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We present the use of OCT for breast cancer imaging. In situ rat mammary and excised human breast tumors were imaged. The feasibility of OCT for applications in surgical oncology of the breast is discussed.

Epidemic, louse-borne typhus persists in the rugged, mountainous areas of Ethiopia and much of northeastern and central Africa as well as in the rural highlands of Central and South America, where the conditions of living favor the harboring of body lice and where antibiotic treatment and effective louse-control measures are unavailable. The historical significance and current epidemiology of typhus, including the reservoir of Rickettsia prowazekii in flying squirrels in the United States, are reviewed, and the clinical presentation, laboratory findings, and hospital course in the cases of 60 patients admitted with epidemic, louse-borne typhus to the St. Paul's Hospital in Addis Ababa, Ethiopia, are described. Treatment of this disease with oral doxycycline, tetracycline, or chloramphenicol prevents complications and results in prompt resolution of symptoms.

Detailed observations on the acquisition and propagation of experimental Rickettsia mooseri infection in two species of fleas are presented. Rickettsia mooseri infection became detectable by means of the direct fluorescent antibody test about 2 days earlier in Leptopsylla segnis than in the putative vector, Xenopsylla cheopis. By the 6th day after the infective feeding, the entire lining and the lumen of the midgut in L. segnis contained masses of rickettsiae and the agent was being passed in the feces of the flea, whereas in X. cheopis these events did not occur until the 8th day. Basic behavioral differences in the two species of flea may explain these discrepancies and also influence their ability to serve as vectors of murine typhus. As a semisessile flea and sustained feeder, L. segnis only rarely attaches to a second individual and thus has an opportunity to acquire a heavy dose of rickettsiae, if feeding on a rickettsemic host. X. cheopis, in contrast, feeds rapidly and intermittently, even on man, and generally leaves its host soon thereafter, later returning to the same or another host to feed again. While L. segnis may not be as efficient a vector as X. cheopis regarding the intramurine cycle or transmission to man of murine typhus, the dense accumulation of infective feces on certain sites on the fur of the host raises the possibility of air-borne infection to man or rodent. Infection with R. mooseri had no effect on the survival of X. cheopis and L. segnis. Furthermore, no visible cytopathological effect was found in the paraffin-embedded sections of infected fleas.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique features of the primary site of rickettsial replication in typhus fevers, i.e., within the endothelial cells of small blood vessels in tissues, suggest that effector mechanisms, other than those dependent on phagocytosis by activated macrophages with enhanced microbicidal properties, most likely are necessary to explain the cell-mediated immune control of intracellular rickettsial replication in these sites. Theoretically, such mechanisms might involve contact between infected endothelial cells and activated T lymphocyte subpopulations or macrophages or immunologically induced soluble factors or lymphokines. Support for the existence of at least one of these alternative effector mechanisms is presented here for Rickettsia prowazekii. Cultures of human blood leukocytes, upon immunologically specific stimulation with R. prowazekii antigen or nonspecific stimulation with the mitogen phytohemagglutinin, produce soluble factor(s) in the supernatant fluid which, in culture, have (a) an intracellular antirickettsial action on R. prowazekii-infected human endothelial cells, fibroblasts, and macrophages, and (b) a specific cytolytic action on R. prowazekii-infected, but not uninfected bystander, human fibroblasts. Neither action is demonstrable in R. prowazekii-infected chicken embryo fibroblasts. The factor(s) has no direct antimicrobial action on extracellular rickettsiae and is inactivated by heating at 56 degree C for 1 h or by acid treatment at pH 2. Expression of the antirickettsial action requires new host cell messenger transcription and protein synthesis, whereas the cytolytic action does not. The circumstances of production and action and the properties of the factor(s) responsible for the intracellular antirickettsial, and perhaps also the cytolytic action are consistent with those of immune interferon (IFN-gamma).

To characterize IgM and IgG antibody responses in Rocky Mountain spotted fever (RMSF), a microtiter enzyme-linked immunosorbent assay (ELISA) using density gradient-purified Rickettsia rickettsii as antigen was developed. Sera of vaccinated individuals and patients with RMSF were tested by ELISA and by indirect fluorescent antibody (IFA) tests. Diagnostic agreement between ELISA and the IFA test was 76% and 52% for IgG and IgM antibody, respectively. Diagnostic agreement between the ELISA for IgG antibody and the IFA test for total immunoglobulins was 84%. The ELISAs for IgM and IgG antibody were as specific (100%) and as sensitive (100%0) as the IFA test (83o%-100%o) in detecting antibody increases in paired sera from persons with RMSF and were superior to the IFA test in detecting seroconversions in vaccinees. The ELISA also detected antibodies in a single convalescent-phase serum with sensitivity and reliability. The ELISA for IgG antibody is appropriate for seroepidemiology and serodiagnosis since it permits measurement of antibody at a single dilution of serum up to a year after illness.

