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Mammalian cells particularly suitable for the study of specialized aspects of rickettsial biology were tested for their ability to support plaque formation by Rickettsia conori. The detection of plaques was substantially influenced by the combination of growth medium and cell type used. Large plaques (2.0 to 3.0 mm in diameter) occurred by 8 days postinfection in WI-38 and DBS-FRhL-2 cells supported by medium 199. Smaller plaques (0.5 to 1.0 mm in diameter) were seen in L-929 and HeLa cells at 8 to 11 days postinfection and were more discernible in cells supported with Eagle minimal essential medium. Chicken embryo cells maintained in Dulbecco's modified Eagle medium exhibited large spherical plaques with a diameter of approximately 1.5 mm by 8 days postinfection.

Three cell surface antigens, protein, lipopolysaccharide, and polysaccharide, were purified from group B and group C strains of Neisseria meningitidis representing a variety of serotypes. Chemical analysis indicated that cross-contamination was on the order of 1%. Sensitization of sheep erythrocytes with these antigens resulted in highly specific passive hemagglutination assays for the three kinds of antigens. Paired human sera from several groups of individuals were tested by hemagglutination for antibody against each of the antigens. Patients with group B or C systemic meningococcal disease showed increases in antibody titer against all three kinds of antigens, but the antibody response to B polysaccharide was low compared with the response to C polysaccharide. Nasopharyngeal carriers of group B meningococci showed significant increases in titer only against the protein antigens, and noncarriers who received a C-polysaccharide vaccine had a specific response to the C polysaccharide. A given protein or lipopolysaccharide antigen reacted on the average equally well with either group B or C convalescent sera. These results suggest that all three antigens may play a role in the broad human immunity following natural infection.

The genes controlling synthesis of Shigella flexneri group- and type-specific antigens were transferred to Escherichia coli K-12 recipients by conjugation with an S. flexneri Hfr. After mating E. coli with an Hfr strain of S. flexneri 2a and selecting for his(+) recombinants, a high proportion of the E. coli hybrids agglutinated in S. flexneri grouping serum. None of these hybrids expressed S. flexneri type-specific antigen II. When an E. coli his(+) hybrid possessing the S. flexneri group antigen was remated with the same Hfr with selection for pro(+) hybrids, a high proportion now expressed the type-specific antigen as well as the previously inherited group antigen. If such crosses were performed in reverse order (i.e., pro(+) followed by his(+) selection), a different pattern of serological behavior was observed. None of the pro(+) hybrids showed the type-specific antigen. Subsequent mating for his(+) resulted in hybrids with both the group- and type-specific antigens. These results show that genes controlling the synthesis of S. flexneri group antigen (linked to the his locus) and type-specific antigen (linked to the pro locus) are widely separated on the chromosome. Expression of the type-specific antigen II depends on the presence of the group antigen.

A method was developed for production of a freeze-dried Western equine encephalomyelitis vaccine from virus propagated in chick embryo cell culture monolayers maintained with a serum-free medium. A sufficient concentration of virus accumulated in the cell culture fluids prior to the occurrence of viral cytopathology to permit the production of a vaccine relatively free from serum and cellular proteins. Inoculation with two mouse ld(50) doses of virus per 100 tissue culture cells was found to yield reproducible high virus titers at a convenient harvest time. These harvests were inactivated at 22 C by 0.05% formalin within 48 hr. Potency test results, as measured by the protection of immunized guinea pigs against an intracerebral virus challenge, indicated that the vaccine produced from the virus propagated in cell culture was equal in potency to a lot of whole chick embryo vaccine used to immunize laboratory and field workers subject to a high risk of infection.

Wohlhieter, J. A. (Walter Reed Army Institute of Research, Washington, D.C.), C. C. Brinton, Jr., and L. S. Baron. Utilization of carbohydrates and metabolic intermediates by piliated and nonpiliated bacteria. J. Bacteriol. 84:416-421. 1962.-The rate of aerobic and anaerobic metabolism of piliated and nonpiliated bacteria was studied in an effort to determine whether the presence of these surface structures altered the metabolic activity of bacteria. Respiration was measured manometrically when various piliated and nonpiliated strains metabolized glucose and several Krebs-cycle intermediates. Spontaneously produced nonpiliated variants of piliated cells were examined, but no difference in the rate of either aerobic or anaerobic metabolism was observed. Piliated and nonpiliated recombinants of a mating of Escherichia coli and Salmonella typhosa had different metabolic rates than the parent strains, but it was shown that these differences were not related to piliation. Clones of piliated and nonpiliated cells isolated from other crosses showed no difference between their respiration rate and that of either parent. Piliated and nonpiliated strains could mutate to higher rates of substrate utilization with no concomitant change in piliation. Since respiration and piliation can mutate independently and segregate independently in genetic crosses, we therefore conclude that there is no direct physiological relationship between them.

