Rocky Mountain spotted fever vaccine in an animal model.

Formalin-killed, purified Rickettsia rickettsii vaccine was evaluated in a guinea pig model of R. rickettsii infection. Vaccinated guinea pigs were partially protected by the vaccine when challenged with virulent, viable rickettsiae. Greater protection was observed when higher doses of vaccine were given and when frequent booster injections were administered. Stimulation of cell-mediated immunity to the vaccine antigens was variable and also appeared to be achieved more reproducibly with booster vaccinations. Serum antibody was elicited by high doses of vaccine and by booster vaccinations. The presence of serum antibody was useful in predicting immunity to challenge with R. rickettsii.     

Hemostatic changes in Rocky Mountain spotted fever and Mediterranean spotted fever.

Rocky Mountain spotted fever and Mediterranean spotted fever are rickettsial infections primarily of endothelial cells that normally have a potent anticoagulant function. As a result of endothelial cell infection and injury, the hemostatic system is perturbed and shows changes that vary widely from a minor reduction in the platelet count (frequently) to severe coagulopathies, such as deep venous thrombosis and disseminated intravascular coagulation (rarely). Changes favoring a hypercoagulable state include endothelial injury and release of procoagulant components, activation of the coagulation cascade with thrombin generation, platelet activation, increased antifibrinolytic factors, consumption of natural anticoagulants, and possibly high levels of coagulation-promoting cytokines. Yet, most studies have been performed on endothelial cell cultures that provide nonphysiologic, reductionistic, experimental conditions. The lack of flow, platelets, and WBCs makes these experiments far from simulating the response of endothelial cells in the human body. Coagulopathies and thrombotic events should be considered as potential complications of severe Rocky Mountain spotted fever and Mediterranean spotted fever.     

Preparation of Rocky Mountain spotted fever vaccine suitable for human immunization.

Rocky Mountain spotted fever vaccine was produced from rickettsiae grown in chicken embryo cells in roller bottle cultures. The rickettsiae were concentrated and purified by passage through a sucrose gradient and inactivated with formalin. This vaccine satisfactorily passed preinactivation and final container testing and is believed to be superior to the presently available yolk sac vaccine.     

Prophylactic treatment of Rocky Mountain spotted fever.

Prophylactic treatment of Rocky Mountain spotted fever with a single dose of oxytetracycline was investigated in guinea pigs. Disease was prevented when treatment was administered shortly before expected onset. Relapses occurred when treatment preceded expected onset by 48 h or more.     

Evaluation of a killed Rocky Mountain spotted fever vaccine in cynomolgus monkeys.

A nonhuman primate model of Rocky Mountain spotted fever infection was developed in cynomolgus monkeys (Macaca fascicularis) infected by the subcutaneous route or by aerosol. Clinical responses, hematology and serum chemistry values, and pathological findings were similar to those found in humans ill with Rocky Mountain spotted fever. The clinical model was then used to test the efficacy of a killed Rocky Mountain spotted fever vaccine grown in chicken embryo cells. Monkeys were immunized with varying dilutions of the vaccine with a two-dose schedule and then challenged at 2 months with virulent Rickettsia rickettsii by the subcutaneous route or by aerosol. The undiluted vaccine totally protected monkeys against both challenges, even at extremely high doses.     

Antibody response to Rocky Mountain spotted fever.

Various techniques were compared to determine the most sensitive method for detection of rocky Mountain spotted fever antibody. A radiometabolic technique for detection of Rocky Mountain spotted fever antibody is also described. In infected monkeys, the fluorescent antibody technique yielded the earliest evidence of seroconversion; with some monkeys the microagglutination procedure was equally effective. The fluorescent antibody and microagglutination measurements showed higher titers than those for complement fixation, Weil-Felix, or the radiometabolic techniques.     

