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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.)

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.

After description of the medical institutions and epidemiological situations of the Austro-Hungarian army in World War I the provisions against spotted fever focused on louse control are discussed. The letter specified for the army had to be adjusted for the local populations. 1915 in the k.u.k. military service in Galicia Edmund Weil and Arthur Felix cultivated Proteus strains from urine of soldiers with spotted fever. As sera of such patients agglutinated these bacteria in considerable titers the investigators developed the reliable diagnostic "Weil-Felix-Test" used still today. In the same military area and time Rudolf Weigl invented the anal infection of lice. This enabled him to harvest a great amount of louse intestines containing the spotted fever Rickettsiae in their epithelial cells. Lots with defined numbers of intestines were homogenized, sterilized and used with success as vaccine for medical staff. This sort of vaccine still was used in World War II.

Rickettsial agents are some of the most lethal pathogens known to man. Among them, Rickettsia prowazekii is a select agent with potential use for bioterrorism; yet, there is no anti-Rickettsia vaccine commercially available. Owing to the obligate intracellular lifestyle of rickettsiae, CD8(+) T cells are indispensable for protective cellular immunity. Furthermore, T cells can mediate cross-protective immunity between different pathogenic Rickettsia, a finding consistent with the remarkable similarity among rickettsial genomes. However, Rickettsia T cell antigens remain unidentified. In the present study, we report an algorithm that allowed us to identify and validate four novel R. prowazekii vaccine antigen candidates recognized by CD8(+) T cells from a set of twelve in silico-defined protein targets. Our results highlight the importance of combining proteasome-processing as well as MHC class-I-binding predictions. The novel rickettsial vaccine candidate antigens, RP778, RP739, RP598, and RP403, protected mice against a lethal challenge with Rickettsia typhi, which is indicative of cross-protective immunity within the typhus group rickettsiae. Together, our findings validate a reverse vaccinology approach as a viable strategy to identify protective rickettsial antigens and highlight the feasibility of a subunit vaccine that triggers T-cell-mediated cross-protection among diverse rickettsiae., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.

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.

We tested sera from 176 homeless people in Houston for antibodies against typhus group rickettsiae (TGR). Sera from 19 homeless people were reactive to TGR antigens by ELISA and IFA. Two people had antibodies against Rickettsia prowazekii (epidemic typhus) and the remaining 17 had antibodies against Rickettsia typhi (murine typhus).

