Human ehrlichiosis: hematopathology and immunohistologic detection of Ehrlichia chaffeensis

Human ehrlichiosis is a recently described zoonosis caused by a rickettsia that infects leukocytes. Most patients have fever, headache, chills, and myalgias and develop leukopenia, thrombocytopenia, anemia, and elevations in serum hepatic aminotransferases. The cause of the peripheral leukopenia and thrombocytopenia is not known. We studied peripheral blood smears, bone marrow aspirates, and bone marrow biopsy specimens from patients with serologically proven ehrlichiosis to characterize the pathologic changes associated with leukopenia or thrombocytopenia, to detect the presence of immunohistologically demonstrable ehrlichiae, and to establish the infected host target cell(s). Specimens were obtained from 12 patients, and immunohistology for Ehrlichia chaffeensis was performed on tissue sections, aspirated bone marrow, and peripheral blood smears. Mean leukocyte and platelet counts available for nine patients were white blood cell count 3,300/microL (range, 1,100 to 10,300/microL) and platelets 61,000/microL (range, 40,000 to 82,000/microL). Findings included myeloid hyperplasia (eight cases), megakaryocytosis (seven cases), granulomas (eight cases), marrow histiocytosis (one case), myeloid hypoplasia (one case), pancellular hypoplasia (one case), and normocellular marrow (two cases). Morulae of E chaffeensis were detected in four of 10 cases examined by immunohistology. Most ehrlichiae were detected within histiocytes, although morulae were rarely present within lymphocytes. Leukopenia, thrombocytopenia, or pancytopenia apparently most often results from peripheral sequestration or destruction; however, hypoplasia of marrow elements is present occasionally. Immunohistologic demonstration of E chaffeensis offers a direct means for establishing the etiologic diagnosis. These observations show the relatively frequent occurrence of bone marrow granulomas and suggest that infection of cells of the reticuloendothelial system may participate in the pathogenesis of human ehrlichiosis.     

Doxycycline hyclate treatment of experimental canine ehrlichiosis followed by challenge inoculation with two Ehrlichia canis strains

These is increasing evidence to suggest that central noradrenergic mechanisms may contribute to the central nervous system manifestations of acute liver failure. To further elucidate this possibility, extracellular brain concentrations of the monoamines, noradrenaline (NA), dopamine (DA), and serotonin, were measured by high-performance liquid chromatography with electrochemical detection in microdialysates from the extracellular compartment of frontal cortex in rats with acute (ischemic) liver failure at various times during the progression of encephalopathy and brain edema, as well as in obligate control groups of animals. In addition, binding sites for the noradrenergic receptor subtype ligands, [3H]-prazosin (alpha1 sites), [3H]-RX821002 (alpha2 sites), and [125]I-iodopindolol (beta sites), were assessed using quantitative receptor autoradiography in regions of the brains of rats at coma stage of acute liver failure and of control groups of animals. Coma stages of encephalopathy in acute liver failure were associated with selectively increased noradrenaline concentrations (P < .05) and a concomitant selective loss of alpha1 and beta1 sites in frontal cortex and thalamus. These findings add to a growing body of evidence that central noradrenergic function is modified in acute liver failure and suggest that alpha1/beta1 receptor-mediated noradrenergic mechanisms may play a role in the pathogenesis of brain edema and encephalopathy in this condition.     

Experimental transmission of Ehrlichia canis (Rickettsiales: Ehrlichieae) by Dermacentor variabilis (Acari: Ixodidae)

Four trials were conducted in which laboratory-reared Dermacentor variabilis nymphs were exposed to Ehrlichia canis by feeding on experimentally infected dogs as soon as classical morulae were detected in peripheral blood monocytes. After molting 25, 50 or 90 adult tick pairs were permitted to feed on 7 Ehrlichia-naive dogs. Transmission occurred in trials 1 (1/1 dog), 3 (1/1 dog) and 4 (2/2 dogs) but not in trial 2 (0/3 dogs), with 4 of 7 dogs becoming infected. Successful transstadial transmission was demonstrated by detection of morulae in peripheral blood lymphocytes and by seroconversion to Ehrlichia canis 30 d post-exposure. Incubation periods ranged between 17 and 22 days (mean = 19). Clinical signs, typical of ehrlichiosis, included mucopurulent ocular discharge, lymphadenopathy and malaise with accompanying pyrexia, leukopenia and thrombocytopenia. Pyrexia, thrombocytopenia and erythrophagocytosis and vacuolization of the cytoplasm of monocytic cells were observed 1-4 d prior to detection of morulae. This is the first demonstration that a tick other than Rhipicephalus sanguineus is capable of transstadial transmission of this important pathogen of dogs.     

