Avidity of IgG to Rickettsia prowazekii and the presence of specific IgM in blood sera for retrospective analysis of the 1998 epidemic typhus outbreak in Russia.

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.

[BRILL-ZINSER DISEASE AS A CONSEQUENCE OF RICKETTSIA PROWAZEKII PERSISTENCE IN PREVIOUSLY ILL WHO HAVE HAD EPIDEMIC TYPHUS (EPIDEMIOLOGIC ASPECTS)].

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.

[Spotted fever and the invention of its serodiagnosis and vaccination in the Austro-Hungarian army in World War I]. / Das Fleckfieber und die Erfindung seiner Serodiagnose und Impfung bei der k. u. k. Armee im Ersten Weltkrieg.

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.

Discovery of novel cross-protective Rickettsia prowazekii T-cell antigens using a combined reverse vaccinology and in vivo screening approach.

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.

Sylvatic typhus associated with flying squirrels (Glaucomys volans) in New York State, United States.

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.

Discovery of a protective Rickettsia prowazekii antigen recognized by CD8+ T cells, RP884, using an in vivo screening platform.

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.

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

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.

Serological evidence of typhus group rickettsia in a homeless population in Houston, Texas.

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).

[The role of Turkish physicians in the vaccination against typhus during the years of World War I]. / I. dünya savasi yillarinda tifüs asisinin uygulanmasinda turk hekimlerinin rolü.

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.

Survey for zoonotic rickettsial pathogens in northern flying squirrels, Glaucomys sabrinus, in California.

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.

Pathogenic rickettsiae as bioterrorism agents.

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.

Detection of a typhus group Rickettsia in Amblyomma ticks in the state of Nuevo Leon, Mexico.

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.

Development of Rickettsia prowazekii DNA vaccine: cloning strategies.

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.

Detection of Rickettsia prowazekii in body lice and their feces by using monoclonal antibodies.

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.

[An indirect hemagglutinin test for Rickettsia prowazekii cultivated by the Weigl method]. / Erytrotsytarnyi diagnostyum dlia RNGA z ryketsii provacheka, kul'tyvovanykh za metodom Veiglia.

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.

[Characteristics of recombinant fragments of the protective antigen SPA of epidemic typhus pathogens]. / Kharakteristika rekombinantnykh fragmentov protektivnogo antigena SPA vozbuditelia épidemicheskogo sypnogo tifa.

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.

Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting.

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.