Ehrlichial Infections Research Articles

TNFα Inhibits the Expression of Germinal Center Phenotype on T-bet+ CD11c+ IgM Memory B Cells without Altering Clonal Diversity

Abstract Our studies have identified a population of T-bet+ CD11c+ IgM memory B cells that arise within 30 days following infection with the intracellular bacterium, E. muris. The memory cells require for their generation T cell help and IL-21; however, they are generated in a GC-independent fashion, because GCs are suppressed during infection. Previous studies have shown that TNFα is necessary for proper GC formation. Given that we detected an increase in serum TNFα following infection, we addressed a role for this cytokine in IgM memory cell development. Relative to wild-type, we observed a substantial increase in the frequency of developing IgM memory. These B cells were positive for GL7+ CD38lo CD95+ on day 16 post-infection. Imaging studies corroborated these findings by demonstrating the appearance of GC-like structures in infected TNFα-deficient, but not in wild-type, mice. Increased GL7 expression on IgM memory cells in absence of TNFα was not correlated with changes in clonal diversity on day 30 post-infection. This suggests that TNFα does not affect B cell selection. To address mechanisms whereby the absence of TNFα may facilitate formation of pseudo-GC structures, we quantitated CXCL13, a chemokine known for its role as a chemoattractant during GC morphogenesis. CXCL13 expression was 10-fold lower in TNFα-deficient mice, indicating its requirement, in part, for splenic disorganization in wild-type mice during infection. Our findings suggest that although GCs are suppressed during ehrlichial and other bacterial infections, GC structures can, when present, magnify IgM memory cell generation. Ongoing studies will address whether changes in lymphoid architecture impact other properties and functions of T-bet+ CD11c+ IgM memory cells.

Prevalence of antibodies against Ehrlichia spp. and Orientia tsutsugamushi in small mammals around harbors in Taiwan.

BackgroundTick-borne ehrlichiosis and mite-borne scrub typhus represent important emerging zoonotic rickettsial diseases. Although scrub typhus has been recognized by the Taiwanese public health system, information on ehrlichial infections is scarce in Taiwan. In this study, the risk of spread of ectoparasites on rodents through aerial and marine transportation was assessed in international and domestic harbors. Here, we report the first systematic surveillance of seroprevalence against Ehrlichia spp. in small mammals on the main island of Taiwan.MethodsIn total, 1648 small mammals were trapped from 8 international ports, 18 domestic fishing harbors, and 7 local public health centers around Taiwan from November 2004 to December 2008. Sera were analyzed using indirect immunofluorescence assays to detect IgG antibodies against Ehrlichia chaffeensis and Orientia tsutsugamushi. A serum titer of ≧1:80 was considered positive.ResultsAntibodies against Ehrlichia spp. and O. tsutsugamushi were detected in 3.28 % and 4.92 % of small mammals active around harbors, respectively. The seropositive rate against Ehrlichia was higher in northern Taiwan from 2005 to 2008. However, O. tsutsugamushi infections increased in southern Taiwan during this period. The serological evidence of ehrlichial and O. tsutsugamushi infections in all international ports were included in the study. No significant differences were found among the seropositive rates of Ehrlichia spp. and O. tsutsugamushi in small mammals trapped between international and local harbors.ConclusionsThe overall prevalence of Ehrlichia spp. and O. tsutsugamushi infections in small mammals active around harbors was 3.28 % and 4.92 %, respectively. The results provided serological evidence supporting the potential risks of transporting pathogens through air and maritime traffic. This study highlights serious issues of the emergence and spread of rickettsial diseases in Taiwan. The incidence of human ehrlichiosis requires further investigation.

The role of CD8 T lymphocytes in rickettsial infections.

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.

