ETIOLOGY OF OROYA FEVER : VIII. EXPERIMENTS ON CROSS-IMMUNITY BETWEEN OROYA FEVER AND VERRUGA PERUANA.

Nine monkeys (Macacus rhesus) and a chimpanzee which had recently recovered from an infection with the Oroya strain of Bartonella bacilliformis were tested for immunity against the verruga strain of Bartonella bacilliformis as well as against the homologous strain. Complete immunity to both strains was demonstrated. The result establishes the identity of the strains and is in agreement with the result of comparative serological study. The criteria of recovery include not only the subsidence of febrile reactions and local lesions but also a negative result of cultural tests of blood and lymphatic gland tissue. Recovery may occur as early as 1 month after inoculation, but in most instances a period of 2 to 5 months is required for the completion of convalescence.     

ETIOLOGY OF OROYA FEVER : IX. BACTERIUM PERUVIANUM,N. SP., A SECONDARY INVADER OF THE LESIONS OF VERRUGA PERUANA.

A minute, pleomorphic, motile, Gram-negative bacterium has been isolated from two specimens of nodular tissue from human verruga. In films and sections of the original tissues the organism in question is difficult to distinguish from Bartonella bacilliformis, with which it was associated, and even in pure culture it has a number of properties in common with that parasite. No sugars are fermented by it, it is an obligate aerobe, the optimum temperature for its growth is 25°C., and it has two to four spiral flagella attached to one end of the body. It is, however, readily cultivated on any ordinary culture medium. Broth cultures contain much mucin, but no hydrogen sulfide is formed. Coagulated serum is liquefied by its growth, and the red corpuscles in a blood agar plate are hemolyzed. Rabbits, guinea pigs, rats, and mice develop acute, fatal septicemia as a result of intravenous or intratesticular inoculation of young cultures. The liver is characteristically affected and shows a general parenchymatous degeneration and necrosis; the entire gastrointestinal tract is intensely congested, and numerous hemorrhagic areas are present; the spleen, dark and soft, is rarely much enlarged; the kidneys are swollen and congested; the adrenals are much swollen and intensely red; the lungs are sometimes congested but otherwise normal. In the case of intratesticular inoculation the scrotum and testicle both undergo rapid gangrene. In monkeys no septicemia has been observed, but a violent local reaction—swelling, congestion, sometimes necrosis—follows intradermal inoculation. Since no microorganism corresponding in character with this one has previously been described, it is regarded as a new species, and because of its presence in material obtained from Peru it has been given the name Bacterium peruvianum. The significance of the association of B. peruvianum with Bartonella bacilliformis deserves further investigation; it is not impossible that the two organisms are introduced into the human body by the same blood-sucking insect.     

ETIOLOGY OF OROYA FEVER : III. THE BEHAVIOR OF BARTONELLA BACILLIFORMIS IN MACACUS RHESUS.

