Early induction of interleukin-10 limits antigen-specific CD4⁺ T cell expansion, function, and secondary recall responses during persistent phagosomal infection.

Diverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4(+) T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4(+) T cells during persistent Ehrlichia muris infection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistent Ehrlichia infection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4(+) T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4(+) T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4(+) T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4(+) T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4(+) T cells may provide a basis to induce a protective immune response against persistent infections.

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis.

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1ß and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.

Isolation, experimental transmission, and characterization of causative agent of Potomac horse fever.

Potomac horse fever, a disease characterized by fever, anorexia, leukopenia, and occasional diarrhea, is fatal in approximately 30 percent of affected animals. The seasonal occurrence of the disease (June to October) and evidence of antibodies to the rickettsia Ehrlichia sennetsu in the serum of convalescing horses suggested that a related rickettsia might be the causative agent. Such an agent was isolated in cultured blood monocytes from an experimentally infected pony. This intracytoplasmic organism was adapted to growth in primary cultures of canine blood monocytes. A healthy pony inoculated with these infected monocytes also developed the disease. The organism was reisolated from this animal which, at autopsy, had pathological manifestations typical of Potomac horse fever. Cross serologic reactions between the newly isolated agent and antisera to 15 rickettsiae revealed that it is related to certain members of the genus Ehrlichia, particularly to Ehrlichia sennetsu. Since the disease occurs in other parts of the United States as well as in the vicinity of the Potomac River, and since it has also been reported in Europe, the name equine monocytic ehrlichiosis is proposed as being more descriptive.

Human ehrlichiosis: a case study.

Ehrlichiosis is an infection of white blood cells that affects various mammals, including mice, cattle, dogs, and humans. It was first reported in dogs in 1935, and the first human case was documented in the United States in 1986. Ehrlichia are obligate, intracellular bacteria that are transmitted by ticks to humans. They grow as a cluster (morula) in neutrophils (Anaplasma phagocytophilum and E. ewingii) and in monocytes (E. chaffeensis). The infection may cause prolonged fever and general aches, and is characterized by leukopenia, cytopenia, and elevated liver transaminases. In the first week of infection, ehrlichiae can be detected by finding intracellular aggregates on the blood/body fluid smears and various other laboratory findings. Immunofluorescent antibodies (IFA) titers and PCR are generally needed for confirmation and a definitive diagnosis. Early diagnosis is necessary as antibiotic treatment with doxycycline is very effective.

The agent of Human Granulocytic Ehrlichiosis resides in an endosomal compartment.

The composition of cytoplasmic vacuoles containing the agent of Human Granulocytic Ehrlichiosis (HGE) was studied to investigate how this pathogen exists within infected host cells. Electron microscopy demonstrated that the HGE organism resides in a membrane-bound compartment within HL-60 cells: early forms of the HGE agent have a round reticular appearance while later structures are small and dense. Vacuoles containing HGE bacteria incorporated endocytosed colloidal gold particles, suggesting that they are part of the endocytic pathway. Antibodies directed to the mannose-6-phosphate receptor labeled vacuole membranes. Antibodies to the transferrin receptor and to the lysosomal membrane glycoprotein LAMP 1 did not. Moreover, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, which normally accumulates in compartments with low pH, was not present inside these vacuoles. These results suggest that vacuoles containing the agent of HGE fail to mature into phagolysosomes. We conclude that the agent of HGE appears to enter and modify part of the endocytic pathway.

Isolation and establishment of the raccoon Ehrlichia-like agent in tick cell culture.