Recombinant Ehrlichia P29 protein, but not P13 protein, induces a protective immune response in a mouse model of ehrlichiosis (P4280)

Abstract Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals. The diseases are caused by obligately intracelluar bacteria belonging to the genus Ehrlichia. Currently, no vaccine is available against any ehrlichial diseases. Several immunoreactive proteins of ehrlichiae identified using immune sera from human patients and animals that include the major outer membrane proteins, ankyrin-repeat proteins and tandem repeat proteins (TRPs). In the present study, we evaluated E. muris P13 and P29 proteins, which are orthologs of E. chaffeensis TRP32 and TRP47, respectively, as subunit vaccines in a mouse model of ehrlichiosis. Immunization of mice with recombinant P29, but not P13, induced significant protection against a challenge infection. The protection induced by recombinant P29 was associated with the induction of a strong IgG antibody response. In contrast to development of IgG antibodies to P13 in all E. muris-infected mice, fewer animals infected with E. muris had IgG antibodies to P29. We hypothesize that polyclonal activation of B cells by CD4+ Th1 cells and subsequent development of hypergamaglobluinemia during persistent E. muris infection impairs formation of antibodies to antigens, such as P29, with low B cell precursor frequencies. In conclusion, our study indicated that Ehrlichia P29, but not P13, induces protective immune response, and induction of antigen-specific antibody responses impaired during persistent ehrlichial infections.

Evaluation of lymphocyte populations in dogs naturally infected by Ehrlichia canis with and without clinical signs

Immune response elicited by the host during ehrlichial infections could influence the clinical signs and laboratory and pathological findings. Twenty-eight dogs naturally infected by Ehrlichia canis were included in this study. Twenty of them presented only laboratory findings traditionally associated with canine monocytic ehrlichiosis (CME), whilst 8 dogs also showed clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). A multiparametric flow cytometric study was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in the peripheral blood. Statistically significant differences between dogs naturally infected by E. canis in a clinical or subclinical stage were not detected when evaluating lymphocyte subsets in peripheral blood samples. Dogs with clinical signs showed lower relative and absolute values of B lymphocytes than dogs without clinical signs, although the differences were not statistically significant.

T-cell receptor repertoire diversity during persistent and fatal ehrlichial infections (40.17)

Abstract Human monocytotropic ehrlichiosis (HME), an emerging tick-transmitted bacterial disease, is caused by Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sub-lethal depending on the dose and route of inoculation. To better define the roles of T cells in protection and pathogenesis, we analyzed TCR Vβ usage in activated T cells from mice infected with E. muris or IOE by TCR-CDR3 spectratyping. Our data indicated that E. muris, but not IOE, induced oligoclonal expansion of CD4+ T cells within the several Vβ families (Vβ1, Vβ2, Vβ8.1, Vβ8.2, Vβ12, Vβ16 and Vβ19) early during the effector phase of T cell response. Further, oligoclonal expansion of CD8+ T cells within the Vβ8.2, Vβ4 and Vβ19 families was observed during E. muris infection compared to oligoclonal expansion of Vβ16 and Vβ19 CD8+ T cells in mice infected with IOE. Functional analysis of T cell subsets by flow cytometry indicated that E. muris induced a strong Th1 response involving several Vβ families compared to a weak Th1 response induced by IOE. The data suggested that unlike IOE, E. muris induces a strong Th1 response which tends to focus on certain Vβ families early during the effector phase of T cell response. Future studies will focus on functional characterization of memory T cell subsets induced following infection with E. muris.

Molecular detection of tick-borne protozoal and ehrlichial infections in domestic dogs in South Africa

A total of 1138 blood specimens were collected over a 6-year period (2000–2006) from domestic dogs in South Africa. Specimens from domestic dogs were obtained from the Onderstepoort Veterinary Academic Hospital (OVAH) in Pretoria, the Society for the Prevention of Cruelty to Animals (Johannesburg, Durban, East London and Bloemfontein) and private practices from four provinces (Gauteng, Mpumalanga, KwaZulu-Natal and Western Cape). All specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/ Anaplasma species using PCR and Reverse Line Blot (RLB) assays. On RLB, 560/1137 domestic dog-specimens were positive for one or more parasites. Of the positive domestic dog-specimens, 420 (75%) were infected with Babesia rossi; 82 (15%) dogs were infected with Theileria sp. (dog); 18 (3%) dogs were infected with Babesia vogeli; 14 (3%) specimens were infected with Ehrlichia canis. Mixed infections were also found: B. rossi and E. canis were detected in 12 (2%) specimens; B. vogeli and E. canis occurred in 7 (1%) specimens; Theileria sp. (dog) and E. canis in 3 (0.5%) specimens; B. rossi and B. vogeli in one specimen. B. rossi, B. vogeli and E. canis occurred simultaneously in one dog. There was also one incidental finding of a dog positive for Trypanosoma congolense. The results indicate that a wide range of tick-borne pathogens are circulating in the canine populations in South Africa.