LaBrec, Eugene H., Herman Schneider, Thomas J. Magnani, and Samuel B. Formal. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol. 88:1503-1518. 1964.-A parent strain of Shigella flexneri 2a and a colonial mutant derived from it were studied in three animal models. Both strains were equally virulent for mice when living cells suspended in hog gastric mucin were injected by the intraperitoneal route. Feeding the parent strain to starved guinea pigs, followed by the intraperitoneal injection of opium, resulted in the formation of ulcerative lesions in the intestinal tract and in the death of these animals. When the colonial variant was fed to similarly prepared animals, the animals survived and the intestinal tract remained normal. The parent produced diarrheal symptoms and intestinal lesions after its oral administration to rhesus monkeys; the variant caused neither symptoms nor pathology in this species. Studies were carried out to define the characteristics present in the parent strain and absent in the colonial mutant, which would enable the parent to produce ulcerative lesions of the bowel and death in the guinea pig model or intestinal lesions and diarrheal symptoms in the monkey. Neither serological studies nor growth studies conducted both in vitro and in vivo offered a clue to explain this difference. The virulent parent strain was shown to penetrate the bowel epithelium and enter the lamina propria; the avirulent mutant did not do this. Entrance to the lamina propria was by way of the epithelial cell of the mucosa. The avirulent mutant did not possess the capacity to penetrate this cell. This observation was extended to show that the virulent parent possesses the ability to infect and multiply within HeLa cells; furthermore, the organisms are able to penetrate epithelial cells of the guinea pig cornea, causing ulcerative lesions. The avirulent variant possesses neither of these capacities. It is suggested that epithelial cell penetration is a major factor in determining the pathogenicity of dysentery bacilli.

Tropical canine pancytopenia (TCP) is a newly recognized infectious disease of dogs in diverse tropical and subtropical areas. The disease is characterized by hemorrhage, pancytopenia, severe emaciation and persistent infection. Dogs with TCP are often presented with epistaxis, which is the most dramatic sign of the disease; however, a large number of affected dogs develop severe pancytopenia and die without manifesting clinical signs of hemorrhage. The disease has been reported most frequently in the German Shepherd. Pathological findings consist of petechial and ecchymotic hemorrhages on serosal and mucosal surfaces of numerous organs. The most prominent histological finding is a perivascular plasma cell infiltrate in most organs. Disease, indistinguishable from the natural disease, has been produced in laboratory dogs inoculated with whole blood from affected dogs. Ehrlichia canis has been consistently recovered from all experimentally infected dogs. Attempts to transmit the disease to other laboratory animals and to propagate the agent in cell cultures and embryonating eggs have been unsuccessful. The tick is the probable vector of the disease.

Falkow, Stanley (Walter Reed Army Institute of Research, Washington, D.C.) and L. S. Baron. Episomic element in a strain of Salmonella typhosa. J. Bacteriol. 84:581-589. 1962.-An episomic element, F(0)-lac(+), has been identified in a strain of Salmonella typhosa isolated from a natural habitat. The F(0)-lac(+) element is transferred at high frequency as a single unit of transmission and replication without linkage to any other genetic character. Cells receiving F(0)-lac(+) are heterogenotes as if F(0)-lac(+) is not integrated as part of the linear structure of the chromosome, but rather replicates autonomously or in some other association with the genome. Evidence from complementation tests and transduction experiments is presented that the lac genes carried by F(0) are identical or at least markedly similar to the lac genes of Escherichia coli K-12. The F(0) transmission factor cannot be precisely identified but it does not appear to be phage or colicin. F(0) does exhibit mutual repression with the sex factor, F, of E. coli, and immunological experiments indicate some relationship between F and F(0).

Two widely separated loci causing the nicotinic acid dependence of wild-type Shigella flexneri 2a were identified by intergeneric mating procedures and found to be closely linked to the gal and fuc chromosomal determinants.