During the years of World War I, several severe typhus epidemics were seen in Erzurum and nearby cities. A total of 164 health officers, 125 of whom were physicians, struggled against the epidemic in the region but also they lost their lives due to typhus. Vaccination against typhus was one of the means of fighting the epidemic. However, there were some claims that a small group of Turkish physicians injected typhus-contaminated serum into Armenian civilians during World War I, and that this should be accepted as a form of biological warfare against Armenian civilians. The purpose of this article is to set out how, by whom, and on whom, and under what conditions the typhus vaccination was applied in order to reveal the truth in terms of the evidence found in historical documents. The typhus vaccine was prepared from blood taken from febrile patients affected by the disease. After the blood of the patients were defibrinated and inactivated at 60 degrees C for an hour, it was used. As the amount of blood needed to prepare the vaccine was so great, the amount of available vaccine was always insufficient to meet the demand. Hence, the prepared vaccine was only applied to those which had the higher risk of contracting typhus such as physicians and nurses. The vaccine prepared by The Third Army Health Commander Dr. Tevfik Salim was first applied to nine officers, five of whom were physicians, and among whom were Dr. Haydar Cemal and Dr. Salahattin on March 28, 1915 in Hasankale, Erzurum. Furthermore, the same vaccine was applied to people in the vicinity by Dr. Alaattin in Erzurum, Dr. Abdulhalim Asim in Bayburt, Dr. Izak in Sivas and Dr. Mihran in Hasankale. Ali Ihsan Sabis and Fevzi Cakmak, who were high ranking officers, were among those who volunteered to have the vaccination. The Third Army Health Commander Dr. Tevfik Salim ordered that the vaccine should not be applied without blood inactivation. Despite this order, Dr. Hamit Osman, who had a mental illness, applied the vaccination without inactivating the blood to some people. Among those were physicians of the Red Crescent Hospital together with soldiers who were nursing in the hospitals in Erzincan. Dr. Hamdi Suat inactivated the blood by leaving it at -16 degrees C for 24-48 hours, and instead of giving a single dose, he applied three-doses with 3-day-intervals, followed by a one more dose, which he called "the vaccine for absolute immunization" to the same people after 10-23 days. This "vaccine for absolute immunization" was actually typhus-contaminated blood which had not been inactivated. It should be noted that he injected himself with the same form of vaccine. In his article published in German in 1916 and in Turkish in 1917, he stated that he injected "the vaccine for absolute immunization" to some subjects 'condemned to death'. Dr. Haydar Cemal claimed, in a newspaper dated December 23, 1918, that the people reported as subjects 'condemned to death' were indeed Armenians, and that the innocent Armenians marked out for deportation were inoculated with the blood of typhus fever patients, and that he eyewitnessed all these events. As a result of his claims, the Interior Ministry demanded an immediate investigation, and at the end of that investigation it was understood that Dr. Haydar Cemal and Dr. Hamdi Suat had never worked together in Erzincan at the time Dr. Haydar Cemal claimed. All the claims were refuted by the investigating committee and nobody was charged. During a severe typhus epidemic, Turkish physicians injected the typhus vaccine for the purpose of "saving a life from the fire". The typhus vaccine was prepared using the available scientific knowledge of the time. No racial or religious discrimination against the people vaccinated had been proved. According to the sources, the claim that some Turkish physicians used the blood of patients with typhus as a means of biological warfare does not reflect the historical truth.

Epidemic typhus, caused by Rickettsia prowazekii, is maintained in a southern flying squirrel (Glaucomys volans) sylvatic cycle in the southeastern United States. The northern flying squirrel (Glaucomys sabrinus) has not been previously associated with R. prowazekii transmission. A second rickettsial pathogen, Anaplasma phagocytophilum, infects dusky-footed woodrats (Neotoma fuscipes) and tree squirrels in northern California. Because northern flying squirrels or their ectoparasites have not been tested for these rickettsial pathogens, serology and polymerase chain reaction (PCR) were used to test 24 northern flying squirrels for R. prowazekii and A. phagocytophilum infection or antibodies. Although there was no evidence of exposure to R. prowazekii, we provide molecular evidence of A. phagocytophilum infection in one flying squirrel; two flying squirrels also were seropositive for this pathogen. Fleas and ticks removed from the squirrels included Ceratophyllus ciliatus mononis, Opisodasys vesperalis, Ixodes hearlei, Ixodes pacificus, and Dermacentor paramapertus.

Because of their unique biological characteristics, such as environmental stability, small size, aerosol transmission, persistence in infected hosts, low infectious dose, and high associated morbidity and mortality, Rickettsia prowazekii and Coxiella burnetii have been weaponized. These biological attributes would make the pathogenic rickettsiae desirable bioterrorism agents. However, production of highly purified, virulent, weapon-quality rickettsiae is a daunting task that requires expertise and elaborate, state-of-the art laboratory procedures to retain rickettsial survival and virulence. Another drawback to developing rickettsial pathogens as biological weapons is their lack of direct transmission from host to host and the availability of very effective therapeutic countermeasures against these obligate intracellular bacteria.

The state of Nuevo Leon, Mexico has had outbreaks of typhus group rickettsiosis, most recently recognized in 1997. Evaluation of the sera of 345 patients with a dengue-like illness revealed that 25.5% had antibodies reactive with typhus group rickettsiae and 16% had antibodies to Rickettsia parkeri. Rickettsiae were detected by PCR and shell-vial isolations in the field-collected Amblyomma ticks. Molecular characterization by DNA sequence analysis of the gltA, ompB, and 17-kDa gene identified the organisms to be R. prowazekii.