The experiments reported here were carried on in the main with passage strains of Bartonella bacilliformis, and the results indicate that the virulence of the organism has been considerably enhanced by passage through susceptible animals. While the animals of the earlier experimental series showed no anemia, some of the present group manifested a definite reduction in the number of red cells and in hemoglobin, and in one instance (M. rhesus 25) anemia was of the extreme type so often associated with Oroya fever in man. The anemic condition appeared to be secondary in character, however, nucleated red cells being few in number. In this animal also Bartonella bacilliformis was readily demonstrated in the erythrocytes by means of stained smears, though the number of cells invaded by the parasites was by no means so great as in the human infection. In most instances of experimental Bartonella infection so far induced the demonstration of the parasites by ordinary routine examination of stained film preparations is possible only when the titer of the blood exceeds 1:1,000. Prolonged search of many slides has not been attempted, however. The number of microorganisms in the blood, as shown by culture tests of ascending dilutions, was in most instances highest (1:100,000 to 1:10,000,000) during the early period of the infection coincident usually with the period of highest fever, falling to a titer of 1:10 during the last half of the disease. In one of the fatally infected monkeys, however, the titer increased from 1:10 on the 4th day to 1:1,000,000 on the 24th day. The titer of the blood was equally great in Monkeys 5 and 6, although the former was inoculated locally, the other intravenously and intraperitoneally. The largest proportion of infected red cells was found in Monkey 25, while the blood titer, as shown by culture test, was highest in Monkey 7. The febrile reaction varied in the animals of this series from a severe continuous fever of 104–105°F., lasting 2 to 3 months, in one instance, with a remittence during the 3rd to 5th weeks, to the acute high fever (106°F.) of 1 day''s duration in the fatally infected monkey, No. 25. The more usual reaction, however, is an irregular course of moderate fever with one or more periods of high temperature (105°). Bartonella bacilliformis was constantly demonstrated, both microscopically and by culture tests, in the lymph glands of animals sacrificed 2 to 3 months after inoculation, and in two of three instances it was present also in the spleen, bone marrow, and heart blood. In the case of M. rhesus 6, which died 26 days after inoculation, the microorganism was obtained also in culture from the lymph glands, spleen, and heart blood taken at autopsy. In the other animal which died, a terminal bactelial infection, while not obscuring the effects of the Bartonella infection, made it impossible to isolate the parasite from either blood or tissues. The skin lesions, whether of the nodular type, induced by introduction of the virus intradermally or by application to the scarified skin, or of the miliary character occurring spontaneously as a result of systemic infection, always yielded cultures of Bartonella bacilliformis, and stained sections of such lesions revealed the parasites in large numbers in their characteristic situation in the endothelial cells. A chronic, systemic infection, in which the lymph glands are enlarged and Bartonella bacilliformis is present in the blood in high titer, may be induced by local inoculation, as shown in the case of M. rhesus 5. The local lesions induced in one instance by introduction of a passage strain, both intradermally and by scarification, attained within 2 months extraordinary size, the nodules arising at adjacent sites of inoculation on the right eyebrow having coalesced into a large pedunculated mass which overhung the eye. This type of reaction had not been observed hitherto in the course of the present study but has been described by earlier investigators as a result of the inoculation of monkeys with human verruga tissues. The striking fact brought out in the present study is the variety of responses to inoculation which animals of the same species may manifest. The clinical features of the infection may be typical of Oroya fever or may resemble those of verruga peruviana, and in M. rhesus 25 we have an instance of a type of infection in which the characteristic phenomena of both conditions are simultaneously present. Whether the appearance will resemble those of the one or the other condition appears to depend on the susceptibility of the individual as well as on the virulence of the organism. Moreover, it seems probable that different degrees of resistance to the invasion of the parasite on the part of the blood cells, internal organs, or skin of a given animal may determine the predominant clinical manifestations of the infection. The factor of variation in susceptibility of different individuals or different tissues of the same individual would account for the variety of types of human Bartonella infection.     

ETIOLOGY OF OROYA FEVER : XIV. THE INSECT VECTORS OF CARRION'S DISEASE.

With a view to determining the mode of infection in Carrion''s disease, a study of the blood-sucking insects found in the districts of Peru where the disease prevails has been carried out, through the cooperation of The Rockefeller Institute and the Rockefeller Foundation. The material studied included ticks, mites, midges, lice, fleas, bedbugs, mosquitoes, buffalo gnats, horse-flies, "sheep ticks," 3 species of Streblidae, and 3 species of Phlebotomus, including Phlebotomus verrucarum Townsend and two new species which have been named Phlebotomus noguchii and Phlebotomus peruensis. The insects were collected without the use of chemicals, were prepared for transportation in such a manner as to prevent drying, and were shipped under conditions of refrigeration to New York, where they were inoculated into monkeys. The plan followed was to inject saline suspensions of the crushed insects intradermally into rhesus monkeys and to make cultures of the blood of the animals at intervals of 1 to 6 weeks after inoculation. The only class of insects in which the presence of Bartonella bacilliformis could be detected were phlebotomi. No cutaneous lesions were induced in monkeys injected with the crushed insects, but in the case of four different lots of phlebotomi the blood of the animals so injected yielded cultures of Bartonella bacilliformis which produced typical verrucous lesions on inoculation into other monkeys. The morphology and cultural characteristics of the Bartonella strains obtained from phlebotomi proved identical with those of strains isolated from human blood and skin lesions. Monkeys which had recovered from infection with the phlebotomus strains resisted inoculation with a human strain of Bartonella bacilliformis, and, conversely, monkeys which had passed through an infection induced by the human strain resisted inoculation with the strains obtained from phlebotomi. The experimental observations described in this paper lead us to conclude that certain phlebotomi act as insect vectors of Oroya fever and verruga peruana. The phlebotomi which have been shown quite certainly to carry the Bartonella bacilliformis are those of the species Phlebotomus noguchii. Phlebotomus verrucarum is also probably a vector, while Phlebotomus peruensis remains doubtful in this respect.     

ETIOLOGY OF OROYA FEVER : XIII. CHEMOTHERAPY IN EXPERIMENTAL BARTONELLA BACILLIFORMIS INFECTION.

The therapeutic effect of several antiparasitic chemicals on experimental verruga peruana is described. The drugs were administered by intravenous injection according as the nodules (1) were already developed to an approximate maximum, or (2) were still in the active period of growth. The effect of the drugs was different under the two circumstances of their administration. When they were given after the maturity of the nodules they hastened the regressive process, but when given during active growth of the lesions no action whatever was detected. Bartonella bacilliformis in culture is acted upon injuriously by a number of the chemicals employed in the therapeutic tests, the most active being formalin and neutroflavine.     

