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 CULTIVATION OF TRICHOMONAS OF THE HUMAN MOUTH (TETRATRICHOMONAS HOMINIS)

Trichomonades from the mouth were studied by Steinberg who proposed to group them into three distinct types; namely, Trichomonas elongata, Trichomonas caudata, and Trichomonas flagellata. Doflein (3) regards them as probably identical with Trichomonas hominis. Opinions differ as to whether or not Trichomonas vaginalis Donné and Trichomonas hominis Grassi are the same species. Lynch, for instance, believes that they are the same species, while von Prowazek (4), Bensen (5), and others (6, 7) insist that they are different types. Bensen''s view seems to be well supported by the difference alleged to be found between the mode of encystment in the two trichomonades, were it not for the fact that our knowledge about the so called cyst of trichomonades is still obscure. According to Alexeieff (8) many of the so called cysts were evidently blastomyces contained in the cell body of the trichomonas. An autogamy alleged to take place in cysts as described by Bohne and von Prowazek (9) has not been confirmed by Dobell (10). And Wenyon (11) contends that it has never been found possible to produce any development of these cysts outside the body on the warm stage as can be done with the cysts of Entamœba coli. Therefore, it is still premature to take the process of encystment into consideration as far as the classification of trichomonas is concerned. On the other hand, Rodenwaldt (12) seems to think that there are many species of trichomonas in the human intestines, and Wenyon has described a new trichomonas from the human intestines (Macrostoma mesnili Wenyon). Further cultural studies in the morphology and biology of these organisms must be carried out in order to solve these problems. In the light of modern investigations there are five subgenera to be included under the genus Trichomonas Donné. They are as follows: (1) Protrichomonas Alexeieff, with three anterior flagella, without an undulating membrane. (2) Trichomastix Biitschli) with three anterior flagella and a trailing flagellum (Schleppgeissel) without an undulating membrane. (3) Trichomonas Donné, with three anterior flagella and an undulating membrane. (4) Macrostoma Alexeieff, Amend, Wenyon (11), with three anterior flagella and an undulating membrane wedged in a deep groove (peristome). (5) Tetratrichomonas Parisi (13), with four anterior flagella and an undulating membrane. As far as our culture trichomonas from the human mouth is concerned, it has been shown that it is not strictly a trichomonas and that it should be classed under the subgenus Tetratrichomonas.     

THE ORIGIN AND FATE OF TWO TYPES OF MULTI-NUCLEATED GIANT CELLS IN THE CIRCULATING BLOOD

1. It has been demonstrated that giant cells of the foreign body and epithelioid types can be induced to appear in the blood stream. 2. Evidence has been presented which indicates that foreign body giant cells are primarily formed by fusion of monocytes and that the fate of these giant cells is accomplished, at least in some instances, by a separation into the constituent elements. 3. Further evidence has been presented which lends support to the hypothesis that "epithelioid giant cells" reach their stage of evolution, not by fusion of monocytes, but by amitotic division of the nuclei of monocytes and epithelioid cells. 4. The presence of giant cells in the peripheral blood as the result of agar injections is almost invariably associated with, or preceded by a marked monocytosis in which the new monocytes are of large size and show evidence of immaturity. 5. Injections of agar into the tissues result in decreased absolute and percentage values of lymphocytes and a diminution of the specific granules in many of the polymorphonuclear leucocytes. 6. It would appear from these studies that a clear differentiation of "epithelioid giant cells" and "foreign body giant cells" in the blood is usually possible, but that on the other hand, a few cells may be present which have some of the characteristics of each type. These latter cells probably represent in their formation both a fusion of individual cells and an amitotic division of the nuclei of monocytes. 7. Clasmatocytes or macrophages have in rare instances been seen to take part in the formation of foreign body giant cells. At least one instance has been noted of the fusion of a clasmatocyte with several monocytes. No evidence is available to demonstrate that macrophages ever play a part in the formation of "epithelioid giant cells."     

DIVISIONS OF THE SO CALLED FLEXNER GROUP OF DYSENTERY BACILLI

From these data it is seen that ill defined divisions of the so called Flexner group exist. The divisions do not appear to be sufficiently distinct to warrant the use of separate names. To avoid confusion all mannitol-fermenting dysentery bacilli should be called Bacillus dysenteriæ Flexner and the subdivision noted. There are two methods for this division, one by the fermentation of carbohydrates, the other by agglutination with monovalent rabbit sera. These do not coincide and one or the other and not both must be adopted. Inasmuch as Murray studied organisms from widely distributed sources, it would seem preferable to adopt his serological classification and to add to it the types that fail to be agglutinated by his V, W, X, Y, and Z sera, as this method is simpler and more rapid. The results of the agglutination reactions of the patient''s serum may be expressed in the same terms as the serological typing of the organism from his stool. Fermentation is less constant and gives rise to more divisions than there are carbohydrates. See PDF for Structure The serological reactions of these type sera, as Murray points out, show cross-agglutination to a greater or less extent, but they indicate that there are five antigens, V, W, X, Y, and Z and probably others, one or more of which predominate in a given strain. Polyvalent diagnostic and therapeutic sera are practically worthless unless they include antibodies for the more common of these types. The diagnostic importance of recognizing that there are five or more antigens in this group is seen from the fact that the sera of some patients react with one, others with another, and that unless several antigens are used, some positive tests may be missed. The therapeutic importance is emphasized by the fact that probably the best polyvalent therapeutic serum at present available has a very low titer for the X antigen, although that type was found in many of these cases (Table V).     

