Search

Over the last decades, rickettsioses are emerging worldwide. These diseases are caused by intracellular bacteria. Although rickettsioses can be treated with antibiotics, a vaccine against rickettsiae is highly desired for several reasons. Rickettsioses are highly prevalent, especially in poor countries, and there are indications of the development of antibiotic resistance. In addition, some rickettsiae can persist and cause recurrent disease. The development of a vaccine requires the understanding of the immune mechanisms that are involved in protection as well as in immunopathology. Knowledge about these immune responses is accumulating, and efforts have been undertaken to identify antigenic components of rickettsiae that may be useful as a vaccine. This review provides an overview on current knowledge of adaptive immunity against rickettsiae, which is essential for defense, rickettsial antigens that have been identified so far, and on vaccination strategies that have been used in animal models of rickettsial infections., Competing Interests: The authors have declared that no competing interests exist.

Arthropod-borne obligately intracellular bacteria pose a difficult challenge to the immune system. The genera Rickettsia, Orientia, Ehrlichia, and Anaplasma evolved mechanisms of immune evasion, and each interacts differently with the immune system. The roles of CD8 T cells include protective immunity and immunopathology. In Rickettsia infections, CD8 T cells are protective mediated in part by cytotoxicity toward infected cells. In contrast, TNF-α overproduction by CD8 T cells is pathogenic in lethal ehrlichiosis by induction of apoptosis/necrosis in hepatocytes. Yet, CD8 T cells, along with CD4 T cells and antibodies, also contribute to protective immunity in ehrlichial infections. In granulocytic anaplasmosis, CD8 T cells impact pathogen control modestly but could contribute to immunopathology by virtue of their dysfunction. While preliminary evidence indicates that CD8 T cells are important in protection against Orientia tsutsugamushi, mechanistic studies have been neglected. Valid animal models will enable experiments to elucidate protective and pathologic immune mechanisms. The public health need for vaccines against these agents of human disease, most clearly O. tsutsugamushi, and the veterinary diseases, canine monocytotropic ehrlichiosis (Ehrlichia canis), heartwater (Ehrlichia ruminantium), and bovine anaplasmosis (A. marginale), requires detailed immunity and immunopathology investigations, including the roles of CD8 T lymphocytes.

The vascular endothelium is the main target of a limited number of infectious agents, Rickettsia, Ehrlichia ruminantium, and Orientia tsutsugamushi are among them. These arthropod-transmitted obligately-intracellular bacteria cause serious systemic diseases that are not infrequently lethal. In this review, we discuss the bacterial biology, vector biology, and clinical aspects of these conditions with particular emphasis on the interactions of these bacteria with the vascular endothelium and how it responds to intracellular infection. The study of these bacteria in relevant in vivo models is likely to offer new insights into the physiology of the endothelium that have not been revealed by other models.

Rickettsiae are obligate intracellular alpha-proteobacteria that primarily target the microvascular endothelium. In the last two decades, new rickettsial pathogens have been associated with human illness around the world. Clinically, the common denominator in all rickettsioses is the development of increased microvascular permeability, leading to cerebral and non-cardiogenic pulmonary edema. With the development of powerful research tools, advances in the understanding of rickettsial pathogenesis have been dramatic. Entry into the host cell is followed by rapid escape into the cytoplasm to avoid phagolysosomal fusion. Spotted fever group rickettsiae induce actin polymerization via a group of proteins called RickA, which promote nucleation of actin monomers via the Arp2/3 complex at one rickettsial pole, propelling the bacteria across the cytoplasm and into neighboring cells. Damage to the host cell is most likely multifactorial. The most extensively studied mechanism is the generation of reactive oxygen species (ROS) and downregulation of enzymes involved in protection against oxidative injury. The significance of ROS-mediated cellular damage in vivo is beginning to be elucidated. The main pathogenic mechanism is increased microvascular permeability leading to profound metabolic disturbances in the extravascular compartment. The underlying factors responsible for those changes are beginning to be elucidated in vitro and include direct effects of intracellular rickettsiae, cytokines, and possibly activated coagulation factors--all of which most likely modify interendothelial junctions. Our knowledge on rickettsial pathogenesis will continue to expand in the near future as new research tools become available.