ETIOLOGY OF OROYA FEVER : I. CULTIVATION OF BARTONELLA BACILLIFORMIS.

A pure culture of a microorganism resembling in morphology and pathogenic action Bartonella bacilliformis has been obtained from blood taken during life from a case of Oroya fever which ended fatally. The blood taken at Lima into citrate solution and transported to New York at refrigerator temperature yielded positive cultures 28 days after its withdrawal from the patient. The strain of Bartonella bacilliformis thus isolated grows well on the semisolid leptospira medium, and also on slant agar containing animal blood. The initial growth is not readily recognizable to the naked eye, but the presence of the organisms can be determined by means of the dark-field microscope and by Giemsa and Gram staining methods. No growth has been obtained on the more ordinary culture media. The organism is an obligate aerobe, is Gram-negative, and under certain cultural conditions motile. All the forms which have been described as occurring in human red corpuscles may be found in the cultures, and in addition many granular and coarsely irregular forms have been met with. The inoculation of cultures of Bartonella bacilliformis into Macacus rhesus produces infection and gives rise to effects which differ with the mode of inoculation. The intravenous injection of the culture into young macaques induces a prolonged irregularly remittent fever. The organism can be cultivated from the blood over a long period, and it has been detected within the red corpuscles of the monkeys, reproducing the precise appearances observed in human cases of Oroya fever. The intradermal injection of the culture into the eyebrow of young macaques gives rise to nodular formations rich in new blood vessels and showing the bacilliform organism within the endothelial cells. From the experimentally induced nodules cultures of the organism are readily recovered.     

ETIOLOGY OF OROYA FEVER : IV. THE EFFECT OF INOCULATION OF ANTHROPOID APES WITH BARTONELLA BACILLIFORMIS.

The inoculation of a chimpanzee with cultures and a passage strain of Bartonella bacilliformis induced local reactions which, while definite and characteristic, progressed less rapidly and were much less striking than those in the control rhesus monkey. Bartonella bacilliformis was demonstrated in the blood corpuscles with difficulty, and the fever was slight compared with the high and persistent fever of the rhesus monkey. In both the swelling of the lymph glands was an early symptom and constantly present. Definite anemia developed in the chimpanzee only after accidental infection with Rocky Mountain spotted fever and may have been due to either one or both infections, though it disappeared when the blood had become negative by culture for Bartonella bacilliformis and the local lesions had disappeared. Incidentally, the chimpanzee was found in this one instance to be less susceptible to the spotted fever than Macacus rhesus and guinea pigs. In the ourang-utan, also, Bartonella bacilliformis induced a mild systemic and local infection. A rise of temperature occurred 10 days after inoculation) and fever continued for a week, though it was decidedly less severe than that in the control rhesus. The lesions induced by scarification were less definite than those which arose at the sites of intradermal inoculation. Bartonella bacilliformis was recovered from the blood on the 9th and on the 16th days after inoculation and from nodules excised on the 33rd and 53rd days. A few erythrocytes containing the organism were demonstrated in stained smears, but prolonged search was required to find them. The symptoms and lesions observed in the chimpanzee and ourangutan as a result of infection with Bartonella bacilliformis are far milder than those of rhesus monkeys and show less resemblance to human Oroya fever or verruga.     

THE ETIOLOGY OF VERRUGA PERUANA

A saline suspension of a subcutaneous nodule excised from a verruga patient, and kept in the refrigerator for 14 days, on inoculation into two young Macacus rhesus monkeys (Nos. 33 and 34) induced irregular febrile reactions and enlargement of the lymph glands, and in one instance a subcutaneous nodule arose, independently of direct inoculation, on the tail. A microorganism has been isolated from the blood of both animals, and from the experimental nodule, which in pathogenic properties and in cultural and morphological characteristics is indistinguishable from the strain of Bartonella bacilliformis isolated from a case of Oroya fever. The spontaneous skin lesion of Monkey 34 and the subcutaneous nodules induced by intradermal inoculation of cultures of the microorganism were histologically typical of experimental verruga lesions in monkeys and identical with the skin lesions induced in monkeys by Bartonella bacilliformis. The organism, like Bartonella bacilliformis, is an intracellular parasite, being found in the cytoplasm of the proliferating endothelial cells of the lesions and in the erythrocytes of the blood. The same variations in the manifestations of disease which have been noted in experimental infection with Bartmetta bacilliformis were observed in the experimental verruga infection. In the second passage (Monkey 41) the infection induced by local inoculation of cultures was severe both locally and constitutionally and was accompanied by marked anemia. The organisms were found in the red cells in large numbers. In the third passage the systemic infection was less severe, but the local lesions were more striking. Detection of the parasites in the blood is far more certain by the cultural method than by microscopic examination, the latter procedure being successful only in rather severe infections. The result of blood culture is therefore the decisive method in the final diagnosis of the disease. Preliminary serological study shows that the organism isolated in the present instance from the skin lesion of a verruga patient and that previously obtained from the blood of a case of Oroya fever belong to the same serological group. The data obtained justify the conclusion that verruga peruana is caused by Bartonella bacilliformis. They also definitely establish the fact that the inoculation of blood or sanguineous exudate from lesions of verruga peruana is capable of inducing in susceptible individuals a severe febrile systemic infection, such as that to which Carrion succumbed. The designation "Carrion''s disease" is therefore the appropriate one for both forms of the infection. Bartonella bacilliformis may be regarded as a bacterium, since it has the essential features of that group of microorganisms.     