THE ORGANOTROPIC,BACTERIOTROPIC, AND LEUCOCYTOTROPIC ACTIONS OF CERTAIN ORGANIC CHEMICALS

We are dealing, as the results show, with groups of chemicals, all of which, whether bacteriotropic or not, greatly inhibit the engulfing of Staphylococcus aureus by leucocytes. Not a sufficiently large number of experiments was performed in attempt to cure experimental staphylococcus infections to warrant any condusion in regard to possible therapeutic activity against this organism. How-ever, as will appear in another paper, the only group out of the seven which definitely possessed an in vivo bactericidal action against pneumococcus is that of the cinchona derivatives. Certain members of the other chemical groups studied, although bactericidal in a very high dilution, —chemicals in which the concentration of a non-lethal dose was many times greater than that required to kill multiple minimal lethal doses of organisms in vitro, —had no certain effect when bacteria and drug were injected simultaneously into the peritoneal cavity of a mouse. In fact, the treated mouse often died before the controls. If we may assume,—leaving out of consideration the practical significance of in vivo chemical destruction and excretion following the injection of the drug into the animal,—that the failure of these chemicals to exhibit a benign influence on a systemic infection in cases in which the drug can be used in a bactericidal dilution, is due to their antiphagocytic property, only one step has been taken in analysis of the factors vital for the defense of the animal against a specific microorganism. Why do these chemicals inhibit leucocytic activity? Is it because of their influence upon complement, opsonin, or the leucocyte itself, or some special one function that determines the ability to ingest bacteria? Only further work can definitely settle this question and also determine whether or not such an analysis would be of practical importance in a rational development of chemotherapy. The ideal chemotherapeutic agent may be one that has an in vivo bactericidal potency and a negligible or stimulatory phagocytic action in doses non-lethal for the experimental animal. However difficult such a drug may be to find, it seems unlikely that the ultimate success in chemotherapy will be so simple. Again, it is conceivable that a secondary action of a drug, although leucocytotropic and not bacteriotropic, may bring about conditions in the animal body that will enable it to throw off the invading organism. Or finally, a drug compatible with the forces necessary to the host''s defense and possessing in vivo bactericidal action to a greater or less degree may be the chemical sought for, the goal toward which we should strive, to achieve a rational chemotherapy for infectious diseases.     

EXPERIMENTS ON THE MODE OF INFECTION IN EPIDEMIC MENINGITIS

The lower monkeys as represented by Macacus rhesus are resistant to a high degree to infection with cultures of the meningococcus introduced into the general blood. The lower monkeys are less resistant to infection when the meningococcus cultures are injected directly into the subarachnoid space by lumbar puncture. Relatively virulent cultures, which have been passed through several monkeys, acquire the power of surviving in the circulating blood of the monkeys for a maximum period of about 72 hours. Nothing has, however, been observed to indicate that the injected meningococci actually multiply in the blood. It has not been found possible to direct the meningococci circulating in the blood into the cerebrospinal meninges of monkeys. In this effort an aseptic meningitis was induced by injecting horse serum, saline solution, or protargol into the subarachnoid space preceding the introduction of the meningococci into the blood. In rabbits the meningococci were able to pass into the spinal fluid from the blood when a physical break in the continuity was made; however, under the conditions of chemical inflammation of the meninges the rabbit reacted just as the monkeys, and the organisms did not pass. Because of the high insusceptibility of the monkey to infection with meningococcus, it is not believed that the experiments throw any new light on the mode of invasion of the body in man by that microorganism. The experiments do not lend any support to the notion that an intraspinal injection of the antimeningococcus serum, early in the course of invasion of meningococcus in man, and possibly at a period at which the meninges do not yet show evidences of inflammation, favors its diversion from the blood stream into the subarachnoid space.     

