Patterns of rickettsemia and antibody response in silvered leaf monkeys (Presbytis cristatus) after inoculation with virulent and avirulent strains of Rickettsia tsutsugamushi.

Most silvered leaf monkeys inoculated with selected strains of Rickettsia tsutsugamushi developed rickettsemia. The time of onset and the duration of rickettsemia were related to the infecting strains, but these parameters could not be correlated with the virulence of the strain. The rickettsemia was not terminated by the appearance of humoral antibody, as detected by the indirect fluorescent antibody test. The specificities of the antibody responses were related to the antigens present in the infecting strains.     

Clinical responses of silvered leaf monkeys to infection with selected strains of Rickettsia tsutsugamushi.

Minimal clinical and hematologic signs were observed in silvered leaf monkeys inoculated intradermally with four strains of Rickettsia tsutsugamushi, both virulent and avirulent for laboratory mice. The clinical response of the monkeys to the infection was related to neither the virulence of the strains in mice nor the antigenic characteristics of the strains.     

Clinical and histological features of inoculation site skin lesions in cynomolgus monkeys experimentally infected with Orientia tsutsugamushi

Cynomolgus monkeys, as animal models of scrub typhus, are typically infected with Orientia tsutsugamushi by intradermal inoculation. However, the clinical and histological features at the O. tsutsugamushi inoculation sites, akin to "eschars" at chigger inoculation sites in humans, have not been fully characterized. We intradermally inoculated one medial thigh of six cynomolgus monkeys with semi-purified O. tsutsugamushi (Karp). Within 7 days, two animals developed scrub typhus-like eschars and four had dusky plaques, accompanied by inguinal lymphadenopathy. Biopsies of eschars and an enlarged regional lymph node resembled human disease and stained positively for O. tsutsugamushi by Giemsa, anti-Karp fluorescent antibody, or streptavidin alkaline phosphatase. O. tsutsugamushi-specific IgM and IgG antibody levels measured in both of two monkeys rose steadily after infection. This pilot study shows that cynomolgus intradermally inoculated with O. tsutsugamushi replicate the localized cutaneous pathogenesis of human scrub typhus infections, strengthening the value of this animal model.     

Blastogenic responses of peripheral blood leukocytes from owl monkeys experimentally infected with Leishmania braziliensis panamensis

The relationship of the progression and regression of cutaneous lesions of 6 owl monkeys (Aotus trivirgatus) to the responses of their peripheral blood leukocytes (PBL) in vitro to mitogens and to leishmanial antigens, as well as their delayed skin test responses (DTH) in vivo to leishmanin antigen, were studied after primary and challenge infections with Leishmania braziliensis panamensis (WR 128 or WR 539). All 6 infected monkeys developed primary and satellite cutaneous leishmanial lesions which were measured for up to 30 weeks in 3 of the monkeys and up to 52 weeks in the other 3 monkeys. Two owl monkeys which had recovered from cutaneous leishmaniasis demonstrated acquired resistance when challenged with an intradermal inoculation of L. b. panamensis (WR 128). Reactivity of PBL from infected owl monkeys to PHA, Con A, and PWM was similar during primary and challenge infections to that observed prior to infection. Reactivity to leishmanial antigens was detected at 20 to 28 weeks post-infection (PI), became statistically significant after 28 weeks and remained elevated up to 52 weeks PI and after challenge infections. During primary infections DTH responses to leishmanin antigen were detected as early as 8 weeks PI, and continued up to 27 weeks PI. After challenge infections DTH reactivity was positive at 25 and 37 weeks, the only times the response was evaluated. The immunological responses of owl monkeys to L. b. panamensis were similar in many respects to those observed in humans with localized cutaneous leishmaniasis. This nonhuman primate model should be useful for future studies involving the immunology and chemotherapy of cutaneous leishmaniasis.     

Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers

The strains of Rickettsia tsutsugamushi found in naturally infected, laboratory-reared Leptotrombidium (Leptotrombidium) arenicola and L. (L.) fletcheri chiggers were characterized by direct immunofluorescence (FA) and by mouse and monkey virulence tests. The strains existing in the L. (L.) arenicola chiggers consisted of different combinations of TA716, TA763, TA686, Karp, and Kato. In addition to these five strains, Gilliam was found in the L. (L.) fletcheri chiggers. Results indicate that individual chiggers can be simultaneously infected with several antigenic strains of R. tsutsugamushi. Although these antigens appear to remain stable within familial lines when several generations were viewed, the antigenic patterns observed in two succeeding generations did not always correlate. This variable expression of antigens was considered to be due to a quantitative fluctuation from one generation to the next in the strains of rickettsiae combined with a lack of sensitivity of the direct FA test in detecting small numbers of antigenically different rickettsiae. Phenotypic variation was considered to be a less probable explanation. Morbidity and mortality were minimal in ICR mice fed upon by individual chiggers of either species, but infection rates were 85-99%. Tissue suspensions prepared from mice infected by L. (L.) arenicola produced higher mortality and longer duration of illness in mice than those prepared from L. (L.) fletcheri-infected mice. Silvered leaf and cynomolgus monkeys were fed upon by the two species of chiggers or inoculated with the mouse tissue suspensions. In both cases, minimal clinical responses were observed.     

Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection

Simian immunodeficiency virus (SIV) infection in cynomolgus macaques leads to severe immunodeficiency with a fatal outcome. In contrast, HIV-2 infects these primates without apparently causing any immunological abnormalities. In this study three cynomolgus monkeys were experimentally infected with HIV-2 strain SBL-K135 and 168 days later challenged with 10-100 animal infectious doses of the closely related SIV strain SM to study protective immunity. At the time of SIV challenge the HIV-2-infected monkeys had neutralizing antibodies against HIV-2, but virus could no longer be recovered from their peripheral blood mononuclear cells (PBMCs) and no clinical symptoms or decrease in CD4+ lymphocytes were observed. Follow-up for 9 months after challenge with SIV showed that the HIV-2-infected monkeys were protected against SIV-induced immunodeficiency (no decrease of CD4+ lymphocytes) and lymphadenopathy. However, they were not resistant to SIV infection since virus could be recovered from their PBMCs and they developed anamnestic antibody responses. Four naive control monkeys which were inoculated with the same dose of SIV became persistently infected and developed a decrease of the absolute numbers of CD4+ cells and showed a marked lymphadenopathy. Two out of four control animals died 58-265 days postinfection with an immunosuppressive disease. Immunohistochemical examination showed abundant viral antigen in lymph-node biopsies from the SIV-infected control monkeys but absence of SIV or HIV-2 antigens in the biopsies from the three HIV-2-preinfected and SIV-superinfected monkeys. The present study demonstrates possibilities for induction of immunity against immunodeficiency induced by a primate lentivirus, a concept with application also to HIV infection and AIDS in man.     

Vaccine protection against HIV-2 infection in cynomolgus monkeys

The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 micrograms protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 micrograms viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as well.     

珠海市恙虫病东方体基因型研究

目的 对广东省珠海市恙虫病东方体进行基因分型。方法 予高热期恙虫病患者静脉采血后,立刻床旁小白鼠腹腔接种,待发病后解剖,取其腹壁刮液涂片、吉姆萨染色镜检;以巢式PCR进行恙虫病东方体DNA检测和序列分析,将结果进行同源性分析。结果 床旁小白鼠腹腔接种,腹壁刮液涂片镜检可见紫红色圆形、椭圆形、短杆状恙虫病东方体菌体。10例恙虫病患者进行特异性的巢式PCR扩增,其中6例扩增出目的基因片段,阳性率为60%。将扩得珠海恙虫病东方体基因片段的DNA序列与基因库序列进行比对,发现其与韩国株Yonchon株同源性最高,大于95%,系统进化树显示其与Karp株在同一分支。结论 珠海市为恙虫病的自然疫源地,该地区恙虫病东方体的基因型与Yonchon株最相近。     

