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A protease from Pseudomonas aeruginosa having collagenase activity exhibited lethal and dermonecrotic properties. It was purified 1500-fold and was capable of hydrolyzing collagen both in vitro and in vivo. The enzyme was assayed in mice by a number of routes and was found to be most toxic when administered into the lungs. This resulted in confluent pulmonary hemorrhage. Five standard methods of extracting endotoxin were employed in an effort to establish its presence in Pseudomonas aeruginosa as well as to make comparative evaluations of its biological and chemical properties. Of the five preparations, aqueous phenol extracted endotoxin exhibited the greatest degree of lethality. The LD50 was 450 microgram dry weight when administered intravenously and 840 microgram intraperitoneally. No lethality was observed when endotoxin was administered intranasally. Lethality appeared to be associated with the core region of the lipopolysaccharide molecule, while no correlation between lethality and lipid content was detected., See also report dated 29 Feb 1972, AD738063.

Korean hemorrhagic fever (KHF) occurred for the first time in Korea, 1951, although it had previously been known to both the Japanese and Russians. After the Korean war, the disease has been fixed in the areas of demilitarized zone (DMZ) as an endemic one, and from 100 to 300 cases have been reported every year. The aims of this project are epidemiologic investigation of the disease in the civilian endemic areas to isolate the causative agent of KHF, and to demonstrate specific antibodies of KHF by fluorescent antibody technique. (Modified author abstract)

Petroleum degradation studies are being done to obtain a seasonal incidence, as well as species distribution of petroleum-degrading microorganisms in Chesapeake Bay. From analysis of water and sediments collected at two stations in Chesapeake Bay it was found that the concentration of petroleum in an oil polluted site in Baltimore Harbor was five times greater than in Eastern Bay. The numbers of petroleum-degrading microorganisms, measured by direct and replica plating, in the water and sediment samples were related to the concentration of oil in each sample. Total yields of petroleum-degrading microorganisms grown on an oil substrate were greater for those organisms exposed to oil in the natural environment. Microorganisms isolated from water and sediment samples collected in Baltimore Harbor grew on substrates representative of the aliphatic, aromatic and refractory hydrocarbons. From analyses of species distribution, it was observed that a hydrocarbon-utilizing fungus, Cladosporium resinae, and actinomycetes were predominant among the hydrocarbon-utilizing isolates.

Studies which have examined various aspects of the physiology and metabolism of Neisseria gonorrhoeae are reported. The principal findings are: (1) Glucose is dissimilated by a combination of the Entner-Doudoroff and pentose phosphate pathways. (2) The dissimilation of glucose is an aerobic process; no growth occurs under anaerobic conditions. (3) Growth temperature markedly affects the dissimilation of glucose. (4) Glucose-limited cultures are susceptible to autolysis; nitrogen limited cultures are not. (5) Glucosamine enhances autolysis in glucose-depleted cultures. (6) N. gonorrhoeae and N. meningitidis are susceptible to growth inhibition by the gonadal steroid progesterone. The site of inhibition appears to be located in the cytoplasmic membrane.

Development and instrumentation of a rapid method of virus and viral antibody detection and identification utilizing the Coated Latex Adsorption Method (CLAM) is presented. Comprised of an inert latex particle 'carrier', adsorbing antibody, and detected virus or antibody, the classic CLAM particle may now be classified into two major types (in vitro and in vivo). The in vitro CLAM particle was developed because of the ability of monodispersed latex particles to adsorb, in vitro, a variety of substances and to produce a complex which could be quickly detected within one minute using present day instrument technology. The in vivo CLAM particle retains the basic descriptive properties of the in vitro particle except that it is applied directly to a living host system by various routes of innoculation. By locating at sites of antibody production, the particle acts as tracer, locator, and quantitator of a variety of dynamic biological processes with the host and can thus permit, for example, determination of the earliest possible time of antibody detection after infection., See also AD-767 041.

