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PubMed2005

Anaplasma phagocytophilum delay of neutrophil apoptosis through the p38 mitogen-activated protein kinase signal pathway.

Human granulocytic anaplasmosis is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium avoids host innate defenses in part by infecting, surviving in, and propagating in neutrophils, as well as by inhibiting neutrophil apoptosis. However, the mechanisms of A. phagocytophilum survival in neutrophils and the inhibition of spontaneous apoptosis are not well understood. In this study, we demonstrated that antiapoptotic Mcl-1 protein (Bcl-2 family) expression is maintained and that inhibition of procaspase-3 processing occurs in A. phagocytophilum-infected human neutrophils. An evaluation of p38 mitogen-activated protein kinase (MAPK) showed evidence of increased phosphorylation with infection. Moreover, antagonism of p38 MAPK by the inhibitor SB203580 reversed apoptosis inhibition in live or heat-killed A. phagocytophilum-infected neutrophils. A role for the autocrine or paracrine production of antiapoptotic interleukin 8 (IL-8) expressed with A. phagocytophilum infection was excluded by the use of IL-8-, IL-8R1 (CXCR1)-, and IL-8R2 (CXCR2)-blocking antibodies. As previously demonstrated, the antiapoptotic effect was initially mediated by exposure to A. phagocytophilum components in heat-killed bacteria. However, an important role for active infection is demonstrated by the additional delay in apoptosis with intracellular growth and the refractory abrogation of this response by the p38 MAPK inhibitor 3 to 6 h after neutrophil infection. These results suggest that the initial activation of the p38 MAPK pathway leading to A. phagocytophilum-delayed neutrophil apoptosis is bypassed with active intracellular infection. Moreover, active intracellular infection contributes more to the overall delay in apoptosis than do components of heat-killed A. phagocytophilum alone.

Anaplasma phagocytophilum↗
PubMed2005

Coinfection with Anaplasma phagocytophilum alters Borrelia burgdorferi population distribution in C3H/HeN mice.

Borrelia burgdorferi, the agent of Lyme disease, and Anaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted by Ixodes sp. ticks and may occasionally coinfect a host. The population distributions of tick-transmitted B. burgdorferi infection were assessed using quantitative PCR targeting the flaB gene of B. burgdorferi in the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected with A. phagocytophilum. Population distributions of Anaplasma infection were assessed by targeting the p44 gene. A. phagocytophilum in blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, but Anaplasma numbers remained constant. Antibody response to A. phagocytophilum, but not B. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection with A. phagocytophilum and B. burgdorferi modulates pathogen burden and host antibody responses. This may be explained by the ability of A. phagocytophilum to functionally impair neutrophils, important cells in the early defense against B. burgdorferi infection.

Anaplasma phagocytophilum↗
PubMed2006

Analysis of involvement of the RecF pathway in p44 recombination in Anaplasma phagocytophilum and in Escherichia coli by using a plasmid carrying the p44 expression and p44 donor loci.

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single p44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the A. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved in p44 gene conversion. The recombination intermediate structure between a donor p44 and the p44 expression locus of A. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor p44 locus connected to the 3' conserved region of the recipient p44 in the p44 expression locus in direct orientation. To determine whether the p44 recombination intermediate structure can be generated in a RecF-active Escherichia coli strain, we constructed a double-origin plasmid carrying the p44 expression locus and a donor p44 locus and introduced the plasmid into various E. coli strains. The recombination intermediate was recovered in an E. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the p44 gene conversion in A. phagocytophilum occurs through the RecF pathway.

Anaplasma phagocytophilum↗
PubMed2006

Porin activity of Anaplasma phagocytophilum outer membrane fraction and purified P44.

Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel beta-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain beta-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.

Amino Acid Sequence↗
PubMed2002

Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet.

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.

Anaplasma↗
PubMed2005

Genetic variants of Anaplasma phagocytophilum infecting dogs in Western Washington State.

Eight dogs from western Washington State suspected of being infected with Anaplasma phagocytophilum because of the finding of morulae in peripheral blood neutrophils were studied for determination of the etiologic agent of disease. All cases were diagnosed between April 2003 and April 2004. Six of the eight dogs had no travel history during the 6 months prior to presentation. Two dogs had traveled within the Northwest United States and Canada. Fever, lethargy, and anorexia were the most common clinical signs in the dogs. Lymphopenia, thrombocytopenia, and an elevated activity of alkaline phosphatase in the serum were the most common laboratory findings. All dogs tested during the acute phase of clinical signs were seropositive for A. phagocytophilum antibodies but negative for Ehrlichia canis antibodies. PCR amplification and direct sequencing of portions of the 16S rRNA gene from the whole blood of all seven dogs that were tested yielded A. phagocytophilum after a comparison to bacterial sequences available in the GenBank database. Five genetic variants were identified based on one or two nucleotide differences in the 16S rRNA gene sequences at nucleotide positions 54, 84, 86, and 120. Individual dogs were infected with more than one variant. Treatment with doxycycline or tetracycline resulted in a rapid resolution of clinical signs. The occurrence of canine granulocytic anaplasmosis in western Washington State suggests that A. phagocytophilum infection should be considered in differential diagnoses of dogs presenting with lethargy, anorexia, fever, and lameness, particularly in the context of lymphopenia, thrombocytopenia, and increased serum alkaline phosphatase. The zoonotic importance of A. phagocytophilum should support an increase in surveillance for horses and people residing in this area.

