Enhancement of murine bone marrow colony formation and L-transformation by Brucella antigen.

Brucella melitensis and Pseudomonas aeruginos antigens, in the form of heat-killed cells, enhanced serum stimulation of colony formation by mouse bone marrow progenitor cells. The antigens also enhanced colony formation in unstimulated medium. Prior sensitization of the C57BL mice by recent infection or by immunization six months earlier increased sensitivity of bone marrow cells to brucella antigen enhancement of colony formation. The immunized mice provided marrow cells more primed to colony formation than infected mice. Evidence is presented that antigen also leads to greater recovery of live brucellae from marrow cells of infected animals. This may be due both to stimulation of the marrow cells and to direct stimulation of the brucellae in those cells. L-forms of brucellae from stimulated marrow cell cultures were isolated. Some degree of stabilization of the L-form was accomplished through incorporation into the marrow culture medium of MgSO4, sucrose and penicillin G. The place in the infection process of L-forms is discussed in terms of the hypothesis that the L-form is a product of immune reactions that involve a step-wise degradation of the brucella cell wall during which the various cell-mediated immune reactions become operative and are themselves the reflection of a general stimulation by the brucellae of the hemopoietic and lymphopoietic systems.

Plague in camels and goats: their role in human epidemics.

In 1976, in a small, remote Libyan village, one apparently sick camel was slaughtered and skinned, and the camel meat was distributed for human comsumption. A few days later, 15 villagers suffered a severe febrile illness. Of the five individuals who had participated in the killing and dispensation of the camel, all were dead within four days. When samples of serum from nine of the remaining patients were examined, seven were found to be positive for plague as determined by the passive hemagglutination test. Another six persons became ill after killing two goats, and the serum of one goat contained antibodies to Yersinia pestis. Because all of the remaining patients except one were treated early enough, they recovered. These incidents confirm previous reports that the camel and the goat are susceptible to naturally occurring plague infection and have a significant role in the dissemination of human plague.

Effect of bacille Calmette-Guérin on the immune response of BALB/c mice to a tumor allograft.

The effect of dosage and route of inoculation of bacille Calmette-Guérin (BCG) on immune response to allogeneic tumor cells was investigated. BALB/c mice were tested 14 and 21 days after injection of EL-4 lymphoma for spleen-cell cytotoxicity against EL-4 cells in vitro and for complement-dependent, antibody-mediated lysis of tumor cells. BCG treatment had no measurable effect on the antibody-mediated lysis of tumor cells, but spleen-cell cytotoxicity was significantly increased in mice treated with 10(4) or 10(8) BCG by the intraperitoneal route; no such increase occurred when BCG was given by the oral or subcutaneous routes. The cytotoxic effector cells were primarily thymus-derived, since treatment of spleens with rabbit antiserum to mouse brain serum decreased cytotoxicity titers by approximately 90%. Within the framework of these experiments, the intraperitoneal route of BCG inoculation resulted in a more effective immune stimulation than the oral or subcutaneous routes.

Scanning electron microscopic study of virulent Yersinia pestis and Yersinia pseudotuberculosis type 1.

Virulent Yersinia pestis was grown on heart infusion blood agar and examined by scanning electron microscopy, exposing the fraction 1 envelope antigen on cell surfaces as a lumpy coating that spilled into the surrounding milieu. The amount of antigen depended on both the duration and temperature of incubation. Comparison of the cultures grown at 37 degrees C for 24, 48, and 72 h with those grown at 22 degrees C for identical periods demonstrated that the antigen increased in amount with the length of incubation, and that the overall production of antigen was much greater of 37 degrees C than at 22 degrees C. These experiments visually confirmed the findings of our previous immunological studies. Moreover, we established that the closely related, virulent organism Y. pseudotuberculosis bears no such antigenic coating at any temperature or incubation period. In addition, the emergence of multiple flagella was noted when Y. pseudotuberculosis was grown at 22 degrees C in a liquid medium, whereas Y. pestis remained without these organelles. These observations preceptibly corroborated the absence of fraction 1 envelope antigen and the presence of flagella, respectively, for distinguishing Y. pseudotuberculosis from Y. pestis.

Immunity in plague: protection of the vervet (Cercopithecus aethips) against pneumonic plague by the oral administration of live attenuated Yersinia pestis.

Protection against pneumonic plague by the oral administration of a live, attenuated Yersinia pestis vaccine, EV76 (Paris) F, was evaluated in the vervet (Cercopithecus aethips). Six animals were vaccinated with a dose of 1.175 X 10(9) colony-forming units (cfu); all tolerated the dose and developed antibodies to Fraction I of Y. pestis. Three immunized animals were challenged with an inhaled dose of 3.2 X 10(6) cfu (160 LD50 [50% lethal dose]) of virulent Y. pestis strain 195/P and two survived; the other three received a challenge dose of 4.9 X 10(6) cfu (245 LD50) and one survived. The three surviving monkeys had high titers of antibody to Fraction I. At necropsy of the animals that succumbed to the challenge infection, almost complete consolidation of the lungs was apparent, and sizable effusions were present in the thoracic cavities. Indeed, the striking pathology indicated the severest form of pneumonic plague. Since it can be assumed that the animal surviving challenge with the larger inoculum would have survived the smaller dose, we conclude that the immunization procedure protected half of the vervets that were exposed to challenge.

Cellular immune response to Yersinia pestis modulated by product(s) from thymus-derived lymphocytes.