Secondary chicken embryo fibroblasts infected in suspension with the Breinl strain of Rickettsia prowazekii and grown in monolayer culture were examined by both transmission and scanning electron microscopy at specific intervals after infection to study the effects of prolonged intracellular growth on the fine structure of the host cell and the rickettsiae. Cytopathological changes in the infected host cells were not apparent until late in the intracellular growth cycle when the cells began to rupture as a result of a large rickettsial burden. The only recognizable changes in heavily infected cells before lysis were the condensation of the intercristal matrix of some mitochondria and the apparent dissociation of ribosomes from the rough-surfaced endoplasmic reticulum. Although the effects of intracellular growth of rickettsiae on the fine structure of the host cell were rather unremarkable when compared with those imposed by Rickettsia rickettsii in a similar cell system, noticeable morphological changes in the rickettsiae were recognized during the intracellular growth cycle. These changes first became apparent about 40 h postinfection and consisted primarily of an increased electron density of the rickettsiae, the appearance of numerous vacuoles in the rickettsial cytoplasm, and a slight reduction in size of the rickettsiae. Changes of this nature may reflect transitional phases of growth characteristically seen in free-living bacterial cell systems.

Rickettsia mooseri infection has been studied in syngeneic guinea pigs inoculated intradermally with the objective of developing a model for the study of immune mechanisms. Characterization of infection included the following: a study of replication, dissemination, and clearance of rickettsiae; measurement of the antibody response with different rickettsial antigens and tests; and attempts to measure the cell-mediated immune response using the correlate of delayed-type hypersensitivity skin reactions. Following intradermal inoculation, rickettsiae replicate locally and then spread to the draining lymph nodes and subsequently cause systemic infection. Spread to draining lymph nodes occurred before the appearance of circulating antibody, whereas systemic infection occurred afterwards. Two distinct patterns of acquired resistance developed. The first was marked by a cessation of rickettsial growth within a given organ and the second by a clearance of rickettsiae. The duration of each of these phases differed markedly from one organ to another. Delayed-type hypersensitivity was not demonstrated by skin testing.

A new formalin-inactivated vaccine prepared by sucrose density gradient centrifugation of tissue culture-grown Rickettsia rickettsii was evaluated for safety and immunogenicity in a placebo-controlled, double-blind study. Most of the 52 seronegative adult vaccinees, 88% after the first and 66% after the second dose, experienced brief, mild (mostly local) reactions, but only 50% exhibited a systemic immune response to vaccination. Six unvaccinated volunteers (controls) and 16 of these vaccinees were challenged with R rickettsii one month after vaccination. Vaccine efficacy was 25%; all six controls and 12 of 16 vaccinees developed typical Rocky Mountain spotted fever. The incubation period was longer, the duration of constitutional symptoms shorter, and the height of fever lower in ill vaccinees than in controls. The vaccine provided only partial protection against Rocky Mountain spotted fever but ameliorated the illness.

The infection cycle of Rickettsia rickettsii, studied in slide chamber cultures of chicken embryo and L-929 cells, was found to be complex and did not conform to a one-step growth cycle. Initial uptake kinetics resembled those established for Rickettsia prowazekii, but subsequent events showed very marked differences. Intracytoplasmic growth commenced exponentially without measurable lag. However, very soon after infection, intracytoplasmic rickettsiae began to escape from the host cell into the medium in large numbers, resulting in (i) failure of large numbers of rickettsiae to accumulate in the cytoplasm, (ii) sustained rapid division of the organisms in the cytoplasm, (iii) substantial accumulation of extracellular rickettsiae, and (iv) rapidly spreading infection in the culture, with most cells infected in 48 to 72 h. In the occasional cell, rickettsiae were found in the nucleus, where they multiplied to form compact masses. Thus, analysis of the growth characteristics of R. rickettsii must consider the entire culture as a unit in which the rickettsiae are distributed among three compartments in which they behave in different ways: (i) intranuclear, (ii) intracytoplasmic, and (iii) extracellular. The rickettsial traffic is bidirectional across the host cell plasma membrane and dominantly monodirectional across the nuclear membranes. The implications of this behavior with respect to location and range of receptors and substrates involved in membrane penetration are discussed. In older cultures, unique intracytoplasmic ring or doughnut colonies were common, indicating a change in the intracytoplasmic environment. The possible significance of the growth characteristics in cell culture to the characteristics of infection in humans and animals is discussed.