Two USA Ehrlichia spp. cause febrile illness in goats

Ehrlichia spp. are not currently recognized as a cause of illness in goats in the USA, but three Ehrlichia are enzootic in lone star ticks ( Amblyomma americanum) in the eastern USA, and related bacteria in other countries cause illness in goats. We exposed naïve goats to Ehrlichia-infected Amblyomma and demonstrated that infection and clinical illness can be caused by two USA species, E. ewingii and the recently discovered Panola Mountain Ehrlichia sp. Clinical features in all five goats are described; ehrlichioses were associated with pyrexia, serous nasal discharge, inappetance, lethargy, decreased alkaline phosphatase, and, in most cases, neutropenia. Goats remained chronically infected for several months following exposure to ehrlichiae and transmitted the pathogens to uninfected ticks. In the eastern USA, undifferentiated febrile illness in goats might be caused by previously unrecognized ehrlichial infections, and pastures housing-infected goats could become infested with a large number of infected ticks.

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.

Treatment and prevention of rickettsial and ehrlichial infections

Rickettsial and ehrlichial infections are both carried by arthropod vectors. Both Rickettsia and Ehrlichia are small intracellular gram-negative coccobacilli. Clinical manifestations of Rickettsia range from spotted fevers to various forms of typhus. Human ehrlichiosis can present as monocytic ehrlichiosis or granulocytic anaplasmosis. Prevention is by avoidance of the responsible vectors. Therapy is usually with doxycycline, but chloramphenicol can also be used.

Seroepidemiological study of canine ehrlichial infections in Yamaguchi prefecture and surrounding areas of Japan

Randomly selected serum samples from 150 dogs from Yamaguchi and neighbouring prefectures were subjected to the indirect immunofluorescent assay to detect antibodies against Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia muris and Ehrlichia from Ixodes ovatus. A total of 30 out of the 150 serum samples reacted with at least one of the antigens at a titer of 1:20 or more. Considerable cross-reactivity was seen and most samples reacted with at least two different antigens. Fifteen (10.0%) dogs had higher titers to E. canis than any of the other antigens. Four (2.7%) dogs had higher titers to Ehrlichia from Ixodes ovatus and one (0.6%) dog had higher titers to E. muris compared to the other antigens. The findings suggest that these five dogs may be infected with the domestic Ehrlichia of Japan. The remaining ten dogs had similar high titers to two or more of the antigens. This is the first serological evidence obtained of canine infection with the domestic Ehrlichia of Japan.

Rickettsial, ehrlichial and Bartonella infections of the myocardium and pericardium.

Myocarditis and pericarditis are uncommon complications of human rickettsial, ehrlichial and Bartonella infections. Myocardial inflammation usually occurs in the setting of acute disseminated infection. Organisms associated with myocarditis include: Rickettsia rickettsii, R. conorii, Orientia tsutsugamushi, Coxiella burnetii, Anaplasma phagocytophila (the causative agent of Human Granulocytic Ehrlichiosis) and Bartonella henselae. Pericarditis has been described in the setting of R. conorii and Coxiella burnetii infections. This article reviews the epidemiology, pathologic characteristics, clinical manifestations, diagnosis and treatment of myocarditis and pericarditis caused by these organisms.

Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle.

The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.

Ocular histopathology of Ehrlichial infections in the dog.

Histologic examination of eyes and brains of 27 dogs experimentally infected with either Ehrlichia canis, E. ewingii, E. chaffeensis, or human granulocytic ehrlichia (HGE) was conducted in the course of several experiments, the primary objectives of which were to investigate the susceptibility of the domestic dog to infection with various ehrlichial species and to assess the ability of ixodid tick species to acquire and transmit those infections. Uveitis and meningitis occurred in each of the dogs infected with E. canis but was not observed in dogs infected with the other Ehrlichia species. The inflammatory infiltrate was predominantly lymphocytic, monocytic, and plasmacytic; granulocytes were notably few. Ocular inflammation was most common and most intense in the ciliary body, becoming less intense in the choroid, iris, and retina, respectively. Meningitis was often accompanied by mild neuroparenchymal vascular cuffing and gliosis. The meningeal inflammatory cell infiltrate included a prominent monocyte population. Ocular and meningeal lesions were present in all E. canis-infected dogs from 22 through 200 days postexposure. Neither ocular nor brain lesions were observed with any of the other ehrlichial infections.