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.

In order to identify Rickettsia prowazekii in lice, we developed a panel of 29 representative monoclonal antibodies selected from 187 positive hybridomas made by fusing splenocytes of immunized mice with SP2/0-Ag14 myeloma cells. Immunoblotting revealed that 15 monoclonal antibodies reacted with the lipopolysaccharide-like (LPS-L) antigen and 14 reacted with the epitopes of a 120-kDa protein. Only typhus group rickettsiae reacted with the monoclonal antibodies against LPS-L. R. felis, a recently identified rickettsial species, did not react with these monoclonal antibodies, confirming that it is not antigenically related to the typhus group. Monoclonal antibodies against the 120-kDa protein were highly specific for R. prowazekii. We successfully applied a selected monoclonal antibody against the 120-kDa protein to detect by immunofluorescence assay R. prowazekii in smears from 56 wild and laboratory lice, as well as in 10 samples of louse feces infected or not infected with the organism. We have developed a simple, practical, and specific diagnostic assay for clinical specimens and large-scale epidemiological surveys with a sensitivity of 91%. These monoclonal antibodies could be added to the rickettsial diagnostic panel and be used to differentiate R. prowazekii from other rickettsial species.

The technology of preparing of a new ready for use diagnosticum for IHAT on the basis of polysaccharide of Rickettsia prowazekii cultivated by the Weight method has been developed. Technological conditions have been worked out, experimental series of the diagnosticum have been made and tested, high stability during the storage was confirmed which allows it to be recommended for the epidemic typhus laboratory diagnostics.

Fragments of a gene for species-specific protective antigen SPA of Rickettsia prowazekii earlier cloned in lambda gt11 were recloned into the in-frame expression vector pQE30. Polypeptides encoded by these fragments were shown to be synthesized in Escherichia coli with a yield of up to 100 micrograms/ml of culture and to be accumulated in the cells as inclusion bodies. The partially purified antigens were used in enzyme immunoassay with the sera of humans convalescing from epidemic typhus, tick-borne rickettsioses, and other infectious diseases. One of two recombinant proteins was shown to react in immunoblotting and ELISA with homologous, but not with heterologous, sera. The immunoreactivities in ELISA of the recombinant antigens and heat-denatured SPA proved to be similar, but substantially lower than that of the native SPA. These data as well as the data of other investigators show that serodiagnostics of epidemic typhus using recombinant antigens remains a problem.

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.

The analysis of the results of prolonged observations on the prophylactic immunization of employees working with R. prowazekii is presented. The necessity of the differentiated approach to the determination of the immunization schedule and the choice of vaccine is shown. The presence of specific antibodies (Ab) and the level of their titers have been found to be related to the degree of anti-infectious protection. The following characteristics indicate the presence of profound immunological transformation in vaccinees: complement-fixing Ab in titers 1:10 and more and/or immunofluorescent Ab in titers not below 1:180, Ab to protein in the hemagglutination test in titers not below 1:1000. These specific Ab and the level of their titers can be registered after the second injection of live combined typhus vaccine E and the third injection of chemical typhus vaccine. Cases of laboratory infection and their relationship to the character of immunization and the intensity of contacts with R. prowazekii virulent strains are discussed. Attention is drawn to the strict observance of professional safety rules.