FURTHER STUDIES ON THE PROPERTIES OF PURE VACCINE VIRUS CULTIVATED IN VIVO

1. The virulence of vaccine virus for the testicular tissues increases until its maximum is finally reached. The selective increase is not associated with any loss, reduction, or modification in its virulence for the skin. A highly potent testicular vaccine is also highly active for the skin. 2. The testicular strain of vaccine virus has no more tendency to localize in various organs than the ordinary skin strain. Both may localize in adjacent lymph nodes when introduced intravenously, subcutaneously, or intratesticularly in sufficiently large quantities, but other organs are not involved. 3. Intravenous inoculation of an excessive amount of a powerful vaccine virus (1 to 2 cc. of undiluted stock emulsion), irrespective of whether it is from the testis or the skin, will result in a generalized eruption over the entire body surface of rabbits. The eruption may be confluent on mucous membranes of the mouth, nostrils, genitalia, etc. Intratesticular or subcutaneous inoculations of the same virus fail to produce this effect. 4. Subcutaneous or intravenous introduction of much smaller quantities of the virus does not cause an appreciable local or general reaction in the rabbit. But the animals which have once received these injections become refractory to a subsequent vaccination as applied to the skin. It seems probable that an active immunity has been conferred. 5. Experiments on the viability and resistance of the testicular strain of vaccine virus indicate that the virus is best preserved when emulsified with Ringer''s solution or 0.9 per cent saline solution. Distilled water, while apparently one of the best diluents, fails to keep the virus active as long as Ringer''s or saline solutions. As would be expected, the lower the temperature is, the longer the virus retains its viability. At 18° or 37°C., the deterioration of the virus proceeds rapidly. However, a small part of the virus survives after many weeks'' standing at 37°C. 6. Of the two most commonly employed chemical agents for the ripening (eliminating bacteria) process of the green vaccine pulp, glycerol and phenol, the latter is the less injurious. Phenol in concentration above 2 per cent destroys the virus within 24 hours at any temperature, but it has almost no injurious effect when used in 0.5 to 1 per cent. On the other hand, glycerol is a powerful vaccinicide. When used in full strength it destroys the virus within 24 hours, even at 4°C. In a concentration of 40 per cent, that ordinarily recommended for the ripening, the virus retains some of its virulence for about half a year at 4°C., while at higher temperatures the same concentration kills the virus within 1 to 2 months. The virus preserved in distilled water or Ringer''s solution under similar temperature conditions remains more active during this period. From this it may be concluded that glycerol is not an indifferent agent, as is assumed by many, but a powerful vaccinicide when used in high concentrations. The injurious effect is markedly accelerated at 18° or 37°C. 7. The vaccine virus retains its virulence better in a sealed tube containing either hydrogen, nitrogen, or air than in an open receptacle. The virus deteriorates when placed in a sealed tube with oxygen or carbon dioxide. 8. Desiccation decreases to a considerable degree the virulence of the vaccine virus. In the dried state the virus retains its viability about as long as does the emulsion, but it is not protected from the deterioration caused by age under various conditions. 9. Iodine is a powerful disinfectant for the vaccine virus, but its sodium and potassium salts have no effect. Various bile salts destroy the vaccine virus when employed in sufficient concentration.     

COMPARATIVE STUDIES OF HERPETOMONADS AND LEISHMANIAS : I. CULTIVATION OF HERPETOMONADS FROM INSECTS AND PLANTS.

Nine strains of herpetomonads have been isolated in pure culture from eight varieties of insects, and three strains from two species of plants. Four of the cultures were derived from latex-feeding insects (Oncopeltus fasciatus, Oncopeltus sp. ?, Lygæus kalmii) and three from latex plants (Asclepias syriaca, Asclepias nivea), two from mosquitoes (Culex pipiens and Anopheles quadrimaculatus), one from the house fly (Musca domestica), and two from bluebottle flies. In addition impure cultures have been obtained from Oncopeltus cingulifer and from its plant host, Asclepias curassavica. The flagellates cultivated, all of which belong to the genus Herpetomonas, have been identified chiefly by their biological relationships, which will be described in detail in Part II of this report. The seven strains from latex-feeding insects and latex plants represent two distinct species, which have been designated H. oncopelti and H. lygægorum. The two strains from mosquitoes proved to be the same organism and have been called Herpetomonas culicidarum. The culture obtained from Musca domestica contained larger individuals than those of any other strain, and the organism is morphologically distinct from either of the Calliphora strains. None of the fly flagellates cultivated could be identified with the. species H. muscæ domesticæ or H. calliphoræ, and hence they have been given new names, Herpetomonas muscidarum, H. media, and H. parva. Blood agar plates were used for initial cultivation of the strains from insects and the semisolid leptospira medium for isolation of the plant flagellates. A number of the strains were purified by plating on acid blood agar, a procedure which reduces considerably the growth of bacterial contaminants. The Barber technique was utilized for isolation of the flagellates from flies, because of the very large number of bacteria found with them in these insects, and, in one or two instances, for the purification of impure cultures. Once they have been obtained in culture, all the strains grow well on leptospira medium, as well as on blood slants. Growth takes place both at 26°C. and at 37°. The morphology of the organisms is considerably modified by cultivation. This is especially true of the plant flagellates. In the latex they have ribbon-like bodies, often twisted, and comparatively short flagella; the protoplasm is clear, almost hyaline. The flagellates seen in the gut and feces of insects are usually large, slender organisms, with flagella as long as or even longer than the body, which contains numerous volutin granules in the cytoplasm. In cultures under parallel conditions the flagellates from both these sources become shorter and thicker, the plant forms no longer appear flat and ribbon-like, and in general the organisms approach one another in morphological features. Even in the case of the least modified insect flagellates, i.e. those from flies, there is never exact correspondence between the natural and the cultivated forms. The morphological features of the cultivated flagellates vary according to the medium on which the organisms are grown and the age of the culture. The flagellates grown on the surface of blood slants are pyriform, with truncated anterior portion, and short flagellum; in the condensation water, however, the individuals are elongated and have long active flagella. On the leptospira medium the slender active forms with long flagella predominate. In the presence of fermentable carbohydrate, or in medium containing considerable acid, peculiar bifurcated or multifurcated individuals are seen. Similar forms have been seen under natural conditions. Cultures of Leishmania behave in the same way under the conditions described. There is a striking difference in rapidity of growth between the organisms isolated by us and the leishmanias, H. ctenocephali, and T. rotatorium. While the stock cultures of the group first mentioned multiply rapidly at 37°C., growth becoming visible within 24 hours, the latter group grow scarcely at all at 37° and only slowly at 25°, 1 to 2 weeks being required for growth to become macroscopically demonstrable. While the flagellum of the leishmanias, as also of H. ctenocephali, is long, serpentine in its movements, and heavy, having the appearance of being enveloped by a sheath throughout its entire length, that of the recently isolated strains is thin, less flexible, and without the sheathlike appearance. The only exceptions to this rule are the flagellates from Musca domestica and Calliphora No. 1, which have a long flagellum not unlike that of the leishmanias. As the foregoing observations indicate, morphological differentiation of the flagellates studied, while not impossible, is subject to error by reason of the variations due to age and cultural conditions. The flagellates of the latex-feeding insects, the plants, the flies, and the mosquitoes can readily be distinguished from Leishmania by their rapid growth at 37°C., but their differentiation from one another is possible only by serological and fermentation reactions.     

