The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. I. Inhibition of Growth of Rickettsia tsutsugamushi,Karp Strain,in Cultured Murine Embryonic Cells and Macrophages by Recombinant Tumor Necrosis Factor-Alpha

Recombinant murine tumor necrosis factor-alpha (TNF-α) inhibited intracellular growth of Rickettsia tsutsugamushi, Karp strain, in the mouse embryo cell line C3H/10T1/2 clone 8 at doses of 100 to 10 U/ml. The growth inhibitory effect of TNF-α was also evident when peritoneal exudate macrophages or bone marrow-derived macrophages were used as the host cell for rickettsial growth. Interferon-gamma (IFN-γ), at doses up to 1,000 U/ml, did not affect the growth of this strain of rickettsiae in the mouse embryo cell line but, as expected, profoundly inhibited rickettsial growth in peritoneal exudate macrophages and bone marrow-derived macrophages. The effect of TNF-α on rickettsial growth in the mouse embryo cell line was not reproducibly enhanced by IFN-γ. Treatment of the cell line with TNF-α delayed rickettsial cytopathic effects, but the rickettsiae ultimately grew to high numbers in the cells and caused cell death. These findings show that, at least in our system, R. tsutsugamushi is resistant to IFN-γ-mediated antirickettsial effects in cells other than macrophages. The results of this study support the suggestion that TNF-α may inhibit rickettsial growth in cells other than macrophages.     

Macrophages in resistance to rickettsial infection: macrophage activation in vitro for killing of Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.     

Incongruent effects of two isolates of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> on the survival of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35–66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.     

Historical and recent evidence for close relationships among Rickettsia parkeri,R. conorii,R. africae,and R. sibirica: Implications for rickettsial taxonomy

Rickettsia parkeri, a member of the spotted fever group rickettsias, was first described in 1939 and was thought to be non‐pathogenic until recently, when it was found to cause a spotted fever‐like illness in humans and areas of necrosis (eschars) at the sites of tick bites. Accordingly, there is currently much interest in this emerging pathogen. In this study, all published articles concerning R. parkeri were reviewed and analyzed for evidence of relatedness among this agent and other spotted fever group (SFG) rickettsiae which also produce similar clinical syndromes and/or eschars, including R. conorii, R. africae, and R. sibirica. A synthesis of the historical (antigenic) and recent (molecular) data supporting a phylogenetic sub‐grouping of these SFG organisms is presented and comments are offered about the taxonomy of rickettsial organisms in general, and R. parkeri in particular.     

Proposal to create subspecies of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> based on multi-locus sequence typing and an emended description of <Emphasis Type="Italic">Rickettsia conorii</Emphasis>

Background  

Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

Demonstration of a Heat-Stable 120-Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group-Common Antigen

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as λRj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and λgt11 lacZ gene amplified the λRj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.     

Identification and characterization of the mammalian association and actin-nucleating domains in the Rickettsia conorii autotransporter protein, Sca2

Establishment of infection by spotted fever group rickettsial species is dependent on the ability of these bacteria to adhere to and invade the host endothelium. Recent studies have attributed these processes to a handful of rickettsial surface proteins from the surface cell antigen (sca) family of autotransporters. A rickettsial autotransporter from Rickettsia conorii, Sca2, has been shown to be sufficient to mediate both adherence and invasion of human endothelial cells and to participate in intracellular actin‐based motility. Here we identify a region of Sca2 capable of interacting with the mammalian cell surface and show that this function of Sca2 is independent and separable from its actin nucleation activity. Furthermore, pre‐incubation of mammalian cells with the Sca2 mammalian association region prior to R. conorii infection can competitively inhibit rickettsial invasion, suggesting that Sca2 plays an important role in the initial interaction with mammalian cells. Together, our results demonstrate that the Sca2 autotransporter protein in R. conorii contains distinct functional domains that likely are involved in mediating cellular interactions at the plasma membrane and the host cytosol.     

The comparison of cell-mediated immunity induced by immunization with porin, viable cells and killed cells of Salmonella typhimurium.