Farrar, W. Edmund, Jr. (Walter Reed Army Institute of Research, Washington, D.C.), Thomas H. Kent, and Van B. Elliott. Lethal gram-negative bacterial superinfection in guinea pigs given bacitracin. J. Bacteriol. 92:496-501. 1966.-Oral administration of a single dose of bacitracin (either 2,000 or 10,000 units) was lethal to more than 80% of guinea pigs. Within the first 12 hr, there was a 2,000-fold fall in the number of gram-positive organisms in the cecum. An increase in the number of coliform bacteria in the cecum was demonstrable within 6 hr, and, by 48 hr, these organisms had increased from the normal level of less than 100 per gram to approximately 1 billion per gram. The changes in intestinal bacterial flora were associated with development of a severe cecitis, mild ileitis, and acute regional lymphadenitis. Bacteremia, primarily due to coliform bacteria, was demonstrated in approximately 40% of the animals killed between 72 and 96 hr after administration of bacitracin. Development of this disease syndrome was suppressed by the administration of neomycin and polymyxin B, nonabsorbable antibiotics effective against coliform bacteria. The lethal disease produced by bacitracin in the guinea pig is similar to that produced by penicillin.

This report describes the pathophysiology, as manifested by hemodynamic and blood volume determinations, in nine acutely quadriplegic patients studied in Vietnam. Four of the nine patients developed fulminating pulmonary edema while in the hospital and showed marked differences in the hemodynamic and blood volume data when compared with five patients who did not develop pulmonary edema. The unique hazard of excessive intravascular volume replacement in quadriplegic patients is documented, and effective diagnostic and therapeutic procedures are suggested. Related reports and physiological studies are discussed.

Formal, Samuel B. (Walter Reed Army Institute of Research, Washington, D.C.), E. H. LaBrec, Amos Palmer, and Stanley Falkow. Protection of monkeys against experimental shigellosis with attenuated vaccines. J. Bacteriol. 90:63-68. 1965.-Two Shigella flexneri 2a strains of reduced virulence were used as oral vaccines to protect monkeys against experimental challenge. One strain, a spontaneous mutant, had lost its ability to cause disease and was unable to penetrate the intestinal epithelium and reach the lamina propria. The other strain was a hybrid obtained by mating virulent S. flexneri 2a with Escherichia coli. This hybrid strain retained the capacity to penetrate the intestinal epithelium but was not able to maintain itself in the lamina propria. Five oral doses of the nonpenetrating mutant strain were required to render monkeys resistant to experimental challenge, but a single dose of the hybrid strain sufficed to protect the animals. There was some evidence that a degree of specificity was involved in the induced resistance, although neither vaccine evoked a consistent serum antibody or a detectable coproantibody response.

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Factors affecting formation of incomplete Vi antibody in mice. J. Bacteriol. 90:635-642. 1965.-Single immunizing doses of purified Vi antigen elicited complete and incomplete Vi antibodies in BALB/c mice, but only incomplete antibody in Cinnamon mice. Three of six other mouse strains tested responded like BALB/c mice; the remaining three, like Cinnamon mice. Varying the quantity of antigen injected or the route of administration failed to stimulate the production of detectable complete Vi antibody in Cinnamon mice. Such antibody was evoked in these animals by multiple injections of Vi antigen or by inoculating them with Vi-containing bacilli or Vi-coated erythrocytes. The early protection afforded by serum from Vi-immunized BALB/c mice coincided with the appearance of incomplete Vi antibody, 1 day prior to the advent of complete antibody. Persistence of incomplete as well as complete antibody in the serum of immunized mice was demonstrated for at least 56 days after injection of 10 mug of Vi antigen. Incomplete Vi antibody was shown to have blocking ability, in vitro bactericidal activity, and the capability of protecting mice against intracerebral as well as intraperitoneal challenge with virulent typhoid bacilli. Production of incomplete and complete Vi antibodies was adversely affected by immunization with partially depolymerized Vi antigens.

Several experiments exploring the effects of certain behavioral procedures were performed on a psychiatric ward for delinquent soldiers. Within the context of a point economy, the behavioral procedures were examined for their applicability to this patient group in a hospital-ward setting. The following procedures were studied: (1) use of points as consequences for specific behaviors compared with demonstration of "model" behavior by a ward officer; (2) punishment by a point-fine to control undesired behavior; (3) use of a chaining-type reinforcement contingency to increase desired behavior; (4) differential reinforcement of the individual versus the group to increase the frequency of a verbal performance; and (5) reinforcement of reports of personal problems versus impersonal problems.