The taxonomy of the typhus biogroup (Rickettsia prowazekii and R. mooseri [R. typhi]) of the genus Rickettsia and Rochalimaea quintana has been studied on the basis of the guanine plus cytosine content of the respective deoxyribonucleic acids (DNAs), the genome size, and the degree of DNA-DNA hybridization. The previously published guanine plus cytosine values for certain strains are confirmed, and the guanine plus cytosine values are described for some additional strains in each species. The genome sizes of four strains of R. prowazekii, three strains of R. mooseri, and two strains of R. quintana were very similar and averaged approximately 109 daltons. The various strains within the species boundaries hybridized at nearly the 100% level. The DNAs of various strains of R. prowazekii hybridized with the DNAs of various strains of R. mooseri rather consistently at the level of 70 to 77%. The DNAs of two strains of Rochalimaea quintana hybridized with those of R. prowazekii Breinl and R. mooseri Wilmington in the range of 25 to 33%.

An electron microscope study of infected human louse gut cells and feces was made to determine whether a valid correlation exists between the increased resistance of Rickettsia prowazeki (in the louse feces) to adverse environmental influences and changes in the organism which might be reflected in its ultrastructure. Upon fine structural examination of this intracellular parasite as it passed from the louse midgut cell to the feces, it was apparent that no such morphological changes had occurred.

During the feeding process, the cat flea, Ctenocephalides felis , acquires Rickettsia mooseri ( R. typhi ), the etiological agent of murine typhus, just as readily as does the putative vector, Xenopsylla cheopis . The course of infection within the species of fleas is similar. Following the logarithmic growth stage of R. mooseri during the first 9 days after the infectious feeding, the rickettsial content of the infected flea remained within 105–106 plaque-forming units/flea. In frozen sections of infected fleas prepared on the 9th day, the midgut epithelial cells were packed with rickettsiae. Rickettsia mooseri was noted within the lumen of the proventriculus and of the foregut in 20% of the cat fleas tested on the 15th–17th days, suggesting that transmission by bite may sometimes occur. Growth of the rickettsiae within the flea proceeded at a much more rapid rate when fleas were maintained under ambient conditions of 24 and 30 °C than when they were kept at 18 °C. The survival rates of infected fleas corresponded with those of uninfected controls throughout the 21 days of experimentation. The pattern and intensity of the laboratory infection of R. mooseri in C. felis , the broad host range and multiple feedings of this flea, and its past history as a suspected vector all suggest that C. felis may play a significant role in transmitting murine typhus infection to man and other mammals.

An investigation of a case of apparent mass psychogenic illness was undertaken in a midwestern electronics assembly plant. The plant employed 500 workers, of whom 80% were female. The illness outbreak involved a total of 90 female first shift workers who reported a variety of nonspecific symptoms such as headache, dizziness, and lightheadedness in response to a strange odor in the workplace. Although environmental testing revealed some localized concentrations of a few airbone contaminants, no environmental toxins were discovered that could account for the continuing outbreaks of illness. An ad hoc sample of affected and nonaffected workers was surveyed to assess the influence of psychological, sociological, and work environment factors in the outbreak. Analysis of the data revealed that affected workers reported more physical discomfort (temperature variations, poor lighting) in the workplace as well as psychological job stress (increase in workload, conflicts with supervisors) than did nonaffected workers. Moreover, affected workers scored significantly higher than nonaffected workers on personality tests measuring extraversion and hysteria traits.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.

