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The authors applied a new methodological approach based not only on the study of IgM/IgG to Rickettsia prowazekii in sera, but also on the estimation of the avidity index of specific IgG. The data allowed the authors to draw new conclusions about the 1998 epidemic typhus outbreak in Russia., Competing Interests: Declaration of Competing Interest Authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background: Resurgence of scrub typhus was reported in Northeast India in 2010 after a gap of 67 years since World War II. However, the presence of other rickettsial infections remained unknown from this region. A seroepidemiological investigation was undertaken in the scrub typhus affected areas from 2013-2015 in Assam, Arunachal Pradesh and Nagaland to assess the exposure to other rickettsial diseases besides scrub typhus., Methods: Samples were collected from people residing in scrub typhus reporting areas. Serology was performed by an indirect ELISA for the three rickettsial agents' viz., scrub typhus group orientiae (STGO), spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). A sample with total net absorbance ≥1.000 was considered as positive. An entomological survey was also carried out in the affected areas., Results: Overall, 1265 human blood samples were collected, of which 30.8% (n=390), 13.8% (175) and 4.2% (53) had antibodies against STGO, SFGR and TGR respectively. Presence of antibodies against more than one of the rickettsial groups was also detected. Among the arthropods collected, chiggers of Leptotrombidium deleinse, fleas belonging to Ctenocephalides felis and Pulex irritans, ticks belonging to Rhipicephalus microplus, Haemaphysalis spp. were predominant. Candidatus Rickettsia senegalensis was detected in C. felis., Conclusions: Our findings confirm wide circulation of rickettsial infections and their probable vectors in the northeast region of India.Accession numbers: KU163367, KU163368, KU499847, KU499848., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Materials, that summarize data of original research and scientific literature on epidemiology and problems of persistence during epidemic typhus, whose causative agent (Rickettsia prowazekii) is reactivated in the organism of the previously ill and is manifested as Brill-Zinser disease, are presented. A retrospective analysis was carried out with the data obtained by Russian (All-Union) Centre for Rickettsioses during study of epidemiologic examination maps of 5705 typhus nidi and results of 19 463 blood sera analysis during study of immunologic structure of population in the territories of the former USSR for the period from 1970 to 1992. A decrease of epidemic typhus morbidity and an increase of the fraction of Brill-Zinser disease took place as a result of pediculosis corporis control. In separate territories specific weight of Brill-Zinser disease was 48% in 1952, up to 80% in 1969, and from 1977 all the ill were previously ill. However, during the perestroika period and afterwards, due to a reduction of economic and hygienic living conditions, appearance of refugees, the immune structure regarding typhus began to change. Due to the buildup of the population migration process and the presence of risk groups (refugees, homeless) among population of regions, where local wars are waged, the enhancement of methods of epidemic typhus and Brill-Zinser disease diagnostics and pediculosis corporis eradication is necessary. Study of R. prowazekii by molecular-genetics methods is necessary for complete understanding of its mechanism of persistence.

Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8(+) T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0.03) and the presence of eschar (P = 0.03), elevated white blood cell (WBC) count (P = 0.007), elevated lymphocyte (P = 0.007) and neutrophil counts (P = 0.015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0.03), the presence of lymphadenopathy (P = 0.033) and eschar (P = 0.03), elevated WBC (P = 0.005) and neutrophil counts (P = 0.0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0.03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar.

Rickettsia prowazekii, the etiologic agent of epidemic typhus, infects vascular endothelium, leading to vasculitis and tissue infiltration of leukocytes. Murine and human endothelial cells (ECs) were infected with R. prowazekii, including the virulent Breinl strain and the attenuated Madrid E strain. The transendothelial migration (TM) of murine and human peripheral blood mononuclear cells (PBMCs) across ECs infected with Breinl organisms was significantly increased compared with that for uninfected ECs or for ECs infected with attenuated organisms, demonstrating that increased TM was related to R. prowazekii virulence. Increased TM was associated with a specific inflammatory pattern. Indeed, only Breinl organisms induced the expression of transcripts for inflammatory cytokines and chemokines by ECs. Murine PBMCs that had transmigrated across ECs infected with Breinl organisms overexpressed inflammatory cytokines and chemokines as well as tissue factor, whereas interleukin-10 expression was down-regulated. The impact of R. prowazekii infection on the TM of PBMCs may play a prominent role in the development of lesions in epidemic typhus.

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1: 40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill-Zinsser disease was prevalent in Korea.