Anaplasma phagocytophilum↗
PubMed2026

Complete genome sequence of the Anaplasma phagocytophilum clinical isolate NCH-1.

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacterium and etiologic agent of human granulocytic anaplasmosis. A. phagocytophilum genomic sequencing has historically been performed via short-read platforms. Our optimized bacterial isolation protocol combined with Nanopore sequencing produced a single, closed 1,481,805 bp circular A. phagocytophilum strain NCH-1 chromosome.

Anaplasma phagocytophilum↗
PubMed1986

A study of the justification for intensive tick control in Kenyan rangelands.

Sixty male weaner cattle at the National Range Research Station, Kiboko, Kenya were placed in four groups of 15 animals under four different tick control regimens for 16 months. The treatment groups were: spraying with acaricide weekly, spraying every three weeks, spraying whenever group mean tick infestations reached more than 200 per animal (once only in the course of the study) and a control unsprayed group. The cattle were weighed monthly. There were no significant differences between the liveweight gains of the groups during a period of severe drought and during the following period of compensatory weight gain. The untreated group gained more weight than the other groups. Cattle died in all groups, but without significant differences between the groups; the long drought and associated malnutrition were the prime cause of death. Rhipicephalus pravus and R pulchellus were the dominant tick species with fewer Amblyomma gemma, Hyalomma truncatum and small numbers of Boophilus decoloratus and R evertsi. These ticks transmit anaplasmosis, babesiosis and cowdriosis in the study area. Total tick counts reached 750 on individual animals but group means rarely reached more than 200 because of high resistance to ticks in some animals. Host resistance ranking within groups was virtually constant throughout the experiment. The study showed that intensive tick control is not required in the semi-arid areas of Africa.

Animals↗
PubMed1987

Clinical trial of halofuginone lactate for the treatment of East Coast fever in Kenya.

A field trial was undertaken to test the efficacy of halofuginone lactate in the treatment of East Coast fever under farming conditions in Kenya. The drug was administered orally at a dose of 1.2 mg/kg bodyweight and treatment was repeated after 48 hours. Of 293 cases treated 236 (80.5 per cent) recovered and 49 (16.7 per cent) died. Five animals were disposed of by the owners and three became chronically infected. No differences were recorded in recovery rate between uncomplicated cases and cases with concurrent anaplasmosis or babesiosis, nor were there any significant differences in recovery rates between animals of different types. However, zebu cattle (Bos indicus) tended to respond more rapidly to treatment than exotic cattle (Bos taurus). Younger animals had a poorer recovery rate than adults. Early treatments were more successful than those administered late. It was concluded that with early detection and treatment, coupled with efficient tick control, halofuginone lactate is effective in the treatment of clinical East Coast fever under field conditions.

Animals↗
PubMed2004

Anaplasma phagocytophilum: an emerging tick-borne pathogen in Hungary and Central Eastern Europe.

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, Ehrlichia equi and Anaplasma phagocytophila) is the causative agent of granulocytic ehrlichiosis (anaplasmosis) in humans, horses, sheep, cattle, dogs and cats. In the present study, 452 European sheep ticks (Ixodes ricinus) collected from 100 red foxes (Vulpes vulpes) in Hungary were tested for the pathogen, as 112 pools each containing five or fewer ticks from one fox. Six of the pools, representing ticks from six different foxes, were found infected in the PCR-based test employed. This is the first time that A. phagocytophilum has been reported in Hungary. A summary of the information available from Central Eastern Europe on the prevalence of A. phagocytophilum in ticks, its seroprevalence in humans, and the number of human cases of granulocytic ehrlichiosis known in the region is presented.

Anaplasma phagocytophilum↗
PubMed2005

Infection with Anaplasma phagocytophilum in a seronegative patient in Sicily, Italy: case report.

BACKGROUND: Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also. CASE PRESENTATION: Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog. CONCLUSION: Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.

Anaplasma phagocytophilum↗
PubMed2005

Detection of a non-pathogenic variant of Anaplasma phagocytophilum in Ixodes ricinus from La Rioja, Spain.