A critical review of the ecology of chigger-borne rickettsiosis (scrub typhus) is presented, based upon a synthesis of both published and unpublished information. All the epidemiological characteristics of chigger-borne typhus are attributable to the habits and other features of the vector chiggers. This rickettsiosis is found in a remarkably broad range of habitats, presumably in all areas where members of the Leptotrombidium deliense -group of chiggers and wild rats of the genus Rattus coexist, ranging from semideserts to alpine reaches in the mountains, and from sandy beaches to dense but disturbed forest. The habitats are characterized by the presence of changing ecological conditions, wrought by man or nature, and expressed by transitional types of vegetation, even if these are in circumscribed foci as along jungle streams. The intimate association between the rats, chiggers and secondary vegetation (which together with Rickettsia tsutsugamushi , the etiological agent, constitute the “zoonotic tetrad”) is an ancient one. We postulate that as Rattus and associated chiggers of the subgenus Leptotrombidium penetrated new areas and habitats, they diversified, and as the chiggers adapted to other hosts in the new regions, such mammals became secondarily involved in the ecology of the rickettsiosis. Chiggers in the L. deliense -group constitute the major vectors of this disease, and we consider that they also serve as the primary reservoir of the infection in nature, perpetuating the cycle by transovarian transmission of the rickettsiae from the naturally infected female mite to the next generation. While chiggers can acquire rickettsiae from a rodent host by feeding, laboratory data suggest that only rarely can this result in infection in another mammal, whether by a chigger that has become infected while feeding and then reattaching to a second host (which is unusual in itself) or by transovarially infected progeny. However, while such an event may be difficult to demonstrate in the laboratory, the same low percentage of success in the field would be significant in the natural cycle because of the vast numbers of chiggers involved, and because of the amplification that would ensue by transovarian transmissionof acquired infection over a period of generations. Small mammals (“theraphions”) like rats, voles, and tree-shrews which are primarily ground-dwelling are important in the ecologyof the rickettsiosis by serving as major hosts of the vector chiggers. Arboreal rodents and commensal rats play little role in the infection, apparently because they seldom come in contact with ground infested with the L. deliense -group. Rickettsia tsutsugamushi differs significantly from other members of the genus, and the similarities between them apparently represent convergence towards existence as intracellular parasites with cycles in both mammals and arthropods. The so-called serotypes of the agent, in contrast, are quite divergent. The widespread range of certain serotypes, and the fact that they may be transmitted by several species of chiggers suggest that infection acquired from theraphions may be significant in the ecology of the rickettsiosis. By using chiggers of the L. deliense -complex and wild rats as indicators of the presence of this infection, “oasis chigger-borne rickettsiosis” has been demonstrated among relict faunae in “ecological islands.” Such faunal data indicate that the infection may be present in areas where it has not yet been demonstrated, as in northern Iran and some southern republics of the U.S.S.R., parts of Tibet, Sinkiang, etc.

Summary In contrast to the focal perivascular collections of mononuclear cells which constitute the classical basic lesion seen in persons dying of typhus fevers, the initial cellular response in man to the presence of typhus rickettsiae, as revealed by the skin window technique, consisted of a typical acute inflammatory reaction. Polymorphonuclear leukocytes appeared within 1.5 hr after introduction of the organisms and increased in numbers during the first 24 hr. Although mononuclear cells began to appear in the lesions between 6 and 12 hr, the polymorphonuclear leukocyte remained the dominant cell throughout the 24-hr observation period. The main difference between the responses of susceptible and immune subjects lay in the more intense polymorphonuclear reaction in the immune subjects. In vivo phagocytosis of living and dead rickettsiae was demonstrable with both polymorphonuclear and larger mononuclear cells in immune as well as nonimmune subjects. The experimental conditions employed reproduced closely the conditions which obtain at the time of natural infection. The implications of these findings with regard to host defense reactions against typhus rickettsiae are discussed.

The presence of scrub typhus infection in rodents or trombiculid mites in the following unusual habitats and geographical areas in West Pakistan is discussed: (1) in alpine terrain at 10,500 ft. elevation in the Kaghan Valley of the Himalayas, where the snow remains on the ground 9 or 10 months of the year; (2) in the mountain deserts of Gilgit Agency, 36°N latitude; (3) in the semi-desert of Multan; and (4) in the plains of Lahore District. In these areas, unlike Sialkot District where Leptotrombidium ( Leptotrombidium ) deliense (Walch 1922) is abundant, known vectors of scrub typhus have not been collected, but related forms of the subgenus Leptotrombidium occur, and several of these are new to science. Natural infection with Rickettsia tsutsugamushi was demonstrated in almost all the habitats, virtually all from tissues of rodents. The highest rate of such positive isolations was from the temperate, coniferous forest zones of the Kaghan Valley (29 of 119, or 24%), primarily from indigenous mice, such as Apodemus, Alticola and Hyperacrius . Notes are included on the possible role in R. tsutsugamushi infection of the following rodents: Alticola roylei, Hyperacrius fertilis, H. wynnei, Millardia meltada, Nesokia indica and Tatera indica . Of the 3 strains recovered from the trombiculid mites (chiggers) 1 was from the endemic area at Sialkot; the others from chiggers from Alticola voles at 8,000 ft. in the Kaghan Valley. In the midst of the desolate mountain deserts of Gilgit occur “ecological islands” atop of peaks or in sheltered valleys watered by glacial streams, where the major elements of the flora and fauna are the same as those in the Kaghan Valley. Many of the species of small mammals and ectoparasites are probably relicts of aeons when the whole area was forested, and hence the infections associated with the fauna of the earlier period may also be present. The “oasis scrub typhus” theory would explain the isolation of R. tsutsugamushi from Apodemus at Gupis, Gilgit Agency.

