5°C. organisms were absent from the cultures histologically and the cultures were non-virulent on and after the 11th day in vitro, in spite of good preservation and growth of the cells. At 32°C. good but slow growth of cells occurred and organisms were found in increasing numbers histologically up to about the 21st day in vitro. At this time, 50 to 99 per cent (approximately) of the cytoplasmic volume of the cultures was occupied by Rickettsiae. From the 21st day to the 51st day (the limit to which cultures have been carried successfully) this condition of unrestricted multiplication remained practically unchanged. Inoculation of guinea pigs with single cultures after varying lengths of time in vitro, (up to the 51st day) always resulted in reproduction of typhus in a characteristic manner. At 27° the cell growth was negligible, but many cells remained alive for 10 days or more. Organisms were absent from the cultures histologically and the cultures were non-virulent on and after the 18th day in vitro. The only histological preparations showing unrestricted multiplication of the organisms (infection of the majority of the cells present) were of cultures incubated at 32°C. It is believed that the detrimental effect of the higher temperatures (37.5° and 41°C.) on the multiplication of the organism is exerted indirectly, by stimulation of the defence mechanism of the cells.     

STUDIES ON TYPHUS FEVER : IX. ON THE SERUM REACTIONS OF MEXICAN AND EUROPEAN TYPHUS RICKETTSIA

1. The blood of guinea pigs convalescent from Old World and New World typhus infection develops agglutinating properties for the tunica and rat Rickettsiae of the New World diseases and for the louse Rickettsia of the Old World disease. 2. The two microorganisms are closely related, though probably not identical. 3. Human convalescents of both varieties of typhus develop agglutinins for both types of Rickettsiae. Such Rickettsia-agglutinating properties are parallel with the Weil-Felix reaction in the human sera. 4. Rabbits immunized with Weigl louse vaccines develop agglutinins for our X-ray rat vaccines and vice versa. In both cases the rabbit sera develop agglutinins for Proteus X-19. 5. These experiments furnish a further and, we believe, conclusive proof of the etiological rôle, in New World typhus fever, of the Rickettsia bodies first seen in the tunica cells of inoculated guinea pigs by Mooser, and obtained in massive amounts by ourselves. 6. The serum reactions also provide a further logical basis for experiments in prophylactic vaccination with these Rickettsiae.     

STUDIES ON THE CULTIVATION OF THE TYPHUS FEVER RICKETTSIA IN THE PRESENCE OF LIVE TISSUE

1. Rickettsia prowazeki can be cultivated for many generations in vitro, without diminution in numbers or virulence, in media similar to those described by Maitland, Rivers, and others for the cultivation of certain viruses. In all probability, such cultures can be maintained indefinitely. 2. It has been impossible, thus far, to cultivate the typhus rickettsia without employing living tissue.     

STUDIES ON TYPHUS FEVER : II. STUDIES ON THE ETIOLOGY OF MEXICAN TYPHUS FEVER

We have adduced strong evidence in favor of identifying the virus of Mexican typhus fever with the Rickettsiae, or Mooser bodies, observed in the tunica lesions of Mexican typhus guinea pigs. Were it not for the possible presence of a few remaining cell fragments in the washings we would consider this evidence crucial proof.     

STUDIES ON TYPHUS FEVER : X. FURTHER EXPERIMENTS ON ACTIVE IMMUNIZATION AGAINST TYPHUS FEVER WITH KILLED RICKETTSIA

1. Vaccines consisting of formalinized Rickettsiae of Mexican typhus fever, obtained by our X-ray rat method, produce definite resistance in guinea pigs to subsequent infection with the virus of this disease. 2. The resistance so produced amounts to complete immunity when the subsequent infectious dose is moderate—that is, consists of typhus blood or of tunica material in reasonable amounts (not more than one-quarter of a tunica—i.e., roughly 100 to 250 infectious doses). When, as in the first experiment, excessive doses of infectious material were given, the vaccination protection was, in two of the three animals, incomplete. 3. Subcutaneous vaccination is fully as effective as intraperitoneal —even when the subsequent infection is intraperitoneal. 4. As in previously reported experiments, the vaccines made with the Mexican organisms conferred only partial and feeble protection against the European virus (Breinl strain).     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : I. CONCURRENT INFECTIONS DURING THE COURSE OF EXPERIMENTAL TYPHUS FEVER IN GUINEA PIGS.