A marked level of cell-mediated immunity (CMI) to Salmonella typhimurium-infection in mice, as determined by acquired resistance, delayed-type hypersensitivity, interleukin-2 production and interferon-gamma production, was induced by immunization with porin or viable cells but not with killed cells of S. typhimurium LT2. When the up-regulation of immune system to each immunogen was studied by comparing increases of Ia-bearing macrophages, the immunization with porin or viable cells, but not killed cells, could stimulate the immune system for more than 14 days. Interleukin-1 (IL-1) production of macrophages to each immunogen was also examined; the result showed that immunization with porin or viable cells could induce a notable level of IL-1 production, while killed cells could not. However, when the abilities to induce these immune responses were compared between UV-killed and heat-killed cells, UV-killed cells were superior to heat-killed cells. These results suggested that the ineffectiveness of immunogen that lacked CMI-inducing ability might be ascribed to the denaturation of antigen and the insufficient inductions of Ia-bearing macrophages and IL-1 production.     

Regulation of macrophage populations. I. Preferential induction of Ia-rich peritoneal exudates by immunologic stimuli

The amounts of Ia-positive and -negative macrophages were studied in peritoneal exudates of normal mice or of mice injected with various inflammatory materials, infected with Listeria monocytogenes, or injected with hemocyanin. Ia-negative macrophages predominated in exudates from normal mice or from mice given mineral oil, peptone, thioglycollate, culture media, or endotoxin. Infection with Listeria caused a very marked increase in Ia-positive macrophages. The induction of Ia-positive macrophages by Listeria inoculation resulted in great part from an immune process. The Ia-positive exudates were more readily generated in immune mice given a secondary challenge with heat-killed organisms. Furthermore, immune T cells transplanted together with heat-killed organisms into normal mice resulted in Ia-rich exudates. Injection of hemocyanin also induced Ia-rich exudates involving an immune process. We conclude that an immune reaction involving T cells regulates the Ia phenotype of the exudate macrophage population. The Ia-positive macrophages were Fc and C3 receptor positive and phagocytized latex particles.     

DETECTION OF RICKETTSIA TSUTSUGAMUSHI FROM VECTOR TROMBICULID MITE BY DNA AMPLIFICATION USING POLYMERASE CHAIN REACTION TECHNIQUE*

Abstract With reference to the Sta 58 major antigen gene of Rickettsia tsutsugamushi (Karp strain), we had designed and synthesized a pair of DNA primers; and a kilobase fragment of Sta 58 is amplified for using in polymerase chain reaction (PCR) with the primers. This is the first report on detection of R. tsutsugamushi in trombiculids by using PCR technique. The experimental results indicated that the time for which R. tsutsugamushi could exist in the adult of Leptotromhidium deliense when inoculated into its abdomenal cavity was 360 days at least and R. tsutsugamushi could be transovarially transmitted to the offsprings for 4 generations; and the time for which R. tsutsugamushi could exist in L. deliense after biting the infective mouse was 270 days at least and R. tsutsugamushi could be transmitted transovarially to the offsprings for 2 generations. The results also showed that the PCK technique was highly specific and sensitive for detection of R. tsutsugamushi; and the amplification of gene outside body to detect the rickettsiae in the body of trombiculid and mouse can be used as a new method for investigation of scrub typhus epidemiology.     

Dual exposure of Rickettsia typhi and Orientia tsutsugamushi in the field‐collected Rattus rodents from Thailand

Field‐collected rodents and fleas from ten provinces covering four regions of Thailand were investigated for possible rickettsial pathogen infections. The 257 trapped‐rodents belonged to 12 species. Five species of Genus Rattus accounted for 93% of the total capture, of which Rattus exulans and Rattus norvegicus were the two major species caught. All flea specimens, removed from trapped rodents, were identified as Xenopsylla cheopis. The PCR technique was performed on ectoparasite specimens to detect the presence of murine typhus pathogen (Rickettsia typhi) and scrub typhus pathogen (Orientia tsutsugamushi). Thirteen flea specimens (2.6 %) were found to be positive for R. typhi but none for O. tsutsugamushi. An ELISA technique was used to detect the rodent's antibodies against R. typhi and O. tsutsugamushi. Sixty‐one rodent serum samples (23.7%) were positive for R. typhi specific IgM, IgG, or both, while 47 of the samples (18.3%) were positive for O. tsutsugamushi. Twenty serum samples from R. norvegicus (7.8%) had detectable antibodies against both R. typhi and O. tsutsugamushi. Our findings revealed the existence of the dual infection of rickettsial pathogens in the same natural hosts.     