Cell walls prepared by mechanically disrupting intact Rickettsia mooseri ( R. typhi ) were examined in an electron microscope and analyzed chemically. Electron micrographs of metal-shadowed and negatively stained rickettsial cell walls revealed no significant differences, except for smaller size, from bacterial cell walls prepared in a similar manner. The chemical composition was complex, and resembled that of gram-negative bacterial cell walls more closely than that of gram-positive bacterial cell walls. R. mooseri cell walls contained the sugars, glucose, galactose, and glucuronic acid, the amino sugars, glucosamine, and muramic acid, and at least 15 amino acids. Diaminopimelic acid, a compound hitherto found only in bacteria and blue-green algae, was demonstrated in rickettsiae for the first time. Teichoic acids were not detected. The compounds identified accounted for about 70% of the dry weight of the cell walls.

Discussion and Summary The rabbit has proved useful for studying certain physiologic changes brought about by R. mooseri toxin. Depending on the dose of toxin employed, either rapid or late death was observed. In general, rapid death was accompanied by moderate to massive progressive hemolysis which resulted in maximum hemoglobinemia at the time of death. Hand in hand with such rapid death and massive hemolysis, a progressive increase in plasma potassium concentration was regularly observed and in the course of 30 to 60 minutes reached extremely high levels. In contrast to such rapid death, an entirely different sequence of events was noted in those rabbits which expired after several hours. Here, although moderate hemolysis did occur, it was maximal long before exitus. Hyperkaliemia developed; however, it did not appear until these animals were prostrate and death was close at hand. Thus, although both hemoglobinemia and hyperkaliemia did appear in these rabbits dying late, they did not bear the same close temporal relationships as were observed in the rabbits dying rapidly. In both types of death electrocardiographic abnormalities were usually observed. These abnormalities were compatible with hyperkaliemia and were associated with the presence of elevated potassium concentrations. In most rabbits a fall in arterial blood pressure appeared soon after the injection of toxin. Subsequently, hypotension became more marked and reached similar low levels irrespective of the time of death. Rapid death would appear to result from the rapidly rising plasma potassium concentration which attained terminal levels of 12 to 15 mEq/L. Such plasma potassium levels are known to be incompatible with life in several experimental animals (16, 17, 18) and man (19). Furthermore, the electrocardiographic changes observed in this study bear a close resemblance to those reported in hyperkaliemic death of dogs (16, 17), cats (18), rabbits (20) and man (21). The rapid massive hemolysis is adequate to account in large part for the profound hyperkaliemia. Thus, based upon hematocrit determinations approximately 30% of the total circulating red blood cell mass of these rabbits underwent lysis prior to death. Theoretical calculations for a rabbit weighing 1,000 grams using 70 ml and 42 ml for the total blood and plasma volumes, respectively, (22) and our observed value of 116 mEq of potassium per liter packed rabbit erythrocytes, indicate that the destruction of 30% of the circulating red blood cell mass in the course of 1 hour would liberate potassium into the plasma at a rate approximating 0.02 mEq/Kg/minute. However, in vivo hemolysis does not appear to proceed at a linear rate which is assumed in the above calculation, but rather increases rapidly to a maximal rate at the time of death. Consequently, at this time the liberation of potassium may well exceed the theoretical values. Normal rabbits are reported to succumb when potassium is infused in excess of 0.025 mEq/Kg/minute (23). It is of interest to note that potassium concentrations comparable to those observed here have been reported to occur in rabbits just prior to death following rapid lysis of a comparable red cell mass by saponin (24). Additional factors, such as the rapid onset of shock or the untoward effect of marked hemoglobinemia on renal function described in man (25), may have served to retard rapid elimination of this cation through normal renal channels. Further evidence, suggesting some impairment of renal function, was found in the elevated terminal plasma phosphate levels in these animals. Presumably, a large amount of this anion was derived from the in vivo destruction of erythrocytes, and, because of altered renal mechanisms, it accumulated as did potassium. Whether the increased potassium in the plasma of these rabbits arose entirely through the mechanism of red cell lysis or in part from damaged tissue cells cannot be stated with certainty at this time. However, we suspect that damaged tissue cells also provided a share of the potassium. Whatever its source, large amounts of potassium did accumulate and reached lethal levels. The role of hemolysis in the pathogenesis of late death in the rabbit is difficult to evaluate. Varying degrees of renal failure, manifested by terminal increases in B.U.N. values, were demonstrable in certain of these animals which expired after a period of 60 minutes or more subsequent to the injection of toxin. Plasma phosphate levels were similarly noted to be increased at the time delayed death occurred. A slow liberation of potassium from erythrocytes as well as other damaged cells, accompanied by increasing renal malfunction, may explain the delayed but definite accumulation of potassium observed. The end result, however, was potassium excess and inevitable exitus. The marked hemoconcentration demonstrated in rats and mice by this study and previously observed in the latter by others (1, 2) suggests that rapid plasma exudation does occur in these species and implies that capillary damage may play an important role in the production of toxic tissue damage. We do not wish to suggest that there is absence of capillary damage in rabbits which die from rickettsial toxin. However, our data permit no statement regarding the presence of such damage in this species of experimental animal. Hypotension was demonstrated to varying degrees in the majority of treated rabbits. The exact mechanism of its production is not clear. Regardless of its origin the severe hypotension might be expected to give rise to numerous secondary changes which could easily be misinterpreted as primary effects of R. mooseri toxin. The results of this study concerning R. mooseri toxin clearly indicate the value of employing several different species of susceptible hosts in defining the general physiologic effects of a given toxin.