The paper discusses the proven and probable role of different tick-borne pathogens and their combinations in the occurrence of zoonotic diseases. The variants of coexistence of various combinations of 7 proved disease agents (3 species of Borrelia, 2 species of Ehrlichia, Babesia, and TBE virus) with different, if might to be, disease agents, such as rickettsiae of two groups, which do not belong to the Borrelia burgdorferi sensu lato group spirochetes, are analyzed. The difference of their development and the time of transmission after of the vector Ixodes persulcatus Schulze attachment are considered. Based on the their own hypothesis that one tick specimen is able to be the vector of only three human pathogenic agents simultaneously, the authors examined the possible number of combinations of pathogens, which might be obtained by a patient after 1 tick attachment. Proper history data are recommended as a tool for making a correct diagnosis, wherein the time of attachment has to be a main factor. For example, it should be taken into account that TBE virus might be transmitted only some minutes after attachment by not only a female tick, but also a male one. Mixed infection of virus plus Borrelia might develop only following some hours of attachment (most probably after 18-24 hours). The very similar situation has to be considered to lead to virus-Borrelia-Ehrlichia (or Rickettsia) coinfection. Any combination with Babesia involvement may appear not earlier than 24-36 hours after mixed infected tick attachment (or even later). It is necessary to consider a possible role of apathogenic microorganisms which are present in the most of vector specimens in nature and which might be injected with infected tick saliva may play a possible role in the manifestation of disease and in the prediction of their possible influence.

Filarial nematodes harbour intracellular, Gram-negative bacteria belonging to the genus Wolbachia. These bacteria have been observed in various species of filariae, including the main filariasis agents of humans and animals. It has been suggested that Wolbachia could play an important role in the biology of filarial nematodes and could be implicated in the pathogenesis of filarial diseases. Wolbachia could thus represent a target for the control of filariasis and key to the understanding of these diseases. Indeed, in various species of filariae, tetracycline treatments have been shown both to reduce/eliminate the Wolbachia population and to determine detrimental effects on the nematodes. In addition, proteins of Wolbachia have been shown to determine specific IgG responses in animals infected by filariae and some Wolbachia molecules (e.g. LPS) have been shown to stimulate innate-immunity responses (e.g. production of cytokines such as IL1, IL6, IL10, TNF-alpha and IFN-gamma by macrophages).

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmonis was consistently concentrated in a visible band within the DMDS density gradient at density of 1.15 to 1.16 g ml(-1). Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Four strains of Orientia tsutsugamushi (KN-1, KN-2, KN-3 and GJ-1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN-1 (identified as Kawasaki type), GJ-1 (identified as Kuroki type) and KN-2 strains were found to be non-lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (10(6) x being the 50% mouse infectious dose). On the other hand, the KN-3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN-1 and KN-2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN-3, Karp and Kato strains. After infection with the GJ-1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.

Ixodid ticks were collected from Connecticut, Massachusetts, Missouri, Pennsylvania, Rhode Island, and British Columbia (Canada) during 1991 to 1994 to determine the prevalence of infection with hemocytic (blood cell), rickettsia-like organisms. Hemolymph obtained from these ticks was analyzed by direct and indirect fluorescent antibody (FA) staining methods with dog, horse, or human sera containing antibodies to Ehrlichia canis, Ehrlichia equi, or Rickettsia rickettsii. Of the 693 nymphal and adult Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Ixodes pacificus ticks tested with dog anti-E. canis antiserum, 209 (32.5%) contained hemocytic bacteria. The prevalence of infected ticks varied greatly with species and locale. In parallel tests of duplicate hemolymph preparations from adult I. scapularis ticks, the hemocytic organisms reacted positively with E. canis and/or E. equi antisera, including sera from persons who had granulocytic ehrlichiosis. In separate PCR analyses, DNA of the agent of human granulocytic ehrlichiosis was detected in 59 (50.0%) of 118 adult and in 1 of 2 nymphal I. scapularis ticks tested from Connecticut. There was no evidence of Ehrlichia chaffeensis DNA in these ticks. In indirect FA tests of hemolymph for spotted fever group rickettsiae, the overall prevalence of infection was less than 4%. Specificity tests of antigens and antisera used in these studies revealed no cross-reactivity between E. canis and E. equi or between any of the ehrlichial reagents and those of R. rickettsii. The geographic distribution of hemocytic microorganisms with shared antigens to Ehrlichia species or spotted fever group rickettsiae is widespread.