Feral animals are reservoirs of emerging human pathogens, as well as carriers of closely related wildlife diseases. The latter may interfere with epidemiologic studies by inducing cross-reactive antibodies, or by providing false positive signals in PCR based tests. We cultured a novel intracellular bacterium from the blood of two raccoons (Procyon lotor): RAC413 and RAC414. RAC413 had been experimentally inoculated with blood from a wild-caught raccoon, and provided the material for a blood passage into RAC414. The microbes grew in Ixodes scapularis (black-legged tick) cells, line ISE6, inoculated either with the leukocyte or erythrocyte fraction of anticoagulated blood. Giemsa-stained cells sampled two and three months after initial inoculation of the cultures revealed inclusions similar to those of Ehrlichia sp., except that individual bacteria commonly were elongated and clustered within endosomes. Electronmicroscopy confirmed the presence of irregularly shaped bacteria with evenly granular bacterioplasm bounded by a unit membrane. 16S rDNA sequencing identified the microbes as the raccoon Ehrlichia-like agent previously detected in feral raccoons from Georgia, United States. In conclusion, the availability of a culture isolate of this agent will facilitate future studies to determine its biology, epidemiologic significance, vector association, and host range. The Ehrlichia-like agent infecting raccoons joins a growing list of tick-borne agents cultivable in tick cells.

Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Ehrlichiosis and rickettsiosis in a canine population of Northern Sardinia.

Ehrlichiosis and rickettsiosis are very common, widespread diseases. These diseases are present in Sardinia year round because its temperate weather permits the survival of many kinds of tick vectors. A thousand dogs were subjected to physical, hematological, biochemical examinations and serological tests. All 1,000 sera were analyzed by indirect immunofluorescence antibody test to detect antibodies against E. canis and R. rickettsii. A high seroprevalence (about 50%) was detected for both etiological agents, without differences in relation to sex, breed, or usage. A high seroprevalence, corresponding to 62.5% for ehrlichiosis and 64% for rickettsiosis, was observed in the age group of 13-60 months. The mortality was greatest in the males in the age group, which manifested the disease in the chronic phase.

Continuous propagation of Ehrlichia sennetsu in murine macrophage cell cultures.

Ehrlichia sennetsu, the etiologic agent of human sennetsu rickettsiosis was successfully propagated in a continuous cell culture using murine cell lines P388D1 and Raw 264. Pleomorphic cytoplasmic inclusion bodies similar to Ehrlichia canis morulae were observed 3-4 days after second post-inoculation split. In the Raw 264 cell line E. sennetsu was not seen until the third passage. Relatively heavier infection was observed in P388D1 than in Raw cell line. The latter reached a maximum of 15% infection whereas P388D1 cell line attained saturation. Structural details of the organism were confirmed by electron microscopy. A unique rippled cell mass surrounding the plasma membrane was observed. Supernatants of cultures were shown to contain infectious organisms. The advantages of propagating E. sennetsu in continuous cell lines are discussed with respect to future physiochemical and immunochemical studies of this organism.

A single tissue culture system for the propagation of the agents of the human ehrlichioses.

Two newly emergent human diseases found in the United States, human monocytotropic ehrlichiosis (HME) and human granulocytotropic ehrlichiosis (HGE), are caused by pathogens of the genus Ehrlichia. The causative agent of HGE can be propagated in HL-60 human promyelocytic leukemia cells. Herein, we report the development of a method to propagate E. chaffeensis, the causative agent of HME, in HL-60 cells, thus providing a common system for the study of both species. The continuous propagation of E. chaffeensis requires the induction of HL-60 differentiation along the monocytic pathway toward phenotypically mature macrophages by the addition of 25-OH vitamin D3 to the growth medium.

In vitro proliferation of a canine granulocytic Ehrlichia.

