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 : 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 : 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 : 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.     

ETIOLOGY OF YELLOW FEVER : III. SYMPTOMATOLOGY AND PATHOLOGICAL FINDINGS IN ANIMALS EXPERIMENTALLY INFECTED.

Studies are reported on the type of disease induced in guinea pigs, dogs, and monkeys by inoculating them (1) with the blood or organ emulsions of guinea pigs or other susceptible animals experimentally infected with Leptospira icteroides, and (2) with a pure culture of the organism. Particular attention has been given in these experiments to the clinical features of the experimental infection in the various animals and to the pathological changes resulting from the infection. The symptoms and pathological lesions induced in guinea pigs are much more pronounced than those observed in dogs or marmosets. The period of incubation is nearly the same in all three species, 72 to 96 hours with intraperitoneal or subcutaneous inoculation, and a day or more longer when the infection is induced percutaneously or per os. The febrile reaction in the guinea pig and marmoset is about the same; in the dog there is less fever. The amount of albumin, casts, and bile pigments in the urine is more abundant in the guinea pig and marmoset than in the dog, and these animals also appear on the whole to become more intensely icteric. The black or bilious vomit, however, though occurring frequently in dogs during life, is observed in the guinea pig and marmoset at autopsy. The hemorrhagic diathesis is most pronounced in guinea pigs, less so in marmosets, and least in dogs. In dogs) for example, subcutaneous hemorrhages almost never occur, and the lungs usually show only a few minute ecchymoses. The pleurse, pericardium, and other serous surfaces of the thorax and abdomen remain free from ecchymoses, which, however, with hyperemia, are very marked along the gastrointestinal tract. The symptoms and lesions observed in animals experimentally infected with Leptospira icteroides closely parallel those of human yellow fever. The pathological changes occurring in human cases of yellow fever are similar to those induced by inoculation in guinea pigs and marmosets and in respect to their intensity stand intermediate between those arising in the two animals mentioned.     

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 : 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 : 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 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.     

ETIOLOGY OF YELLOW FEVER : VII. DEMONSTRATION OF LEPTOSPIRA ICTEROIDES IN THE BLOOD,TISSUES, AND URINE OF YELLOW FEVER PATIENTS AND OF ANIMALS EXPERIMENTALLY INFECTED WITH THE ORGANISM.

Examinations of fresh blood from yellow fever patients by means of the dark-field microscope, made in more than twenty-seven cases, revealed in three cases the presence of Leptospira icteroides. In no instance was a large number of organisms found, a long search being required before one was encountered. The injection of the blood into guinea pigs from two of the three positive cases induced in the animals a fatal infection, while the blood from the third positive case failed to infect the guinea pigs fatally. Careful but by no means exhaustive dark-field searches for the leptospira with fresh specimens of blood from the remaining cases of yellow fever ended without positive findings, although four of the specimens, when injected into guinea pigs, caused a fatal leptospira infection. Stained blood film preparations from the corresponding cases were also examined, but the percentage showing the leptospira in the blood was no greater than that found by examination in the fresh state with the dark-field microscope. In fact, owing to the defective stains that were available at the time of the investigation a great many slides did not take the proper coloration with Giemsa''s or Wright''s stain and could not be relied upon. Regarding the presence of Leptospira icteroides in various organs both dark-field and stained films were examined. In only one instance so far a few organisms were detected in the emulsion of liver taken shortly after death from a case dying on the 4th day of yellow fever. This part of the work will be reported later upon completion. Examinations of the urine from different cases of yellow fever were made both by dark-field microscope and by inoculation into guinea pigs. The results were totally negative in thirteen cases, including many convalescents, but in one case one of the guinea pigs inoculated with 10 cc. of the urine came down on the 15th day with suggestive symptoms (suspicion of jaundice, and some hemorrhagic and parenchymatous lesions of the lungs and kidneys). This specimen showed no leptospira by dark-field examination. In experimental infection of guinea pigs with Leptospira icteroides the blood became infective in many instances 48 hours after inoculation, and was always infective after 72 hours. The liver and kidney become infective simultaneously with the blood. Detection of the organism by means of the dark-field microscope has seldom been accomplished before the 5th day. The organisms are most abundant on the 6th to the 7th day, but become fewer or completely disappear before death. In the meanwhile the number of organisms increases in the liver and kidney, from which they disappear as the jaundice and other symptoms become aggravated. When death occurs these organs seem to have lost most of the leptospira) and positive transfer by means of them is less certain. At the later stage of the disease the blood is often free from the organisms and ceases to be infective. Positive transmission with blood obtained from moribund animals is not impossible, however, even when no leptospira can be detected under the dark-field microscope.     