ETIOLOGY OF OROYA FEVER : XV. EFFECT OF IMMUNE SERUM ON THE COURSE OF BARTONELLA BACILLIFORMIS INFECTION IN MACACUS RHESUS

Experiments are reported on the effect upon the course of experimental verruga peruana in Macacus rhesus of the injection of (1) small quantities of rabbit immune serum simultaneously with living cultures, (2) one large dose of convalescent monkey serum 24 hours prior to inoculation, (3) a similar preliminary dose followed by three subsequent injections of the serum, (4) three large doses of convalescent serum, following the inoculation. The convalescent serum was found (1) to prevent the multiplication of Bartonella bacilliformis in the blood in most instances, and (2) to delay the development of the skin lesions for considerable periods, when given before inoculation. When the serum treatment was not begun until after the appearance of the skin lesions, it had no effect on the progress of the nodules, although the blood became free from Bartonella bacilliformis. Since the severe effects of verruga peruana (Carrion''s disease) are believed to be due to the multiplication of Bartonella bacilliformis within the blood, the injection of convalescent serum in cases of Carrion''s disease in man would appear to offer promise.     

ETIOLOGY OF OROYA FEVER : VI. PATHOLOGICAL CHANGES OBSERVED IN ANIMALS EXPERIMENTALLY INFECTED WITH BARTONELLA BACILLIFORMIS. THE DISTRIBUTION OF THE PARASITES IN THE TISSUES.

The pathological changes observed in the organs in Macacus rhesus monkeys which have succumbed to severe infection with Bartonella bacilliformis are similar to those found in human organs in persons dying of Oroya fever. The characteristic changes in the liver are the zonal necrosis of the cells around the hepatic veins, involving active macrophagocytosis of invading polymorphonuclear leucocytes in the necrotic areas, and a marked endothelial hyperplasia in the sinusoids or around the portal veins. In some instances there is fatty infiltration of hepatic cells. In the spleen persistent hyperplasia of the endothelial cells of the capillaries leads to the formation of minute foci of infarction, owing to occlusion of the lumina. The follicles are dispersed or reduced, and there is an active macrophagocytosis of cellular débris, polymorphonuclear leucocytes, and erythrocytes. In some specimens an increase in normoblasts is noted. Pigment is sometimes present. The lymphatic system shows general progressive endothelial hyperplasia, with active invasion of macrophages which contain polymorphonuclear leucocytes, erythrocytes, and greenish or dark pigments. In the bone marrow there is increased activity of macrophagocytes. Numerous normoblasts are found in some instances. In one monkey, sacrificed during the course of infection, small, verruga-like nodules were found in the lungs and spleen. Bartonella bacilliformis has been detected microscopically, though in small numbers, in all tissues showing histological changes. Parallel cultural determinations of the presence of Bartonella bacilliformis in the blood, liver, spleen, lymphatic glands, bone marrow, and local lesions established the relationship between the pathological conditions and the presence of the parasite. The organism seems to persist longest in the lymphatic glands. Cultural methods offer a simple and conclusive means for the determination of the presence or absence of the infecting organism.     

ETIOLOGY OF OROYA FEVER : VII. THE RESPONSE OF THE SKIN OF MACACUS RHESUS AND ANTHROPOID APES TO INOCULATION WITH BARTONELLA BACILLIFORMIS.