Barbash strain spotted fever group rickettsia is a strain of Rickettsia conorii and differs from Rickettsia sibirica

The Barbash strain of spotted fever group rickettsia was reexamined in this study by the microimmunofluorescence test with mouse antisera and with monoclonal antibodies. Protein immunoblotting was performed for comparison of purified antigens of R. rickettsii, R. sibirica, R. conorii and Barbash strain. Comparison of Barbash strain, R. rickettsii (Sheila Smith strain), R. conorii (Malish 7 strain), and R. sibirica (strains 232, 246 and Jinghe-74) of the spotted fever group in the microimmunofluorescence test of Philip et al. revealed that Barbash strain has antigens that yield homologous titers with the R. conorii strains and differ from R. sibirica and R. rickettsii. Monoclonal antibodies specific for R. conorii react at identical titres with the Barbash strain, and a monoclonal antibody specific for R. sibirica does not react with the Barbash strain. Likewise, T-cell hybridomas reactive with R. conorii but not R. sibirica yield a strong response when stimulated by Barbash strain antigens. Western immunoblotting with the same polyclonal and monoclonal antibodies confirmed the presence of specific protein antigens of R. conorii and different protein antigenic composition of R. sibirica when compared with Barbash strain. Thus, Barbash strain is a strain of R. conorii.     

Laboratory diagnosis of Rocky Mountain spotted fever by immunofluorescent demonstration of Rickettsia in Cutaneous lesions.

Direct immunofluorescent staining for Rickettsia rickettsii was performed on cryostat sections of skin biopsies from 27 patients suspected of having Rocky Mountain spotted fever. In nine of the 17 patients whose final diagnosis was Rocky Mountain spotted fever, coccobacillary forms of R. rickettsii were identified in endothelium and vascular walls within the dermis. Facotrs recognized as contributing to false-negative results were prior treatment with tetracycline or chloramphenicol for 24--48 hours or longer and failure to obtain a section through the focus of vasculitis. No false-positive result was obtained in the ten patients whose final diagnoses were not Rocky Mountain spotted fever. The laboratory test offers an immediate, positive laboratory diagnosis for this treatable, life-threatening disease.     

Rapid immunoperoxidase demonstration of Rickettsia rickettsii in fixed cutaneous specimens from patients with Rocky Mountain spotted fever

Immunofluorescence (IF) of skin biopsies for detection of Rickettsia rickettsii (RR) has proven useful as a rapid test for confirmation of Rocky Mountain spotted fever (RMSF). However, IF lacks sensitivity, requires special equipment and training, and is difficult to interpret. The authors have developed an indirect avidin-biotin immunoperoxidase (IP) technique to detect RR in fixed and frozen tissue sections. The technique was evaluated on fixed cutaneous specimens from patients dying of RMSF and compared to specimens from control patients dying of an acute febrile illness with skin manifestations and vasculitis. IP correctly identified RR in 9 of 12 cases with probable identification in 2 additional cases. Of 11 controls, 10 were negative and one was uninterpretable. RR was easily visualized within cytoplasm and nuclei of endothelial cells in association with perivascular lymphocytic infiltrates and less frequently with vasculitis or non-inflamed vasculature. IP is rapid, amplifies small quantities of antigen, gives excellent histologic detail as compared with IF, and is easily adapted for use in hospitals with immunoperoxidase capabilities.     

Humoral immune response to Rocky Mountain spotted fever in experimentally infected guinea pigs: immunoprecipitation of lactoperoxidase 125I-labeled proteins and detection of soluble antigens of Rickettsia rickettsii.