Cytoplasmic incompatibility (CI) in Drosophila is related to the presence of Wolbachia, an intracellular microorganism found in many species of insects. In order to study the intracellular localization of Wolbachia in eggs and embryos, we have purified the bacteria from fly embryos and subsequently generated a monoclonal antibody (Mab Wol-1) specific for Wolbachia. Indirect immunofluorescence staining using Wol-1 reveals that during mitosis, Wolbachia are localized near spindle poles and centrosomes. Double label immunofluorescence experiments using anti-tubulin and anti-Wolbachia antibodies show that Wolbachia co-localize with centrosomal microtubules throughout the cell cycle. Direct interactions between the bacteria and centrosome-organized microtubules are implied from seven observations: (1) throughout the mitotic cycle, the position and movement of Wolbachia precisely mimic the behavior of the centrosome and apparently associated with centrosome-organized microtubules; (2) Wolbachia segregate equally to each spindle pole during mitosis; (3) Wolbachia do not associate with spindle microtubules during mitosis; (4) Wolbachia located in the egg cortex localize to the domains of cytoplasm organized by microtubules during blastoderm formation; (5) polar body nuclei that lack centrosomes but contain associated microtubules do not contain Wolbachia; (6) Wolbachia no longer associated with yolk nuclei, following differentiation and loss of centrosomes; (7) during pole cell formation, Wolbachia co-localize with the centrosome on the apical side of the nucleus as pole cells form. Quantitative data indicates that no Wolbachia growth occurs during the preblastoderm period even though rapid nuclear, and subsequent cellular, proliferation takes place during this same period. This indicates that Wolbachia are under strict growth regulation by the host suggesting that host factors play a role in regulating growth of Wolbachia in the egg. Further cellular and molecular studies of the extensive, global interactions between host and symbiont observed in this egg should provide important new insights into the evolution of host/symbiosis and the cell biology of cytoplasmic incompatibility.

Rochalimaea henselae, a recently described pathogen thought to cause syndromes as varied as bacillary angiomatosis, parenchymal bacillary peliosis, fever with bacteremia, and cat-scratch disease, is associated with CNS diseases including cerebral and retinal bacillary angiomatosis, as well as cat-scratch-related encephalitis, myelitis, cerebral arteritis, and retinitis. We used a newly developed enzyme immunoassay and the polymerase chain reaction to investigate the association of R henselae infection with HIV-related CNS disease and found that whereas seroprevalence rates in HIV-positive patients unselected for neurologic disease were 4% to 5.5%, those with neurologic disease had seroprevalence rates of 32%. The ratio of organism-specific antibodies in CSF compared with serum suggested intra-blood-brain-barrier synthesis of these antibodies. CSF specimens containing only R henselae IgM had 16S rDNA specific for R henselae. Stored serum from one of these patients indicated he had developed R henselae-reactive IgM antibodies 10 months prior to the onset of neurologic disease. In the 14 patients for whom clinical data were available, evidence of CNS invasion by R henselae was accompanied by acute and subacute mental status changes including hallucinations, disorientation, and rapidly progressive dementia.

BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.

Three patients with atypical manifestations of cat-scratch disease (CSD) had the diagnosis supported by a newly available serologic assay for antibodies to Rochalimaea henselae. Previously, CSD has been a diagnosis of exclusion because no confirmatory test was readily available. Atypical or severe cases of CSD have often required detailed (and expensive) clinical and laboratory investigation. The use of Rochalimaea-specific serologic tests may help avoid extensive diagnostic testing or invasive procedures in such cases.

An immunocompetent 9-year-old boy with disseminated cat scratch disease involving spleen, cervical and abdominal lymph nodes, skull, and one clavicle is reported. Antibodies to Rochalimaea quintana and R. henselae were detected, at increasing, then decreasing concentration. DNA extracted from the biopsied skull lesion was amplified by polymerase chain reaction and hybridized with species-specific oligonucleotides proving the presence of R. henselae in affected tissue. Our findings suggest that R. henselae plays a pathogenic role in cat-scratch disease.

Background: Although cat scratch disease is commonly diagnosed in patients who have unexplained regional lymphadenopathy after encounters with cats, its epidemiology and the risk factors for disease are not clearly defined, and there is no generally accepted diagnostic test., Methods: We conducted a physician survey to identify cases of cat scratch disease occurring over a 13-month period in cat owners in Connecticut. We interviewed both the patients (or their parents) and controls matched for age who owned cats. Serum from the patients was tested for antibodies to Rochalimaea henselae with a new, indirect fluorescent-antibody test., Results: We identified 60 patients with cat scratch disease and 56 age-matched subjects. Patients were more likely than controls to have at least one pet kitten 12 months old or younger (odds ratio, 15), to have been scratched or bitten by a kitten (odds ratio, 27), and to have had at least one kitten with fleas (odds ratio, 29). A conditional logistic-regression analysis found that in kitten-owning households, patients were more likely than controls to have been scratched or bitten by a cat or kitten (odds ratio, 12.4; 95 percent confidence interval, 1.0 to 150). Of 45 patients, 38 had serum samples with titers of 1:64 or higher for antibody to R. henselae, as compared with 4 of 112 samples from controls (P < 0.001). The positive predictive value of the serologic test was 91 percent. Of 48 serum samples from patients' cats, 39 were positive for antibodies to R. henselae, as compared with positive samples from 11 of 29 control cats (P < 0.001)., Conclusions: Cat scratch disease is strongly associated with owning a kitten, and fleas may be involved in its transmission. The serologic test for rochalimaea may be useful diagnostically, and our results suggest an etiologic role for this genus.