Bartonella bacilliformis failed to induce lesions when merely rubbed on the surface of the intact skin of a chimpanzee, an ourang-utan, and numerous Macacus rhesus monkeys, although when applied to the scarified skin of the same animals it gave rise to extensive lesions. Application of infectious material to the scarified skin did not always induce verruga lesions, but intradermal inoculation almost invariably gave rise to nodule formation. The localization of Bartonella bacilliformis in the skin is not, in experimental animals, determined by mechanical factors, since scarification of the skin or intradermal injection of foreign substances in monkeys infected with Bartonella bacilliformis does not give rise to verruga formation. The degree of susceptibility of the skin tissues appears to be considerably diminished during the course of experimental infection with Bartonella bacilliformis. Inoculation of the scarified skin of infected animals gave uniformly negative results, and intradermal inoculation induced only a mild local reaction. In a few exceptional instances, however, of animals previously infected with the strain of Bartonella bacilliformis derived from a human verruga nodule, reinoculation with the same strain gave rise to unusually marked reactions. The evolution of the skin lesion induced in experimental animals by Bartonella bacilliformis may be divided into four stages, the period of incubation, the initial stage, the mature and vascular stage, and the regression. In the initial stage the lesion is a pure angioendothelioma, but in the stage of full development the histological picture is complicated by connective tissue proliferation and occasionally also by penetration of epidermis into the lesion. The demonstration of Bartonella bacilliformis in the endothelial cells distinguishes the lesion from others which simulate it. The cutaneous lesions known as verruga nodular, verruga mular, and verruga miliar have been reproduced in monkeys.     

ETIOLOGY OF YELLOW FEVER : I. SYMPTOMATOLOGY AND PATHOLOGICAL FINDINGS OF THE YELLOW FEVER PREVALENT IN GUAYAQUIL.

The clinical and pathological features of the yellow fever prevalent in Guayaquil conform with those described by other investigators of this disease as it has occurred elsewhere, both epidemically and endemically.     

ETIOLOGY OF OROYA FEVER : V. THE EXPERIMENTAL TRANSMISSION OF BARTONELLA BACILLIFORMIS BY TICKS (DERMACENTOR ANDERSONI).

Experiments are reported in which Bartonella bacilliformis was transmitted from infected to normal rhesus monkeys by the bite of the tick, Dermacentor andersoni. A long period of feeding, both on the infected animal and on the normal animal subjected to infection, was required in order to secure positive results. The infection transmitted by the ticks was mild, but definite, as shown by the recovery of Bartonella bacilliformis from the lymph nodes and blood.     

ETIOLOGY OF OROYA FEVER : XI. COMPARISON OF BARTONELLA BACILLIFORMIS AND BARTONELLA MURIS. CULTIVATION OF BACTERIUM MURIUM,N. SP.

Bartonella muris appeared in the blood of all white rats, wild rats, Chinese hamster, and mice, from which the spleen was removed, but did not appear in that of splenectomized monkeys, rabbits, or guinea pigs. It has not been possible to transmit B. muris to normal rats, monkeys, rabbits, or guinea pigs, by intraperitoneal, intradermal, or intravenous injection of blood containing B. muris from splenectomized rats. In two instances an acute orchitis was induced in normal rats by injection directly into the testicle of blood or saline suspensions of the liver of splenectomized rats. The intracorpuscular elements occasionally identified as B. muris or as having appeared as a result of the inoculation. The acute orchitis of rats was transferable to normal rats in series. From the testicular tissue, as well as directly from the blood of a splenectomized rat, there was isolated in pure culture a microorganism which induced in the testicles of normal rats an acute orchitis such as resulted from inoculation of the blood or liver suspensions of splenectomized rats. While a few inclusions were found in the erythrocytes of some of the animals, their number was so small and their occurence so infrequent that they could not be definitely identified as B. muris. In morphological features the cultural forms of the microorganism isolated resemble B. muris. The organism found in the testicular tissues, however, is considerably coarser than B. muris and takes a deeper stain. Immunological study failed to settle the question of the relation between B. muris and the cultivated organism, which is provisionally called Bacterium murium. Bartonella muris, Bartonella bacilliformis, and Grahamella talpæ have characteristic individual morphological features.     