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, purified from infected L-929 cells by density gradient banding were extrinsically radioiodinated with lactoperoxidase. Immunodominant 125I-labeled antigens were identified by radioimmunoprecipitation of detergent-solubilized antigens with protein A-Sepharose and anti-R. rickettsii sera collected 0, 3, 7, 11, 32, and 163 days after infection of guinea pigs. The average fever greater than or equal to 40 degrees C was detected by days 3 and 4 after infection with 6 X 10(7) and 6 X 10(6) PFU, respectively. By microagglutination and complement fixation assays, anti-R. rickettsii antibodies were detected as early as day 3 after infection, with titers increasing markedly between days 7 and 163. Convalescent sera, collected on day 163, from infected guinea pigs were used to identify seven 125I-labeled antigens with apparent molecular sizes of 186,000 (I), 145,000 (II), 49,000 (III), 32,000 (IV), 27,500 (V), 17,500 (VI), and 16,500 (VII) daltons. Differences in antibody reactivity and specificity against the seven antigens were demonstrated with serially obtained sera. Sera from a guinea pig infected with 6 X 10(7) PFU exhibited antibody-antigen interactions with all seven 125I-labeled antigens by day 7, whereas the same antibody activity required 32 days for an animal infected with 6 X 10(6) PFU. Prominent antibody activities toward proteins II and IV were demonstrated both early and late after infection. The fluids obtained from infected L-929 cells contained three soluble antigens which were detected with the 11-, 32-, and 163-day sera by an immunodiffusion assay. The soluble and 125I-labeled antigens of R. rickettsii identified in this study may be important candidates for vaccines against Rocky Mountain spotted fever.     

Routine argyrophil techniques detect Rickettsia rickettsii in tissues of patients with fatal Rocky Mountain spotted fever

Rickettsia rickettsii, a bacterial tickborne pathogen that causes Rocky Mountain spotted fever (RMSF), stains poorly or not at all with conventional tissue Gram techniques, and contemporary visualization of the pathogen in formalin-fixed, paraffin-embedded tissues has relied almost entirely on immunohistochemical staining methods that are generally limited to specialized research laboratories or national reference centers. To our knowledge, previously described argyrophil-based histochemical techniques have not successfully detected rickettsiae in formalin-fixed, paraffin-embedded tissues. To investigate the ability of standard silver impregnation techniques to demonstrate the occurrence and distribution of R. rickettsii in tissues of patients with RMSF confirmed by molecular and immunohistochemical methods, three widely recognized and commercially available silver impregnation methods (Warthin–Starry, Steiner, and Dieterle’s) were applied to various tissues obtained at autopsy from 10 patients with fatal RMSF. R. rickettsii bacteria were demonstrated in one or more tissues of all patients, using each of the argyrophil-based methods, and appeared as small, dark brown-to-black lanceolate rods, often in pairs and occasionally surrounded by a faint halo. Rickettsiae were identified most consistently in small arteries and arterioles of liver, kidney, and leptomeninges, and were localized predominantly to the cytoplasm of endothelial cells and less often within the internal elastic lamella and smooth muscle of the media. This validation of argyrophilic techniques to detect R. rickettsii demonstrates the utility of inexpensive core histochemical methods in the diagnosis of infectious agents in pathology specimens and may have utility in certain resource-limited settings where RMSF is endemic.     

Rocky Mountain spotted fever. Gastrointestinal and pancreatic lesions and rickettsial infection

Recent clinical studies have shown a high incidence of nausea, vomiting, diarrhea, and abdominal pain in Rocky Mountain spotted fever (RMSF), and case reports have documented rickettsial infection and vascular injury in the small intestine, appendix, and gallbladder. To determine the incidence and distribution of Rickettsia rickettsii and rickettsial lesions that might be the basis for these clinical manifestations of RMSF, tissues that were available from the stomach, small intestine, colon, and pancreas in fatal cases of RMSF were examined. Lesions were identified in pancreatic tissue in 91% of cases and in tissue obtained from the stomach, small intestine, and colon in all cases. Most tissues were judged to be only moderately injured. Organisms of R rickettsii were demonstrated by immunofluorescence in 14 (50%) of 28 cases and, when identified, correlated topographically with the location of vascular injury. These observations support the concept of rickettsial vascular injury of the gastrointestinal (GI) tract and pancreas leading to GI signs and symptoms in RMSF.     

Detection of the Rocky Mountain spotted fever agent, Rickettsia rickettsii, in dead ticks, Dermacentor andersoni.