Rickettsia prowazekii, the etiologic agent of louse-borne typhus, is listed as a category B agent under the select agent list of the United States Centers for Disease Control and Prevention. R. prowazekii was placed on the select agent list due to its potential to cause epidemic, high mortality in untreated and/or misdiagnosed cases, and ease of spread in vulnerable populations. Historically, R. prowazekii vaccines using crude antigen and/or inactivated rickettsia were partially protective but have been accompanied with undesirable toxic reactions and difficulties in standardization. The availability of the genome sequence of R. prowazekii allowed us to select genes that encode proteins with potential in immuno-protection against this human pathogen. We successfully PCR-amplified a group of genes involved in invasion (invA), cell division (fts), protein secretion (sec gene family), and virulence (ompA and ompB, virB gene family, cap and tlyA and tlyC). The generated PCR products were cloned into the Gateway cloning system and the cloned products will be introduced into Vical VR 1020-DV and VR 1012-DV DNA vaccine plasmids. Twenty-four target genes from R. prowazekii have been PCR amplified, of which fifteen have been introduced into the pENTR/SD/D-TOPO entry cloning vector.

A total of 157 sera from febrile patients in the Philippine General Hospital in Manila, Luzon, and the Northern Samar Provincial Hospital, the Philippines, were used. Serum antibodies against spotted fever group Rickettsia (SFGR) and typhus group Rickettsia (TGR) were detected by indirect immunofluorescence test. Antibody positive rates were 1.3% for SFGR (Rickettsia japonica) and 2.5% for TGR (R. typhus), respectively. Rickettsial antibodies in humans in the Philippines were found for the first time. These results underscore the need for further epidemiological study of clinical rickettsioses in the Philippines.

A mouse model of typhus rickettsiosis that reproduces the hematogenous dissemination to the critical target organs, including brain, lungs, heart, and kidneys, primary endothelial and, to a lesser degree, macrophage intracellular rickettsial infection, and typical vascular-based lesions of louse-borne typhus and murine typhus was established. Intravenous inoculation of C3H/HeN mice with Rickettsia typhi caused disease with a duration of the incubation period and mortality rate that were dependent on the infective dose of rickettsiae. Lethal infection was associated with high concentrations of R. typhi in the lungs and brain, despite a brisker humoral immune response to the rickettsiae than in the sublethal infection. Gamma interferon and CD8 T lymphocytes were demonstrated to be crucial to clearance of the rickettsiae and recovery from infection in experiments in which specific monoclonal antibodies were administered to deplete these components. Death of animals depleted of gamma interferon or CD8 T lymphocytes was associated with overwhelming rickettsial infection demonstrated by titers of infectious rickettsiae and by immunohistochemistry. An effective antirickettsial immune response was associated with elevated serum concentrations of IL-12 on Day 5 and increased secretion of IL-12 by concanavalin-A-stimulated spleen cells on Day 5. Evidence for transient suppression of the immune response consisted of marked reduction in the secretion of IL-2 and IL-12 by concanavalin-A-stimulated spleen cells on Days 10 and 15. This model offers excellent opportunities for study of attenuation and pathogenetic mechanisms of typhus rickettsiae, which are established biologic weapons of potential use in bioterrorism.

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.

A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.

Typhus and other louse-transmitted bacterial infections in Peruvian sierra communities are known to occur but have not recently been assessed. In this study, 194 of 1,280 inhabitants of four villages in Calca Province in the Urubamba Valley were included. Thirty-nine (20%) of the 194 volunteers had antibodies to Rickettsia prowazekii, whereas 24 (12%) had antibodies to Bartonella quintana and 2 against Borrelia recurrentis. There was a significant correlation between the presence of infesting ectoparasites and antibodies to R. prowazekii, as well as between antibodies to R. prowazekii and ectoparasite infestation and fever in the previous 6 months. The proportion of inhabitants infested with ectoparasites was significantly higher in the highest-altitude village than in the other three villages. Two volunteers' antibody levels suggested a recent typhus infection, but only B. quintana DNA was amplified from lice. Epidemic typhus remains extant in the area, and B. quintana infections were encountered and documented for the first time in South America.