THE ETIOLOGY OF RAT-BITE FEVER

The similarity in the cases of rat-bite fever recorded in the literature establishes it as a definite clinical entity. The same symptomatology occurs in cases from Asia, Europe, and America. The greater frequency of the disease in Japan than elsewhere is probably due to the housing conditions and habits of the people resulting in the more frequent occurrence of rat-bites. It does not seem necessary to consider that cases occurring in Europe and America are due to the bites of rats that have been imported from Japan. The clinical picture and course of the disease indicate that it is infectious in origin. Until Schottmüller''s case appeared in 1914, the etiology had been undiscovered. He isolated from his case in eight consecutive blood cultures a streptothrix which he has designated Streptothrix muris ratti. His work has been confirmed by the isolation of an identical streptothrix from the blood during life and at autopsy in the case here reported. Further confirmation of the etiological relationship of this organism to the infection in our patient is found in the production of powerful agglutinins for the organism in the blood serum of this case and in the demonstration of the organism in the vegetation on the mitral valve. It is not unreasonable to suppose that Proescher (13) observed the same organism in the sections of the excised wound in his case. Although it is fully realized that Koch''s postulates have not been fulfilled in the absence of successful animal experimentation, nevertheless the accumulated evidence here presented leaves little reason to doubt that the specific cause of rat-bite fever is Streptothrix muris ratti. The pathology of rat-bite fever has hitherto been largely a matter of surmise. One autopsy only has been recorded in the literature (Miura (22)), and nothing abnormal was noted other than injection of the pial vessels. The autopsy in the case here reported has proved of considerable interest in the extent and character of the lesions found. A streptothrix septicemia with the localization of the organism in the mitral valve producing an acute ulcerative endocarditis is the most striking feature of the case. The infarcts of the spleen and kidney are a natural sequence of the endocarditis. The subacute lesions of the myocardium, liver, adrenal, and kidneys, glomerular and interstitial, are all of a similar nature, consisting of areas infiltrated with leukocytes, lymphocytes, plasma, and endothelial cells with varying degrees of degeneration of the normal cells of the affected area. In no instance has the presence of the streptothrix in these lesions been demonstrated, and it is not unreasonable to assume that they are toxic in origin. The data here presented may be correlated with the clinical features of rat-bite fever to give us a clear understanding of the course and nature of the disease. The patient is inoculated by the bite of a rat with Streptothrix muris ratti. After a variable incubation period a non-suppurative inflammatory reaction occurs at the site of the wound with extension to the neighboring lymphatics and lymph nodes. Invasion of the blood stream follows, accompanied by the onset of severe toxic symptoms. Clinically the nervous system and frequently the kidneys seem to be especially involved. That the myocardium, liver, and adrenals may also suffer is shown by the autopsy findings in the case reported above. Ulcerative endocarditis is probably a rare occurrence. In the majority of cases after a more or less prolonged course, the disease terminates spontaneously and so may be considered a self-limited infection. This is presumably brought about by the development in the body of a protective mechanism against the streptothrix. That such a process does occur is evidenced by the demonstration of agglutinins in our case. Whether a permanent immunity is acquired after one attack of rat-bite fever is not known. No instances of a second infection are recorded in the literature. Although rat-bite fever varies somewhat in its symptomatology in individual cases, the picture is sufficiently characteristic to make the diagnosis not a difficult matter. The history of a rat-bite, latent incubation period with subsequent non-suppurative inflammatory reaction of the wound, lymphangitis, and enlarged lymph nodes, severe chill at onset, high fever of the relapsing type, intense muscular pain and nervous symptoms, and the characteristic bluish red exanthem, present a symptom-complex not easily overlooked. The disease is frequently complicated by a severe nephritis, and prolonged cases develop a high grade of anemia and cachexia. In the case here reported ulcerative endocarditis occurred. In the large majority of cases the prognosis is favorable for a successful termination. The patients, however, are often incapacitated for a considerable period of time The mortality is about 10 per cent, death usually occurring in the first febrile period apparently from a profound toxemia, or at a later stage due to the development of a severe nephritis. Until recently treatment has been entirely symptomatic and has been of little avail in altering the course of the disease. Miyake has found immediate treatment of the wound by cauterization or with carbolic acid highly efficient as a prophylactic measure. Hata (30) in 1912 introduced salvarsan therapy and reported eight cases so treated, seven of which showed marked and rapid improvement. One case was apparently unaffected. Two of the cases receiving only small doses had a subsequent relapse. Surveyor (31) and Dalal (18) also have reported success with salvarsan injections. It is to be hoped that further experience with this method of treatment will yield equally favorable results.     

ETIOLOGY OF YELLOW FEVER : IX. MOSQUITOES IN RELATION TO YELLOW FEVER.