The work reported in this paper relates to the bacteria which can be cultivated from the blood and spleen of guinea pigs at different stages of infection with the virus of typhus fever. The studies show that during the period of incubation and before the onset of fever no ordinary bacteria appear in the cultures, while on the 1st day of the febrile reaction different bacteria were found in 6 of 26 guinea pigs cultured; on the 2nd day, in 10 of 16; on the 3rd day, in 3 of 4; and on the 4th day in cultures of all of the 4 guinea pigs observed. The findings indicate that the virus of typhus fever is distinct from ordinary cultivable bacteria, and, as the disease set up by the virus progresses, the infected guinea pigs become subject to invasion by secondary or concurrent bacteria which thus induce a mixed infection. The bacteria which under the influence of the virus of typhus fever thus invade the body of the guinea pig are of several kinds, and vary not only among themselves, but also with the day of the fever on which the examination is made. Thus, on the 1st day of the fever Plotz'' bacilli were recovered twice and anaerobic streptococci, proteus bacilli, aerobic diphtheroids, Gärtner type bacilli, and Staphylococcus aureus each once. On the 2nd day Plotz'' bacilli were found four times, anaerobic streptococci three times, Gärtner type bacilli, aerobic diphtheroids, Bacillus welchii, aerobic Gram-positive diplobacilli, and Staphylococcus aureus each once. On the 3rd day Plotz'' bacilli were recovered once, as were anaerobic streptococci and Grtner type bacilli. On the 4th day Staphylococcus aureus was found twice and Plotz'' bacilli and Bacillus proteus each once. This variation in the kind of bacteria as well as the lack of predominance of one kind over another during the different stages of the febrile reaction in guinea pigs leads us to infer that they occur concurrently with the typhus virus. And since the more unusual of these organisms, the Plotz bacillus, the anaerobic streptococcus, the aerobic diphtheroid, and the diplobacillus are non-pathogenic for guinea pigs, while the more common bacteria such as the Gärtner type bacillus, Welch''s bacillus, the proteus bacillus, and the staphylococci induce distinctive effects, and since all the bacteria could be suppressed without their reappearance in guinea pig passages of the virus containing them, we believe that they are independent and unrelated to the true virus of typhus fever.     