Background: Fever of intermediate duration (FID), characterized by a febrile syndrome lasting from 7 to 28 days, is a frequent condition in clinical practice, but its epidemiological and etiologic features are not well described. Murine typhus (MT) is a worldwide illness; nevertheless, to our knowledge, no studies describing its epidemiological and clinical characteristics have been performed in the south of Spain. Also, its significance as a cause of FID is unknown., Objective: To determine the epidemiological features, clinical characteristics, and prognosis of MT and, prospectively, its incidence as a cause of FID., Design: Prospective study of cases of MT over 17 years (1979-1995) and of all cases of FID treated in a tertiary teaching hospital in Seville, Spain., Results: One hundred and four cases of MT were included, and MT was the cause in 6.7% of 926 cases of FID. Insect bites were reported in only 3.8% of the cases of MT previous to the onset of illness. Most cases (62.5%) occurred in the summer and fall. A high frequency of rash (62.5%) was noted. Arthromyalgia (77%), headache (71%), and respiratory (25%) and gastrointestinal (23%) symptoms were also frequent. Laboratory findings were unspecific. Organ complications were uncommon (8.6%), but they were severe in 4 cases. The mean duration of fever was 12.5 days. Cure was achieved in all cases, although only 44 patients received specific treatment., Conclusions: Murine typhus is prevalent in the south of Spain and is a significant cause of FID. Clinical signs are benign, but some patients may develop severe complications. A high degree of clinical suspicion is required for diagnosis.

We recently investigated a suspected outbreak of epidemic typhus in a jail in Burundi. We tested sera of nine patients by microimmunofluorescence for antibodies to Rickettsia prowazekii and Rickettsia typhi. We also amplified and sequenced from lice gene portions specific for two R. prowazekii proteins: the gene encoding for citrate synthase and the gene encoding for the rickettsial outer membrane protein. All patients exhibited antibodies specific for R. prowazekii. Specific gene sequences were amplified in two lice from one patient. The patients had typical clinical manifestations, and two died. Molecular techniques provided a convenient and reliable means of examining lice and confirming this outbreak. The jail-associated outbreak predates an extensive ongoing outbreak of louse-borne typhus in central eastern Africa after civil war and in refugee camps in Rwanda, Burundi (1), and Zaire.

The structure of the O-specific polysaccharide chain of Proteus vulgaris OX19 lipopolysaccharide which determines the O1 specificity of Proteus and is used in the Weil-Felix test for diagnostics of rickettsiosis was established. On the basis of 1H- and 13 C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), it was found that the polysaccharide consists of branched pentasaccharide repeating units containing D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc, two residues), which are connected to each other via a phosphate group (P): [formula: see text]. The polysaccharide is acid-labile, the glycosyl phosphate linkage being cleaved at pH 4.5 (70 degrees C) to give a phosphorylated pentasaccharide with a galactose residue at the reducing end. Structural analysis of the oligosaccharide and a product of its dephosphorylation with 48% hydrofluoric acid using 1H- and 13C-NMR spectroscopy and electrospray ionization mass spectrometry confirmed the structure of the polysaccharide.

Correlation between the rate of seeding of organs by rickettsiae and duration of the exciter persistence and condition of immunological reactivity was established using the experimental rickettsial infection as a model. When using the preparation which stimulates the immunity indices, we have revealed that the number of rickettsiae in organs was less and the release from them was faster than under immunodepression.

It was shown in the experiments on the model of typhus infection in guinea-pigs that the pathogenic organism can be inherited by foetus from the mother's organism in the period of acute infection. It is confirmed by the presence of positive seroconversion in 86% of born offsprings and the character of the immunity response after the control infection that is the initial formation of immunoglobulins of form G (Ig G). At the same time there was no such a transfer of pathogenic organisms in 14% of offsprings. This fact is testified by the absence of seroconversion and the initial formation of immunoglobulins of form M (Ig M) with following formation of immunoglobulins of form G (Ig G) after control infection rickettsia Prowazekii.

Microimmunofluorescence and Western immunoblotting were compared with the classical complement fixation reaction and the Weil-Felix test to study the serological responses of patients to Rickettsia prowazekii and both Proteus vulgaris OX19 and OX2 during primary and recrudescent typhus infections. The serological response to R. prowazekii was found to be similar during primary and recrudescent typhus, and all sera examined contained antibodies to the same R. prowazekii cell structures. Immunoglobulin G (IgG) and IgM were found to be the dominant anti-R. prowazekii immunoglobulins in all sera tested and were found to be directed against the 100-kDa protein and the lipopolysaccharide. IgA antibodies, when present, were mainly against the 100-kDa protein. For P. vulgaris, IgG antibodies recognized the proteins and lipopolysaccharides of both OX19 and OX2 serotypes; IgM antibodies were directed against the P. vulgaris OX2 lipopolysaccharide. In addition, donor blood sera, which were negative by microimmunofluorescence, were found to contain IgG immunoglobulins reacting with R. prowazekii protein antigens of 135, 60, and 47 kDa by western immunoblotting.

