Mechanisms of immunity in typhus infection: analysis of immunity to Rickettsia mooseri infection of guinea pigs.

To study the mechanisms of immunity to Rickettsia mooseri (R. typhi) infection, sera and splenic cells collected from nonimmune and immune guinea pigs were inoculated separately into syngeneic nonimmune recipients which were subsequently challenged intradermally. Protection was measured by comparing the course of the challenge infections of recipients with infections initiated with the same rickettsial inocula in nonimmune animals. Recipients of splenic cells collected 21 days after donor infection were protected from lesion development at sites of intradermal challenge and showed fewer rickettsiae in their kidneys. Cells obtained from nonimmune donors did not protect against either skin lesion development at sites of challenge or kidney infection. Antibody-containing sera collected 21 days after donor infection, but not normal sera, reduced levels of kidney infection, but immune sera did not protect against the development of lesions at sites of intradermal challenge. It was concluded that both immune sera and immune splenic cells possess capacities to effect a partial control of the systemic phase of R. mooseri infection in guinea pigs, but that immune splenic cells possess a capacity not shared by immune sera, i.e., the capacity to protect from infection at local sites of intradermal inoculation.     

Mechanisms of immunity in typhus infection: adoptive transfer of immunity to Rickettsia mooseri.

When nonimmune guinea pigs are inoculated intradermally (i.d.) with Rickettsia mooseri (R. typhi), the rickettsiae replicate at the site of inoculation, leading to the development of a grossly observable lesion. In contrast, guinea pigs which have recovered from R. mooseri infection are resistant to challenge and prevent both rickettsial growth and the formation of lesions. To study the mechanisms of this immunity, sera or splenic cells collected from nonimmune or immune guinea pigs were inoculated separetely into nonimmune recipients. Splenic cells collected from immune donors protected R. mooseri-naive recipients from i.d. challenge as measured by control of rickettsial growth and by prevention of development of lesions at i.d. sites of inoculation. In contrast, serum from immune and nonimmune doners failed to protect nonimmune recipients by either criterion.     

Mechanisms of immunity in typhus infection: some characteristics of Rickettsia mooseri infection of guinea pigs.

Rickettsia mooseri infection has been studied in syngeneic guinea pigs inoculated intradermally with the objective of developing a model for the study of immune mechanisms. Characterization of infection included the following: a study of replication, dissemination, and clearance of rickettsiae; measurement of the antibody response with different rickettsial antigens and tests; and attempts to measure the cell-mediated immune response using the correlate of delayed-type hypersensitivity skin reactions. Following intradermal inoculation, rickettsiae replicate locally and then spread to the draining lymph nodes and subsequently cause systemic infection. Spread to draining lymph nodes occurred before the appearance of circulating antibody, whereas systemic infection occurred afterwards. Two distinct patterns of acquired resistance developed. The first was marked by a cessation of rickettsial growth within a given organ and the second by a clearance of rickettsiae. The duration of each of these phases differed markedly from one organ to another. Delayed-type hypersensitivity was not demonstrated by skin testing.     

Effect of immunosuppression on Rickettsia rickettsii infection in guinea pigs.

The role of the immune response in the pathogenesis of Rickettsia rickettsii infection in guinea pigs was investigated by immunosuppression, using antilymphocyte serum. Twenty guinea pigs were inoculated with R. rickettsii, Sheila Smith strain, on day 0. Fifteen animals received antilymphocyte serum on days --1, 0, 2, 4, and 6. Five animals received normal rabbit serum on the same schedule. At necropsy, specimens were collected for histological examination, rickettsial immunofluorescence, rickettsial titration, and antirickettsial antibody titration. All normal rabbit serum recipients and 12 of 15 antilymphocyte serum recipients developed typical disease. Comparison of animals in terminal stages of disease revealed the same clinical course and gross lesions, but differing rickettsial burden and cellular response. Immunosuppressed animals had higher titers of splenic rickettsiae and greater numbers of immunofluorescent rickettsiae. Thus, although antibody was undetectable in both groups, there appeared to be an inhibition of antirickettsial immunity. Microscopic vasculitis was similar quantitatively, but differed qualitatively, with immunocompetent animals having the typical monouclear/lymphocytic inflammation and immunosuppressed animals having neutrophilic predominance. This study demonstrates that immunopathological mechanisms are not necessary for the pathogenesis of experimental Rocky Mountain spotted fever. The rickettsiae themselves seem capable of causing cellular and tissue damage.     