Studies on the life cycle of spirochetes; the life cycle of the Nichols pathogenic Treponema pallidum in the rabbit testis as seen by phase contrast microscopy

A series of observations with the phase contrast microscope on the occurrence of a complex life cycle in the pathogenic Treponema pallidum as it occurs in the syphilitic rabbit testis has been presented and it seems likely from these observations that there are two means of vegetative reproduction, consisting of (1) transverse division (the most important under usual conditions); and (2) the production of gemmae or buds which eventuate into unispirochetal cysts comparable to those described for saprophytic forms, within each of which single spirochetes develop and differentiate, and from which they subsequently emerge. In addition preliminary evidence is presented which suggests that a more complex process is involved in which multispirochetal cysts develop following aggregation of two or more organisms. Within each of these larger cysts numerous organisms develop and subsequently emerge as tangled ropes. Following emergence, they subsequently undergo transverse division and gemmae formation, and so reproduce vegetatively. Subsequent papers will elaborate upon these processes.     

Transfusion Classics

Dr. Croone told me, that at the meeting of Gresham College tonight, which it seems they now have every Wednesday, there was a pretty experiment of the blood of one dog let out till he died, into the body of another on one side, while all his own run out on the other side. The first died upon the place, and the other very well and likely to do well. This did give occasion to many pretty wishes, as of the blood of a Quaker to be let into an Archbishop, and such like; but, as Dr. Croone says, may, if it takes, be of mighty use to man's health, for the amending of bad blood by borrowing from a better body.     

THE COMPARATIVE RESISTANCE OF BACTERIA AND HUMAN TISSUE CELLS TO CERTAIN COMMON ANTISEPTICS

The comparative resistance of bacteria and human tissue cells to antiseptics and other chemicals may be easily tested by tissue cultures under conditions which approximate those found in the living body. A comparative study shows that while human cells (connective tissue and wandering cells) are highly resistant to many antiseptics, they are in general more easily killed than bacteria (Staphylococcus aureus). Of the antiseptics tested, which include mercuric chloride, iodine, potassium mercuric iodide, phenol, tricresol, hydrogen peroxide, hypochlorites (Dakin''s solution), argyrol, and alcohol, the one which approaches most closely the ideal disinfectant is iodine, which kills bacteria in strengths that do not seriously injure connective tissue cells or wandering cells.     

FREE ANTIGEN AND ANTIBODY CIRCULATING TOGETHER IN LARGE AMOUNTS (HEMAGGLUTININ AND AGGLUTINOGEN IN THE BLOOD OF TRANSFUSED RABBITS)

In rabbits transfused almost daily with the whole citrated blood of other rabbits, an extraordinary condition often develops, which manifests itself in an almost immediate clumping together of all the red cells in specimens of the shed blood. This clumping is due to one or more true agglutinins, of which the strength may be such as to cause clumping in a 1: 2,800 plasma dilution. The agglutinating principle circulates with the corpuscles against which it is effective; but under ordinary circumstances intravascular clumping fails to occur because the union of antigen and antibody can take place only at a temperature several degrees below that of the body. If the temperature is sufficiently lowered, as when a tourniquet is applied to the rabbit''s ear, intravascular clumping ensues. In defibrinated blood, gradually cooled, clumping is first noted as the temperature of 35°C. is approached; and at room temperature (22°) the corpuscles will often come together in a short time into a single, solid mass. At 0°C. the agglutination is still more marked. The reaction seems to be completely reversible, for when the blood is warmed again, the clumps break up and disappear at between 35° and 36°C. Cooling and warming with the resultant clumping and dissociation can be carried out many times on the same blood specimen without apparent change in the corpuscles or in the rapidity of the reaction. The response to temperature changes is extremely prompt. Once it has been elicited, the agglutinating principle may persist for a long time after the transfusions are stopped, in one instance it was still strong 133 days after the last transfusion. During this period the plethora was succeeded by a severe anemia, which in turn was recovered from. In many rabbits no agglutinin develops, and a continuance of the transfusions will not elicit it. Indeed, when present it tends to disappear if the transfusions are persisted in. In several of the animals in which the agglutinin was strongest, the plethora was suddenly succeeded by severe anemia, despite continued transfusions. The character of the temperature control of the agglutination, which somewhat resembles that of the hemolysin in paroxysmal hemoglobinuria, has led us to consider whether the blood destruction might not be due to accidental chilling of the animal. Efforts to induce a fall in the hemoglobin by placing the rabbit''s ear in ice water have as yet been unsuccessful. Thus far no adequate search for an hemolysin has been made. The object of the present paper has been to describe a condition in which large amounts of free antigen and antibody circulate together in the organism, and to demonstrate the factor which prevents their union, the results of which could easily be fatal. The causes of the condition will be dealt with in a subsequent communication.     