The immunogenic properties and protective activity of basic protein I and tris-soluble antigens isolated from R. prowazekii were analyzed in comparison with those of chemical typhus vaccine on the model of anti-infectious immunity in guinea pigs. The analysis revealed that purified protein I has protective activity for guinea pigs, which is less pronounced in protein I from strain E with weak pathogenicity than protein I from strains EVir and Breinl. The activity and immunogenic properties of tris-soluble antigens, especially antigen I, are comparable with those of chemical typhus vaccines in the parameters under study.

Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants expressed complete 60 kDa polypeptide, and others expressed beta-galactosidase fusion polypeptides containing different epitopes of 134 kDa protein of the R. prowazekii outer membrane (OM). Amplified genomic library was screened with monospecific antibodies directed against abundant 31 kDa and 29.5 kDa OM proteins (OMPs). Several recombinant clones expressing full or part of 29.5 kDa polypeptide, and none expressing 31 kDa polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in Western blot with recombinant 29.5 kDa protein.

A 58-year-old man developed continuous fever of around 40 degrees C and a macular rash on the trunk and limbs one week after an eight-day visit to the island of Rhodos. Typhus was diagnosed on the basis of the clinical findings, the characteristic rash and a rise in antibody titre against Rickettsia prowazekii and mooseri (1:1280). The fever subsided and the patient's general state markedly improved within three days of starting antibiotic treatment with doxycycline. Five days after hospital admission renal function deteriorated, serum creatinine rising from 1.7 to maximally 8.7 mg/dl. Renal biopsy on the eighth day of illness revealed, in concordance with the diagnosis of a rickettsial infection, acute granulomatous interstitial nephritis. Doxycycline administration was continued for a total of 18 days and renal function improved. Two weeks after discharge serum creatinine had fallen to 1.8 mg/dl and to 1.2 mg/dl after 6 months. The antibody titre against Rickettsia prowazekii had fallen to 1:20 about 12 weeks after discharge. Early antibiotic treatment of typhus is of importance to avoid irreversible renal damage.

The protein antigens of two distinct lines of genetically related strains, namely the nonpathogenic strain E and its virulent revertant EVir and of the standard virulent strain Breinl were compared in SDS-PAGE and immunoblot assay using typhus patient sera and immune rabbit sera. No differences in the polypeptide pattern as detected in SDS-PAGE were found between strain E and EVir; the Breinl strain differed in a 30 kD protein. The high immunogenicity of the protein antigens of E, EVir and Breinl strains was demonstrated by immunoblot assay with human sera, which did not show any differences between the strains studied. Immunoblot analysis with immune rabbit sera to the strain E, EVir, and Breinl showed differences in immunological response to the 70 kD and 60 kD polypeptides of low virulent strain E and those of virulent strains EVir and Breinl.

A model of experimental typhus infection has shown that not only specific protection factors but also nonspecific ones are very important to remove Rickettsia prowazekii from macroorganisms. A complex application of antibiotics and drugs increasing immunobiological reactivity of the organism (prodigiosan, methyl uracil) noticeably decreases the rickettsia persistence level in the organism of experimental animals, the indices of specific and nonspecific protection being increased.