Canine granulocytic Ehrlichia sp., an agent which parasitizes the neutrophilic leukocytes in dogs, was transiently propagated in vitro. Dogs were experimentally inoculated with blood containing canine granulocytic Ehrlichia. Bacteremias in experimentally infected dogs varied from 1.2 to 9.3% granulocytes infected. Granulocytes from experimentally infected dogs were harvested and cultured in the presence of RPMI 1640 medium supplemented with fetal bovine serum, conditioned medium, and HEPES buffer. The percentages of granulocytes containing ehrlichial morulae increased significantly with time for 2 to 4 days, with at least one culture from each dog achieving 20% of granulocytes infected. Granulocytes taken from infected dogs early in bacteremia yielded cultures with the greatest percentage of infected cells. By 5 days post-infection the percentage of infected granulocytes decreased as did leukocyte viability. Attempts to maintain the in vitro cultures for prolonged periods by addition of uninfected granulocytes failed to increase the number of infected host cells, suggesting that no new infections were initiated and that observed increases in the percentage of infected cells in in vitro cultures were due to growth of the organism in granulocytes that were infected in vivo.

In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever.

Cytoecetes phagocytophila, a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37 degrees C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever.

Functions of neutrophils in sheep experimentally infected with Ehrlichia phagocytophila.

Ehrlichia phagocytophila infection in sheep is characterized by persistent neutropaenia, indicative of decreased phagocytic capacity. This predisposes infected animals to other infections. A whole blood flow cytometrical method was used to document the degree and extent of reduced phagocytic and respiratory burst activity in phagocytes during an experimental infection with E. phagocytophila, and monitored until 56 days post-infection. Six sheep at 5 months of age were inoculated with an intravenous injection of infected blood. Six age-matched sheep were used as controls. A period of reduced respiratory burst lasting up to Day 17 post-infection was recorded. The population of cells showing phagocytic activity without respiratory burst was larger in the infected animals compared to controls up to Day 45 post-infection.

Detection of antigenemia by enzyme-linked immunosorbent assay in horses with experimental Ehrlichia risticii infection.

Four horses were inoculated with Ehrlichia risticii contained in either infected murine P388 D1 cells or heparinized blood from an infected horse. All 4 horses produced serum antibody, plasma antigen, and clinical signs of the disease. An enzyme-linked immunosorbent assay was used to detect antibody in the serum and was also used in conjunction with an anti-E. risticii monoclonal antibody to detect antigenemia. These laboratory and clinical findings were correlated to determine the efficiency of the antigen detection method for discerning E. risticii infection.

The first isolation, in vitro propagation, and genetic characterization of Ehrlichia canis in Israel.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, was isolated in Israel from a naturally infected dog with acute signs of the disease. The organism designated E. canis 611, was passaged experimentally to a beagle, from which it was propagated in primary canine monocytes. The organism was then grown in vitro in a continuous canine cell line, DH82. Nine beagles subsequently injected with whole E. canis-infected blood all developed typical symptoms of ehrlichiosis. An indirect immunofluorescence antibody test to E. canis was developed and compared with a commercial kit, revealing a good correlation between the two assays. Transmission electron microscopy of DH82 cells infected with the Israeli strain of E. canis (611), revealed organisms similar to those described in the literature: two different forms of morulae appeared, one tightly, the other loosely, packed. The 16S rRNA gene sequence obtained from the Israeli Ehrlichia isolate was compared with other isolates, E. canis Oklahoma and E. canis Florida. The Israeli strain 16S rRNA had three nucleotide differences from the Oklahoma isolate, and four nucleotide differences from the Florida isolate, in addition to one nucleotide gap in each. The Israeli isolate was found to be 0.54% different from the Oklahoma strain, and 0.61% different from the Florida strain. There are the same magnitudes of differences displayed by the other most closely related group in the phylogenetic tree, namely Ehrlichia equi, Ehrlichia phagocytophilia and the human granulocytic ehrlichia.

Humoral antibody and lymphocyte blastogenesis responses in BALB/c, C3H/HeJ and AKR/N mice following Ehrlichia risticii infection.