Experimental cholera vaccines were prepared from suspensions of cholera vibrios which were extracted with Genetron-113. These vaccines, when compared with a control preparation which was not treated with Genetron, had a lower nitrogen content and were less toxic for mice but were as effective in protecting mice against infection and in inducing agglutinating and vibriocidal antibody responses in rabbits. This procedure may permit the preparation of cholera vaccines with a higher antigenic content without the corresponding increase in toxic reactions.

A behavioral measurement system was designed around a point economy for a psychiatric ward of delinquent patients. The characteristics of the system were: (1) the records of points earned for appropriate patient behavior formed the primary data. (2) After the points were recorded in a data matrix, graphs were prepared to show the behavior of individual patients, the participation of the patient group in the various ward activities, and an overall index of ward operation. (3) Several techniques were devised for reviewing the graphs so that the ward staff could evaluate the success of their procedures over substantial time periods.

Methods have been developed for the pilot-scale production of group A and group C meningococcal polysaccharide immunogens based on the meningococcal strains, the medium, and the basic processing procedures used by Gotschlich et al. Physical and chemical assays on the final products obtained on a pilot-scale level indicate that these purified polysaccharides are entirely comparable to the Gotschlich preparations which proved to be immunogenic in man.

The primary step in the pathogenesis of bacillary dysentery is the penetration of intestinal epithelial cells by shigellae. Lacking this capacity, Shigella flexneri becomes avirulent. By means of intergeneric conjugation between various Escherichia coli K-12 Hfr strains and S. flexneri 2a virulent recipients and by reciprocal transduction analysis with phage P1 vir, we established a locus on the genome of S. flexneri 2a which is necessary for the ability of this strain to penetrate epithelial cells as measured by the Sereney test for keratoconjunctivitis. This locus, termed kcpA (in reference to its involvement in provoking keratoconjunctivitis), has been positioned between the lac and gal chromosomal markers and is contransducible with the purE allele.

Four antigenically related transmissible agents were recovered from canine fecal specimens. The agents produced cytopathic effects in a continuous dog cell line developed in this laboratory. Increased antibody titers were demonstrated in three of the four dogs which provided the isolates. The virus did not produce cytopathic effects in primary canine kidney or thymus cell cultures, or in cell cultures of human, simian, porcine, bovine, feline, and murine origin. The agent is resistant to ether, chloroform, and heat treatment, and the growth of the virus is inhibited by 5-iodo-2-deoxyuridine. After acridine orange staining, green fluorescent intranuclear inclusions are seen in infected cell cultures. By electron microscopy, the virions measure approximately 20 to 21 nm in overall diameter and are present in the nuclei of infected cells. These properties are consistent with membership in the parvovirus or picodnavirus group. The agent hemagglutinates rhesus red blood cells at 5 C and by hemagglutination-inhibition tests could be readily distinguished from H-1, rat virus, and the minute virus of mice. Canine gamma globulin contains high titers of neutralizing antibody and neutralizing antibody was found in a high percentage of military dogs and in beagles of a breeding colony.

Falkow, Stanley (Walter Reed Army Institute of Research, Washington, D.C.), Robert Rownd, and L. S. Baron. Genetic homology between Escherichia coli K-12 and Salmonella. J. Bacteriol. 84:1303-1312. 1962.-Recombinant analysis and interrupted mating procedures, in conjunction with molecular hybridization experiments, demonstrated that the genetic homology between Escherichia and Salmonella is incomplete. The most likely explanation of this incomplete homology is imperfect pairing between the deoxyribonucleic acid molecules of the two species. Despite the inhomologies, there is ample evidence that the order and distance of the genetic characters on the Salmonella chromosome are identical to those of Escherichia.

The chemotherapeutic efficacy of quinapyramine (antrycide) could not be demonstrated in hamsters experimentally infected with a virulent strain of serotype bataviae.

A bactericidal assay is described which allows identification of distinct serotypes within a serogroup of Neisseria meningitidis. Antisera produced in rabbits against seven group C strains by two intravenous inoculations of live organisms were found to contain two types of bactericidal antibodies. One, directed against the group-specific polysaccharide, caused various degrees of killing of all strains. Absorption of this antibody by purified group C polysaccharide revealed the presence of the second bactericidal antibody. This antibody was directed against antigenically distinct factors associated with serotype specificity. Extensive cross-absorption yielded antisera with activity directed against four separate factors. The presence of a factor in a strain was indicated by its susceptibility to killing by antisera containing antibody to that factor. A serotype was defined by the particular combination of factors. Six different serotypes, containing one or two factors, were identified among 16 group C strains examined.