A total of 500 subjects (288 males and 212 females) were tested in Addis Ababa, Ethiopia, in Virology and Rickettsiology Division of the National Research Institute of Health, in 1987, for anti R. prowazekii using Complement Fixation Test (CFT). Out of these 58 subjects (41 males and 17 females) were also tested for anti R. typhi using the same test. The study population included three groups. Group I included 200 patients referred to the National Research Institute of Health (NRIH) for the Weil-Felix test for the diagnosis of typhus. Group II consisted of 200 patients with febrile illness visiting the Outpatient Department (OPD) of St. Paul's Hospital. Group III included 100 blood donors' serum samples included from previous collections. The blood donors had no sign of febrile illness during the collection of the blood samples. The results showed that anti R. prowazekii was detected in 38 subjects (7.6%). The sex ratio among the positive subjects indicated that there were 32 males (22%) and 6 females (2.8%). From the 58 subjects who were also tested for anti R-typhi only 7 (5 males and 2 females) (12%) were found to be positive. Only one person was found to be positive both for anti R. prowazekii and anti R-typhi. From 200 samples (Group-I) tested both by the Weil-Felix test and by Complement Fixation Test for anti R. prowazekii only 4 samples were positive by both test, thus showing very low percent agreement.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-free extracts of disrupted Renografin-purified Rickettsia typhi and R. prowazekii were evaluated as antigens in lymphocyte transformation assays for cell-mediated immunity to typhus group rickettsiae in 19 individuals with and 9 without histories of exposure to these organisms. Exposure consisted of clinical disease, vaccination with epidemic typhus vaccine, or occupational exposure to these agents. Both the soluble and membrane fractions of disrupted purified rickettsiae were used, and transformation of peripheral blood lymphocytes (PBL) was determined in microcultures by incorporation of [(3)]thymidine. Of the antigen concentrations tested (1 to 400 mug/ml), 10mug/ml appeared to be the most satisfactory. At this concentration, PBL transformation was highly reproducible and correlated well with donor exposure and the presence of enzyme-linked immunosorbent assay anti-typhus group immunoglobulin G. At higher concentrations, PBL from both exposed and control donors often responded to a lipopolysaccharide-like component present in these preparations. Specific transformation responses to rickettsial fractions were detected in several individuals decades after infection or vaccination, indicating that both fractions contained antigens associated with persisting cell-mediated immunity in humans. Generally, stimulation indexes with the soluble fraction were slightly greater than those obtained with corresponding concentrations of the membrane preparation, and in three individuals transformation was observed only with the soluble fraction. PBL transformation to soluble fractions also appeared to have some species specificity, since PBL from individuals with documented R. typhi infections were more responsive to the homologous soluble preparation than to the soluble fraction of R. prowazekii. PBL transformation also correlated well with homologous but only poorly with heterologous enzyme-linked immunosorbent assay immunoglobulin G titers.

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.

Rickettsia prowazeki, pretreated with typhus immune human serum, readily infects, and grows in, chicken embryo cells in culture. This finding is similar to those of previous studies which showed that typhus rickettsiae, pretreated with immune serum, grow in cells of the yolk sac of embryonated hen eggs and in the cells of the midgut of the human body louse. In contrast, identically treated typhus rickettsiae were destroyed by human macrophages in culture. Collectively, these observations seem to support an emerging concept that the fate of antibody-sensitized typhus rickettsiae depends upon the presence or absence of certain specialized properties of the host cell into which they gain entrance-nonphagocytic cells or "nonprofessional" phagocytic cells versus certain kinds of "professional" phagocytes. The phenomena involved probably have an important bearing on the mechanisms of the persisting infection and the nonsterile immunity which characterizes convalescence from typhus fever in man. They also form the basis for certain practical technical innovations in the laboratory.

The effect of lymphokine treatment of mouse and human fibroblast cell lines on the growth of Rickettsia prowazekii within the fibroblasts was studied. Treatment of mouse L929 cells with concanavalin A- or antigen-induced mouse lymphokines both before and after infection with R. prowazekii led to clearance of the rickettsiae from a substantial proportion of the cells and suppression of rickettsial growth in those cells which remained infected. Similar but less dramatic anti-rickettsial effects were observed in L929 cells treated with mouse lymphokines either only before or after infection with rickettsiae. Mouse lymphokine treatment of L929 cells had similar anti-rickettsial effects on the avirulent E strain and the virulent Breinl strain of R. prowazekii. Addition of cycloheximide or emetine to L929 cells at the same time as the lymphokines markedly suppressed the inhibition of rickettsial growth by the lymphokines. Mouse lymphokine treatment inhibited rickettsial survival and growth in mouse 3T3-A31 cells as well as in mouse L929 cells, but had no effect on rickettsial survival and growth in human foreskin fibroblasts. Conversely, concanavalin A-induced human lymphokines inhibited rickettsial survival and growth in human foreskin fibroblasts but had no effect on rickettsial survival and growth in mouse L929 cells. The rickettsia inhibitory activity in concanavalin A-induced mouse lymphokines was destroyed by heating the lymphokines at 80 degrees C for 10 min or by holding the lymphokines at pH 2 for 24 h but was retained after heating at 56 degrees C for 30 min.