Coxsackievirus B3-Induced Chronic Myocarditis in Mouse: Use of Whole Blood Culture to Study the Activation of TNFα-Producing Cells

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNFα secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5 × 10⁵ pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 μl) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 μl RPMI in the presence or the absence of activators (LPS + PHA). The TNFα levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNFα was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNFα was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNFα was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNFα was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNFα level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNFα level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.     

OmpA‐mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin

Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant s urface c ell a ntigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB‐mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non‐phagocytic human endothelial cells. A recombinant soluble C‐terminal OmpA protein domain (954–1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA‐mediated attachment to mammalian cells is in part dependent on a conserved non‐continuous RGD motif present in a predicted C‐terminal ‘pertactin’ domain in OmpA.Our results demonstrate that multiple adhesin–receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.     

Warmer Weather Linked to Tick Attack and Emergence of Severe Rickettsioses

The impact of climate on the vector behaviour of the worldwide dog tick Rhipicephalus sanguineus is a cause of concern. This tick is a vector for life-threatening organisms including Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, R. conorii, the agent of Mediterranean spotted fever, and the ubiquitous emerging pathogen R. massiliae. A focus of spotted fever was investigated in France in May 2007. Blood and tissue samples from two patients were tested. An entomological survey was organised with the study of climatic conditions. An experimental model was designed to test the affinity of Rh. sanguineus for biting humans in variable temperature conditions. Serological and/or molecular tools confirmed that one patient was infected by R. conorii, whereas the other was infected by R. massiliae. Dense populations of Rh. sanguineus were found. They were infected with new genotypes of clonal populations of either R. conorii (24/133; 18%) or R. massiliae (13/133; 10%). April 2007 was the warmest since 1950, with summer-like temperatures. We show herein that the human affinity of Rh. sanguineus was increased in warmer temperatures. In addition to the originality of theses cases (ophthalmic involvements, the second reported case of R. massiliae infection), we provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. sanguineus, leading to increased human attacks. From a global perspective, we predict that as a result of globalisation and warming, more pathogens transmitted by the brown dog tick may emerge in the future.     

Intracytoplasmic Localization of Antigenic Heat-Stable 120- to 130-Kilodalton Proteins (PS120) Common to Spotted Fever Group Rickettsiae Demonstrated by Immunoelectron Microscopy

Immunoelectron microscopy demonstrated antigenic heat-stable 120- to 130-kilodalton proteins (PS120) of spotted fever group (SFG) rickettsiae with antiserum against recombinant PS120 of Rickettsia japonica. In the case of R. japonica, a major part of the protein was shown to be localized outside the electron-lucent nucleoid-like region in the cytoplasm of the organisms. The other SFG rickettsiae represented a similar localization of the PS120 antigens cross-reactive to that of R. japonica. On the other hand, a typhus group rickettsia demonstrated no antigens cross-reactive to the PS120 of SFG rickettsiae.     

Cross-Reactivity of Rickettsia japonica and Rickettsia typhi Demonstrated by Immunofluorescence and Western Immunoblotting

Cross-reactivity between Rickettsia japonica and R. typhi was observed by immunofluorescence tests using sera from patients with Oriental spotted fever (OSF), from whom the causative agent was isolated and identified as R. japonica. Western immunoblotting with these sera revealed that only the 120-kilodalton surface polypeptide, i.e., rickettsial outer membrane protein (rOmp) B, has a common antigenicity with the 105-kilodalton surface polypeptide of R. typhi. In some cases, antibodies specifically reactive with R. typhi were detected in acute-phase sera followed by a significant rise in titers, possibly because of an anamnestic response to a previous infection with an R. typhi-like agent; the sera retained reactivity to R. typhi even after absorption by a homologous strain. A lipopolysaccharide (LPS)-like antigen of R. typhi was found to be reactive with some sera of OSF patients. The ladder bands on Western immunoblot of rickettsial organisms were confirmed to be polysaccharide in nature, which was demonstrated by comparing them with the pattern of silver-stained gel of proteinase K-treated rickettsial specimens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A Complex Interaction between Rickettsia conorii and Dickkopf-1 – Potential Role in Immune Evasion Mechanisms in Endothelial Cells

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.