Evaluation ofprimary blood monocyte and bone marrow cell culture for the isolation of Rickettsia rickettsii.

Rickettsia rickettsii was isolated from experimentally infected guinea pigs by culture of blood monocytes and bone marrow cells, and from experimentally infected rhesus monkeys by blood monocyte culture. Rickettsiae were identified in monocyte-macrophage monolayers stained by Giménez or flourescent antibody techniques. A total of 78 culture attempts were made from 20 guinea pigs and 16 monkeys. The success of isolation of R. rickettsii in culture was positively correlated with the numbers of rickettsiae present in the blood and bone marrow. in cultures derived from infected guinea pigs, rickettsiae were usually observed after 5 to 7 days of culture, and in monkeys monocyte cultures they were usually observed within 3 to 5 days. Positive cultures were derived from guinea pigs and monkeys as early as the first day of fever and 1 to 3 days before the appearance of other clinical signs. Monocyte cultures became negative with the resolution of rickettsemia and concomitantly with the appearance of serum antibody. Monocyte culture isolation of R. rickettsii may be as sensitive for the detection of rickettsiae in blood and marrow as the intraperitoneal inoculation of guinea pigs or the plaque assay technique. Because of the simplicity of the method and because rickettsiae were often identified within 3 to 5 days after initiation, the monocyte culture technique may be useful in the early diagnois of human rickettsial disease.     

Immunity to foot-and-mouth disease virus in guinea pigs: clinical and immune responses.

Clinical and immune responses were determined for guinea pigs infected with different doses of foot-and-mouth disease virus (FMDV) type A12, strain 119, administered by different routes. Vesicles developed on the tongue or heel pad 1 day after these areas were intradermally inoculated with FMDV. However, vesicles did not develop on the feet and tongue until 3 to 4 days after the intradermal inoculation of FMDV in the flank skin or after intracardiac or subcutaneous inoculation. Infected guinea pigs developed neutralizing antibody, immediate skin reactivity of the Arthus type (4 h), and delayed skin reactivity. In addition to a delayed skin response, the presence of a cell-mediated immune response to FMDV was shown by the specific production of macrophage migration inhibition factor by peritoneal exudate cells in response to FMDV. Kinetic studies showed that neutralizing antibodies were detected at 3 days postinfection, and Arthus and delayed skin reactivity were detected at 4 days postinfection. Some guinea pigs developed either mild or subclinical infections. Regardless of the dose of infectious virus, the route of inoculation, the severity of disease, or the time of clinical onset of disease, infected guinea pigs developed similar immune responses.     

Hylan G-F 20 induces delayed foreign body inflammation in Guinea pigs and rabbits

Recent clinical evidence suggests that hylan, a modified hyaluronan, and related products potentially elicit foreign body granulomatous inflammation in human soft tissue. We investigated the biocompatibility of hylan G-F 20 (Synvisc) for up to 28 days after intradermal injection in guinea pigs and intramuscular injection in rabbits. Compared to saline and unmodified hyaluronan, hylan induced definitive macroscopic changes in guinea pigs by day 14 or later and in rabbits by 28 days after injection. Histologically, at the injection sites, there was severe granulomatous inflammation in guinea pigs and acute inflammation with minimal infiltration of macrophages and foreign body giant cells in rabbits. Furthermore, specific antibodies against hylan were demonstrated in guinea pigs by passive cutaneous anaphylaxis, and substantial deposits of IgG on hylan were evident by immunohistochemistry. The present results contradict previous reports on biocompatibility of hylan and suggest that hylan may potentially induce similar unfavorable reactions in humans.     

Establishment of cutaneous Leishmania enriettii infection in hamsters.

A model of a self-healing type of cutaneous leishmaniasis was established in hamsters using the guinea pig parasite Leishmania enriettii. L. enriettii was passaged several times in hamsters without losing its infectivity for guinea pigs or for hamsters. The course of the infection in hamsters resembled that of guinea pigs, with the exception that the lesion at the site of parasite inoculation did not ulcerate and no metastatic lesions developed spontaneously. Moreover, unlike guinea pigs, infected or recovered hamsters were skin test unresponsive to various preparations of L. enriettii antigens. However, histological examination of draining lymph nodes showed features of a cell-mediated immune response, and in vitro inhibition of macrophage migration was demonstrable using peritoneal exudate cells from recovered animals and specific leishmanial antigen. Antibody was demonstrable by indirect immunofluorescence starting 1 week after infection. Recovered animals were immune to reinfection; however, the passive transfer of peritoneal exudate cells or serum from recovered animals did not confer protection against L. enriettii infection in normal animals.     