To determine the current prevalence of antibody to arboviruses, rickettsiae, and hantaan-like viruses, a survey was carried out in the Nile River Valley of Egypt, one of the principal foci of the 1977-1978 Rift Valley fever (RVF) outbreak. Blood specimens were obtained from 915 persons representing 190 study households. Enzyme immunoassay testing showed that the overall prevalence of IgG antibody was 4% to sand fly fever Sicilian (SFS), 2% to sandfly fever Naples (SFN), 15% to RVF, 20% to West Nile, and 4% to Hantaan (HTN) viruses. Antibody was demonstrated among 32% of the same study subjects to Coxiella burnetii, 58% to Rickettsia typhi, and 32% to R. conorii. The prevalence of agent-specific antibody tended to increase with age. Particularly notable was the low prevalence of RVF infection in children born after the height of the RVF outbreak. No detectable antibodies were found in the population less than seven years of age and in only 3% of those 7-12 years old. In contrast, 26% of the study population 13-19 years old, who were young children and infants at the time of the outbreak, were found to have RVF antibodies, suggesting that the level of intensity associated with transmission decreased considerably following the documented 1977-1978 outbreak. Geometric mean titers (GMT) ranged from 139 for C. burnetii to 1,305 for RVF, and did not vary significantly by age, except for high titers for RVF in the 20-49-year-old age group. A significant upward trend in GMT was also noted when antibody was detected in the specimen for more than one phlebovirus.(ABSTRACT TRUNCATED AT 250 WORDS)

From 1987 to 1991, a seroepidemiologic survey for antibodies to hantaviruses, leptospira, rickettsiae, and Borrelia was conducted in selected Italian population groups. In the mountainous areas of northeastern Italy, the prevalence of antibody to hantaviruses, as detected by indirect immunofluorescent antibody (IFA) assay, was 7.1%, 4.8%, 4.3%, and 4% in 265 forestry workers, 82 rangers, 395 farmers, and 75 hunters, respectively. Among 299 Alpine soldiers, the prevalence was lower (0.7%). Of those with Hantaan antibody, the reactivity pattern using Hantaan, Puumala, and Fojnica viruses suggested a prevalence of antibody to Hantaan virus, with titers reaching levels of 128. The presence of leptospiral antibodies (by microagglutination test), which included the prevalence of antibodies to Leptospira icterohaemorrhagiae, L. bratislava, and L. saxkoening serotypes, was observed in 10-12% of the farmers and forestry workers in these Alpine mountain regions. Only a few sporadic clinical cases of leptospirosis have been reported from these regions. Antibodies to Borrelia burgdorferi (by IFA) were observed in 19% of the rangers and forestry workers, with lower values in farmers (10%) and hunters (8%). These data suggest the presence of a large number of asymptomatic infections with B. burgdorferi and the leptospires in the densely wooded areas of the Alpine Italian regions. Furthermore, the recent identification of a case of Hantaan acute nephropathy in a man living in the mountainous northeastern area of Italy confirms the presence of hantavirus in the Italian Alpine zones, especially those near the Slovenian border.

Rochalimaea henselae causes persistent bacteremia, bacillary angiomatosis, and parenchymal bacillary peliosis. Detection of a specific antibody response to R. henselae infection may represent an alternative to cultivation as a means of diagnosis. We assessed the specificity of induced murine antibodies for antigens from R. henselae and the closely related species R. quintana. Groups of CD-1 mice were inoculated with whole organisms of six strains of R. henselae and two of R. quintana. Pre- and postinoculation blood specimens were collected. Enzyme immunosorbent assays were performed by using as antigens preparations of immunogenic proteins from one isolate of R. henselae or from the R. quintana type strain. These demonstrated high specificity of R. henselae-induced antibodies for proteins of R. henselae and of R. quintana-induced antibodies for proteins of R. quintana. Protein preparations extracted from all of the strains were separated electrophoretically. After their transfer to membranes, immunoblots were performed by using 1:1,000 dilutions of all of the pre- and postinoculation sera in combination with proteins from all of the strains. Preinoculation sera had minimal reactivity. All of the R. henselae-induced immune sera reacted with numerous proteins of all of the R. henselae strains but cross-reacted minimally with proteins of R. quintana. Immune sera from R. quintana-inoculated mice had similar species specificity. An immunofluorescence assay was developed by using antiserum to one strain of R. henselae. A 1:1,000 dilution yielded fluorescence with all strains of R. henselae but with none of R. quintana, Bartonella bacilliformis, or Afipia felis. Acinetobacter calcoaceticus subsp. anitratus was also unreactive with a dilution of 1:500. A 1:10 dilution yielded weak fluorescence with R. quintana but none with Staphylococcus epidermidis.