STUDIES ON MEXICAN TYPHUS FEVER. I

The preceding studies on typhus fever, chiefly done with a Mexican strain obtained from Dr. Mooser, concern themselves largely with reinvestigations of some of the fundamental problems of this disease. Filtration experiments carried out with methods almost regularly successful with true filterable viruses, in regard to material, suspension fluid, reaction, nature of filters and pressure employed for filtration, indicate that the virus is not filterable in the ordinary sense in which this expression is employed. It is probably smaller than bacteria and the results of filtration experiments suggest that its magnitude is consistent with the tunica bodies observed by Mooser. Negative filtrates did not immunize, a result consistent with the previous work of Olitsky. The virus is present in blood plasma, hardly if at all in leucocytes, and becomes closely associated with the red blood cells, though we do not believe that it is contained in them. It becomes firmly associated with normal red blood cells when these are exposed to infectious plasma, a result similar to that obtained in Rocky Mountain spotted fever by Spencer and Parker. In tissue culture, tunica material with Mooser bodies remains alive and virulent for about 10 days, but so far we have not been able to determine that it can keep alive without the presence of living cells. These results do not carry this subject any further than it has been carried for European typhus in tissue cultures with the same method by Wolbach, Schlesinger and Pinkerton (12). Within glass capsules in the peritoneum of guinea pigs, the virus may remain alive for about the same length of time as in the tissue cultures. Rough comparative virulence estimations between blood plasma in which it would be hardly possible to find a limited number of Mooser bodies, even though they were present, showed the blood plasma to be less infectious than the tunica material, in which considerable numbers of Mooser bodies were visible. The testicular swelling characteristic of Mexican typhus and showing the above mentioned bodies—probably Rickettsia—may be absent in individual guinea pigs under ordinary conditions and in guinea pigs inoculated by other than the intraperitoneal route. On re-inoculation into the peritoneum after non-orchitic passages, the swelling reappears. Whenever it did not so reappear, we found that the strain had either degenerated in virulence or it had been contaminated by intercurrent infection. Though we can not prove it at the present time, we believe that the tunica lesion is an integral part of this disease in guinea pigs, and not an accidental accompaniment. Convalescent blood from Mexican typhus guinea pigs mixed in the test tube with virus affords protection if the blood is taken between the first to the tenth day after defervescence. After the third week, the blood no longer contains protective bodies although the guinea pigs may still be immune. In one case a serum was obtained which was both protective in such a test but at the same time seemed still to contain virus, a result which we cannot explain. No complement-fixing antibodies were found when virus serum was used as antigen and convalescent serum as antibody. The low concentration of the virus in the serum may account for this. In a limited number of observations guinea pigs which were negatively inoculated with virus-serum mixtures proved on re-inoculation to be immune. In one of these cases the protective serum mixture with the virus was taken 1 day, in the other 5 days after temperature had returned to normal and the re-inoculations were done 36 and 40 days after the primary injection. This recalls similar experiences of Nicolle and encourages further immunological study in this direction. In a number of experiments active immunization with formalinized tunica material containing large numbers of the Mooser bodies seems to have modified the course of subsequent inoculations in the direction of protection. A single accidental human infection seemed particularly associated with tunica material, although this cannot be positively asserted. All that part of our work which has bearing on the infectious agent is consistent with the assumption that the small, Giemsa-staining bodies observed by Mooser in the tunica of Mexican typhus guinea pigs represent the virus of the disease.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : VI. SKIN LESIONS IN EXPERIMENTAL TYPHUS FEVER OF GUINEA PIGS.

The skin of guinea pigs in which the virus of typhus fever is propagated, when mildly irritated in advance of the febrile reaction, shows a characteristic exanthem during the height of the experimental disease. More drastic methods of irritation, however, cause a dermatitis which obscures the rash but produce in the corium more marked specific histopathological changes. The exanthem may aid the study of the specific incitant of typhus fever in the lesions.     

STUDIES ON TYPHUS FEVER : XI. A REPORT ON THE PROPERTIES OF THE SERUM OF A HORSE IMMUNIZED WITH KILLED FORMALINIZED RICKETTSIA

The experiments recorded above demonstrate that the systematic treatment of a horse with formalin- and phenol-killed Rickettsiae obtained from the Mexican virus by our X-ray rat technique induces the development of properties in the horse''s serum which may be described as follows: 1. An original agglutinating potency for Proteus X-19 (Weil-Felix reaction) not exceeding dilutions of 1–40 was enhanced to a potency of 1–160 and, feebly, 1–320. 2. The horse''s serum, after immunization, exerts distinct protective action against the Mexican virus, whether mixed with the virus before injection, administered 1 week or somewhat longer before the virus or given 24, 48 or 72 hours after infection with virus. Experiments of the last category have been most successful when the serum was given subcutaneously. 3. The serum of the immunized horse agglutinates the Weigl louse vaccines, containing Rickettsia prowazeki of the European disease in dilutions comparable in potency to the Weil-Felix reaction. Since the serum described exerted protective effects when given 7 and 13 days before infection with the virus, there is some prospect of prophylactic usefulness on the part of this serum. Since the serum also protected when subcutaneously administered 24, 48 and 72 hours after intraperitoneal infection of guinea pigs, and somewhat modified the disease even when given 96 hours after infection—that is 1 day before the control came down with a typical reaction—, it is at least logical to investigate its possible therapeutic value by early administration in the human disease of the New World or Mexican type. The time intervals governing the experiments of the last two categories in guinea pigs were of course dependent upon the balance between the amounts of virus and of serum used. The failure of similar effects upon the European virus is not easily explained. This is especially difficult to understand in view of the unquestionable agglutinating potency of the serum for the Weigl vaccines. Whether this difficulty is purely a quantitative one, or depends upon other factors, can be determined only by further investigation.     