Background: After a 12-year absence, epidemic typhus has re-emerged among the displaced population of Burundi. Following the outbreak of civil war in 1993, over 760000 people now inhabit refugee camps, under appalling conditions. A typhus outbreak occurred among prisoners in a jail in N'Gozi in 1995. At the time, the disease was not recognised, and was referred to as sutama. Reports of sutama among the civilian population date back to late 1995 and, in association with body-louse infestation, the disease has subsequently swept across the higher and colder regions of the country., Methods: During a field study in February, 1997, 102 refugees with sutama underwent clinical examination and interview. Serum samples were collected and infesting body lice removed. Microbiological analysis included antibody estimations and specific PCRs aimed at diagnosis of Rickettsia prowezekii, Bartonella quintana, and Borrelia recurrentis. Between January and September, 1997, nationwide epidemiological data on the prevalence and distribution of sutama was obtained through liaison with local health services. A second field study in March, 1997, entailed the collection of further serum samples from suspected cases of sutama in different regions of Burundi., Findings: Most of the 102 patients with sutama during initial assessment presented with manifestations similar to those previously described for typhus in Africa, though skin eruptions occurred in only 25 (25%) cases. Microbiological testing revealed evidence of R prowazeki infection in 76 (75%) patients, confirming that most cases of clinically-diagnosed sutama were epidemic typhus, and supporting the reliability of clinical diagnosis as a basis for the nationwide surveillance of the disease. Up to September, 1997, 45558 typhus cases were clinically diagnosed, most of which occurred in regions at an altitude of over 1500 m. Serological testing of 232 individuals from different regions of Burundi provided microbiological evidence to support clinical diagnoses in seven provinces, confirming the widespread nature of the outbreak. Serum from 13 of the original 102 patients and 19 (8%) of the 232 suspected cases had raised antibody titres against B quintana. A fatality rate of 15% among jail inmates fell to 0.5% after administration of a single dose of 200 mg doxycycline to suspected cases., Interpretation: A gigantic outbreak of R prowazekii-induced typhus and B quintana-induced trench fever is continuing in Burundi. Transmission of both diseases to such a large number of people has followed a widespread epidemic of body-louse infestation. Diagnosis of typhus could be reliably made by means of clinical criteria, and the disease could be efficiently and easily treated by antibiotics. This epidemic highlights the appalling conditions in central-African refugee camps and the failure of public-health programmes to serve their inhabitants. Louse-associated disease remains a major health threat in this and other war-torn regions of the world.

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-D-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (beta-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.

Species-specific antigenic erythrocyte diagnosticum (SAED) for the detection of antibodies to R.prowazekii was developed. The cells used as the basis for the preparation of the antigenic erythrocyte diagnosticum were formulated and tanned sheep red blood cells (FT-SRBC). Original indication antigens, solubilized with Triton X-100 from common R.prowazekii antigen for the complement fixation test, the antigen neutralization test and the fluorescent analysis, were used as sensitin. The optimum parameters for sensitizing FT-SRBC were established. The SAED thus obtained was stabilized by the method of lyophilization. In a separate series of experiments the specificity and activity of SAED were studied in comparison with those of preparations obtained with the use of different sensitins. The newly developed SAED was shown to be highly specific.

In this article materials on obtaining rickettsial liposomal preparations on the basis of R.prowazekii phospholipid antigen and chemical typhus vaccine are presented. Electron microscopic studies revealed the formation of liposomes in this system. Liposomal preparations were not hemotoxic and produced no toxic action in experiments on guinea pigs and white mice. Phospholipid antigens incorporated into liposomal preparations were found to produce a potentiating effect on the immunogenic properties of chemical typhus vaccine. The adjuvant properties of rickettsial phospholipids incorporated into liposomes on the basis chemical typhus vaccine were registered.

A monoclonal antibody directed against an epitope on the lipopolysaccharide of typhus-group rickettsiae was developed for the purpose of detecting this heat-stable, proteinase-resistant antigen in formalin-fixed, paraffin-embedded tissues. Rickettsia prowazekii organisms were identified in endothelium and macrophages in sections of the brains of three Egyptian men who died of epidemic louse-borne typhus in Cairo during World War II and in the brain from a recent case of typhus fever acquired in Burundi. R. typhi organisms were identified in endothelial cells from a fatal case of murine typhus and in experimentally infected mice. This approach is applicable not only to the study of archival tissues and experimental animal models but also could be used to establish a timely diagnosis of typhus-group rickettsiosis by immunohistochemical examination of cutaneous biopsies of rash lesions during the acute stage of illness.

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.