Experimental infection and immune response of guinea pigs with varicella-zoster virus.

An immune response (fluorescent antibody to membrane antigen) was detected in guinea pigs inoculated with varicella-zoster virus (VZV) adapted to guinea pig embryonic cells, including the Oka vaccine strain, even when inoculation was by an external route, i.e., nasal or corneal. Live or UV-inactivated virus having the same virus titer before irradiation was administered to guinea pigs by the corneal route, and antibody induction was detected only with live virus. The transmission of VZV from infected guinea pigs to noninfected ones was suggested by the appearance of antibody in the serum of the latter, who were kept in the same cage. The time course of the appearance of humoral and cellular immune responses in guinea pigs was examined by the fluorescent antibody to membrane antigen test and the skin reaction, with varicella antigen representing delayed-type hypersensitivity. When VZV was injected subcutaneously, skin reaction appeared as early as 4 days after inoculation, which preceded the appearance of detectable antibody by 2 to 6 days. In in vitro studies, the Oka vaccine showed a higher adsorption rate and better growth in guinea pig embryonic cells than did other wild-type strains when assayed by the infectious center assay. These results suggest that a system of VZV adapted to guinea pig cells and guinea pigs provides a good animal experimental model for immunological study of VZV infection.     

Characteristics of Rickettsia mooseri infection of normal and immune mice.

Rickettsia mooseri infection initiated by subcutaneous injection has been studied in BALB/c mice with the objective of developing a model for the study of immune mechanisms. Characterization of infection included the following: measurement of the replication, dissemination, and clearance of rickettsiae; measurement of correlates of the immune response, including humoral antibody, hypersensitivity to subcutaneously inoculated rickettsial antigen, and activation of nonspecific macrophage microbicidal capacity; and measurement of resistance to a second homologous challenge. Local infection at the site of subcutaneous injection progressed through day 5 and was controlled by day 7. Systemic infection as determined by the presence of rickettsiae in spleen was first detected on day 7 and progressed through day 14; however, rickettsiae persisted in this organ at reduced numbers through at least day 28. Control of the local infection at the site of subcutaneous injection occurred at about the time humoral antibodies and hypersensitivity reactions to subcutaneously injected rickettsial antigens became demonstrable and was paralleled by a capacity to resist homologous subcutaneous challenge at a site distant from that of the primary infection. Systemic infection progressed in spite of this acquired immune capacity and was controlled in the spleen in parallel with the development of enhanced macrophage microbicidal capacity in the liver. The results show that an acquired immunity is capable of restricting rickettsial growth at subcutaneous sites at a time when rickettsiae are increasing in titer in deep organs.     

Humoral and cell-mediated immune mechanisms in the production of pathology in avirulent Semliki Forest virus encephalitis

Seven days after peripheral inoculation with an avirulent strain of Semliki Forest virus, the brains of CBA and nude mice exhibited a mononuclear inflammation and spongiform degeneration. Mice that had received cyclophosphamide (150 mg/kg) 24 h after infection showed no pathology until day 11. However, immunofluorescence studies of the brains of immunosuppressed, infected mice demonstrated viral antigen within the soma and processes of neurons at earlier periods. The brain lesions could be reconstituted on day 7 in immunosuppressed, infected recipients with 6-day immune spleen cells. Immune spleen cells depleted of T lymphocytes, the non-immunoglobulin-bearing population deficient in B lymphocytes, or immune sera plus nonimmune bone marrow cells could also reconstitute the lesions. However, inflammation and spongiform changes were reduced when donor immune cells were depleted of either T or B lymphocytes. When both T and B lymphocytes were removed from the donor immune population, recipient brains did not show pathology. The results demonstrate that either antibody or immune T cells can trigger pathology, but there is also participation of nonimmune bone marrow-derived mononuclear cells, probably of the monocyte-macrophage lineage.     

Enhancement of experimental ulcerative colitis by immunization with Bacteroides vulgatus.