The Mddle Salt Lagoon, adjacent to the Chukchi Sea, at Barrow, Alaska, is the depository for sewage from the Naval Arctic Research Laboratory. It is covered with ice for most of the year (9-10 months), and during 1972 the salinity reached a maximum of 106.50 degrees/00; concentration of other solutes and solids was also accompanied by ice growth. Minimum temperatures were -5C, the lowest of any of the coastal bodies of water in the vicinity. During the period of ice-cover, microbial populations were of a low order. During the summer when free-water was present, increases in numbers were observed particularly among marine and halophilic bacteria. Enteric indicator organisms for the most part were low, except at the sewage outfall and in bottom sediments. Although the lagoon, interacting with the nearby Sea, would appear to be an ideal, two-cell, sewage disposal system, very little of the sludge and other material concentrated near the bottom by ice growth found their way for dilution into the Sea.

Contents: Development of an effective and practical vaccine against experimental and human syphilis; Mechanism(s) of the immune response in experimental and human syphilis.

Work during the project period focused on microbial adaptive enzyme synthesis under pressure, on the role of adenosine triphosphate supply in determining microbial barotolerance, on cell death caused by 1000 atm pressure and on growth optimization in the pressure-temperature domain. It was found that some types of adaptive enzyme synthesis were no more pressure sensitive than was protein synthesis in general. But derepression of the lac operon of Escherichia coli or the penicillinase genes of Bacillus licheniformis was more sensitive to pressure than was growth or general protein synthesis. Therefore, under pressure E. coli cells than had to adapt to lactose utilization or B. licheniformis cells that had to adapt to penicillin were more barosensitive than were previously adapted cells or cells of constitutive mutants. Thus, the pressure sensitivities of the adaptive processes compromised survival capacities under pressure., See also report dated 29 Dec 73, AD-771 469.

The research program consists of two interrelated areas of work, both aimed at understanding the genetic basis of variation in scrub typhus organisms. It was felt that a combined program, studying both nucleic acid synthesis and genetics, was the most logical approach to this problem. Once the mechanism of variation was revealed, it might be possible to influence changes in the rickettsiae by chemical mutagenesis and develop strains particularly suitable for vaccine development which contain multiple major surface antigens. (Modified author abstract)

The studies undertaken in the contract are fully counter to the recent and much-quoted contention that ticks and domestic animals play a significant role in the ecology of epidemic typhus and, instead, strongly suggest that the serological data that had been presented in support of such a hypothesis are based upon artifacts. Thus, out of 861 Ethiopian livestock sera and 2,849 sera from Egyptian domestic animals tested by complement fixation (CF) in our program, none of the samples contained antibodies specific for rickettsiae of either epidemic typhus or murine typhus. Moreover, 821 of 822 sera from Egyptian donkeys were anticomplementary and therefore nonspecifically 'positive' in CF tests. It is our opinion that the identical or a similar anticomplementary substance, identified by us as IgM, accounted for the results reported as 'positive' by earlier workers. (Modified author abstract)

It is the purpose of this contract to investigate the mechanisms used by soil bacteria to oxidize, ortho-, meta- and para-xylene. The progress report describes significant accomplishments including: The isolation of a strain (Pxy) of Pseudomonas that will oxidize p- and m-xylene to carbon dioxide and water; The isolation of a mutant strain of the above organism that accumulates p-toluic acid from p-xylene; The isolation of a second mutant strain that accumulates 3- and 4-methylcatechol from m- and p-xylene; The elucidation of the metabolic sequences used by Pseudomonas pxy to degrade p- and m-xylene to aliphatic products; The isolation and identification of intermediates in the metabolism; The isolation of the first bacterium reported to grow with o-xylene as the sole source of carbon and energy; and, Identification of 3,4-dimethylcatechol as an intermediate in o-xylene metabolism.

During the course of the contract the authors have defined the physical components in relation to the animals tested, determined the efficacy of jet injection in relation to the biological effectiveness of live antigen on a host animal, compared the relative efficacy of jet injected antigens with those administered by needle and syringe and made recommendations on improvements of existing equipment. (Modified author abstract), See also rept. no. 8, AD-767 399.