THE MECHANISM OF THE ABDERHALDEN REACTION : STUDIES ON IMMUNITY. I.

1. The Abderhalden reaction is specific. 2. The properties of serum on which it depends develop in experimental animals simultaneously with antibodies during the process of immunization. 3. It is impossible to observe by direct methods the presence of digesting ferments in the blood of immune animals. 4. The Abderhalden test may be resolved into two phases. A dialyzable substance appears in the second phase and is the result of the autodigestion of serum. 5. The autodigestion of serum in the Abderhalden test is due to the removal of antitrypsin from the serum by the sensitized substratum.     

FURTHER STUDIES ON TYPHUS FEVER : ON HOMOLOGOUS ACTIVE IMMUNIZATION AGAINST THE EUROPEAN STRAIN OF TYPHUS FEVER

1. Guinea pigs can be actively immunized against European typhus fever with homologous formalinized Rickettsia tissue cultures, provided sufficient amounts are injected. The method is suggested for practical application in man. 2. Serovaccination against European typhus fever can be successfully applied to guinea pigs by a variety of methods, the simplest of which consists of the injection of mixtures of virulent defibrinated guinea pig blood and convalescent guinea pig serum taken from 3 to 5 days after defervescence. Similar results can be obtained with mixtures in which tissue culture virus, either with convalescent guinea pig serum or with antimurine horse serum, is used. There is no indication so far that such animals become carriers. Possible application of these methods to typhus epidemics is discussed.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : III. FILTRATION EXPERIMENTS.

We have presented experiments to show that the typhus virus in the tissues of the guinea pig during the height of reaction to the experimental disease does not lose its infecting power when the cells of the brain or of the spleen are disintegrated by repeated freezing and thawing, or by freezing and desiccating, or by crushing by mechanical means, or by grinding into a homogeneous pulp with sand. The virus after such treatment is as actively infective as in the same tissue not subjected to the disintegrating influences. The possibility exists, therefore, of an extracellular condition of the typhus virus. Fourteen attempts to filter through Berkefeld V and N candles the virus contained in the disintegrated tissue have all resulted in failure.     

STUDIES ON TYPHUS FEVER : V. ACTIVE IMMUNIZATION AGAINST TYPHUS FEVER WITH FORMALINIZED VIRUS

We have adduced evidence that guinea pigs can be completely or partially protected by three injections of typhus tunica material in which there are moderate numbers of Rickettsiae, treated for from 24 to 48 hours with a 0.2 per cent formalin solution. We believe that the immunization is due to the presence of the Rickettsiae, since in our preceding experiments we have satisfied ourselves that these organisms are the true etiological factors of the disease. For the reasons stated above, we believe that the formalinized vaccine does not contain living, but attenuated organisms, and that the immunizing effect is the result of treatment with formalin-killed Rickettsiae. This point, however, we admit, is not absolutely determined. These experiments, together with the results obtained in the concentration of Rickettsia material by the diet method of reducing resistance as described in the paper which follows, furnish a hopeful method and a reasonable theoretical basis for a procedure of active immunization against this disease in human beings.     

STUDIES ON THE MECHANISM OF PENICILLIN-INDUCED FEVER

Rabbits immunized to benzylpenicillin G responded with fever when challenged with a penicillin-serum protein conjugate, but not with penicillin itself. After one or two challenges with conjugate, the rabbits became unresponsive (tolerant) to further injections. This form of hypersensitivity was transferable with plasma of immunized donors to normal rabbits. Blood leukocytes of immunized rabbits incubated with penicillin-protein conjugate and hypersensitive serum released endogenous pyrogen in vitro. Spleen cells from the same animals, on the other hand, were inactive when incubated with this antigen in vitro. These experiments appear to be the first to demonstrate in vitro a possible mechanism of drug-induced fever.     