Previous studies with the guinea pig model for ulcerative colitis have shown that the inducing agent, carrageenan, does not provoke ulcerations in germfree guinea pigs, but animals monoassociated with Bacteroides vulgatus develop cecal ulcerations when fed carrageenan. In an effort to define the role of B. vulgatus in this model system, conventional guinea pigs were immunized with B. vulgatus before carrageenan treatment. Immunized animals developed both circulating antibody and positive skin tests for the homologous antigen. A comparison of B. vulgatus-immune and nonimmune carrageenan recipients after 30 days of carrageenan treatment revealed ulcerations of the ceca and large intestine in both groups, but the immune group developed more severe lesions than the nonimmune group. The most severe intestinal lesions were detected in B. vulgatus-immune animals fed both carrageenan and daily doses of viable B. vulgatus. Immune recipients of B. vulgatus alone did not develop ulcerations, but showed histological evidence of intestinal inflammation. Lesions observed in B. vulgatus-immune animals were most severe in the colon and rectum, in contrast to more severe cecal lesions detected in nonimmune carrageenan recipients. Similar experiments with Bacteroides fragilis as an antigen for immunization showed no difference between immune and nonimmune groups. These results suggest that immunization with B. vulgatus enhances the severity of carrageenan-induced colitis.     

Interaction between Coxiella burnetii and guinea pig peritoneal macrophages.

The phagocytosis and subsequent degradation of phase I and II Coxiella burnetii by macrophages obtained from immune and nonimmune guinea pigs were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. There was no significant difference in the percentage of phagocytosis of either phase of rickettsiae by macrophages from immune or nonimmune animals. After ingestion, phase I and II organisms pretreated with normal serum multiplied and destroyed normal macrophages as well as macrophages obtained from guinea pigs immunized with phase II rickettsiae. In contrast, only phase I organisms were degraded by macrophages from phase I-immunized animals in the presence of normal serum. Immune serum rendered rickettsiae more susceptible to phagocytosis and also potentiated the destruction of organisms by all types of macrophages. The specificity of macrophages from phase I animals to degrade only phase I rickettsiae was demonstrated by the ability of Rickettsia rickettsii to replicate in these macrophages.     

Immunologic Events in Experimental Hypersensitivity Granulomas

Polyacrylamide beads were covalently linked to proteins and injected intravenously into normal or immune guinea pigs. The beads trapped in the lung produced very mild foreign body granulomas in nonimmune guinea pigs. In immune guinea pigs, severe granulomas developed which progressed through the several characteristic histologic stages with time. Severe granulomatous reactions developed only upon recognition of carrier determinants of hapten-protein conjugates; thus guinea pigs immune to dinitrophenyl hapten (DNP)-hemocyanin developed characteristic granulomas only upon injection of beads coated with hemocyanin or DNP-hemocyanin, but not with DNP on another unrelated antigen. Granulomatous reactivity was passively transferred into normal animals by lymph node cells but not by serum antibody from sensitized guinea pigs.     

A humoral immune response confers protection against Haemophilus ducreyi infection

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Neither naturally occurring chancroid nor experimental infection with H. ducreyi results in protective immunity. Likewise, a single inoculation of H. ducreyi does not protect pigs against subsequent infection. Accordingly, we used the swine model of chancroid infection to examine the impact of multiple inoculations on a host's immune response. After three successive inoculations with H. ducreyi, pigs developed a modestly protective immune response evidenced by the decreased recovery of viable bacteria from lesions. All lesions biopsied 2 days after the first and second inoculations contained viable H. ducreyi cells, yet only 55% of the lesions biopsied 2 days after the third inoculation did. Nearly 90% of the lesions biopsied 7 days after the first inoculation contained viable H. ducreyi cells, but this percentage dropped to only 16% after the third inoculation. Between the first and third inoculations, the average recovery of CFU from lesions decreased approximately 100-fold. The reduced recovery of bacteria corresponded directly with a fivefold increase in H. ducreyi-specific antibody titers and the emergence of bactericidal activity. These immune sera were protective when administered to na?ve pigs prior to challenge with H. ducreyi. These data suggest that pigs mount an effective humoral immune response to H. ducreyi after multiple exposures to the organism.     

Mechanisms of immunity in typhus infections. VI. Differential opsonizing and neutralizing action of human typhus rickettsia-specific cytophilic antibodies in cultures of human macrophages.