THE BEHAVIOR OF CHICKEN SARCOMA IMPLANTED IN THE DEVELOPING EMBRYO

The direct inoculation of a sarcoma of the fowl into the developing chick embryo or its membranes has yielded growths in many cases. The best results have been obtained with grafts of the living tumor tissue, but, as in the adult, growths can be engendered with dried tissue or with the Berkefeld filtrate of a tumor extract. When living tumor tissue is used, an actual transplantation occurs. The neoplasms developing are spindle-celled sarcomata, remarkably uniform in structure, and similar to those in the adult fowl, except that in the embryo the neoplastic cells are often extremely long and slender, and the structure of the growth is very loose. The membranes adapt themselves in a remarkable way to the support of the tumors. In them, the growth is seldom invasive; and while regional metastases are occasionally seen, none occur by the bloodstream, despite the predilection of the growth for this path of distribution in adult hosts. In the more resistant structures of the embryo itself, an invasive extension of the sarcoma occurs. Growths originally in the yolk-sac outside the chick may be carried into the latter during the course of development. Secondary growths in the viscera may cause the death of the host some weeks after hatching. In order to produce tumors in the embryo, the sarcoma cells or the agent engendering the growth must be brought into a direct association with the mesodermal tissues. This necessity is responsible for interesting differences in the location of the growths in the various membranes. The sarcoma will grow in the membranes of pigeon or duck embryos, whereas in adults of these species it will not do so; and in chicken embryos of different varieties, it grows uniformly well, a finding not obtained in adults. In embryo hosts of all the sorts mentioned, there is a total absence of the cellular reaction which in adults indicates resistance to the tumor''s development. Relatively speaking, the embryo seems much more favorable than the adult as a host for the sarcoma.     

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.     

THE INTERRELATION OF THE SURVIVING HEART AND PANCREAS OF THE DOG IN SUGAR METABOLISM

The experiments indicate that the pancreas, when perfused aseptically with Locke''s solution containing physiological concentrations of dextrose, does not alter the reducing properties of the perfused solution. The pancreas, however, seems to supply something to the Locke''s solution circulating through its arteries which in some way brings about a utilization of sugar by the living heart to an extent that does not occur with the heart alone. This pancreatic substance possesses some of the characteristics of an enzyme. It is inactivated by boiling; it is unstable, rapidly becoming inactive on standing; it acts in small amounts; it causes a great acceleration in the rate of a reaction which otherwise proceeds slowly, and the rate of reaction diminishes as the reaction proceeds. Thus this substance has more of the characteristics of an enzyme than of a stable internal secretion like that of the adrenal glands. The disappearance of sugar was dependent upon the presence of living heart tissue, and it ceased as soon as the perfusate was removed from the heart-pancreas circulation and did not occur at all when a pancreatic perfusate was passed through a non-beating heart. This result indicates that the reaction is not similar to that obtained when muscle and pancreas extracts act on more concentrated solutions of dextrose. The living heart in the presence of the pancreatic factor and dextrose, is responsible for two effects. First, a condensation of the sugar to a non-reducing form that yields again a simple sugar on hydrolysis or by simply standing, with a preservative, at 37°C. for 24 hours. Second, a disappearance of sugar which is probably due to its destruction by hydrolysis or oxidation. After deducting the reducing sugar in the heart-pancreas perfusions which could be recovered by hydrolysis, the amount of sugar which had actually disappeared exceeded that which was used by the heart when perfused with dextrose alone. As to the fate of this portion of the sugar, no definite evidence was obtained. The question arises as to whether this substance obtained from the perfused pancreas is identical with the hypothetical internal secretion of the pancreas so essential in sugar metabolism. That there is an internal secretion of the pancreas which can be obtained by this method, and that in some way it accelerates the utilization of sugar by the living heart, seems evident. Though the conclusions are based on the heart and pancreas isolated from the numerous interrelating factors occurring in the body, the evidence suggests, at least, that the substance or substances obtained by perfusing the pancreas may be concerned in the normal activity of the pancreas upon sugar metabolism.     