Carbamazepine hypersensitivity and rickettsiosis mimicking Kawasaki disease

We report on two patients whose clinical presentation resembled that of Kawasaki disease. The first patient was a boy with epilepsy, whose symptoms first appeared following treatment with carbamazepine. The second boy had Mediterranean Spotted Fever. The significance of medical history in avoiding overdiagnosis of Kawasaki disease is emphasized.     

Dogs infected with a human granulocytotropic Ehrlichia spp. (Rickettsiales: Ehrlichieae)

In contrast to the anticipation that in sepsis granulocyte colony-stimulating factor (G-CSF) would overactivate the nonspecific immune system by recruiting and priming leukocytes with consequent aggravation of inflammatory tissue lesions, recombinant (r) G-CSF pretreatment was protective in various experimental non-neutropenic models of inflammation. The mechanisms of protection, however, are not fully understood. Using intravital fluorescence microscopy, we show that rG-CSF enhances leukocyte endothelial cell interaction within the microvasculature of normal rat livers, whereas rG-CSF pretreatment of animals exposed to lipopolysaccharide (LPS) attenuates the LPS-induced leukocytic response, including stasis in sinusoids as well as rolling and adherence in postsinusoidal venules with subsequent tissue infiltration. Moreover, rG-CSF, which did not affect Kupffer cell activity in normal rat livers, reduced the immediate activation of Kupffer cells on LPS exposure, as indicated in vivo by the delayed adherence/phagocytosis of intra-arterially administered latex particles associated with attenuation of proinflammatory cytokine release (tumor necrosis factor alpha and interleukin-6). Finally, rG-CSF reduced LPS-induced nutritive perfusion failure and hepatocellular excretory dysfunction. This study provides evidence for a distinct, possibly tumor necrosis factor alpha-dependent modulation of LPS-induced cellular response within the liver by rG-CSF, thereby achieving protection against microcirculatory perfusion failure and hepatic dysfunction.     

Lethal effect of Rickettsia rickettsii on its tick vector (Dermacentor andersoni)

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88. 3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4 degreesC had a lower mortality (80.9%) than did those held at 21 degreesC (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.     

Spotted fever group rickettsiosis and vectors in Kanagawa prefecture

Primer pairs for PCR were designed from the gene encoding the 17,000-molecular-weight genus-common antigen of Rickettsia japonica, Rickettsia rickettsii, Rickettsia conorii, Rickettsia typhi and Rickettsia prowazekii. Primers R1, R2 were designed for amplifying the genomic DNA from spotted fever group (SFG) rickettsiae and epidemic typhus rickettsiae. Primers Rj5, Rj10 were designed for amplifying the genomic DNA from only R. japonica. Using the primers R1, R2, about a 540-bp fragment was observed by amplifying the genomic DNA from R. japonica, R. rickettsii, R. conorii, Thai tick typhus TT-118, Rickettsia sibirica, Rickettsia montana, Rickettsia askari, R. typhi, R. prowazekii and Katayama strain isolated from the patient infected with SFG rickettsiae. Using the primers Rj5, Rj10, the 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and Katayama strain. Therefore, the Katayama strain was identified to belong to R. japonica. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp bands were amplified from blood of the patients infected with SFG rickettsiae in Kanagawa prefecture. These findings indicate that the causative agent of SFG rickettsiosis in these two patients was R. japonica. The ticks, Ixodes ovatus and Haemaphysalis flava, were collected by out field research in Kanagawa prefecture. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp were amplified from these ticks. This indicates that I. ovatus and H. flava were the vector of R. japonica in Kanagawa prefecture. Also, with the primers R1, R2, about a 540 bp fragment was amplified but with primers Rj5, Rj10, no fragments were amplified from I. ovatus and H. flava. Therefore, these ticks may have SFG rickettsiae other than R. japonica and epidemic typhus rickettsiae.     

Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Minimum infection rate of Ambylomma americanum (Acari: Ixodidae) by Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in southern Indiana

OBJECTIVE: This study examined the 30-month stability of axis II conditions. METHOD: One hundred eight depressed outpatients received comprehensive, semistructured personality disorder assessments at baseline and at follow-up. RESULTS: The diagnostic stability of personality disorders ranged from low to moderate at the categorical level and was generally moderate at the dimensional level. Most disorders exhibited good discriminant validity, in that the association between a disorder at baseline and at follow-up was greater than the associations between that disorder at baseline and the other 11 axis 11 disorders at follow-up. Two variables, sex and lifetime history of substance abuse or dependence, were significantly related to change in level of personality disorder features over time. CONCLUSIONS: Personality disorders have low to moderate stability over a 30-month period in depressed outpatients.     

Case presentation: bovine leukocyte adhesion deficiency (BLAD) in Switzerland

Bovine Leukocyte Adhesion Deficiency (BLAD) Syndrome is a lethal congenital immunodeficiency caused by the strong reduction in the expression of leukocyte integrins (beta 2 integrins) on the surface of leukocytes. Therefore, neutrophils from BLAD animals lack the capacity to adhere to the endothelium, a necessary step in their emigration into foci of infection. Due to the virtual absence of neutrophil-mediated host defense, animals suffer from recurrent infection of the respiratory and gastrointestinal tracts and finally succumb to infections. A 14 days old Holstein-Friesian calf showing omphalophlebitis and leukocytosis, was referred to our clinic. It was found to suffer from several febrile episodes of infection. The tentative diagnosis BLAD could be confirmed for the first time in Switzerland by flow cytometry, pedigree analysis and by restriction fragment length polymorphism.     

Antimicrobial action of rabbit leukocyte CAP18(106-137)

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.     

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI)

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

Ehrlichia chaffeensis and Rochalimaea antibodies in Kawasaki disease

Sera from 38 patients with Kawasaki disease were tested for immunofluorescent antibodies to Ehrlichia chaffeensis, Rochalimaea henselae, and R. quintana Oklahoma. Only 2.5% of the patients tested positive for E. chaffeensis, and 5% were positive for R. henselae and R. quintana Oklahoma. Our data suggest that Ehrlichia and Rochalimaea spp. do not play a unique role in the etiology of Kawasaki disease.     

Prevalence of antibodies to Rickettsia conorii Ricketsia africae, Rickettsia typhi and Coxiella burnetii in Mauritania

A 24-year-old male developed cytogenetic relapse of chronic myeloid leukemia (CML) four years after allogeneic BMT. After a year of treatment with IFN-alpha, he achieved a partial cytogenetic response. Treatment with donor leukocyte infusions (DLI) was given (total dose 1 x 10(8) T lymphocytes/kg). Two months later, he developed acute GVHD (skin and liver), that improved with CsA and methylprednisolone and resulted in cytogenetic remission with complete donor chimerism. One month later he developed rhinocerebral mucormycosis and was successfully treated with surgical debridement and liposomal amphotericin B (total dose 12 g). This is the first case of mucormycosis described after DLI.     

3-(Alkylthio)-N-hydroxysuccinimide derivatives: potent inhibitors of human leukocyte elastase

A series of 3-(alkylthio)-N-hydroxysuccinimide derivatives was synthesized and their inhibitory activity towards human leukocyte elastase (HLE) was investigated. The interaction of the compounds having a 3-alkylthioether side chain (compounds 1 and 2) with HLE was found to involve rapid acylation of the enzyme, followed by total regain of enzymatic activity within 3 h. Interestingly, compounds 3-8, having an oxidized thioether side chain, were found to be highly effective, time-dependent, irreversible inhibitors of the enzyme. The k(obs)/I values for compounds 3-8 ranged between 890 and 24,000 M-1 s-1. These findings demonstrate that, unlike the physiological inhibitor of HLE (alpha-1-proteinase inhibitor), which is inactivated upon oxidation, low-molecular-weight compounds retain and/or show enhanced inhibitory activity towards HLE upon oxidation of the thioether side chain and lay the groundwork for the development of compounds that embody proteinase inhibitory and antioxidant activity.