The incidence of rickettsioses in Togo was examined by means of the microagglutination-technique according to Giroud. Out of 559 sera obtained from different parts of the country the following percentages of positive reactions were found: R. prowazeki 0.2%, R. mooseri: 4.7%, R. conori: 0.4%, R. burneti: 2.0%. It was proved that 1) R. mooseri-infections were more prevalent in adults than in children and adolescents and 2) R. mooseri-infections were found more frequently in the southern regions of the country, whereas R. burneti-infections were more widely distributed in the north of Togo.

The authors infected lambs with R. prowazeki and R. canada to ascertain their possible role in the natural infection of the animals. The lambs were infected subcutaneously with increasing doses; rickettsiemia was recorded with the aid of tests on guinea pigs and Ixodidae and Argasidae ticks fed on the lambs. Dynamics of antibody formation was ascertained in the infected animals in the agglutination reaction and in the complement fixation test. The antigenic affinity of R. canada and rickettsia of the typhoid group and the presence of common antigenic determinants with the Proteus OX19 was confirmed. The absence of any clinical manifestations, the character of antibody formation, impossibility of inducing the generalized infection and of the isolation of the causative agent from the blood pointed to the low susceptibility of lambs to R. prowazeki and R. canada; thus a possibility of circulation of the causative agents of typhius among the domestic animals scarcely probable.

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.

From January 1976 through January 1979 serum specimens from 1,575 individuals were received at the Center for Disease Control and tested for antibodies to rickettsiae. Of these, sera from eight persons gave serological results indicative of recent infections with epidemic typhus rickettsiae (Rickettsia prowazekii). Five of the persons were from Georgia, and one each was from Tennessee, Pennsylvania and Massachusetts. The illnesses occurred during the winter, chiefly in persons living in a rural environment. The clinical picture was compatible with louse-borne epidemic typhus. There was no apparent contact with human body or head lice, and no cases occurred in patient contacts, indicating that infection was not associated with the classic man-louse-man cycle of epidemic typhus. Two of the eight patients had contact with flying squirrels suggesting that they became infected from this known extrahuman reservoir of R. prowazekii.

Asymptomatic typhus infection was regularly modelled in guinea pigs with increased resistance to Rickettsia prowazeki; this infection was characterized by the presence of immunological shifts in the blood and immunity to reinfection, persistenc of rickettsia in the organs of animals in the absence of pyretic reaction and periorchites. Modelling in guinea pigs of asymptomatic infection is also possible against the background of passive immunity created by specific immune serum obtained during convalescence. Investigations demonstrated that rickettsiocidal activity (in experiments on chick embryos) and antiinfectious activity (in experiments on guinea pigs) of the sera was caused by 7S-antibodies.

The avirulent Madrid E strain of Rickettsia prowazekii cultivated in chicken yolk sacs could be purified successfully with a Renografin density gradient method developed previously for Rickettsia typhi. Recovery during purification, viability, and lack of contamination with host cell components were similar for the two species, although yields of R. prowazekii per yolk sac were lower. Purified typhus rickettsiae provided satisfactory antigens in the complement fixation, Ouchterlony double-diffusion, and microagglutination tests. The retention of the typhus soluble group antigen during purification was readily demonstrated by complement fixation tests. However, removal of the soluble group antigen by ether treatment was not always adequate for the demonstration of type-specific particulate antigens. Heat-killed R. prowazekii cells gave higher serum microagglutination titers than untreated or formalized cells, a difference was noted for R. typhi cells. Although the protein profiles of whole cells and extracts of R. typhi and R. prowazekii on sodium dodecyl sulfate-polyacrylamide gels were relatively similar, a small but reproducible, difference in the electrophoretic mobilities of their malate dehydrogenases was detected. Purification of typhus rickettsiae on Renografin gradients has no apparent adverse effects on their metabolic or antigenic properties.

Three men native from Northern Africa, aged more than 40 years, but by their profession in France with narrow contacts to rats, present clinical signs of typhus. Serology (FCT and FAT) gives positive results with R. prowazeki and R. typhi which have a strait antigenic community. Following assays of absorption of the sera by the two antigens and light differences of titers we conclude that two cases are Brill-Zinsser illnesses, but one is really an autochtone case of murin typhus. The last affirmation is confirmed by the detection of specific IgM-antibodies.

The function of peritoneal macrophages from Rickettsia prowazeki-infected guinea pigs at various intervals of postinfection immunity was studied. The activity of macrophages in immunized animals was higher than in non-immunized ones; it was the highest in the period of the highest level of immunity and high levels of complement-fixing antibodies.