THE FROST RESISTANCE OF HOOP PINE

Drought deaths on shallow soils have been a feature of the forest during arid cycles deficient in winter rainfall. These sites have been free of deaths since 1952.

Deaths have occurred more or less continuously since early 1952 in gullies, soaks, and flats throughout the forest, mostly in very vigorous forest, which is equal to the best development of P. radiata in the Adelaide Hills.

Analysis of foliage samples indicates significant amounts of chloride, the upper limit of apparently unaffected needles being 0.5 per cent, chloride. Affected foliage has a characteristic scorched appearance due to browning or death of the top of the needle.

Affected trees cover a range of ages from 5 to 39 years over several geological formations with various aspects and the conclusion is that there has been a climatic upset. Rainfall data indicate a very high water-table in 1951, one condition necessary to salt uptake.

Abnormal salt concentration in that water-table has been possibly caused by three factors concentrating the annual accession of cyclic salt which is present in all the rainfall over southern Australia. Other factors may have been contributory.

The “accident” of early 1952 appears to be a coincidental combination of several factors which may never occur again. Similar deaths in all the forest reserves of the Adelaide Hills indicate the widespread nature of the climatic upset and the necessity for further investigation.

Afforestation schemes on poorly drained country near the coast of southern Australia may well have to consider the relation between ever-present cyclic salt and fast-growing conifers, the large water requirements of which tend to exert a concentrating effect upon that salt in the soil water.     

ON THE FORMATION OF PRECIPITATES AFTER THE INTRAVENOUS INJECTION OF SALVARSAN

The results of these experiments are definite. There is, in the first place, a very striking difference with regard to precipitate formation between the acid and alkaline solutions of salvarsan when injected intravenously. Intravenous injections of alkaline solutions of salvarsan produce no precipitate in the blood, while injections of the acid solution nearly always give a precipitate. Furthermore, after injections of the acid solution, there is a striking difference between the blood from the right side of the heart and that from the left side. At the end of injections of an acid solution of salvarsan, a precipitate was seldom present in the arterial blood. Blood taken from the left ventricle at this time (at autopsy) also showed no precipitate in a large majority of cases; in eight experiments there was no precipitate, in three a doubtful trace of precipitate, and in one a definite small amount. On the other hand, blood obtained from the right ventricle and the lungs showed a very different condition. In ten out of twelve animals (rabbits and dogs), blood from the right ventricle contained a definite precipitate, and in a number of these cases the amount of precipitate was large. Blood squeezed from the lungs showed in eight out of ten cases at least as much precipitate as was found in the blood from the right ventricle. The results of injections of alkaline solutions of salvarsan, as pointed out before, are quite different from those produced by the acid solutions. In thirteen experiments upon dogs and rabbits, no trace of a precipitate was found in the arterial blood, the blood from the left ventricle, the right ventricle, or the lungs. There is no apparent difference in the process of precipitate formation whether salvarsan solutions and the blood are mixed in vivo or in vitro. In both mixtures the acid solutions produce a precipitate, while the alkaline solutions of salvarsan do not. These experiments have demonstrated the fact that a precipitate is present in the blood after an injection of an acid solution of salvarsan. One would expect that such a precipitate, consisting as it usually does of rather coarse particles, would, if brought to the medulla, cause immediate death by producing emboli. However, the freedom from such occurrences may be explained by the fact that the precipitate, which is abundantly present in the right ventricle, is only rarely seen in blood taken from the carotid or femoral arteries or even from the left ventricle itself. The fact itself, however, is quite difficult to interpret. It might perhaps be assumed that the precipitate is filtered out during its passage through the lung capillaries. If this is the case, we might expect intravenous injections of salvarsan to produce embolism in the pulmonary vessels with consequent fatal results. As a matter of fact, we have in the recent literature an instance which seems to point to such a result. Miessner (6) tried the effects of salvarsan in cattle which had foot and mouth disease. He used at first the acid solution, and though the dose was small, seven milligrams per kilo of body weight, all the animals (four) died in from ten hours to two days after the injection. They all showed labored respiration during or soon after the injection of salvarsan. He then decreased the dose to five milligrams per kilo of body weight, and repeated the experiments. He used also normal animals as controls upon those which had the foot and mouth disease. Both the sick and normal (control) animals showed labored respiration. One died after four days. At autopsy all organs except the lungs appeared to be normal. The lungs presented the following appearance: There were grayish yellow spots scattered irregularly over the surface. On the cut surface these were seen as grayish yellow spots the size of a pea, which appeared in groups and which sometimes filled a lobule completely. Other spots were surrounded by a small area of dark red lung parenchyma. The affected portion contained no air and felt solid. In adjacent parts the tissue seemed normal. A microscopic examination showed that the larger and smaller pulmonary arteries were filled with uniform, homogeneous, yellow masses. About the vessels there was a serous exudate. In brief, the changes seen indicated, he believed, that there was a thrombosis of the blood-vessels with inflammatory exudative changes of the lung parenchyma. Miessner states that a similar pathological condition was found in a normal control animal that died. He suggests that the acid solution of salvarsan might lead in man to a thrombosis of the pulmonary arteries. In support of this suggestion, he mentions a case reported to him by Ehrlich of a man who died following the injection of an acid solution. The lung picture in this case was somewhat similar to that which he had found in cattle. It may be mentioned in passing that Miessner found that alkaline solutions of salvarsan were far less toxic than the acid solutions. Animals (cattle) which received in an alkaline solution 400 milligrams of salvarsan per kilo of body weight did not show the least symptom of disturbance. In contrast to Miessner''s results seem to stand my observations and those of Auer described in the introduction of this paper. Auer (5) found (in 8 rabbits) that no evident harmful effects followed the injections of very large doses of the acid solution, if they were given in a highly diluted form (one tenth per cent.). In my own experiments, it was found that a one fifth per cent. acid solution (3 rabbits) and even a one half per cent. solution (1 rabbit) produced no ill effects. The experiments described in this paper make it certain that the doses of the acid solution given to these last mentioned four animals must have produced a precipitate in the right ventricle and in the lungs, and yet the animals survived and showed no symptoms whatever of disturbance following the injection. This difference between our observations and those of Miessner might perhaps be explained by the assumption that the action of salvarsan in acid solution is more deleterious to cattle than to rabbits. Furthermore, Miessner seems to have injected the salvarsan in high concentrations. In one instance, in which figures are given, the drug was administered in a five per cent. solution. As mentioned before, Auer has shown the importance of the concentration. While in a one tenth per cent. solution twenty and thirty milligrams per kilo of body weight of the acid solution may be injected with impunity, even six or seven milligrams per kilo may prove rapidly fatal when injected in a one half per cent. solution. Our own results, however, leave us with two puzzling questions : First, if the acid solution of salvarsan causes such a coarse precipitate in the right ventricle and in the lungs, how does it happen that this precipitate does not bring about the death of the animal? Second, what is the real cause of the remarkable fact that this precipitate does not pass over into the arterial side of the circulation? Does the precipitate undergo a profound chemical or mechanical change while it passes through the lung capillaries? In future investigations we may try to answer these interesting questions. For the present, it is necessary to be content with the establishment of the bare facts as they are presented in the conclusions.     