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.     

ETIOLOGY OF YELLOW FEVER : XI. SERUM TREATMENT OF ANIMALS INFECTED WITH LEPTOSPIRA ICTEROIDES.

The use ot a polyvalent immune serum ot nign potency in tne treatment of an experimental infection of guinea pigs with Leptospira icteroides was found to be of definite advantage in checking the progress of the infection. When administered during the period of incubation the serum was found capable of completely preventing the development of the disease, although on subsequent examination hemorrhagic lesions of greater or less number and extent were found in the lungs of the guinea pigs which survived. Moreover, the serum modified the course of the disease and when used in the early stages of infection prevented a fatal outcome. Employed at a later stage, however, when jaundice and nephritis had been present for several days and the animal was near collapse, the serum had no perceptible beneficial effect. This was, of course, to be expected in view of the incidence of various pathological phases of this disease—nephritis, hepatitis, and other toxic symptoms in succession. In man the clinical manifestations are more gradual and distinct than in the guinea pig, yet the yellow fever patient whose temperature is sub-normal, and who has reached the stage of hemorrhages from the gums, nose, stomach, and intestines, and of uremia and cholemia, would seem to have little or no chance of deriving benefit from the use of a specific immune serum. This latter assumption would probably hold irrespective of the relation which Leptospira icteroides proves to have to the etiology of yellow fever.     

ETIOLOGY OF OROYA FEVER : XVI. VERRUGA IN THE DOG AND THE DONKEY

In the experiments here reported, definite verruga lesions, in which the presence of Bartonella bacilliformis was established by culture or by passage to rhesus monkeys, were produced in a dog and in a donkey by inoculation of cultures or monkey passage strains. The reaction induced in these animals was entirely local, however; blood cultures were sterile. Histologically, the lesions produced were similar to those obtained in monkeys by inoculation of Bartonella bacilliformis, except for the presence of a marked polynuclear leucocytic exudate. In another donkey a lesion histologically suggestive of verruga was produced, while in one donkey and a horse the results of inoculation were negative or indefinite. The intravenous injection of a filtrate or of heat-killed cultures of Bartonella bacilliformis into two donkeys was followed by the appearance of large, soft, subcutaneous swellings, on various parts of the body, not resembling in any way verruga lesions.     

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 : 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 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.     

STUDIES ON THE ETIOLOGY OF HEARTWATER : I. OBSERVATION OF A RICKETTSIA,RICKETTSIA RUMINANTIUM (N. SP.), IN THE TISSUES OF INFECTED ANIMALS.

A Gram-negative, intracellular, coccus-like microorganism was found in cases of heartwater in the three species which are susceptible to the disease; namely, goats, sheep, and cattle. It was absent in the case of control animals, both normal ones and those dying of some. other diseases. The presence of this microorganism was definitely related to the febrile reaction. It was most easily detected in the renal glomeruli and in the small capillaries of the cerebral cortex but probably occurred throughout the body. The microorganism was a typical endothelial parasite, being restricted in distribution to the endothelial cells of the smaller blood vessels and to portions of such elements which had broken off into the blood stream. It was never observed to cause injury to the cells other than those incident to mechanical distention through accumulation within them of many individuals in large densely packed masses which were characteristically spherical. A typical attribute was the presence of several of these masses within the cytoplasm of a single endothelial cell. In view of the association of this microorganism with heartwater, a disease of ruminants, and thus far the only one in which microorganisms resembling Rickettsiœ have been reported, the designation Rickettsia ruminantium is proposed.     