Fatal infection of silvered leaf monkeys with a virus-like infectious agent (VLIA) derived from a patient with AIDS

Four silvered leaf monkeys, inoculated with a virus-like infectious agent (VLIA) derived from transformed NIH/3T3 cells (sb51) transfected with Kaposi's sarcoma DNA of an AIDS patient, showed wasting syndromes and died in 7-9 months. Two monkeys had a transient lymphadenopathy in earlier stages. Two moribund animals showed lymphopenia. Although 3 of the VLIA inoculated monkeys had persistent low grade fever early in the infection, the animals became afebrile in the later stages. One VLIA inoculated animal had a prominent antibody response, which occurred 7 months after VLIA inoculation. The other 3 monkeys had a transient or poor antibody response in the later stages. These 3 animals revealed periodic VLIA antigenemia during the course of the experiment. A control monkey was killed 8 months after the last VLIA inoculated monkey succumbed and showed neither an antibody response nor evidence of antigenemia. VLIA-specific DNA could be directly detected in necropsy tissues of all 4 monkeys inoculated with VLIA using the polymerase chain reaction method. VLIA infection was identified in all 4 spleens, 2 of 4 livers, 1 of 2 kidneys, and all 3 brains tested from these 4 animals, but not in the tissues from the control monkey. The necropsy examination of the 4 VLIA inoculated animals revealed no opportunistic infections, acute inflammatory lesions, malignancy or cause of death other than VLIA infection. We believe that the VLIA caused a fatal systemic infection in these monkeys.     

The first case of tsutsugamushi disease in Ehime Prefecture

The first case of tsutsugamushi disease in Ehime Prefecture was experienced in December 1987 with successful isolation of the causative agent. The patient was taken ill twelve days after infection. Immunofluorescent antibody tests using the isolate, Yamazaki strain, and Gilliam, Karp, Kato, Irie and Shimokoshi strains as antigens revealed that the specific antibodies against these antigens appeared and increased in the blood of the patient during the course of the disease. And the antibody titers to the Yamazaki antigen were the highest of these antigens. Agglutinin for Proteus OXK did not appear in the blood of the patient. The immunofluorescent antibody test using type-specific monoclonal antibodies to Gilliam, Karp, Kato, Irie and Shimokoshi strains and these five strains and the Yamazaki strain as antigens revealed that the Yamazaki strain was identified as Karp type of Rickettsia tsutsugamushi.     

Single and repeated infections of grivet monkeys with Schistosoma mansoni: parasitological and pathological observations over a 31-month period

Groups of 6 to 8 grivet monkeys (Ceropithecus aethiops aethiops), each of which had been exposed to 600 cercariae of Schistosoma mansoni, were killed 3, 6, and 31 months after exposure. Other groups of monkeys were repeatedly challenged with cercariae beginning 4 or 23 months after the initial infection, and were killed 31 months after the first exposure. Peripheral blood leukocytes, eosinophiles, and schistosome eggs passed in the feces were determined during the course of the infection. At autopsy, gross and histopathologic observations were made, worms were recovered by perfusion, and the number of eggs in the tissues was determined by digestion. Monkeys became acutely ill about 8 weeks after the initial exposure, and death was attributed to acute'toxemic' schistosomiasis in 4 of 46 infected animals. The symptoms and signs of the acute phase did not recur with reinfection. Most subsequent natural deaths were caused by Klebsiella infection which occurred in both infected and control monkeys and did not appear to be related to the schistosome infection. Relatively stable infections were found after a single exposure to cercariae, with only a slight decrease in worm numbers and moderate decrease in egg passage after 31 months. With repeated reinfection, only 19% of challenge cercariae developed into adult worms. Oviposition by these worms was delayed and the rate of oviposition was substantially less than in monkeys infected only once. Eggs deposited in the tissues were rapidly destroyed. The calculated rate of oviposition varied from 658 eggs/day per female 3 months after a single infection to 299 eggs/ day per female 31 months after repeated infections. Although very heavy infections were present in repeatedly infected monkeys, only slight hepatic fibrosis was seen and no portal hypertension or portal-systemic collateral circulation developed. Severe intestinal disease was present in only two monkeys. The remaining animals had little diarrhea after the acute phase, and there was only slight loss of plasma protein into the feces 31 months after exposure.     