Human lymphocytes stimulated with PHA are capable of supporting the replication of many viruses, including 17-D, in vitro. Lymphocytes removed from 17-D virus vaccinated human volunteers, 7-11 days following immunization, failed to support 17-D virus replication in vitro; by day 25, lymphocytes from most vaccines had regained completely their capacity to replicate 17-D virus. The results of our studies suggest that the 17-D virus refractory state may be attributable to the release of cytotoxic substances by certain lymphocytes in culture which in turn affect the ability of other cells to support virus replication. The substantiation of these results would suggest a similar mechanism for the control of viral infections by lymphocytes in vivo. (Author)

An infrared photomicrographic technique useful in providing differentiation among 10 different closely related bacterial pathogens is described. The theoretical considerations resulting in the construction of an infrared iso-adiabatic calorimeter are presented. (Author)

The report summarizes studies done on the immunochemistry and immunizing properties of a polysaccharide slime layer material synthesized by strains of S. aureus when cultured under appropriate conditions on a modified Staphylococcus 110 medium. It was found that the polysaccharide was composed of a polymer of galactose and galacturonic acid, but also contained significant amounts of glycine, proline, glutamic acid,and alanine. Neither active nor passive immunization studies with a number of recently isolated or laboratory strains of S. aureus consistently demonstrated any protection inducing antigenic properties in slime layer against experimental challenge of mice with the Smith, diffuse strain of S. aureus or with homologous non-Smith strains. It was also demonstrated that the antigen(s) present in slime layer material adsorbed spontaneously to erythrocytes from several species and sensitized them for passive hemagglutination in the presence of staphylococcal antibody. (Modified author abstract), See also report dated 16 Jan 73, AD-754 435.

Branched and straight chain hydrocarbons are metabolized by Brevibacterium erythrogenes by means of two distinct pathways. Normal alkanes are degraded, after terminal oxidation, by the beta-oxidation system operational in fatty acid catabolism. Branched alkanes like pristane and 2-methylundecane are degraded as dicarboxylic acids, which also undergo beta-oxidation. Pristane-derived intermediates are observed to accumulate, with time, as a series of dicarboxylic acid pathway is not observed in the presence of normal alkanes. Release of (14)CO2 from 1-(14)C-pristane is delayed, or entirely inhibited, in the presence of n-hexadecane, while CO2 release from n-hexadecane remains unaffected. These results suggest an inducible dicarboxylic acid pathway for degradation of branched chain alkanes. (Author)

An antigen has been extracted from T1 phase of Neisseria gonorrhoeae which has been characterized very crudely as a nucleoprotein. Preparations of this antigen were labeled biosynthetically with 14C and used in a radioassay for serodiagnosis of gonorrhea in humans. The results of the assays with this antigen may be summarized as follows: 90% of male and 98% of female sera were correctly diagnosed as positive (combined 93% correct); 32% of male and 82% of female sera were correctly diagnosed as negative (combined 60% correct); 5% of male and 2.5% of female sera could not be assigned positive or negative values. Preliminary results with an 125I labeled gonococcal antigen were similar to those described above. (Author)

Current studies indicated that mice receiving either sub-cutaneous or intradermal injections of viable P. aeruginosa cells exhibited systemic infections after several days. The primary target organs appeared to be the kidneys and lungs. Administration of an equal number of dead cells did not kill the mice although approximately 30% of the animals exhibited black necrotic lesions similar in appearance to ecthyma gangrenosum. Attempts to establish pulmonary infections in mice receiving per-oral administration of P. aeruginosa directly into the lungs was unsuccessful. However, per-oral administration of viable cells into the stomach of mice resulted in fatal systemic infections. Chronic systemic infections of rabbits were established in order to study the sequence of events leading to severe kidney damage. Renal lesions were detected by the 5th to 7th day post-infection along with progressive azotemia., See also report dated 15 Jan 1974, AD773549.

Five standard methods of extracting endotoxin were employed in an effort to establish its presence in Pseudomonas aeruginosa as well as to make comparative evaluations of its biological and chemical properties. Of the five preparations, aqueous phenol extracted endotoxin exhibited the greatest degree of lethality. The LD50 was 450 micrograms dry weight when administered intravenously and 840 micrograms intraperitoneally. No lethality was observed when endotoxin was administered intranasally. Lethality appeared to be associated with the core region of the lipopolysaccharide molecule, while no correlation between lethality and lipid content was detected., See also report dated 28 Feb 1973, AD755745.