THE ACTION OF THE LETHAL DOSE OF STROPHANTHIN IN NORMAL ANIMALS AND IN ANIMALS INFECTED WITH PNEUMONIA

The results of these experiments permit a comparison of the action of strophanthin in the normal and the infected animals. In cats the percentage of deaths following the injection of 0.1 mg. of strophanthin per kilo of body weight was the same in normal and in pneumonic cats. The number of recoveries in this series was larger than was expected. This result was due to the fact that the dose injected was not the lethal dose, but the average lethal dose (0.1 mg.) determined by Hatcher and Brody (7) and by Eggleston (8). The doses which the latter actually injected ranged from 0.085 rng. to 0.16 mg.; from these the average minimal lethal dose was calculated. In adopting the average dose as the standard one to inject, those of our cats that required more than 0.1 mg. naturally survived. The death rate was therefore low. The plan employed in dogs differed from that used in cats. A lethal dose was injected in each dog. Death occurred when an average of 0.12 mg. of strophanthin per kilo of body weight was injected. The same dose was required in normal and pneumonic dogs. The effect of strophanthin in both groups of infected and non-infected cats and dogs is, therefore, identical. Whether the uniformity of strophanthin action in the two experimental groups may serve as the basis for assuming a like uniformity of action in normal individuals and in pneumonia patients is a subject which requires further analysis. The difficulty in transferring the experimental results to patients lies in the question of whether the type of pneumonia produced in animals is the same as that found in man. Clinically, the two diseases present both resemblances and differences. The animals become definitely ill and show the symptoms already described. The illness, however, is of short duration and apparently reaches its height in the majority of animals in twenty-four to seventy-two hours. Before the expiration of this time, the temperature frequently returns to normal. Many of the infected animals, when they survive, recover in three to five days. The mortality in dogs infected with pneumococci is given by Lamar and Meltzer as 16 per cent. In the present series of twelve infected cats, the mortality was also 16 per cent. These findings differ from human pneumonia in the following particulars: The infection is not so severe; the temperature, though elevated at first, soon falls; the duration of the disease is short; and convalescence is rapid. The mortality is slightly lower. Musser and Norris (12) give the human mortality at 21.06 per cent. Pathologically the two diseases also show differences. The gross appearance of the lungs is not dissimilar, but in the animals the consolidated portions are somewhat dry and they fail to show a stage of gray hepatization (5). The amount of fibrin present is small. There is comparatively slight congestion of the alveolar walls and of the walls of the bronchi. The relation of experimental pneumonias to the human disease has been discussed by a number of investigators. Almost all believe that the two types are similar, if not identical. Among the first to express this opinion was Sternberg (13) ; and later Gamaleia (14), Prudden and Northrup (15), Kinyoun and Rosenau (16), Wadsworth (17), Lamar and Meltzer (5), Wollstein and Meltzer (9) coincided with his view. Lamar and Meltzer, especially, have insisted on the identity of the two processes. On the other hand, Welch (18) in his study of experimental pneumonia, says: "Many inoculations of cultures of virulent pneumococci into the trachea and lungs of dogs have been made in my laboratory by Dr. Canfield and myself, but in no instance were we able to produce an inflammation of the lungs which we were willing to identify with acute lobar pneumonia as found in human beings." But he adds that, in the majority of experiments, there was no demonstrable consolidation and that pleurisy and more or less extensive areas of pneumonia were produced only in a few animals. The inference consequently cannot be drawn that an effect obtained with strophanthin in the experimental disease may be anticipated in man. The striking similarity in action in infected and uninfected animals renders it likely, however, that the usual action of the drug in man may be expected in the presence of pneumonia. We have accumulated evidence, to be published later, which shows that this action actually takes place in the human disease. As far as evidence obtained electrocardiographically is concerned, our experiments show that strophanthin causes the same electrical changes in the heart when the animals are infected as it does under normal conditions.     

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

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

THE SUSCEPTIBILITY OF INFANT RHESUS MONKEYS TO POLIOMYELITIS VIRUS ADMINISTERED BY MOUTH : A STUDY OF THE DISTRIBUTION OF VIRUS IN THE TISSUES OF ORALLY INFECTED ANIMALS

Although rhesus monkeys have been generally regarded as refractory to infection with poliomyelitis virus administered by the oral route, two of seven infant rhesus developed paralytic poliomyelitis when fed murine-adapted strains of virus. Preliminary intranasal treatment with zinc sulfate and negative serial sections of the olfactory bulbs of the positive animals ruled out the possibility that infection occurred by way of the olfactory pathway. Studies on the distribution of virus in the tissues of the infected animals yielded positive results in one animal only. In this instance, virus was widely distributed throughout the body being isolated from spinal cord, buccal mucosa, duodenal wall, colon contents, superficial lymph nodes, spleen, heart, and adrenals.