THE PRESERVATION OF LIVING RED BLOOD CELLS IN VITRO : I. METHODS OF PRESERVATION.

The erythrocytes of some species are much damaged when handled in salt solutions, as in washing with the centrifuge after the ordinary method. The injury is mechanical in character. It may express itself in hemolysis only after the cells have been kept for some days. It is greatest in the case of dog corpuscles, and well marked with sheep and rabbit cells. The fragility of the red cells, as indicated by washing or shaking them in salt solution is different, not only for different species, but for different individuals. It varies independently of the resistance to hypotonic solutions. The protection of fragile erythrocytes during washing is essential if they are to be preserved in vitro for any considerable time. The addition of a little gelatin (⅛ per cent) to the wash fluid suffices for this purpose, and by its use the period of survival in salt solutions of washed rabbit, sheep, and dog cells is greatly prolonged. Plasma, like gelatin, has marked protective properties. Though gelatin acts as a protective for red cells it is not preservative of them in the real sense. Cells do not last longer when it is added to the fluids in which they are kept. Locke''s solution, though better probably than Ringer''s solution, or a sodium chloride solution, as a medium in which to keep red cells, is ultimately harmful. The addition of innocuous colloids does not improve it. But the sugars, especially dextrose and saccharose, have a remarkable power to prevent its injurious action, and they possess, in addition, preservative qualities. Cells washed in gelatin-Locke''s and placed in a mixture of Locke''s solution with an isotonic, watery solution of a sugar remain intact for a long time,—nearly 2 months in the case of sheep cells. The kept cells go easily into suspension free of clumps, they pass readily through paper filters, take up and give off oxygen, and when used for the Wassermann reaction behave exactly as do fresh cells of the same individual. The best preservative solutions are approximately isotonic with the blood serum. If the cells are to be much handled gelatin should be present, for the sugars do not protect against mechanical injury. Different preservative mixtures are required for the cells of different species. Dog cells last longest in fluids containing dextrin as well as a sugar. The mixture best for red cells is not necessarily best for leukocytes. A simple and practical method of keeping rabbit and human erythrocytes is in citrated whole blood to which sugar solution is added. In citrated blood, as such, human red cells tend to break down rather rapidly, no matter what the proportion of citrate. Hemolysis is well marked after little more than a week. But in a mixture of 3 parts of human blood, 2 parts of isotonic citrate solution (3.8 per cent sodium citrate in water), and 5 parts of isotonic dextrose solution (5.4 per cent dextrose in water), the cells remain intact for about 4 weeks. Rabbit red cells can be kept for more than 3 weeks in citrated blood; and the addition of sugar lengthens the preservation only a little. The results differ strikingly with the amount of citrate employed. Hemolysis occurs relatively early when the smallest quantity is used that will prevent clotting. The optimum mixture has 3 parts of rabbit blood to 2 of isotonic citrate solution. In the second part of this paper experiments are detailed which prove that cells preserved by the methods here recorded function excellently when reintroduced into the body.     