Surveys designed to study spread of typhus infection (persistent form and Brill's disease), and state of laboratory diagnosis in Ukraine showed an important role the laboratory diagnosis plays in the system of epidemiological surveillance aimed at preventing epidemic typhus. Main trends of activities on perfection of the system of laboratory control of the infection under present-day conditions are outlined.

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.

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were synthesized on derivatized polyethylene pins. A modified enzyme-linked immunosorbent assay was used to identify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epitopes were mapped by this method in three regions. Four of the epitopes, which were located in the carboxyterminus of mature processed SPA, were strongly competitively inhibited by native folded SPA but not by intact rickettsiae, suggesting that they were on the SPA surface but not exposed on the rickettsial surface. Three of these epitopes were present on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further characterized by synthesizing modified peptides. Glycine substitution experiments determined the critical residues in the epitopes. The dependence of binding of the peptide epitopes to the polyclonal antisera was mapped to single residues. The limited number and weak reactivity of linear peptide epitopes observed with human and rabbit sera, possibly due to a lack of the methylated amino acids which are present in rickettsia-derived SPA, suggest that the present approach will not provide useful synthetic antigens for diagnosis of typhus infections.

The prevalence of rickettsial antibodies in north-western part of Bosnia and Herzegovina was studied. Among 231 sera tested by complement fixation (CF) positive were: 61.5% for Rickettsia typhi, 4.3% for R. prowazekii, 1.7% for R. conorii and 19.0% for Coxiella burnetii. Of 183 sera tested by indirect immunofluorescence assay (IFA) 37.7% reacted with R. typhi, 1.6% with R. conorii and 22.4% with C. burnetii. The results show that at least R. typhi and C. burnetii are highly endemic in this area.

An unusual pattern of seroreactivity to antigens of rickettsial organisms (Rickettsia rickettsii, R rhipicephali, R montana, and R bellii), particularly to R bellii antigen, was detected in 3 dogs during a 2-month period. Thus, studies were initiated to clarify the pathogenic potential of the more distantly related rickettsial organisms (R canada and R prowazekii) in dogs. Because R bellii are nonpathogenic rickettsiae that share numerous common properties with spotted fever-group and typhus-group rickettsiae, and because closely related pathogenic relatives of R bellii have not been identified, we examined the pathogenic potential of these typhus-group rickettsiae by testing stored serum samples, by attempting rickettsial isolation from febrile dogs, and by experimentally inoculating dogs with R canada and R prowazekii. Evaluation of results of a serosurvey of acute and convalescent serum samples from 80 dogs in which Rocky Mountain spotted fever had been considered as a differential diagnosis, but seroconversion to R rickettsii had not been documented, identified 1 dog with a fourfold increase in antibody titer to R rhipicephali and 3 dogs with fourfold increases in antibody titer to 1 or more antigens of typhus-group rickettsial organisms. A study of 15 dogs that were febrile during summer months failed to identify serologic or tissue culture evidence of typhus-group rickettsial infection or typhus-group rickettsemia, but did result in isolation of R rickettsii and Ehrlichia canis, respectively, from 1 dog each. In our final study, after experimentally inoculating 6 dogs with R canada and R prowazekii, all dogs seroconverted to the respective rickettsiae, but rickettsemia or clinical and hematologic evidence of disease was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper provides an overview of the development of typhus vaccines between the first and second world wars. It is shown that there was a shift in the classification of the causal Rickettsiae from being classed as bacteria to being conceptualised as a type of virus. This 'paradigm switch' stimulated interest in the possibility of producing an effective medicine.

Two strains of Coxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time from Ixodes ricinus ticks collected in the Alpine region of Tirol, Austria. The C. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes with C. burnetii and R. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.