Human peripheral blood monocytes were incubated in vitro for 6 days to allow time for transformation into macrophage-like cells. Cytophilic antibodies in typhus convalescent human serum were demonstrated by addition of Rickettsia mooseri or Rickettsia prowazeki to passively sensitized human peripheral blood monocyte-derived macrophages that were held at 4 degrees C. Rosettes of rickettsiae were found around macrophages sensitized with immune serum but not around macrophages that had been incubated with normal serum. Inhibition of rosette formation occurred if the macrophages were maintained in normal human serum before addition of immune human serum. Rosettes of R. mooseri were also formed around monocytes obtained from an individual infected with R. mooseri. If the antibody-sensitized macrophages were maintained at 34 degrees C, enhanced phagocytosis of R. mooseri or R. prowazeki occurred as compared with macrophages exposed to normal human serum before infection. However, the cytophilic antibody did not significantly inhibit the subsequent growth of R. prowazeki within the macrophages. This is in contrast to results obtained when R. prowazeki was mixed with immune serum before addition to the macrophage. In the latter case, growth of R. prowazeki was largely inhibited. The significance of antibody cytophilic for macrophages in typhus infections is discussed.     

Mechanisms of Immunity in Typhus Infections II. Multiplication of Typhus Rickettsiae in Human Macrophage Cell Cultures in the Nonimmune System: Influence of Virulence of Rickettsial Strains and of Chloramphenicol

Monocytes from the peripheral blood of nonimmune human subjects transformed in cell culture into macrophages with increased phagocytic capacity for killed typhus rickettsiae. When such cells were exposed to living virulent Rickettsia mooseri (Wilmington strain) or R. prowazeki (Breinl strain), or to the attenuated E strain of R. prowazeki, in the presence of medium containing normal human serum, all three strains readily entered the macrophage, but the subsequent fate varied according to strain and its virulence. Thus, R. mooseri grew readily to attain very high intracellular populations which eventually destroyed the macrophage in 3 to 5 days and escaped to infect other cells. Virulent R. prowazeki also grew at about the same rate for the first 2 to 3 days but then often abruptly ceased to multiply. Circumstantial evidence suggests a toxic effect on host cells by smaller numbers of R. prowazeki organisms than with R. mooseri. The attenuated E strain of R. prowazeki failed to grow in most cells and eventually disappeared, but did grow to substantial numbers in the very rare cell in some cultures, suggesting the presence of a few cells which may not be typical macrophages. The growth of R. mooseri in the macrophage cytoplasm was inhibited by chloramphenicol in the culture medium. When the drug was removed after 3 days, growth began after a lag period and assumed a normal rate.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Infection of guinea pigs with vesicular stomatitis New Jersey virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae)

Intrathoracically inoculated Culicoides sonorensis Wirth & Jones were capable of transmitting vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) during blood feeding on the abdomen of six guinea pigs. None of the guinea pigs infected in this manner developed clinical signs of vesicular stomatitis despite seroconversion for VSNJV. Guinea pigs infected by intradermal inoculations of VSNJV in the abdomen also failed to develop clinical signs of vesicular stomatitis. Three guinea pigs given intradermal inoculations of VSNJV in the foot pad developed lesions typical of vesicular stomatitis. Transmission by the bite of C. sonorensis may have facilitated guinea pig infection with VSNJV because a single infected C. sonorensis caused seroconversion and all guinea pigs infected by insect bite seroconverted compared with 50% of the guinea pigs infected by intradermal inoculation with a higher titer VSNJV inoculum. The role of C. sonorensis in the transmission of VSNJV is discussed.     

Pathogenesis of Herpes simplex virus infections in guinea pigs.

The pathogenesis of herpes simplex virus types 1 and 2 has been studied in guinea pigs after inoculation by various routes (subcutaneous and intradermal infection in footpads and vaginal infection). Clinical observations as well as virus isolation studies are reported. Herpes simplex virus type 2 infection by all three routes of inoculation led to acute primary and recurrent lesions. Virus persisted in the nervous system, particularly in sensory ganglia, and locally at the site of inoculation. Herpes simplex virus type 1 infection induced no or very mild primary symptoms. Recurrent lesions were only observed after intradermal inoculation. Invasion of the nervous system and consequent establishment of latent ganglionic infection was less efficient than after herpes simplex virus type 2 infection. Peripheral persistence was, however, equally common.