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PubMed2006

Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway.

Anaplasma phagocytophilum, an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro. The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL-60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome-associated membrane protein-1 (LAMP-1), whereas, VirB9-undetectable bacteria were not colocalized with LAMP-1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosome-lysosome pathway.

Anaplasma phagocytophilum↗
PubMed2006

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel.

The two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.

Anaplasma phagocytophilum↗
PubMed2006

Anaplasma phagocytophilum delays spontaneous human neutrophil apoptosis by modulation of multiple apoptotic pathways.

Anaplasma phagocytophilum infects human neutrophils and inhibits the intrinsic pathway of spontaneous neutrophil apoptosis by protecting mitochondrial membrane integrity. In the present study, we investigated the molecular signalling of the extrinsic pathway and the interaction between the intrinsic and extrinsic pathways in the inhibition of spontaneous human neutrophil apoptosis by A. phagocytophilum. Cell surface Fas clustering during spontaneous neutrophil apoptosis was significantly blocked by A. phagocytophilum infection. The cleavage of pro-caspase 8, caspase 8 activation and the cleavage of Bid, which links the intrinsic and extrinsic pathways, in the extrinsic pathway of spontaneous neutrophil apoptosis were inhibited by A. phagocytophilum infection. Inhibition of this pathway was active as the cleavage of pro-caspase 8 and Bid in anti-Fas-induced neutrophil apoptosis was also inhibited by A. phagocytophilum infection. Likewise, A. phagocytophilum infection inhibited the pro-apoptotic Bax translocation to mitochondria, activation of caspase 9, the initiator caspase in the intrinsic pathway, and the degradation of a potent caspase inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP), during spontaneous neutrophil apoptosis. These data point to a novel mechanism induced by A. phagocytophilum involving both extrinsic and intrinsic pathways to ensure to delay the apoptosis of host neutrophils.

Anaplasma phagocytophilum↗
PubMed2006

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.

Adhesins, Bacterial↗
PubMed2005

Immune-mediated haemolytic anaemia and thrombocytopenia associated with Anaplasma phagocytophilum in a dog.

A 10-year-old, crossbreed dog was presented with a history of severe lethargy, pyrexia and inappetence of several days' duration. Clinical examination revealed pallor of the mucous membranes, petechiae, generalised lymphadenopathy and effusions in multiple joints. Laboratory evaluation showed severe anaemia and thrombocytopenia, with positive in-saline agglutination and the presence of antiplatelet antibodies. The DNA of Anaplasma phagocytophilum, an endemic granulocytic rickettsial parasite, was detected by PCR. A poor response to doxycycline and immunosuppressive therapy with corticosteroids was seen. Euthanasia was performed after the development of disseminated intravascular coagulation. Postmortem examination demonstrated changes consistent with the development of disseminated intravascular coagulation and infection with granulocytic ehrlichiosis. This case documents the presence of canine granulocytic ehrlichiosis caused by A phagocytophilum in the U.K., and highlights the range of clinical signs and clinicopathological abnormalities that may be observed in infected dogs.

Anaplasma phagocytophilum↗
PubMed1998

Development of a non-radioactive DNA probe and in situ hybridization for detection of Anaplasma marginale in ticks and cattle.

A non-radioactive DNA probe was developed for detection of Anaplasma marginale in ticks and cattle. The probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction. The probe was tested on bovine blood and was found to be a sensitive and specific detection method for A. marginale in cattle. The DNA probe was then adapted for in situ hybridization (ISH) of A. marginale in Dermacentor andersoni and D. variabilis ticks infected either as nymphs or adults. One-half of each tick was studied with ISH while the other half was examined with light and electron microscopy. In male ticks infected as adults, tick gut cells first became infected with A. marginale while ticks fed on an infected calf, and they remained infected as they transmission fed on a second, susceptible calf. At the onset of transmission feeding, salivary glands became infected with A. marginale. During transmission feeding infection was also observed in interstitial, reproductive, skeletal muscle, fat body and Malpighian tubule tissue, resulting in a generalized A. marginale infection. When adult ticks that acquired infection as nymphs were examined with ISH and microscopy, gut tissues of both D. andersoni and D. variabilis became infected with A. marginale. However, salivary gland infection was seen only in D. variabilis, even though both species of ticks transmitted A. marginale to susceptible calves. A. marginale was not seen with ISH or microscopy in hemocytes collected from both species of ticks and, thus, hemocytes do not appear to play a role in the development of A. marginale in ticks.

Anaplasma↗
PubMed2003

Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophila.

Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. DNA was extracted from a subset of these ticks, and PCR and DNA sequencing of the 16S rRNA gene were used to determine the ratio of Anaplasma phagocytophila-human agent (AP-ha) to a genetic variant not associated with human disease (AP-Variant 1). The remaining ticks were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks, and blood samples drawn from each mouse at one-week intervals, were evaluated by PCR and DNA sequencing for the presence of AP-ha and Variant 1. Although a high percentage of the infecting ticks harbored AP-Variant 1, only AP-ha was amplified from the mouse blood samples. Because the A. phagocytophila variant did not establish an infection either in the natural reservoir of AP-ha, the white-footed mouse, or in a common research laboratory mouse (DBA/2), AP-Variant 1 may have an alternative natural reservoir, possibly the white-tailed deer.