Summary and ConclusionsThe spotted fever group complement fixing antibody response was used as a measure of the immunogenicity of a commercially available killed Rocky Mountain spotted fever vaccine in human subjects. A single-dose primary course of vaccine elicited a detectable antibody response in only about 22% of 68 subjects, whereas a 3-dose primary course stimulated the development of complement fixing antibodies in about 63% of eight subjects. The low antibody titers attained may be a function of the relatively low complement fixing antigen content of the vaccine.A booster dose of vaccine given between 1 and 6 months after the primary course failed to elicit an antibody response in the majority of subjects. A second booster dose, given a year after the 6-month booster dose, also failed to cause a significant response. On the other hand, three subjects who had last received RMSF vaccine several years prior to this study developed a typical secondary antibody response upon revaccination. These same s...

Summary 1. Experiments involving parastized and control fowls were made to test the tolerance of growing chickens to moderate infections of ascarids and tapeworms. 2. The criteria for judging the tolerance included growth, blood sugar level, and hemoglobin content of parasitized fowls comparable to those of the control chickens. The criteria for the tapeworm tests included also differential blood counts. 3. Chickens 23 days of age parasitized with 200 infective eggs of the fowl ascarid, Ascaridia galli, were able to tolerate infections of from one to 46 worms without manifesting definite harm from the worms in four weeks. The average of 17.9 ascardis per fowl is above the average infection in range fowls. 4. Growing chickens parasitized at 40 days of age with 200 larval tapeworms (Raillietina cesticillus) were not significantly harmed by infections of from two to 172 tapeworms during a period of eight weeks. The average of 61.5 tapeworms per fowl is more than an average infection found under conditions of general poultry production.

Summary Immunologic studies were performed in mice, guinea pigs and man with cell wall and cytoplasmic fractions of Rickettsia mooseri obtained by mechanical disruption. On a weight basis, cell walls were more effective antigens than either whole rickettsiae or cytoplasmic fraction in the following tests: typhus group complement-fixation and hemagglutination tests and stimulation of antitoxic immunity in mice and anti-infectious immunity in guinea pigs. Cell walls also elicited a delayed type skin reaction in hypersensitive human subjects and a toxic skin reaction in nonsensitive subjects. It was concluded that the antigens involved in these tests were located in or on the cell wall.

Summary Intravenous administration of lethal quantities of murine typhus toxin to white rats is followed by a characteristic and reproducible pattern of changes in the meso-appendiceal vascular bed. After a latent period of about ½ hr, the large and small arterioles and precapillaries undergo a constriction that gradually increases in intensity until the animal dies. At the same time, capillary blood flow diminishes progressively finally leaving the capillary bed markedly ischemic. Epinephrine sensitivity of the terminal arterioles and precapillaries does not change throughout the course of the toxemia. Hemoconcentration is generally present terminally. Despite this, systemic arterial blood pressure in the unanesthetized animal remains essentially unchanged until near death. These observations suggest: a) capillary vascular permeability is increased in a remarkably specific manner that leaves unaltered the capacity of terminal arterioles and precapillaries to react to epinephrine or to participate in certain reflex compensatory mechanisms and b) the humoral vasoexcitor mechanism (VEM) usually operative in acute blood loss does not function. The systemic arterial blood pressure in the anesthetized rat falls sharply at about the same time that the visible peripheral arteriolar constriction and slowing of blood flow commence. Unaltered hematocrit values at the time of rapid fall in arterial blood pressure stand as evidence against the occurrence of hemoconcentration at this early stage. These observations strongly suggest that cardiac output, at least in the anesthetized animal, is appreciably diminished early in the course of the toxemia prior to, and initially independent of, a reduction in total blood volume from the loss of plasma into the tissues through excessively permeable vessels. The distinctive pattern of peripheral vascular changes caused by lethal doses of murine typhus toxin in white rats differs in important details from the effects of lethal amounts of Salmonella typhosa endotoxin and from acute blood loss by hemorrhage.