Contents: Development of an effective and practical vaccine against experimental and human syphilis; Mechanism(s) of immune response in experimental and human syphilis; The 'in vitro' cultivation of Treponema pallidum in tissue culture monolayers., See also report dated 31 Mar 1973, AD757429.

Challenge infection with V. cholerae in the ligated ileal loop of the normal adult rabbit indicated that the unwashed loop was significantly more susceptible than the washed loop. It is suggested that this was due to the potentiating effect of mucin in the former. Such challenge of actively immunized animals in which the local immunity was a function of either antitoxic or complement-dependent vibriocidal antibody showed that both kinds of immunity were protective to ca 2 logs in the washed loop, but no significant protection was observed in the washed loop. This was taken to suggest the primary importance of intraluminal antibody. Comparison of these results with those of challenge with cell-free toxin in which protection appeared to be a function of tissue-contained antitoxic 11S IgA indicated that intraluminal 11S IgA had a complement-independent antibacterial activity as well as antitoxic activity.

The report describes the interaction of selected respiratory viruses and Eaton's agent. For example, enhancement of rhinovirus-RNA synthesis in Mycoplasma pneumoniae treated KB cells was demonstrated, and in contrast the inhibition of poliovirus-RNA synthesis in similar treated cell systems. In addition, basic studies concerning the action of rhinovirus on cell systems are explained in terms of nucleotide phosphorylation and RNA incorporation of 3H- uridine. Because of the importance of other selected respiratory viruses (adenovirus and influenza virus) on the military population these agents were investigated. In particular, the effect of mycoplasma on adenovirus 3H- thymidine uptake and influenza virus infections of mice. For comparison with adenovirus-mycoplasma systems another DNA virus, herpes simplex virus, was employed., Supported in part by Grant DAHC19-70-G-0009.

The report describes work on the polysaccharide and teichoic acid cell wall antigens of the streptococci. It is shown that the chemical modification of such antigens provides them with the ability to sensitize red blood cells, and such cells can be used to measure antibody levels in blood sera. Methods for the extraction of polysaccharide and teichoic acid antigens from the streptococci have been developed, and the chemical composition and serological specificity of these antigens has been defined. The role of the streptococcal cell wall in the uptake of DNA and subsequent genetic transformation has been investigated.

Within the framework of a study on the oil biodegradation potential of the sea the ability of a Flavobacterium sp. and Brevibacterium sp. to metabolize a paraffinic crude oil and a chemically defined hydrocarbon mixture was investigated. Major components of the crude oil were identified by combination gas chromatography and mass spectrometry. The rate and extent of total hydrocarbon biodegradation was measured. Degradation started after a 2 to 4 day lag period, and reached its maximum within two weeks. At this time up to 60% of the crude oil was degraded. n-Paraffins were preferentially degraded as compared to branched chain hydrocarbons. Biodegradation and mineralization of petroleum, added at 1% (v/v) to freshly collected sea water, were measured using gas-liquid chromatographic, residual weight, and CO2-evolution techniques. Only 3% of the added petroleum was biodegraded and 1% was mineralized in unamended sea water after 18 days of incubation. Added nitrate, phosphate supplements in combination increased petroleum biodegradation and mineralization. Attempts to clean up oil spills with the aid of microorganisms should take into consideration the nutritional deficiencies of sea water.

Isotopic and enzymatic evidence shows that growth of Pseudonomas methanica on methane or methanol proceeds by a variant on the pentose phosphate cycle into which the carbon enters by addition of formaldehyde to ribose 5- phosphate giving allulose 6-phosphate. The metabolism of C-1 growth substrates by Pseudomonas AM1 has been studied by measurement of activities of enzymes involved in serine metabolism during growth on methanol or succinate, and by isolation of auxotrophic mutants. Studies have been made of regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalatious. (Author)

Partial Contents: Carrier Rates in Hospital Patients and Personnel and Their Relation to Infection, Clinical Pathology and Iatrogenic Diseases, Hemodynamic Interpretations of Cardiovascular Sound.