Anaplasma phagocytophilum↗
PubMed2003

Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan.

In the present study, three canine blood samples from Japan, that were suspected to be ehrlichia positive were examined. After sequencing the 16S rRNA genes, each dog was found to be infected either with Ehrlichia canis (Kagoshima 1), Anaplasma platys (Okinawa 1), or Wolbachia sp. (Okinawa 2). Phylogenic analysis was performed on these sequences. The nearly entire 16S rRNA sequence of Kagoshima 1 was found to be most similar to the sequences from Oklahoma and Venezuela E. canis strains (1 base pair difference out of 1,387, 99.9% sequence identity). The 16S rRNA gene sequence of Okinawa 1 showed the closest DNA identity to the French strain of A. platys (1 base deletion out of 1,385 bp, 99.6% sequence identity). The 16S rRNA gene sequence of Okinawa 2 illustrated the closest DNA identity to that of a Wolbachia sp. from Dirofilaria immitis (98.9% sequence similarity). These data imply a low diversity within E. canis strains and within A. platys strains, including those strains reported in this study. This is also the first demonstration of Wolbachia DNA in dog blood, suggesting the involvement of Wolbachia sp. in canine febrile illnesses.

Anaplasma↗
PubMed1982

The Australian brown dog tick Rhipicephalus sanguineus as an experimental parasite of cattle and vector of Anaplasma marginale.

Experiments were done to explore the possible relationship between cattle, Australian dog ticks (Rhipicephalus sanguineus) and Anaplasma marginale. Calves' ears were exposed to larval, nymphal and adult ticks on 8, 9 and 7 occasions, respectively. The immature instars fed readily, but the adults attached very poorly to calves. Transtadial transmission of A. marginale was achieved on 6 occasions from 9 attempts: ticks infected as larvae or nymphs were able to transmit at the subsequent stage or stages. Transovarial transmission was not achieved. Six calves supported more than one infestation of ticks. Attached ticks caused the calves no apparent discomfort and calves developed no noticeable skin reactions. An abattoir survey of 200 hides detected no R. sanguineus.

Anaplasma↗
PubMed1986

A microplate enzyme-linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum.

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.

Anaplasma↗
PubMed1997

Effect of breed of cattle on innate resistance to infection with Babesia bovis, B bigemina and Anaplasma marginale.

OBJECTIVE: To assess the innate resistance of naive Bos taurus, Bos taurus cross Bos indicus and Bos indicus cattle to virulent Babesia bovis, B bigemina and Anaplasma marginale parasites. DESIGN: Groups of 10, pure B indicus, 1/2 B indicus cross, 1/4 B indicus cross and pure B taurus steers were infected with virulent B bovis, B bigemina and A marginale parasites [corrected]. PROCEDURE: Sequential infections were carried out by intravenous inoculation of infected blood containing 1 x 10(8) parasites of B bovis, followed by B bigemina and then A marginale. To assess resistance, measurements were made of parasitaemia, rectal temperature, packed cell volume and the number within a group requiring chemotherapy to control infection. There was a recovery period between each infection. RESULTS: Infection with B bovis showed that pure B indicus steers were significantly more resistant to B bovis infection than the other groups, with none of this group requiring treatment. There was no significant difference between 1/2 B indicus cross and 1/4 B indicus cross with 30% and 20%, respectively, of steers in these groups requiring treatment [corrected]. The pure B taurus steers were significantly more affected then those in the other three groups with 80% requiring treatment. Infections of B bigemina produced a mild response in comparison to that of B bovis and none of the steers required treatment. However, the pure B taurus group was significantly more affected than the other three groups for all other measurements. After the A marginale infection, B indicus steers were moderately affected with 50% requiring treatment, whereas 70% of the 1/2 B indicus group, 80% of the 1/4 B indicus cross group and 100% of the pure B taurus group required treatment [corrected]. CONCLUSIONS: All breeds of cattle, ranging from pure B indicus to pure B taurus may be at risk of severe disease if exposed to virulent A marginale. The results confirm that pure B indicus cattle are relatively resistant to B bovis, but there could be a significant risk of severe mortalities if cross-bred herds are exposed to virulent infection.

Anaplasma↗
PubMed2006

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia.

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.

Anaplasma↗
PubMed2004

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog.

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.

Anaplasma phagocytophilum↗
PubMed2003

Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographic areas in the United States.

We tested the antibiotic susceptibilities of eight strains of Anaplasma phagocytophilum (the agent of human granulocytic ehrlichiosis) collected in various geographic areas of the United States, including Minnesota, Wisconsin, California, and New York. The results are homogeneous and show that doxycycline, rifampin, and levofloxacin are the most active antibiotics against these strains in vitro.

Anaplasma phagocytophilum↗
PubMed2004

Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR.

We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffeensis were only partly susceptible. Beta-lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.

Anaplasma phagocytophilum↗
PubMed2003

Characterization of Anaplasma marginale isolated from North American bison.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.

Amino Acid Sequence↗
PubMed2003

Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep.

Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at -70 degrees C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 x 10(6) neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.

Anaplasma phagocytophilum↗