SummaryDiaminopimelic acid is present in whole cells of R. prowazekii, R. mooseri, R. quintana, and C. burneti in concentrations only slightly lower than those reported for Gram negative bacteria. In each species studied meso DAP is the only isomer present.

Preincubation of Rickettsia mooseri with human typhus convalescent serum, which is not rickettsiacidal but which confers passive protection to animals, opsonizes the rickettsiae for enhanced phagocytosis by monocyte-derived human macrophages in cell culture and renders them susceptible to destruction within the macrophages. Nonspecific opsonization by preincubation of the rickettsia with methylated bovine serum albumin enhances phagocytosis, but the rickettsiae are not prepared for intracellular destruction. Instead, they grow within the macrophages and eventually destroy these cells. Thus, immune serum and macrophages, neither of which is capable of killing these rickettsiae alone, act in concert to destroy the virulent organisms. In this system, immune serum appears to exert two distinct, possibly dissociable, actions on the rickettsiae: enhancement of phagocytosis and preparation for intracellular destruction. Complement is not required for this action but, when present with immune serum, markedly enhances phagocytosis of the rickettsiae, often leading to rapid destruction of the macrophage.

The passive permeability properties of Rickettsia mooseri to both inorganic and organic solutes have been examined. Visual observations by phase-contrast microscopy of rickettsiae in macerated yolk sacs taken directly from heavily infected eggs revealed plasmolysis with hypertonic NaCl and KCl as well as with sucrose solutions. In contrast, similar visual studies of rickettsiae which had been subjected to freezing or to a purification process, or both, were plasmolyzed by hypertonic sucrose but not by hypertonic NaCl and KCl. These primary observations were extended to a variety of solutes and were placed on a quantitative basis by use of optical density and radioisotope dilution methods. Intracellular Na + and K + concentrations in processed rickettsiae, measured by flame photometry, closely paralleled the concentration of these ions in the suspending medium. It was concluded that R. mooseri appears to possess an osmotically active, functional, and structural membrane distinct from the cell wall, located at the surface of a structure analogous to the bacterial protoplast. In the intact organism, this membrane is passively impermeable to sucrose, NaCl, and KCl. However, altered permeability properties, especially to inorganic electrolytes, may be expected in rickettsiae which have been stored in the frozen state and subjected to a lengthy purification process.

Summary 1. A cave harboring the tick Ornithodorus turicata, is reported from Denton County, Texas. 2. The cave was identified as the Sam Bass Cave studied previously by Kemp and his associates. 3. For the first time experimental evidence is presented that many of these ticks are naturally infected with the spirochete of relapsing fever and that they are able to transmit the infection to man and white rats. 4. An attempt has been made to indicate the possible mechanisms through which the infection in this area apparently disappeared and reappeared.

Summary Purified preparations of yolk sac-grown virulent (Breinl strain) and attenuated (Madrid E strain) Rickettsia prowazeki and the Wilmington strain of Rickettsia mooseri were found to exert a positive chemotactic effect upon migrating human polymorphonuclear leukocytes in an in vitro test system. Normal yolk sac material failed to influence leukocyte migration.

Summary The preparation of purified suspensions of Rickettsia mooseri in a metabolically active state has been described. A rough correlation was found to obtain between total nitrogen content of the suspensions and (a) rate of glutamate oxidation, (b) amount of complement-fixing antigen, and (c) toxin titer. An attempt has been made to estimate the relative amount of certain host cell contaminants in the rickettsial suspensions by assay of an enzyme, cytochrome oxidase, which is associated with specific particulate fractions of the yolk sac material. These particles are concentrated by differential centrifugation along with the rickettsiae and are reduced in amount by subsequent procedures. The residual cytochrome oxidase measured is probably not a constituent part of the rickettsiae. Catalase and adenosinetriphosphatase are present in both rickettsial and control preparations. The former is reduced in amount during the purification process at roughly the same rate as cytochrome oxidase, whereas the latter appears in both normal and infected preparations in amounts greater than would be anticipated were it entirely associated with the same particulate matter as cytochrome oxidase. Studies in the analytical centrifuge have demonstrated the presence of a particulate contaminant in the final rickettsial suspensions. Optimal concentrations of substrate, PO≡4, Mg++ and Mn++ as well as optimal hydrogen ion concentration have been determined for the oxidation of glutamate by these suspensions. The gross effects on glutamate oxidation of a number of antimicrobial agents and of certain enzyme inhibitors have been studied. The antibiotic substances with marked therapeutic value in rickettsial diseases do not affect appreciably this metabolic function. On the other hand, certain common enzyme inhibitors, such as, arsenite, malonate, cyanide, fluoride and selenite, produced variable degrees of inhibition.