Our aim was to identify variants of Anaplasma phagocytophilum 16S rRNA gene sequences among products amplified from Ixodes ricinus collected in La Rioja, Spain. A. phagocytophilum AP-variant 1, reported as non-pathogenic, was detected in 12 samples (two adults and ten nymphs). This finding could justify the low incidence of human anaplasmosis in our area, despite the high prevalence of A. phagocytophilum in ticks.

Anaplasma phagocytophilum↗
PubMed2005

Platelet dysfunction after association with Anaplasma phagocytophilum in vitro.

Granulocytic anaplasmosis is a tick-borne, zoonotic disease caused by an obligate intracellular granulocytotropic bacterium, Anaplasma phagocytophilum. A. phagocytophilum and other Anaplasma and Ehrlichia spp. have been variably implicated with an associated hematologic alteration, platelet dysfunction, that can result in potentially serious clinical sequelae including hemorrhage. The objective of this work was to define an in vitro model to assess alterations in platelet function associated with A. phagocytophilum.

Anaplasma phagocytophilum↗
PubMed2005

Anaplasma phagocytophilum infection reduces expression of phagocytosis-related receptors on neutrophils.

Anaplasma phagocytophilum is a tick-transmitted obligate intracellular bacterium of neutrophils that causes human granulocytic anaplasmosis. Previous data confirm that in vitro infection by A. phagocytophilum modifies neutrophil functions, including a 50% or greater reduction in phagocytosis and shedding of neutrophil cell surface adhesion molecule receptors. If these receptors are downregulated or shed from the surface of neutrophils with A. phagocytophilum infection, it may prevent neutrophils from effective phagocytosis. We hypothesized that diminished phagocytosis in neutrophils is partly associated with the loss of surface phagocytic receptors. To address this, we assayed the expression of these receptors after 24 h of A. phagocytophilum infection in human neutrophils.

Anaplasma phagocytophilum↗
PubMed2006

Highly variable year-to-year prevalence of Anaplasma phagocytophilum in Ixodes ricinus ticks in northeastern Poland: a 4-year follow-up.

Anaplasma phagocytophilum is transmitted mainly by Ixodes ricinus ticks in Europe. We followed-up A. phagocytophilum infection rate in I. ricinus in three selected collection sites in northeastern Poland during a four-year period. Overall infection rate was 14.1% (208/1474) with highest infection rate among females (36.8% verses males 8.2% and nymphs 0.9%). We noted a very big year-to-year variation of infection prevalence in each collection cite every year reflecting changeable granulocytic anaplasmosis risk for humans and animals.

Anaplasma phagocytophilum↗
PubMed2006

New findings on members of the family Anaplasmataceae of veterinary importance.

Members of the family Anaplasmataceae are obligate intracellular Gram-negative bacteria that naturally infect a variety of wild and domestic animal species, the spillover of which may lead to zoonosis. I discuss new findings on members of the family Anaplasmataceae of veterinary importance and therefore, I will describe the recent findings on Neorickettsia risticii in the trematode and related Neorickettsia species. I also will review the recent progress on Aegyptianella pullorum and other Aegyptianella sp., "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum strains in various hosts. The whole genome sequences of two important veterinary pathogens-Anaplasma marginale, the bovine anaplasmosis agent, and Ehrlichia (formerly Cowdria) ruminantium, the agent of heartwater of ruminants-have been published. Taken together, these advances in research of the family Anaplasmataceae in the veterinary field provide us with insights into the evolution, reservoir, and transmission of these organisms in nature and their pathogenesis in natural and accidental hosts. It is through this work that surveillance, diagnosis, preventive measures, and treatment of ehrlichioses of both animals and humans can be improved.

Anaplasmataceae↗
PubMed2007

Experimental infection of C3H/HeJ mice with the NY18 isolate of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections.

Anaplasma phagocytophilum↗
PubMed2003

The Lyme Wars: time to listen.

Lyme disease represents a public health threat of major proportions. The murky science and acrimonious politics of Lyme disease have created barriers to reliable diagnosis and effective treatment of this protean illness. Two major clinical problems with the disease are the absence of a therapeutic end point in treating Borrelia burgdorferi, the spirochetal agent of Lyme disease, and the presence of tick-borne co-infections, such as babesiosis, anaplasmosis and bartonellosis, that may complicate the course of the illness. From a pathophysiological standpoint, the affinity of B. burgdorferi for multiple cell types and the presence of non-replicating forms of the spirochete have contributed to persistent infection and failure of simple antibiotic regimens. Newer approaches to the treatment of Lyme disease should take into account its clinical complexity in co-infected patients and the possible need for prolonged combination therapy in patients with persistent symptoms of this potentially debilitating illness. The risk and prevention of human transmission of Lyme disease merit further study.

Animals↗