A novel spotted fever group rickettsia has recently been isolated from Amblyomma hebraeum ticks in Zimbabwe. In a survey of 172 goat sera collected throughout Zimbabwe the highest prevalence of antibodies reactive with this rickettsia was in the south of the country, the area where A hebraeum is most commonly found. Nine goats were infected using male and female A hebraeum taken from a tick line shown to be infected with the novel rickettsia. By week 3 after infection, seroconversion occurred in all nine goats but no clinical signs of disease could be detected. A leucocytosis due to a mature neutrophilia one to two weeks after infection was the only abnormality. Rickettsaemia was detected only on day 3 after exposure to infected ticks. Immunosuppression failed to induce recrudescence of the rickettsaemia.

Dogs orally infected with Neorickettsia helminthoeca developed immunoglobulin G titers against Erlichia risticii, Erlichia sennetsu, and Erlichia canis similar to those against N. helminthoeca antigen, as determined by immunofluorescence. Western immunoblotting showed that the major common antigens shared among the microorganisms were 80- or 78-kDa and 64-kDa polypeptides. In contrast, horse anti-E. risticii and anti-E. sennetsu and dog anti-E. canis sera reacted more weakly to N. helminthoeca antigen than to homologous antigens in both immunofluorescence and Western immunoblotting. Antisera raised in other species of animals, i.e., mouse anti-E. canis and rabbit anti-E. risticii and anti-E. sennetsu sera, however, all reacted with the 64-kDa antigen of N. helminthoeca. This strong antigenic cross-reactivity and similarity in Western immunoblotting reaction profiles indicate that N. helminthoeca is antigenically closely related to E. risticii and E. sennetsu and less so to E. canis. In both immunofluorescence and Western immunoblotting, E. canis shared fewer common antigens with E. risticii and E. sennetsu than N. helminthoeca did. It is reasonable to conclude that these results may have both diagnostic and taxonomic significance.

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.

Antigenic diversity in five stocks of the tick-borne rickettsia Cowdria ruminantium, the causal agent of heartwater disease of ruminants, was studied by cross-immunity trials in goats and sheep. Complete absence of cross-protection was found only between the Kümm and Kwanyanga stocks, and in all other combinations there were various degrees of cross-immunity. Immunological strain differences were more pronounced in goats than in sheep.

The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non-infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle.

Lyophilized tissues of mice and blood of sheep, infected with either the Kümm, the Welgevonden or the Ball 3 stock of Cowdria ruminantium, remain infective to mice and sheep after storage at 4 degree C for 90 days. Freeze-dried tissues stored at -18 degrees C and -28 degrees C are still infective after 6 months and 2 years, respectively.

Giemsa-stained, peripheral blood smears of 67 dogs, showing clinical signs typical of babesiosis or reminiscent of concurrent babesiosis and ehrlichiosis, were examined for the presence of Babesia canis and Ehrlichia canis. Since Cowdria ruminantium cross-reacts with Ehrlichia, the sera of these dogs were also subjected to the indirect fluorescent antibody (IFA) test in which C. ruminantium was used as antigen. Fifty-five per cent of these dogs had mixed infections of B. canis and E. canis, as judged by blood smear examination and serology. The serum of 32% of these dogs with mixed infections reacted positively in the IFA test. Six out of 9 dogs, the blood smears of which were negative for both B. canis and E. canis, were serologically positive for E. canis. Furthermore, sero-conversion from a negative in the initial serum sample to titres of up to 1:160 in a subsequent sample was recorded in 9 out of 13 dogs with suspected mixed infection on blood smear.

Neither sheep immune to the Ball 3, the Kwanyanga or the Mara stocks of Cowdria ruminantium nor those immunized with combinations of these 3 stocks were protected against challenge with the Mali stock. Against challenge with the Welgevonden stock, however, immunization with each of the 3 combinations and with single stocks effected a protective immunity.

Human ehrlichiosis, an acute febrile illness caused by Ehrlichia canis or a closely related rickettsial organism, was first identified in 1986. From 1986 through 1988, sera from 85 patients demonstrated a fourfold rise or fall in antibody titer to E. canis. Seven (22%) of 32 patients initially tested during the first week after onset of illness. 17 (68%) of 25 tested during the second week, and all 18 tested during the third week had titers that exceeded the minimum positive titer of greater than or equal to 80. Of the 85 confirmed ehrlichiosis patients, 31 (36.5%) also had indirect fluorescent antibody titers considered diagnostic of infection with Rickettsia rickettsii, Rickettsia typhi, or Coxiella burnetti, but in most these diagnoses were not supported by epidemiologic, clinical, or serologic evidence. These results emphasize that patients suspected of having a tick-borne infection should be tested for antibodies to E. canis as well as for those to other rickettsiae.