The solution sought in this research project is the discovery of the mediators of disease, anemia, splenomegaly, petechial hemorrhage and nephritis in human malaria. The approach is based on the concept that disease is mediated by in vivo immunologic reactions rather than by toxic substances elaborated by the parasites. This concept has received general acceptance in cases of most of the infectious diseases caused by fungi, bacteria, rickettsiae or the viruses. However, in the cases of malaria, the immunologic mechanisms are obscure. Thus in this project, research must be directed at discovering and defining the immune mechanisms, and then to seek for a causal relationship between the allergic state and/or the antibodies of immunity and the diseases associated with malaria. Keywords: Immunopathology, Malaria, Hemosporidian infections, Medical research, U/A Reports. (JG)

New methodology for the study of rickettsiae has opened the way for an enormous range of new studies of a quantitative nature. Most important among these is the routine application of modificatons of the plaquing technique refined by wike et al., the simple method for counting the absolute number of the RLB described by Silberman and Fiset and various modifications of the tissue culture technique, including the slide chamber method. These methods have permitted the recognition of extraneous agents in most yolk sac seeds of rickettsiae, plaque-purification and cloning of extraneous agents rickettsial lines, definition of the intracellular growth cycle of Rickettisia prowazeki, investigations of hose cell range of those rickettsiae and of the rickettsia-host cell interaction, methods for determining accurately the antibiotic sensitivity of rickettsiae. Reproducible, predictable in vitro systems appear to be possible. Clinical data from Burundi has confirmed our original observation that a single 100 mg dose of doxycycline will cure louse-borne typhus and has established for the first time that minocycline is equally effective. A field trail of the living attenuated E strain typhus vaccine in Bolivia confirmed its acceptability in this population.

A variety of bacteria which degrade pyruvate by the 'phosphoroclastic reaction' were examined with respect to the role of CoA in this reaction. This strictly anaerobic bacteria, which cleave pyruvate by the phosphoroclastic reaction characteristic of Clostridia, required catalytic levals of CoA for the Co2-pyruvate exchange and acetoin forming portions of the phosphoroclastic reaction. These reactions were reversibly inhibited by the CoA analogue, desulpho-CoA. In contrast, using cell-free extracts of bacteria which degrade pyruvate by the coliform phosphoroclastic reaction (pyruvate formate-lyase), no requirement for CoA could be observed for the formate-pyruvate exchange reaction. It is suggested that CoA serves a regulatory function in the early portion of the clostridal type of phosphoroclastic reaction. (Author)

A phagocytosis-promoting factor (PPF) has been isolated from conventional and germfree guinea pig and rat peritoneal exudate fluid. The factor was found to resemble a globulin type of serum protein. It contained no hemolytic complement or gamma globulin. Electrophoresis under conditions of low pH and histochemical techniques indicated the PPF was a glycoprotein. Divalent cations were required for enhancement produced by the PPF. The terminal sialic acid group of the glycoprotein was shown to be indispensible for opsonic activity. The PPF appears to promote adsorption of bacteria to phagocyte but not ingestion. The PPF was inactive when used with polystyrene latex particles or with heterologous cells. (Author)

Two types of microorganism were isolated which grow readily and abundantly on natural and synthetic rubber as well as on various polyisoprenoid substrates. The cultures include a black yeastlike organism and a pinkish Gram-negative bacterium. Filtrates from cultures of each of these organisms possess heat labile rubber-degrading activity as determined by assay procedures using vulcanized rubber bands. In attempts to obtain cell-free culture filtrates with rubber-degrading activity, it was found that the activity was associated with particulate matter consisting of protein and partially degraded polyisoprene. (Author)