Patterns of parasitaemia, antibodies, complement and circulating immune complexes in drug-suppressed simian Plasmodium knowlesi malaria

Rhesus monkeys were inoculated intravenously with 1 x 10(4) P. knowlesi infected erythrocytes. After about three days prepatency, peripheral smears were found positive and the animals were cured with chloroquine phosphate when parasitaemia reached about 15-25 per cent. The monkeys were repeatedly exposed with three bouts of infection. The first and second bouts were cured but after the third challenge, all 10 monkeys showed a longer prepatent period, lower parasitaemia and then self-recovery. Sera were collected in different phases of infection to assess immune responses. Antimalarial IgG and IgM responses were measured by ELISA. The presence of IgM antibody was associated with every bout of infection. With repeated infections until self-recovery, a substantial amount of IgG was found in circulation. A significant level of schizont-infected cell agglutination antibody was also detected in the animals after survival from challenge infection. Antigen-specific circulating immune complexes, both of IgG and IgM types, appeared in various phases of infection, but their appearance did not coincide with the acquired immune responses of the animals. During the self-recovery phase, almost all monkeys had an elevated level of serum C3 and C4.     

Attempts to immunize monkeys against Plasmodium knowlesi by using heat-stable, serum-soluble antigens.

Thirty-six Macaca mulatta monkeys were immunized with different concentrations and regimens of heat-stable, serum-soluble (S-) antigens of Plasmodium knowlesi prior to challenge with the homologous parasite via sporozoite inoculation. Fewer deaths and reduced maximum parasitemias occurred in those animals inoculated with 10 to 40 mg of S-antigen compared to nonimmunized monkeys or those receiving only Freund's adjuvant. Protection was incomplete, however, suggesting that atibodies to S-antigens may have a limited role in protection of hosts to malarial infection.     

Antibody responses to experimental Brugia malayi infections in patas and rhesus monkeys

Humoral antibody responses in experimental infections with Brugia malayi (subperiodic strain) were compared in two primate species. Erythrocebus patas and Macaca mulatta. Antibody responses were related to the infection protocol and the duration and magnitude of microfilaremia. Patas monkeys were uniformly susceptible to infection and characteristically exhibited prolonged microfilaremia; infections in Rhesus monkeys produced low and usually microfilaremia. Antibody, measured by enzyme linked immunosorbent assay with extracts of adult Brugia and microfilariae as antigens, declined at patency in Patas monkeys and there was an inverse relationship between serum antibody concentration and the number of circulating microfilariae. Rhesus monkeys generally had high, sustained antibody levels relative to Patas monkeys, but antibody levels were comparable in the two species when the numbers of circulating microfilariae were similar. By fluorescent antibody technique, antibodies reactive with somatic antigens of microfilariae were detected in all infected monkeys; antibodies reactive with the cuticle of infective larvae were also present in both primates and were consistently detected in monkeys receiving multiple infections. Antibodies (IgG, IgM) reactive with the sheath of microfilariae were detected only in certain Rhesus monkeys which were essentially amicrofilaremic and sera with antibodies specific for microfilarial sheath promoted in vitro microfilarial agglutination and leukocyte adherence.     

Antigenic types of Orientia tsutsugamushi in Malaysia

The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.     