Summary Studies of the skin reactions produced by formalin-killed suspensions of Rickettsia mooseri and Rickettsia prowazekii in normal human subjects and in subjects with various kinds of previous experience with typhus organisms have yielded the following observations: Relatively concentrated rickettsial suspensions produce local “toxic” reactions in the skin of normal subjects which are compatible with an endotoxic component in the organisms.Subjects with previous typhus experience exhibit a markedly enhanced local and systemic reactivity to the typhus antigens. Thir reaction is compatible with a delayed type hypersensitivity to typhus antigens.Histologic studies of skin biopsies taken from different subjects at various times after injection of test material revealed an early inflammatory response in normal subjects compatible with that induced by endotoxins and a delayed inflammatory response in hypersensitive subjects compatible with a reaction of delayed type hypersensitivity.The skin reaction did not distinguish between murine and epidemic typhus, suggesting that the major antigen(s) involved is common to both species of organism.The delayed type hypersensitivity to typhus antigens developed with regularity following vaccination with the attenuated living E strain of R. prowazekii, a positive reaction often appearing as early as 7 to 10 days following vaccination.

Summary Infection of human subjects with virulent or attenuated (Strain E) Rickettsia prowazekii was followed within 2 weeks by the appearance of antibodies which would enhance phagocytosis of homologous rickettsiae by mouse peritoneal leukocytes. Although an injection of conventional killed epidemic typhus vaccine appeared to cause more rapid appearance of antibodies (by the end of 1 week), the titers attained at this time were low and, on the average, did not increase significantly in later specimens. Serum from all groups also enhanced phagocytosis of R. mooseri but for the most part these antibodies occurred in lower titers than the antibodies involved in the opsonization of R. prowazekii. The cross-reactions observed with the murine typhus antigen tended to be lower relative to the homologous reaction in the group which received the living attenuated (E strain) vaccine than in the other groups studied.

The Considerable progress which has been made in the study of cell population kinetics2 in proliferating cell systems has been due primarily to the use of labeled DNA precursors, particularly tritiated thymidine, and the development of high resolution autoradiography (14, 36, 62, 75). These technics have provided elegant methods for kinetic analysis of cellular proliferation which heretofore have been unavailable with the classical methods based on mitotic indices (19, 37, 38, 61). Following administration in vivo, TdR-3H is available to a proliferating cell population for a short interval relative to the duration of DNA synthesis, and all cells synthesizing DNA will incorporate the label (14, 30, 32, 36, 55, 62, 69). Analyses of the quantitative relationships between labeled cells and mitoses and the intervals following injection of TdR-3H provide data which relate to the cell cycle and the cell population and concern: (a) the sites of cellular proliferation; (b) the size of the proliferating populations...

Summary Neutralizing and hemagglutination-inhibiting antibodies have been found to persist 16 to 19 years following a single dose of 17D strain yellow fever vaccine in retiring Navy and Marine personnel who gave no evidence of other Group B arthropod-borne virus exposure.

THE semi-conservative replication of DNA of Gram-positive bacteria is specifically inhibited by 6-(p-hydroxyphenyIazo)-uracil (HPUra; obtained from ICI) in an apparently novel mechanism1–4. We have attempted to characterize the HPUra-sensitive site in replication using in vitro preparations of drug-sensitive bacteria. In particulate and soluble preparations of sensitive bacteria, however, HPUra at high concentration does not significantly inhibit polymerization of deoxyribonucleotides2,4. Since these systems may not accurately represent the process of DNA replication as it occurs in vivo, we have examined the effect of HPUra on a more suitable, toluene-treated preparation of Bacillus subtilis described by Matsushita et al.5. In this preparation, DNA replication is ATP-dependent, utilizes deoxyribonucleotides to give biologically active DNA, semi-conservatively and sequentially in the proper gene order. HPUra can inhibit DNA replication by this system. We describe here the characteristics of HPUra inhibition and the conditions necessary for it to occur.

The present work is part of a broad study on quantitative aspects of cell proliferation in normal and neoplastic human tissues. Normal and tumor growth in vivo has been estimated quantitatively in experimental animals by a variety of methods; the most valuable for analyzing cell population kinetics has been the incorporation of tritiated thymidine (TdR-3H) (1, 4). Thymidine is a specific DNA precursor, and is incorporated into cells synthesizing DNA and preparing for division. The resulting nuclear labeling permits identification of sites of cell proliferation and provides information on the proliferative capacity of the cell population. A method has now been developed whereby surgical and biopsy specimens may be labeled with TdR-3H in vitro but under conditions in which the incorporation of the label occurs only in those cells which were synthesizing DNA in the patient, thereby providing a pattern of labeling similar to that obtained by in vivo methods. Materials and Methods Small tissue samples (1 × 3 m...