ON THE RELATION OF THE ORGANISMS IN THE TUNICA VAGINALIS OF ANIMALS INOCULATED WITH MEXICAN TYPHUS TO RICKETTSIA PROWAZEKI AND TO THE CAUSATIVE AGENT OF THAT DISEASE

Healthy lice became infected with Rickettsia prowazeki after feeding on monkeys inoculated with a strain of Mexican typhus. The same result was obtained in 100 per cent of lice by rectal inoculation of an emulsion of tunica vaginalis of guinea pigs reacting to the same strain. In the tunica vaginalis of guinea pigs and rats inoculated intraperitoneally with an emulsion of lice containing Rickettsia prowazeki the intracellular organism constantly associated with the passage strain appeared regularly. Rickettsia prowazeki found in lice and the organism constantly present in the tunica of guinea pigs and rats reacting to our strain of tabardillo are indistinguishable morphologically and tinctorially and their mode of intracellular multiplication is alike in every respect. It is concluded that they are identical. This organism is constantly associated with the causative agent of Mexican typhus, both in mammals and in lice, and all of our attempts to separate them have failed.     

ON THE PRESERVATION OF TYPHUS FEVER RICKETTSIAE IN CULTURES

1. Cultures of typhus fever rickettsiae were as a rule found to remain viable and virulent after being stored at 37° and –20°C. for several months, whereas they failed to survive when stored at the intermediate temperatures of 20° and –4°C. for 4 weeks and 10 days respectively. In one instance cultures were stored at 37°C. for 8 months without having been transferred, and were subsequently found to be viable and infectious. 2. The conditions influencing such long survival of an organism which seems to require living tissue for multiplication (as filterable viruses do in general) are discussed. 3. Typhus-infected tissues (minced guinea pig tunica) suspended in a serum-Tyrode mixture in sealed flasks remained infectious at 37°C. for at least 10 weeks. 4. Additional evidence for the etiological significance of the rickettsiae in typhus fever is obtained from the experiments described.     

STUDIES ON TYPHUS FEVER : III. STUDIES OF LICE AND BEDBUGS (CIMEX LECTULARIUS) WITH MEXICAN TYPHUS FEVER VIRUS

Our experiments have shown that the Mooser bodies or Rickettsiae derived from guinea pigs with Mexican typhus fever can survive in bedbugs after intra-coelomic injection for 10 days, remaining capable of infection. We have also succeeded in similarly infecting bedbugs by allowing them to feed on benzolized rats in whose blood Rickettsiae had been shown to be present. Injection of the organs of such bedbugs 5 days after the last, 9 days after the first infectious feeding into guinea pigs produced typical Mexican typhus fever. Some of the guinea pigs infected with such bedbug organs and passing through a typical typhus proved to be immune to subsequent inoculation with the European disease. Attempts to infect normal guinea pigs by allowing infected bedbugs to feed on them or by rubbing the feces into the uninjured skin have, so far, been unsuccessful. We have not, therefore, completed the cycle proving that bedbugs can transmit the disease, but we have shown that this is a possibility when dealing with man, obviously more susceptible to the disease than any of our experimental animals. The ease with which the Rickettsiae seem to survive in the bedbugs suggests the desirability of investigating other common insects for a similar capacity of harboring the typhus Rickettsiae-experiments which we have not yet had the time to carry out.     

STUDIES ON TYPHUS FEVER : VII. ACTIVE IMMUNIZATION AGAINST MEXICAN TYPHUS FEVER WITH DEAD VIRUS

Guinea pigs can be immunized against Mexican typhus virus by peritoneal injections of formalinized Rickettsia material, provided sufficient amounts of the organisms are used. Our results in this respect are analogous to those of Spencer and Parker with carbolized virus of Rocky Mountain spotted fever. The Rickettsia suspensions appear to possess considerable toxicity. We do not wish to be misunderstood as implying that the results in guinea pigs offer anything more than a demonstration of the principle of active immunization with killed Rickettsiae. Application to man will have to be worked out, and preliminary to this, we are now attempting to apply the methods to a limited number of monkeys.