Resistance to infection with Yersinia pestis was found to depend on whether the macrophage can inactivate and withstand the cytotoxic effects of phagocytized Y. pestis. Serum from mice immunized with antigens of Y. pestis enhanced the resistance of monolayers of normal cultures to the cytotoxic effects of Y. pestis and increased the capacity of peritoneal exudate cells from immune mice to inactivate these bacteria. The enhancing component of the serum was not removed by absorption with heat-killed Y. pestis. Similar enhancement was provided by supernatant fluids of spleen cultures from immunized mice but not by those from unimmunized mice. Pretreatment of spleen cells with rabbit hyperimmune antiserum to mouse brain theta-antigen plus complement caused a reduction in the enhancing capacity of the spleen cell culture fluids. Removal of the glass-adherent cell population from suspensions of primed spleen cells prior to in vitro antigenic stimulation resulted in a loss of activity from the subsequently harvested culture fluids. Thus, the enhancing component of the serum appears to be a product of thymus-derived lymphocytes (T-cells). Furthermore, splenic macrophages seem to be required for the interaction of primed T-cells with heat-killed Y. pestis.

Priming of Macaca cynomolgus philippinensis with purified antigen of Brucella melitensis before injection of Rev. I vaccine.

In experiments designed for study of bacteremia and serologic response of Macaca cynomolgus philippinensis to vaccination with a soluble antigen (fraction I) of Brucella melitensis and the live Rev. I vaccine strain, fraction I and Rev. I were administered both four weeks apart and simultaneously. One of three monkeys given the vaccines four weeks apart and two of three monkeys vaccinated with fraction I and Rev. I in a single injection developed transient bacteremia, as shown by blood cultures. Both methods of vaccination induced significant titers of antibody to B. melitensis, and vaccinated monkeys were protected against subsequent challenge with a virulent strain of B. melitensis.

Isolation of Brucella melitensis phage of broad biotype and species specificity.

A Brucella melitensis phage was isolated. Stocks of phage were established which produced early large plaques. The phage host range included all smooth Brucella species.

Yersinia pestis: correlation of ultrastructures and immunological status.

Ultrastructural identification and localization of the fraction 1 "envelope" antigen in the plague bacillus Yersinia pestis were the primary objectives of this brief study. The antigenicity of extra-cellular material between the bacilli in undisturbed cultured colonies and that of the pathogen per se were measured and correlated by means of the semi quantitative complement fixation method after incubation for 72 h at 37 C. When the amount of extracellular substance in wild-type T1 (virulent) bacteria was compared by electron microscopy with that in avirulent strains of Y. pestis, with and without passage through guinea pigs, we found that the material of interest was greatly attenuated or even absent in colonies that had not been passed through animals, whereas passage markedly augmented production of the material. We also explored the requirement for larger quantities of Ca(2+) and Mg(2+) in the culture media and discovered that without these ions production of the extracellular material was limited. These observations support the hypothesis that this extracellular substance between cultured Y. pestis bacilli of various strains represents the source of the fraction 1 envelope antigen.

Variations in properties of L-forms of Pseudomonas aeruginosa.

In a study of the pathogenic potentials of Pseudomonas L-forms, three unstable L-forms were derived by carbenicillin inductionfrom a mouse virulent strain of Pseudomonas aeruginosa, Rosenthal 180. One L-form, induced on a sucrose-stabilized medium, grew more slowly and differed in a number of properties from two other L-forms induced on a medium supported with polyvinylpyrilidone. After adaptation to a common liquid medium, the three L-forms differed with respect to colonial shape on solid medium, growth rate, certain biochemical properties, antibiotic sensitivities and antigenic surface, and virulence for mice. The L-form may revert in vitro to a serotype different from that of the parent culture. The revertant may acquire new antibiotic resistances and sensitivities in the absence of previous exposure to the drugs and enhanced resistance to the L-inducing agent. The three L-forms showed a characteristically lower, but wide, range of virulence than did the parental form. Though death of mice was accompanied by reversion of the L-forms in vivo to the bacterial form, reversion in vivo was not necessary for virulence of L-forms. Modification of residual cell wall antigens accompanied the induction of each L-form as determined by type-specific antisera.

Surface morphology of peritoneal macrophages during the attachment of Brucella melitensis.

When macrophages from immunized rabbits are in the act of phagocytosis of the homologous organism, the macrophages are seen by scanning electron microscopy to possess undulating membranes and microvilli at various stages of formation and spatial relationships to the organisms.

Sensitization and recall of anti-Brucella immunity in Guinea pig macrophages by attenuated and virulent Brucella.

Peritoneal macrophages from guinea pigs vaccinated with strain Rev I of Brucella melitensis were only moderately activated thereby to limit, in an in vitro system, the intracellular growth of Rev I bacilli. Nevertheless, the appropriate memory cells had been primed, as demonstrated by the observation that reinfection of animals with virulent B. melitensis followed by intraperitoneal inoculation of mineral oil called forth macrophages in immunized guinea pigs which inhibited strongly the intracellular growth of brucellae. These macrophages slowed the growth of brucellae in the absence of immune serum. The intensity of the recall response was related to the challenge route and to the virulence of the challenge strain. After equal doses of attenuated or virulent brucellae, resistance was highest in macrophages recalled by the virulent strain. An important basis for the attenuation of the Rev I strain may lie in its initially low degree of macrophage activation during primary infection, although still retaining the capacity to prime stem cells. This property is associated with a protein found in fraction I, because 600 mug/ml in Freund's adjuvant primed guinea pigs so that challenge by strain 6015 evoked activated macrophages. This was seen microscopically as a reduced spread of infection in and amongst the macrophage population. Immune serum further reduced this spread and limited the number of viable intracellular brucellae.