Five strains of Rickettsia prowazekii different in origin, biological and genetic properties were compared in protein and LPS patterns by the polyacrylamide gel electrophoresis and in antigenic properties by immunoblotting with specific sera of infected white mice. Three virulent strains Breinl, G and Katsinjan had identical protein patterns and differed from isogenic pair of strains E and EVir in the electrophoretic properties of 29-30 kDa proteins. Silver-strained LPS patterns were different in five compared strains. Strain G and strain Katsinjan had the longest O-chaines of LPS. Polyclonal mouse antisera contained specific antibodies which mainly directed against LPS and 25-60 kDa proteins. Strains E and EVir were identical in all performed immunoblotting reactions and separated from three virulent strains. Out of virulent strains, whole cell antigen of strain Katsinjan and LPS antigen of strain G had different reactions in comparison with correspondent antigen of the standard strain Breinl.

The article substantiates epidemiological expediency of complex (associated) immunization of servicemen and population against typhoid, typhus and plague in polyetiological zones of these infections, and also in cases of simultaneous proliferation of these diseases. For simultaneous preventive vaccination against these infections a complex immunization scheme was experimentally substantiated and clinically approved. It is based on national commercial vaccines and ensures a simultaneous administration of 2-3 vaccine preparations by hypodermic syringe or jet injection. Typhoid and typhus vaccines are injected under one shoulder-blade, and plague vaccine is injected under another shoulder-blade. This complex vaccine is harmless, moderately reactogenic, develops expressing immunity which have the same protective features as monovaccines alone. This scheme is recommended for use in anti-epidemic practice.

Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species of the spotted fever group (SFG) rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragments of R. prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsugamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization patterns with TG species and R. akari. PBH11, PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intraspecies patterns were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification.

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.

The immunogenicity of the Coxiella burnetii corpuscle antigen and two its components, obtained after the treatment with chloroform-methanol or extraction by trichloroacetic acid, was investigated. All the studied antigens were highly immunogenic. A strong correlation was discovered between the immunogenic and immunomodulating properties of the C. burnetii antigens. Cells of the mononuclear phagocytic system are the targets for stimulating action of the studied antigens. A cyclic increase of antibacterial activity and oxygen-dependent metabolism of the peritoneal cells was noted, in addition to the increase of the natural splenocyte cytotoxicity caused by the action of these antigens. The role of cytokines in rising the activity of peritoneal cells and splenocytes, caused by immunization of mice with the above antigenic substances, is discussed.

With the use of the unified indirect solid-phase fluorescent enzyme immunoassay the combined evaluation of the antigenic and immunogenic properties of experimental whole-virion inactivated virus vaccines against Venezuelan and eastern equine encephalomyelitides, as well as of dried chemical typhus vaccine, has been made; their safety was determined indirectly by the content of ovalbumin. The protective role of antibodies evaluated by the solid-phase fluorescent enzyme immunoassay in typhus immunity has been shown.

The data of the comparative study of the immunomodulating action of donor interferon and reaferon in experiments with typhus vaccine, used as an experimental model, on monkeys of different species and guinea pigs are presented. One of the main properties of interferons, besides their antiviral activity, is their capacity for producing an immunomodulating effect.

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.

A serological study of 92 patients with rickettsiosis, revealed in the blood serum of 19 patients antibodies to the pathogens of Q-fever or typhoid fever. In three patients the character of distribution of immunoglobulins of different classes corresponded to the chronic course of rickettsia infection. Patients of the therapeutic profile from infections foci should be examined for rickettsiosis.

Blood of 56 guinea pigs with experimental Q rickettsiosis was studied cytochemically (lysosomal cationic test) to measure the level of cationic proteins in neutrophil granulocytes. Development of Q rickettsiosis resulted in a decrease in the killing ability of neutrophils, depending on infection dose introduced. However, by day 7 of the disease, the level of cationic proteins in blood neutrophil granulocytes returned to the initial range. Similar situation was noted after subcutaneous injection of Coxiella burnetti corpuscular antigen. Subcutaneous infection with the living culture stimulus induced the wave-like decrease of the cationic proteins content. Infection of pre-immunized animals led to smaller decrease in the cationic proteins levels and to their more rapid recovery. Aspects of antimicrobial activity of neutrophil granulocyte cationic proteins in experimental Q rickettsiosis is discussed.