The factor structure of the twelve item General Health Questionnaire (GHQ-12): the result of negative phrasing?

Background

The 12-item General Health Questionnaire (GHQ-12) is used routinely as a unidimensional measure of psychological morbidity. Many factor-analytic studies have reported that the GHQ-12 has two or three dimensions, threatening its validity. It is possible that these 'dimensions' are the result of the wording of the GHQ-12, namely its division into positively phrased (PP) and negatively phrased (NP) statements about mood states. Such 'method effects' introduce response bias which should be taken into account when deriving and interpreting factors.

Methods

GHQ-12 data were obtained from the 2004 cohort of the Health Survey for England (N = 3705). Following exploratory factor analysis (EFA), the goodness of fit indices of one, two and three factor models were compared with those of a unidimensional model specifying response bias on the NP items, using structural equation modelling (SEM). The hypotheses were (1) the variance of the responses would be significantly higher for NP items than for PP items because of response bias, and (2) that the modelling of response bias would provide the best fit for the data.

Results

Consistent with previous reports, EFA suggested a two-factor solution dividing the items into NP and PP items. The variance of responses to the NP items was substantially and significantly higher than for the PP items. The model incorporating response bias was the best fit for the data on all indices (RMSEA = 0.068, 90%CL = 0.064, 0.073). Analysis of the frequency of responses suggests that the response bias derives from the ambiguity of the response options for the absence of negative mood states.

Conclusion

The data are consistent with the GHQ-12 being a unidimensional scale with a substantial degree of response bias for the negatively phrased items. Studies that report the GHQ-12 as multidimensional without taking this response bias into account risk interpreting the artefactual factor structure as denoting 'real' constructs, committing the methodological error of reification. Although the GHQ-12 seems unidimensional as intended, the presence of such a large response bias should be taken into account in the analysis of GHQ-12 data.

     

A FURTHER NOTE UPON THE EXPERIMENTAL PRODUCTION OF LEPROSY IN THE MONKEY (MACACUS RHESUS), WITH A CRITICAL STUDY OF THE CULTURE EMPLOYED

Fatal leprosy, with all its clinical and pathological manifestations in man, may be experimentally induced in the monkey (Macacus rhesus) with a pure culture of the acid-fast bacillus cultivated by one of us (Duval) from a leprous lesion in man. To produce the disease experimentally, it seems necessary to give the animal repeated injections of large numbers of leprosy bacilli at given intervals for a period of months. That the infection is more likely to follow where sensitization is first established is definitely proven by the specific experiments that we have carried out upon a variety of laboratory animals. The first injection, we assume, sensitizes the animal and may consist of either killed or viable lepra bacilli. The necessity of first sensitizing the monkey and then giving repeated doses of viable organisms over a long period might explain the relative infrequency of the disease in man; at least, it offers an explanation of the fact that man rarely, if ever, contracts leprosy, although intimately associated for an indefinite period with those afflicted with the disease. The leprous lesions in the monkey are histologically indistinguishable from those in man and do not essentially resemble the specific lesion of tuberculosis, blastomycosis, or the lesions experimentally produced with saprophytic acid-fast species, since the appearance of large lepra cells and the arrangement of the bacilli in dense packets within these cells to form the so-called globi is a constant and characteristic feature for the experimental as well as the human lesion (figures 17, 18, and 19). The production of leprosy in the monkey proves conclusively that the acid-fast bacillus cultivated by one of us (Duval) from the human lesion is the Hansen bacillus and not some extraneous saprophyte, and that it is the etiological factor in human leprosy. In our experience, it has been extremely difficult to produce, in the lower animals, more than a transient localized lesion with human leprous material rich in the specific bacilli, unless the animal is first sensitized, when lesions histologically identical with those produced by pure cultures are easily induced. Therefore, it is natural to expect that cultures of Bacillus leprœ which are many generations removed from the parent stem are less likely to infect, unless given in larger doses on the ground of loss in virulence. When experimental leprous lesions occur in the internal organs, they are more often found in the liver and spleen, while the experimental lesions occasionally produced in the lower animals with some of the saprophyte species, such as the bacillus of timothy hay, Moeller''s grass bacilli, etc., rarely, if ever, occur in these organs (Abbott and Gildersleeve). These authors did not find lesions in the liver and spleen in a single instance after inoculating forty-five rabbits intravenously with large doses of the "confusing group." Furthermore, the cell picture and the appearance and arrangement of these bacilli in the lesions in no way resemble experimental leprosy (Hölscher). It is no indication that a given culture is not the Hansen bacillus because the individual organisms differ in size and shape from those in the tissues, since it is a well known fact that marked variations in morphology are common for many bacterial species under natural and artificial conditions. One of us (Couret) has already pointed out that there is a wide variation in morphology for Bacillus leprœ under different environments. The experimental work serves not only to emphasize this fact, but is proof that a transformation from the slender beaded rods of the tissues to solidly staining diplococcoid forms of culture does occur for Bacillus leprœ; and, conversely, that the coccoid forms of culture may again assume the slender beaded appearance by passage through warm-blooded animals.