The foregoing experiments show that symptoms and lesions closely resembling those of yellow fever in man may be induced in guinea pigs by the bite of female stegomyias that have previously sucked the blood of a yellow fever patient or of an animal experimentally infected with Leptospira icteroides. With mosquitoes infected directly from a yellow fever patient the infectivity seems to become manifest after a longer period of incubation than with those infected with the animal blood. In the former, at least 12 days are said to be necessary before they become infectious, and this hypothesis seems to be borne out by the present experiment. On the other hand, the mosquitoes which were engorged with the infected blood of the guinea pig were found to be capable of transmitting the disease within 8 days after the feeding. This discrepancy may be explained by the fact that the number of leptospira existing in experimentally infected guinea pigs is far greater than that in human blood. The frequency with which positive transmission by the stegomyia was obtained in both instances was very small indeed, in view of the number of mosquitoes employed. It appears that even under natural circumstances the percentage of mosquitoes that eventually become infected with the yellow fever microbe by sucking the blood may be very small. It has already been shown by previous investigators that to transmit yellow fever from a patient to a non-immune person requires from 0.1 to 2 cc. of blood at the height of disease. According to my estimate a female stegomyia may take up 0.01 cc. or even less. Apparently a mosquito occasionally becomes infectious by taking up the one or two organisms which happen to be circulating in the peripheral blood of man, and it is these occasionally infected few which carry the disease. It is not difficult to realize the extent of ever increasing danger from a constant supply of the microbic virus which an endemic center or an epidemic of yellow fever can provide. One infected mosquito may mean many patients, and the life of such a mosquito is usually longer than that of the persons whom it fatally infects. Finally, it is of interest to note that the development and maintenance of Leptospira icteroides are indispensably associated with the blood constituent, the serum, and this is amply supplied by the bloodsucking insect. The organism is one of the most fragile of all the pathogenic parasites and cannot survive the concurrence of other less fastidious organisms such as bacteria. The comparatively aseptic body cavity of the stegomyia furnishes a secure shelter for the parasite, which undoubtedly penetrates the zone of safety as soon as it is taken into the stomach of the insect. Unlike many other parasites this organism is capable of penetrating the intact skin or a bacteriaproof filter, and hence it is probably an easy matter for it to pierce the tissue of the visceral organs of the mosquito. Whether or not Leptospira icteroides can survive and multiply only in the body of Stegomyia calopus and not in other varieties or genera is yet to be determined. Another interesting fact with regard to the extrinsic life of this organism is that it can multiply steadily at a temperature from 18–37°C. The optimum temperature, at which it remains viable for many months, is 26°. The climate in most of the tropical countries offers optimum conditions both for Leptospira icteroides and for the mosquito which carries and nourishes it.     

STUDIES UPON THE ETIOLOGY OF DENGUE FEVER : II. CULTIVATION AND NATURE OF THE VIRUS.

We have described the cultivation of a minute organism, upon the special media devised by Noguchi for growing spirochetal organisms, that gives rise to pyrexial and leucocytic reactions in the guinea pig. The reaction in the animal following the injection of culture filtrate is identical with that induced by the inoculation of human dengue blood. While it is possible, it is hardly probable that the infection of animals with cultures of this microorganism several generations removed from the original is due to a mechanically transferred virus with which the visible microorganism cultivated became accidentally associated. Furthermore, it is unlikely that such a virus would remain viable and be carried over to subcultures in sufficient numbers to infect the animal. The microorganisms appear in culture as globoid bodies measuring from 0.1 to 0.3µ in diameter and are arranged singly, in pairs, and in short chains. They readily pass through the Berkefeld filter (N and V) and the filtrate gives rise to a characteristic reaction in the inoculated guinea pig. The filtrate yields in subplants the same globoid bodies of the original culture. Initial cultures have been obtained directly from the blood of the human case as well as from the blood of guinea pigs reacting to the human material. However, only early generations retain the degree of virulence necessary to cause the experimental reaction, the culture of remote generation failing to infect. As far as known this minute organism has characters in common with the globoid bodies of Flexner and Noguchi obtained from cases of poliomyelitis. In plasma semisolid medium the striking feature indicative of growth is colonization, which in itself serves to differentiate the opalescence of the medium occasioned by disintegration of the contained tissue. As regards the presence of spirochetal organisms in the blood of dengue fever, we may state that examination was made of the material from human cases and from. a large number of inoculated animals. Despite the most careful search with the dark-field microscope and repeated examinations of blood specimens stained by the best methods, we have been unable to find any demonstrable spiral organism in the blood of dengue patients or in the experimentally inoculated animal. The minute organism herein described is frequently present in cultures of the blood of human dengue and of animals inoculated with dengue material. The inoculation of the culture into guinea pigs produces a response comparable to that occurring in the human case, and that induced by the injection of human dengue blood in guinea pigs. While we are of the opinion that the anaerobic globular bodies described may bear etiological relationship to dengue, we realize that further proof and confirmation of our work are required to establish the connection.     

THE ETIOLOGY OF COLORADO TICK FEVER

1. The infectious agent of Colorado tick fever filtered through 181 mµ membranes caused infection in hamsters and one volunteer. 2. In five out of seven instances, hamsters were infected initially with serum filtered through 24 mµ membranes or the tick fever appeared on serial transfer. 3. We could not infect two volunteers with 24 mµ filtrates, though these filtrates did cause infection in hamsters as shown by development of the fever on serial transfer. In one instance, the serum of the hamsters rendered ill in this way was injected into the volunteer who had remained well after inoculation with the original material and it caused Colorado tick fever. 4. Normal hamster serum was passaged through 10 groups of animals and then two human volunteers were injected with it. They did not come down with Colorado tick fever, although both of them were susceptible to the disease as shown by the fact that they fell ill of it on later inoculation with serum from a natural instance of the disease. 5. The infectious agent of Colorado tick fever passes through 24 mµ membranes. On this basis, it is classified as a virus.     