Rapidly Fatal Infection withEhrlichia chaffeensis

To the Editor: Human ehrlichial infections are increasingly being recognized as common tick-borne diseases in the United States. Clinical characteristics of ehrlichiosis include fever, headache, and malaise with leukopenia, thrombocytopenia, and elevated levels of hepatic aminotransferases.1 In rare instances, infection may result in multiple organ failure and death, particularly in immunosuppressed patients.2 Despite the potential severity of disease, death is uncommon in normal human hosts. We report two cases of rapidly fatal Ehrlichia chaffeensis infection in patients who presented to our institution in early June. A 22-year-old man was hospitalized with hypotension after a one-week illness characterized by fever, headache, . . .

The incidence of ehrlichial and rickettsial infection in patients with unexplained fever and recent history of tick bite in central North Carolina.

We examined the clinical and laboratory findings of a consecutive series of patients from central North Carolina presenting with fever and a history of tick bite within the preceding 14 days. Evidence of a tick-transmitted pathogen was detected in 16 of 35 patients enrolled over a 2-year period. Nine patients were infected with Ehrlichia chaffeensis, and 6 were infected with a spotted fever group rickettsia; 1 patient had evidence of coinfection with E. chaffeensis and a spotted fever group rickettsia. Four patients had detectable antibodies against the human granulocytic ehrlichiosis agent; however, only 2 had a 4-fold antibody titer rise without detectable antibodies against E. chaffeensis. The other 2 were thought to have cross-reacting antibodies to E. chaffeensis. We conclude that ehrlichial infections may be as common as spotted fever group rickettsial infections in febrile patients from central North Carolina with a recent history of tick bite.

Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut.

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.

Potential value of major antigenic protein 2 for serological diagnosis of heartwater and related ehrlichial infections.

Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae.

Serological evidence of Ehrlichia infection in Swedish Lyme borreliosis patients.

We studied sera from patients who had participated in a prospective study of borreliosis in Sweden and had acquired tick bites in areas of the country with a high prevalence of granulocytic ehrlichial infections in animals. The sera were examined for IgG anti Ehrlichia antibodies by an indirect immunofluorescence assay using a locally isolated bovine Ehrlichia antigen. Confirmation of the serological results was done at the Unité des Rickettsies, Marseille, France. Three out of 37 of the investigated patients and 1 out of 100 investigated healthy blood donors had significant antibody titres to granulocytotropic Ehrlichiae. No patient or blood donor had specific antibody titres to Ehrlichia chaffeensis. These data suggest that Scandinavian Ehrlichia species can infect and evoke immunological response in tick-exposed humans.

Isolation of Ehrlichia canis from dogs following subcutaneous inoculation.

Subcutaneous inoculation of dogs with Ehrlichia canis was investigated as a more appropriate model of canine ehrlichiosis, which is naturally transmitted by arthropod vectors. A dose-dependent response occurred following subcutaneous inoculation of seven groups of dogs with log concentrations of E. canis-infected canine-origin cells. Ehrlichial infection in dogs was defined as concurrence of an increased titer of anti-E. canis immunoglobulin G (IgG) antibody in serum, a decreased platelet concentration, and isolation of E. canis by blood culture. In dogs administered the two lowest doses, no changes were detected. In seven of nine dogs administered three intermediate doses, the only change detected was a transient and mild increase in the anti-E. canis IgG antibody titer in serum. Only two of nine dogs inoculated with the intermediate doses developed an ehrlichial infection. Five of six dogs administered the two highest dose of E. canis developed an ehrlichial infection. These dogs had the highest IgG antibody titers in serum and the earliest isolation of E. canis from blood. In dogs that developed an ehrlichial infection, thrombocytopenia occurred by 28 days after inoculation, while increased IgG antibody titers in serum and blood cultures positive for E. canis occurred as early as 14 days postinoculation. Thrombocytopenia and seroconversion occurred later in the course of infection than previously reported for ehrlichial infections induced by intravenous inoculation. The route of administration and E. canis inoculum size can influence the course of ehrlichial infection and should be regarded as important variables in experimentally induced canine ehrlichiosis.