The studies undertaken are fully counter to the recent and much quoted contention that ticks and domestic animals play a significant role in the ecology of epidemic typhus and, instead, strongly suggest that the serological data that had been presented in support of such a hypothesis are based upon artifacts. Thus, out of 861 Ethiopian livestock sera and 2,849 sera from Egyptian domestic animals tested by complement fixation (CF) in the program, none of the samples contained antibodies specific for rickettsiae of either epidemic typhus or murine typhus. Moreover, 821 of 822 sera from Egyptian donkeys were anticomplementary and therefore nonspecifically positive in CF tests. The identical or a similar anticomplementary substance, identified as IgM, accounted for the results reported as 'positive' by earlier workers. These results indicate that such domestic animals could not serve as a reservoir or source of infection of ticks with R. prowazeki in nature. (Modified author abstract), See also report dated 30 Sep 73, AD-783 069.

By fermentation using Streptomyces longisporus ruber 3762 and S. sp. Y-42 the authors prepared and purified 500 mg each of 3 basic pigments for antimalarial testing. One of these and two prepared earlier were active against malaria in mice. (Author), See also report dated 15 Aug 72, AD-758 178.

A serological survey of 659 human sera from the British Solomon Islands Protectorate using 11 antigens representing groups A, B and Bunyamwera arboviruses failed to reveal any evidence of infection. No arboviruses were isolated from mosquitoes, bird bloods or rat organs. Suggestive evidence of a mite-borne rickettsiosis has been obtained. This may be widespread in the group. The carrier rate for hepatitis B antigen is very high, particularly among children. (Author)

Results of experiments to define the ways in which pressure and temperature interact to affect growth of psychrophilic, mesophilic and thermophilic bacteria indicated that barotolerance is greatest at optimal growth temperatures. Below the optimum, pressure and temperature acted antagonistically; while at higher temperatures, they generally acted synergistically to restrict growth. Other experimental results indicated that pressure markedly narrows pH ranges for bacterial growth and, at extreme pH, causes leakage of potassium ions from cells without also causing massive RNA breakdown. The adverse effects of low, but not high, pH under pressure could be partially reversed with divalent cations. It was found also that part of the basis for low growth yields commonly obtained with pressurized cultures is the enhanced sensitivity to metabolic acids that build up in enclosed pressure vessels during growth. (Modified author abstract)

Primary atypical pneumonia, caused by Mycoplasma pneumoniae, is a significant health problem among military recruits. The current study was designed to yield information on the mechanism of pathogenesis in this disease. Special processes for the cultivation of the causative agent have been developed. Organisms were collected and membrane fractions were prepared. When membranes were evaluated for cytotoxic potential in the hamster tracheal ring/organ culture system, they displayed active biological activity. The necrosis induced in the ciliated respiratory epithelium was grossly equivalent to that induced by viable cells, and was dose-dependent. The data indicate that the cell membrane of this pathogen may be the site of the toxic factor. Further biological and biochemical identification of this toxic factor could ultimately play a key role in the development of a suitable vaccine to reduce the high morbidity rate associated with this disease. (Author) Portions of this document are not fully legible.

In depth evaluation of the jet injector for use in large animal medicine was made. The study encompassed both the physical and biological properties of the jet injector gun. As presently engineered, the gun is entirely satisfactory for use in a number of different species of animals, both food and companion, including equine, bovine, caprine, ovine and porcine, certain changes in the combination of nozzle, orifice diameter, intensifier and spring size are required depending on the species under study. These optimal combinations have been detailed in the body of the report. (Modified author abstract)

Previous research has found crude Newcastle disease virus (NDV) - induced interferon produced in L-929 cell cultures to be highly effective in protecting these cells against Mengovirus but totally incapable of protecting mouse-passaged Ehrlich ascites tumor (EAT) cells. This anamolous intraspecies specifity was due to the presence in the crude material of an interferon antagonist. Adsorption on CM Sephadex C25 and elution with a rising pH gradient effected isolation of semipurified interferon, fully protective of EAT cells, as well as separation of the antagonist. The latter eluted in two positions - one just after the loading volume and the other just prior to interferon elution. This report further characterizes the antagonist by biochemical and biological parameters., Report of a Study of a Mouse Cell Interferon Antagonist Effective in Ehrlich Ascites Tumor Cells.