ETIOLOGY OF OROYA FEVER : X. COMPARATIVE STUDIES OF DIFFERENT STRAINS OF BARTONELLA BACILLIFORMIS,WITH SPECIAL REFERENCE TO THE RELATIONSHIP BETWEEN THE CLINICAL TYPES OF CARRION'S DISEASE AND THE VIRULENCE OF THE INFECTING ORGANISM.

Through the cooperation of Dr. Sebastian Lorente, Director of the National Department of Public Health of Peru, nine strains of Bartonella bacilliformis have been isolated, by means of the semisolid leptospira medium, from nine of twelve specimens of blood withdrawn from cases of verruga and forwarded from Peru under conditions of refrigeration. The cultural titer of the blood specimens immediately after their arrival (2 weeks after withdrawal) varied from 1:10 to 1:100,000. Blood from the severe anemic type of the disease, in which there was no eruption, had the highest titer. Blood agar slants yielded irregular results, but some strains grew well on these media. Morphologically the strains differed very little in fresh preparations examined by dark-ground illumination. In stained preparations some strains appeared coarser, others finer than the average. Special staining indicated that the flagella were characteristically unipolar and varied in number from one to four, some strains showing distinctly more wavy and heavier flagella than others. Young cultures grown on the surface of horse blood agar for 3 to 6 days show individuals with fairly sharp contours, short rods, often varying in thickness toward one or both ends, being intermingled with smaller oval or coccoid elements. Some strains show a predominance of bacillary, some of coccobacillary forms. It is not known whether these features are inherent or are due to conditions of growth, which, though identical, may react differently upon different strains. Definiteness in outline disappears with the age of the culture. More striking variations are found in the virulence of the different strains for the monkey (Macacus rhesus). Three of the nine strains isolated proved to be non-pathogenic for the monkeys. All three of these were derived from cases of benign verruga. The remaining six strains all gave rise to local lesions when intradermally inoculated and were recovered in culture from the blood of the animals. So far, severe anemia has not developed in any of the monkeys. It is significant that most of the severe cases yielded virulent strains, while some of the strains from benign verruga were non-pathogenic. It appears highly probable that the severe form of Carrion''s disease is, in general, caused by a virulent strain, while the benign forms are due to a strain of low virulence. On the other hand, a virulent strain may cause benign verruga in unusually resistant persons and a weak strain may give rise to severe blood infection in unduly susceptible individuals. The form of Carrion''s disease is probably determined primarily by the inherent virulence of the strain of Bartonella bacilliformis and is modified secondarily by individual predisposition in a given case. An interesting phenomenon brought out by the present investigation was the failure of the nine human blood specimens to induce local verruga in the same monkeys in which the corresponding cultures, inoculated simultaneously at separate sites, gave rise to typical lesions. Yet the original blood samples were shown by cultivation to have contained live bartonellas at the time they were inoculated, and blood culture revealed the presence of the microorganisms in the blood of monkeys which showed no other signs of infection after inoculation with the human blood. Whether this striking difference is merely a quantitative one or is due to some factor still unknown—such as, for example, a biological phase of the microorganism—has not been determined. The uniformly negative results of transmission experiments with blood by previous investigators is explained by an actual inability of the blood to induce skin lesions and the lack, until now, of a reliable method of detecting Bartonella bacilliformis in the monkeys'' blood. The strains isolated showed similar serologic properties, as tested by complement fixation.     

EXPERIMENTAL STUDIES ON THE ETIOLOGY OF TYPHUS FEVER : II. SURVIVAL OF THE VIRUS IN AEROBIC AND ANAEROBIC CULTURE MEDIA.

The results of the foregoing experiments show that the typhus virus, found in the blood of guinea pigs during the height of typical experimental typhus fever, does not survive at 37°C. in anaerobic media for as long a period as in the same media under aerobic conditions. In media from which oxygen is excluded, the viability period is 24 to 48 hours; in the same media having no barrier to atmospheric oxygen, the period is usually 5 days, in one instance, 3 days. The dead virus fails to induce not only the typical experimental disease but also an immunity to further injections of typhus virus. That the death of the virus is due to exclusion of oxygen from the medium, rather than to a change in the hydrogen ion concentration therein is inferred from the fact that media with varying hydrogen ion concentrations, such as broth (pH 7.4), horse serum (pH 7.8), and human ascitic fluid (pH 8.0) have the same comparative effect on the virus, when under aerobic and anaerobic conditions. That is, in all, the anaerobic state causes a shortening of the viability period of the typhus virus. In the Smith-Noguchi tissue, ascitic fluid, sealed medium in which bacteria resembling Plotz'' bacilli grow luxuriantly and remain viable for several weeks, the typhus virus does not increase in virulence, and even dies after 24 hours. This evidence supports the conclusion previously presented that the Bacillus typhi exanthematici of Plotz is not identical with the active agent of typhus virus.