The prevalence of antibodies to Ehrlichia equi in horses from the foothill regions of northern California and from the Sacramento valley (non-foothill area) was determined, using an indirect fluorescent antibody test. Horses from foothill regions had a higher prevalence of seropositivity (10.4%) and higher titer (1:10 to 1:80) than did those from non-foothill regions (3.1%; titer less than or equal to 1:10). Fifty percent of healthy horses on a foothill farm enzootic for E equi had titer to E equi, suggesting that infection with E equi can be subclinical. Six veterinarians surveyed from northern California diagnosed clinical E equi infection in 38 horses during 1985-1986 based on clinical signs of infection and observation of E equi inclusion bodies in neutrophils on blood smears.

Antibody titer to Ehrlichia risticii was determined, in 2,549 equine serum samples, using an indirect fluorescent antibody assay. During 1986, samples were obtained from the Minnesota State-Federal Equine Infectious Anemia Diagnostic Laboratory, the Minnesota Racing Laboratory, from horses admitted to the University of Minnesota Veterinary Teaching Hospital, and as a result of field investigations of horses with acute diarrhea. Results of the study revealed antibody prevalence of 33, 24, 47, and 25% for the respective groups. There was no statistical association between seropositive status and age, sex, breed, or clinical problem of horses referred to the teaching hospital. There was an increase in the total percentage of seropositive samples over the duration of the sample collection period, suggesting a seasonal exposure pattern, and E risticii was associated with clinical and subclinical infections in horses of Minnesota.

Major advances made in the last few years in the knowledge of heartwater have not yet resulted in significant improvement of its prevention and control. Acaricide resistance and the high cost of chemical vector control stimulate research on alternative methods of vector control, such as immunization against the tick and the use of pheromones. Recently developed in vitro methods for the culture of Cowdria ruminantium may make it possible to arrive at safe recombinant or molecular vaccines. Specific chemotherapy may be improved by new laboratory screening methods, and studies on the pathogenesis of heartwater could lead to better non-specific treatment as well. Studies on the heritability of natural resistance to heartwater should determine whether or not practical selection and transfer of resistance is feasible. More research is also needed on the immunology and diagnosis of the disease and the factors involved in the resistance of young animals.

The results of a serosurvey of Oklahomans for the presence of antibody to Ehrlichia canis is reported. Paired serum specimens, from patients lacking the serologic criteria for diagnosis of Rocky Mountain spotted fever (RMSF), were tested. A four-fold increase in E. canis-IFA antibody was found in 16/144 (11 percent) of these paired serum samples. Patients with serologic evidence of E. canis infection had a mean age of 34 years, 69 percent were male, and 63 percent lived in a town less than 10,000 population. Signs and symptoms included: fever 94 percent, headache 94 percent, fatigue 94 percent, anorexia 81 percent, nausea 60 percent, and rash 44 percent. When compared to control patients, whose sera were submitted for RMSF testing but did not meet serologic criteria for RMSF or E. canis, case-patients were more likely to have had leukopenia (OR = 4.9, 95 percent Cl = 1.2, 19.0) and tick exposure (OR = 9.5, 95 percent Cl = 1.4, 62.7). The results suggest E. canis, or a closely related agent, is a cause of human illness. Ticks are probable vector.

A cell line of bovine endothelial cells (E5), infected with 3 different stocks of Cowdria ruminantium, was used as antigen in an indirect fluorescent antibody test for the serodiagnosis of heartwater. These antigens were compared to peritoneal macrophages from mice infected with the Kümm stock and to caprine neutrophils in primary cultures from goats infected with 4 different stocks of Cowdria. The use of endothelial cell cultures proved to be superior in all respects. The antigens can be produced in large quantities at a low cost, contrary to the other types. The reaction is easily and quickly read, compared to the laborious reading of neutrophil or macrophage antigens which often contain few and small colonies of Cowdria. Moreover, not all stocks are suitable for the preparation of neutrophil antigens, while macrophage antigen can only be obtained with the Kümm stock. Endothelial cell antigens also distinguish serotypes in C. ruminantium, but these differences seem to be less pronounced than those found with neutrophil antigens. Finally, the specificity of endothelial cell antigens appears to be better than that of Kümm antigen and comparable to that of neutrophil antigens. The use of Kümm antigen may have been responsible to a large extent for past unexplained positive serological results on certain Caribbean islands where it has not been possible to isolate Cowdria and where no clinical evidence of the disease has been found.