Rickettsia rickettsii remained viable and retained chromophilic properties for not more than 24 h after Dermacentor andersoni were killed by freezing. Antigenic reactivity was detected for at least 71 days by direct immunofluorescence. However, rickettsiae in ticks suffocated with mineral oil remained pathogenic for at least 14 days. Accordingly, ticks removed from a host by mineral oil or dying from desiccation in transit are still suitable for rickettsial examination.     

A focus of Rocky Mountain spotted fever within New York City

In the spring and summer of 1987, four persons acquired Rocky Mountain spotted fever within New York City, an area in which the disease had not previously been known to be endemic. Three of the four patients were residents of the Soundview area of the Bronx. All diagnoses were confirmed by indirect fluorescent-antibody tests. Environmental investigation revealed that the tick vector for Rickettsia rickettsii, Dermacentor variabilis, was present in a local park. Of the 66 specimens of D. variabilis collected, 5 (8 percent) were positive for rickettsiae from the spotted fever group. Of an additional 96 specimens of D. variabilis, 5 (5 percent) were found positive for rickettsiae by a more specific monoclonal antibody assay. Eight additional New York City parks in all five boroughs were searched for ticks. D. variabilis was found in only one other park; of the 147 ticks collected there, none were positive for rickettsiae. These findings emphasize the focal nature of Rocky Mountain spotted fever and the need to consider that disease in the differential diagnosis of any obscure acute febrile illness, even in the absence of a history of travel to known endemic areas.     

Laboratory-acquired Rocky Mountain spotted fever. The hazard of aerosol transmission.

Nine patients with laboratory-acquired Rocky Mountain spotted fever were seen during the period 1971 to 1976. Investigation of each case revealed either definite or probable exposure to an aerosol containing infectious rickettsiae; in no case was there evidence of parenteral exposure either by accidental self-inoculation or by tick bite. These illnesses are believed to represent infection acquired via the respiratory route. This report emphasizes the aerosol hazard of Rickettsia rickettsii in the laboratory and discusses the possibility of respiratory transmission of Rocky Mountain spotted fever in nature. The illness occurred only in personnel who had received either no vaccination or the primary series of the commercial (Lederie) vaccine against this infection. Other personnel who had received the primary series with multiple booster vaccinations demonstrated increased immunity as measured by humoral antibody titers and rickettsial antigen-induced lymphocyte transformation; no cases of clinical disease developed in these multiply-vaccinated personnel.     

New assay of protective activity of Rocky Mountain spotted fever vaccines.

Areas under the fever curves of guinea pigs inoculated with Rocky Mountain spotted fever vaccine over a restricted dose range and infected with a standardized dose of Rickettsia rickettsii varied linearly with log10 dose of vaccine. A calculator was programmed to plot fever curves and calculate the vaccine dose that reduced the fever of infected animals by 50%.     

钩端螺旋体DNA疫苗pcD-flaB在豚鼠体内的免疫保护性研究

目的观察钩端螺旋体DNA疫苗peD-flaB是否具有抗钩体交叉感染的保护作用。方法将纯化后的钩体DNA疫苗pcD-flaB100μg200μl肌肉注射豚鼠，共免疫2次，间隔7d，初次免疫后的第21天分别攻击感染赖型、临海型和波摩那型钩体，观察保护作用。结果该疫苗对3型钩体的保护率分别是88．89％、100％和100％，总保护率为96．3％，与对照组差异具有显著性。结论DNA疫苗pcD-flaB在豚鼠体内不但具有抗同型钩体(临海型)感染的作用，也具有较好的交叉保护作用。     

Discrepancies in Weil-Felix and microimmunofluorescence test results for Rocky Mountain spotted fever.

Only 4.2% of 284 single specimens and 17.6% of 51 pairs of sera reactive in Weil-Felix agglutination tests for Rocky Mountain spotted fever were confirmed by a specific Rickettsia rickettsii microimmunofluorescence test.