Three strains of mice, two susceptible to Ehrlichia risticii induced disease (BALB/c and C3H/HeJ) and one resistant (AKR/N), were evaluated for the development of humoral and cell mediated immune responses following infection with E risticii. The production of serum antibody was determined by indirect fluorescent antibody testing of sera from mice of each strain challenged with one of three different dose levels of E risticii. Antibody was assayed on days 7, 9, 12, 15 and 20 after inoculation. Cell mediated immune responses were evaluated by measuring the blastogenesis response of spleen cells from E risticii infected mice 28 days after inoculation. All three strains of mice at the high challenge level responded with the production of antibody by day 9 after inoculation. Overall, the antibody response occurred earlier and was of greater magnitude in the susceptible BALB/c and C3H/HeJ strains. A marked blastogenesis response occurred in splenocytes from E risticii infected mice of all three strains upon re-exposure to ehrlichial antigen. The findings of this study indicate that susceptibility to E risticii induced disease was not the result of deficient or delayed humoral immune responses and that E risticii infection induced the development of strong cell mediated immunity.

Immunosuppression in sheep experimentally infected with Ehrlichia phagocytophila.

The effect of tick-borne fever (TBF) on antibody formation and lymphocyte proliferation in sheep was studied following experimental infection with Ehrlichia phagocytophila. All infected sheep developed fever within three to four days. The sheep recovered clinically within eight days. Both infected and non-infected control sheep were immunised twice with different antigens, that is, on days 9 and 35 following the experimental infection. The levels of antibodies produced against tetanus toxoid and influenza virus in the infected sheep were significantly lower than in the control animals. The findings indicated that a TBF-infection may impair both primary and secondary antibody responses for up to six weeks. Immunisation with Actinomyces pyogenes resulted in significantly higher antibody titres in the TBF-infected group than in the control group, as measured by an enzyme-linked immunosorbent assay (ELISA). It is believed that TBF-induced neutropenia may lead to increased exposure to A pyogenes-antigens and thereby enhance antibody production. Antibodies to E phagocytophila were measured by the indirect fluorescent antibody test and by an ELISA. The inoculated sheep responded with the formation of antibodies to E phagocytophila at one week (P < 0.025), and showed a peak response at four weeks (P < 0.0005) after inoculation. The antibody titre decreased between four and six weeks, but was still high at six weeks (P < 0.0005). The lymphocyte responses to phytohaemagglutinin (PHA) concanavalin A (Con A) and pokeweed mitogen (PWM) were lower than in the control group and this difference was significant at most time points after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Survey of tick infestation and tick-borne ehrlichial infection of dogs in Ishigaki Island, Japan.

Twelve (54.5%) of 22 free-roaming dogs in Ishigaki Island had tick infestation identified as Rhipicephalus sanguineus. There were 121 ticks recovered and consisted of 28 females, 58 males, 22 nymphs and 3 larvae. Infection of dogs possibly with canine ehrlichial pathogens was examined by both indirect immunofluorescence assay and polymerase chain reaction (PCR). Two dogs of the 13 examined were sero-positive for the human granulocytic ehrlichia agent, and one of two dogs was PCR positive for Ehrlichia platys. This dog had platelet numbers slightly lower than normal value, however, no morulae were found within platelet on peripheral blood smear stained with Giemsa.

Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever).

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.

Effect of canine immune serum on the growth of Ehrlichia canis within nonimmune canine macrophages.

The effect of canine immune serum (CIS) on the growth of Ehrlichia canis was studied in macrophage cultures derived from peripheral blood monocytes of normal dogs. Ehrlichiae treated with canine normal serum and then introduced into normal macrophage cultures maintained in canine normal serum multiplied within the macrophages and destroyed them. Immune serum, collected from E canis carrier dogs, suppressed ehrlichial growth in normal macrophages. An inverse relationship existed between the amount of CIS to which ehrlichiae were exposed and the growth of the organism in normal macrophages. The anti-E canis activity was most prevalent in the 7-S fraction of immune serum. Fresh CIS had a greater suppressive effect on the growth of E canis than did heat-inactivated CIS. Antibody cytophilic for canine monocyte-derived macrophages was not detected in CIS.