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.

Ten stocks of Cowdria ruminantium (Ball 3, Breed, Comoro, Germishuys, Kümm, Kwanyanga, Mali, Mara, Nonile and Welgevonden) were compared from a cross-immunity, serological and mouse pathogenicity point of view. They were found to differ in varying degrees. Except for the Ball 3, Comoro and Germishuys stocks that were similar but not identical, there was no pattern in the antigenic diversity of the 10 stocks. The Welgevonden stock emerged as the stock that elicits an immunity against most of the South African stocks. The inability of the reference Ball 3 stock to protect sheep against no fewer than 6 other stocks questions the advisability of retaining this stock as the vaccine stock. The antigenic diversity of the 10 stocks could not be correlated with the antibody levels detected with the indirect fluorescent antibody test, since the sera against all 10 stocks reacted positively to the Kümm stock antigen and the variation in titres was not stock-related.

A sensitive and reliable enzyme-linked immunosorbent assay for the detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum nymphae is described. For the screening of antibodies, C. ruminantium from A. hebraeum nymphae, partially purified by wheat-germ lectin affinity chromatography, was used as antigen. To screen nymph populations, sera from either Ball 3 strain-infected sheep or Kumm-strain infected mice were used. By using appropriate controls the assays were rendered specific with respect to C. ruminantium.

The antigenic relationship between some rickettsiae of arthropods, which do not transmit germs pathogenic to vertebrates was investigated by agglutination and immunofluorescence techniques. The comparison of R. melolonthae, R. tipulae, R. cetonidarum, R. grylli and of the rickettsiae of the scorpion Buthus occitanus shows that the Wolbachiae are divided into three serological groups. The insect crystallogen rickettsiae belong to the first group, R. grylli and the scorpion Rickettsia are antigenically distinct (tables I and II). No antigenic relationship was found between these arthropod microorganisms and the following agents of vertebrate rickettsioses and chlamydioses : historical typhus, murine typhus, pustulous fever, Q-fever, epizootic absortion of sheep and psittacosis (tables III and IV). Bacteria of OX2 and OX19 strain of P. vulgaris when mixed with anti-Wolbachiae serums of I, II, III groups, give a negative Weil a Felix reaction. The OXK Proteus are however agglutinated by 4 out of 6 rabbit anti-Rickettsiella serums of the group. This fact calls for a further investigation regarding the comparison of these Rickettsiella with various strains of R. tsutsugamushi.

The conglutinin titres of year-old Bonsmara-cross cattle infected with Cowdria ruminantium were inversely proportional to the severity of the reactions elicited by the infection. There was no correlation, however, between conglutinin levels of 8-month-old calves of the same breed, sex and origin and their susceptibility to heartwater. The role possibly played by conglutinin in the non-specific resistance of cattle to heartwater and in the epidemiology of the disease is discussed.

No bactericidal effect was produced when Rochalimaea quintana was exposed for 1 h to a combination of high-titered anti-R. quintana rabbit serum and guinea pig complement.

Investigation into the presence of C. ruminantium antigen, using an enzyme-linked immunosorbent assay (ELISA) in various tick tissues and haemolymph of adult Amblyomma hebraeum ticks revealed that the organism invades a number of body parts and can be demonstrated in A. hebraeum. In females, the gut, salivary glands, hypodermis and synganglion and in males, the salivary glands and gut showed the highest concentration.

The isolation of Cowdria ruminantium by means of wheat germ lectin affinity chromatography as described in this paper permits the recovery of partially purified viable organisms under mild conditions in short time. These conclusions are based upon results of analyses of column fractions by intravenous inoculation into sheep, protein determination, electronmicroscopy and enzyme-linked immunosorbent assay. The entire purification procedure could be completed in 4-5 hours using only either infected sheep tissue or nymphae as starting material.