Long-term observation of baboons, rhesus monkeys, and chimpanzees inoculated with HIV and given periodic immunosuppressive treatment

Baboons, rhesus monkeys, and chimpanzees were injected with the human immunodeficiency virus (HIV) and monitored for up to 4 years. Various immunosuppressive regimens were used during this time in attempts to induce development of the acquired immune deficiency syndrome (AIDS). No infectious virus was recovered or anti-HIV antibodies detected in the baboons and rhesus monkeys. Virus has been recovered from lymphocyte cultures of all five of the chimpanzees at intermittent periods following inoculation. The chimpanzees developed anti-HIV antibodies from 1 to 5 months after virus inoculation and had circulating antibodies that neutralized HIV. All the infected animals were capable of in vitro lymphocyte blastogenic responses to recombinant envelope and core HIV antigens. Despite immunosuppressive therapies and evidence of some immunologic abnormalities, none of the five chimpanzees has yet developed AIDS or a related disorder.     

Development of antigen-specific cell-mediated immune responses after infection of cynomolgus monkeys (Macaca fascicularis) with Rickettsia tsutsugamushi

Cynomolgus monkeys were evaluated for cellular immune responses after infection with the Karp strain of Rickettsia tsutsugamushi. Antibody and clinical signs of localized and systemic infection were also evaluated. Animals challenged with homologous or heterologous strains at various times after a primary infection were also followed up. Naive monkeys developed eschars, lymphadenopathy, rickettsemia, and elevated body temperatures. Antibody in these animals was IgM followed by IgG. Lymphocyte proliferation and production of gamma-interferon by peripheral blood mononuclear leukocytes also were demonstrated. If challenged six years after the initial infection, clinical signs and cellular responses were indistinguishable from naive animals but an anamnestic IgG antibody response was noted. If challenged eight months after the initial infection, complete resistance was noted, but if challenged at one year, a localized cutaneous lesion developed. The majority of animals infected previously had preexisting lymphocyte activity, a characteristic suggesting long-term immunologic memory that was not protective against rechallenge.     

Analysis of immunological characteristics of newly isolated strains of Rickettsia tsutsugamushi using monoclonal antibodies

Since 1975, there has been an increase in the number of patients with tsutsugamushi disease in Japan, and marked antigenic heterogeneity has been found among newly isolated strains of Rickettsia tsutsugamushi. For antigenic analysis of these strains, we produced monoclonal antibodies against the Irie strain isolated in 1971, and the Hirano and Shimokoshi strains isolated in 1980. In all, 34 monoclonal antibodies were produced and their reactivities were determined by the immunofluorescent antibody test. The serological reactivity of the antibodies against these three strains and classic representative strains (Gilliam, Karp and Kato) showed varied reactive characteristics, i.e., serotype-specific, species-specific and intermediate reactivities. It was revealed that these strains are antigenically different from the classic ones. Moreover, by using the serotype-specific monoclonal antibodies, nine strains newly isolated in Miyazaki Prefecture were classified into the Irie and the Hirano types. The antigenicity of the Shimokoshi strain differed from those of the other strains used in this study. From these results, the strains of R. tsutsugamushi used in this study fell into six serotypes including the classic strains. SDS-PAGE and immunoblotting were performed to determine the molecular sizes of the antigenic polypeptides. The results revealed that the serotype-specific antigens belong to the 60-kDa class whereas the species-specific antigens belong to the 61-kDa, 60-kDa or 44-kDa class.     

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.     

Protective antibodies against erythrocytic stages of Plasmodium falciparum in experimental infection of the squirrel monkey, Saimiri sciureus

Serum and ascitic fluid from squirrel monkeys ( Saimiri sciureus ) inoculated with erythrocytic stages of Plasmodium falciparum were collected at different periods of the infection. Protection against P. falciparum was achieved by passive transfer of the sera or fluid recovered from animals after spontaneous or drug-induced cure. Purified immunoglobulins from the ascitic fluid also conferred protection. In contrast, protective antibodies directed against erythrocytic stages of P. falciparum could never be demonstrated during the acute phase of infection in spite of the high titres of malarial antibodies detected by immunofluorescence. The comparative immunochemical analysis of antigens recognized by protective and non-protective antibodies revealed quantitative differences which may be of use for the identification of antigens inducing protection.