Ehrlichia risticii has a close antigenic relationship to E. sennetsu. Sera of ponies experimentally infected with E. risticii, the etiologic agent of Potomac horse fever, consistently reacted with E. sennetsu, a human pathogen, in indirect fluorescent-antibody (IFA) testing, while human E. sennetsu convalescent serum reacted with E. risticii by IFA testing and immunoferritin labeling of cells infected in vitro. Two ponies injected intravenously with live E. sennetsu did no develop clinical illness. Subsequent injection with live E. sennetsu did not develop clinical illness. Subsequent injection with live E. risticii also did not induce any disease, in contrast to two control ponies given E. risticii without prior exposure to E. sennetsu. Both controls developed fever, anorexia, depression, dehydration, and diarrhea, which are typical clinical signs of Potomac horse fever, and had characteristic lesions of enteritis and lymph node histiocytosis at postmortem examination. E. sennetsu-exposed ponies had normal gastrointestinal morphologies and lymph node hyperplasia. Ponies primed with E. sennetsu before E. risticii challenge developed high titers of immunoglobulin G antibody which reacted against both E. sennetsu and E. risticii antigens by IFA testing. The most prominent antigenic polypeptide in Western (immuno-) blot analysis of sera collected from ponies primed with E. sennetsu before subsequent challenge with E. risticii was present in lysates of both Ehrlichia species and had an apparent molecular mass of 44 kilodaltons. This band was not prominent in Western blots performed with sera of ponies injected with E. risticii alone. Thus, injection of E. sennetsu protects ponies from clinical and pathological manifestations of the disease induced by injection with E. risticii. Immunologic cross-reactivity of the two organisms with IFA testing and strong immunologic recognition by ponies of the 44-kilodalton antigen common to the two organisms may be related to the development of protective immunity against E. risticii.

Competitive enzyme-linked immunosorbent assay (CELISA), mediated by a monoclonal antibody designated HybI, was developed for the diagnosis of equine monocytic ehrlichiosis. Inhibition of binding of HybI by the horse antibodies to Ehrlichia risticii was optimum at dilutions of 1:20 for serum and 1:10,000 for HybI. Mean optical densities (ODs) of positive and negative sera were 0.158 and 0.855, respectively. A comparison of ODs obtained by CELISA and indirect enzyme-linked immunosorbent assay (ELISA) indicated a marked tendency of positive and negative samples to cluster separately with respect to CELISA ODs, whereas the ELISA results displayed a continuum of ODs from negative to positive. Analysis of diagnosis by indirect fluorescent-antibody test (IFA), ELISA, and CELISA for 66 field-collected serum samples indicated that CELISA was superior to IFA and ELISA. Among 11 acute-phase serum samples negative by IFA which were obtained from horses that subsequently seroconverted, CELISA clearly demonstrated antibodies in 8 of these acute-phase sera, whereas 5 were borderline positive by ELISA. The presence of agent-specific humoral antibodies could be demonstrated conclusively by 14 days after infection. The results suggest that CELISA is more sensitive than IFA and ELISA and, owing to the marked differences between positive and negative samples, can be easily adapted for use in the field for detection of horse antibodies to E. risticii.

Hybridomas producing monoclonal antibodies to Ehrlichia risticii were developed to provide a means of molecular investigation of the biochemical and immunopathologic characteristics of the organism. All of 6 stable monoclonal antibodies obtained were IgG isotypes. The ascitic fluid titers induced by the hybridomas ranged from 10(2) to 10(7). Competitive binding experiments conducted by ELISA and binding of labeled protein A to antigen-antibody complexes indicated competition among monoclonal antibodies. Two monoclonal antibodies (HybI and 14D4) were reactive in an indirect fluorescent antibody test; these antibodies also bound a maximum of labeled protein A, indicating recognition of epitopes on the surface of the ehrlichia. Protein specificity of monoclonal antibodies could not be demonstrated with western blot procedure. HybI monoclonal antibody, however, did precipitate the 28 kD protein from 125I-surface-labeled ehrlichiae and was shown to be specific to E risticii on the basis of nonreactivity with E sennetsu, using the indirect fluorescent antibody test. By use of the different monoclonal antibodies as probes, more definitive molecular studies now will be feasible.

Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group (SFG) rickettsiae were detected by indirect immunofluorescence in sera from 27 ill individually owned thrombocytopenic dogs (platelet concentrations less than 200,000 platelets/microliters) and 59 healthy kenneled dogs located in southern Louisiana. Platelet concentrations less than 100,000 platelets/microliters were detected in 63% of ill thrombocytopenic dogs and 6.8% of healthy kennel dogs. One ill thrombocytopenic dog had intracytoplasmic E platys morulae detected within platelets. The prevalence of increased serum antibody titers to E canis and E platys was 25.9% and 40.7% for the ill thrombocytopenic dogs and 20.3% and 54.2% for the healthy kennel dogs, respectively. All dogs with seropositivity to E canis had increased antibody titers of greater than or equal to 1:100 to E platys. Simultaneous examination of increased serum antibody titers (greater than or equal to 1:64) to four SFG rickettsiae indicate that Rickettsia rhipicephali and Rickettsia montana accounted for the majority of the antibodies detected in these dogs. Of 86 dogs tested, 44.2% were seronegative to E canis, E platys, and SFG rickettsiae.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.