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PubMed2004

Molecular and serologic evidence of Anaplasma phagocytophilum infection in cats in North America.

Anaplasma phagocytophilum DNA was detected in blood of clinically ill cats from Massachusetts (n = 4) and Connecticut (1) by use of polymerase chain reaction assay and DNA sequencing. All 5 cats were allowed outdoors, and Ixodes scapularis were found on 3 cats. Clinical signs of fever, anorexia, and lethargy resolved quickly after treatment with doxycycline or tetracycline. Serum samples from each cat reacted with A. phagocytophilum morulae via an indirect fluorescent antibody assay; positive antibody titers persisted even after 21 to 30 days of treatment with tetracycline. To the authors' knowledge, this is the first report of A. phagocytophilum infection of domestic cats in North America. Results suggest that infection with the organism may be associated with clinical illness in some cats.

Anaplasma phagocytophilum↗

PubMed2006

Prevalence of DNA of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum,' Anaplasma phagocytophilum, and species of Bartonella, Neorickettsia, and Ehrlichia in cats used as blood donors in the United States.

OBJECTIVE: To identify the prevalence of DNA of Mycoplasma haemofelis; 'Candidatus Mycoplasma haemominutum'; Anaplasma phagocytophilum; and species of Bartonella, Neorickettsia, and Ehrlichia in blood of cats used as blood donors in the United States. DESIGN: Prospective study. ANIMALS: 146 cats that were active blood donors. PROCEDURES: Environmental history was requested for each blood-donor cat from which a blood sample (mixed with EDTA) was available. Polymerase chain reaction assays capable of amplifying the DNA of the microorganisms of interest following DNA extraction from blood were performed. RESULTS: Overall, DNA of one or more of the infectious agents was detected in blood samples from 16 of 146 (11%) feline blood donors. Twenty-eight laboratory-reared cats housed in a teaching hospital had negative results for DNA of all organisms investigated. The DNA of at least 1 infectious agent was amplified from blood samples collected from 16 of 118 (13.6%) community-source cats; assay results were positive for 'Candidatus M haemominutum,' M haemofelis, or Bartonella henselae alone or in various combinations. Of the community-source cats allowed outdoors (n = 61) or with known flea exposure (44), DNA for a hemoplasma or B henselae was detected in 21.3% and 22.7%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: When community-source cats, cats allowed outdoors, or cats exposed to fleas are to be used as blood donors, they should be regularly assessed for infection with M haemofelis, 'Candidatus M haemominutum,' and Bartonella spp, and flea-control treatment should be regularly provided.

Anaplasma phagocytophilum↗

PubMed2003

Seasonal dynamics of Anaplasma phagocytophila in a rodent-tick (Ixodes trianguliceps) system, United Kingdom.

We investigated the reservoir role of European wild rodents for Anaplasma phagocytophila using polymerase chain reaction (PCR) analysis of blood collected from individually tagged rodents captured monthly over 2 years. The only tick species observed in the woodland study site was Ixodes trianguliceps, and ruminant reservoir hosts were not known to occur. A. phagocytophila infections were detected in both bank voles and wood mice but were restricted to periods of peak nymphal and adult tick activity. Most PCR-positive rodents were positive only once, suggesting that rodent infections are generally short-lived and that ticks rather than rodents may maintain the infection over winter. Bank voles were more likely to be PCR positive than wood mice, possibly because detectable infections are longer lived in bank voles. This study confirms that woodland rodents can maintain A. phagocytophila in Great Britain in the absence of other reservoir hosts and suggests that I. trianguliceps is a competent vector.

Anaplasma phagocytophilum↗

PubMed2004

Anaplasma phagocytophilum, Babesia microti, and Borrelia burgdorferi in Ixodes scapularis, southern coastal Maine.

Ixodes scapularis (deer ticks) from Maine were tested for multiple infections by polymerase chain reaction and immunofluorescence. In 1995, 29.5%, 9.5%, and 1.9% of deer ticks were infected with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. In 1996 and 1997, the number of A. phagocytophilum-infected ticks markedly declined. In 1995 through 1996, 4 (1.3%) of 301 were co-infected.

Anaplasma phagocytophilum↗

PubMed2005

Anaplasma phagocytophilum in white-tailed deer.

We examined the reservoir potential of white-tailed deer for Anaplasma phagocytophilum. Results suggest that white-tailed deer harbor a variant strain not associated with human infection, but contrary to published reports, white-tailed deer are not a reservoir for strains that cause human disease. These results will affect surveillance studies of vector and reservoir populations.

Anaplasma phagocytophilum↗

PubMed2005

Anaplasma phagocytophilum-infected ticks, Japan.

We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.

Anaplasma phagocytophilum↗

PubMed2005

Human granulocytic anaplasmosis and Anaplasma phagocytophilum.

Human granulocytic anaplasmosis is a tickborne rickettsial infection of neutrophils caused by Anaplasma phagocytophilum. The human disease was first identified in 1990, although the pathogen was defined as a veterinary agent in 1932. Since 1990, US cases have markedly increased, and infections are now recognized in Europe. A high international seroprevalence suggests infection is widespread but unrecognized. The niche for A. phagocytophilum, the neutrophil, indicates that the pathogen has unique adaptations and pathogenetic mechanisms. Intensive study has demonstrated interactions with host-cell signal transduction and possibly eukaryotic transcription. This interaction leads to permutations of neutrophil function and could permit immunopathologic changes, severe disease, and opportunistic infections. More study is needed to define the immunology and pathogenetic mechanisms and to understand why severe disease develops in some persons and why some animals become long-term permissive reservoir hosts.

Anaplasma phagocytophilum↗

PubMed2001

Highly conserved regions of the immunodominant major surface protein 2 of the genogroup II ehrlichial pathogen Anaplasma marginale are rich in naturally derived CD4+ T lymphocyte epitopes that elicit strong recall responses.

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4(+) T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-gamma, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.

Amino Acid Sequence↗

PubMed2002

Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation.

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.

Anaplasma phagocytophilum↗

PubMed2005

Insights into pathogen immune evasion mechanisms: Anaplasma phagocytophilum fails to induce an apoptosis differentiation program in human neutrophils.

Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phagocytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial survival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis.

Anaplasma phagocytophilum↗

PubMed2003

Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China.

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.

Anaplasma phagocytophilum↗

PubMed1986

Experimental anaplasmosis in American bison: persistence of infections of Anaplasma marginale and non-susceptibility to A. ovis.

Blood collected 314 and 496 days after experimentally infecting splenectomized and spleen-intact American bison (Bison bison) with Anaplasma marginale was infective for splenectomized bovine steers. The pathogenesis was identical to that seen in bovine studies using bovine blood inoculations. A splenectomized bison remained normal clinically, hematologically and serologically for 10 mo after repeated inoculation of ovine blood infected with A. ovis.

Anaplasma↗

PubMed1989

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana.

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.

Agglutination Tests↗

PubMed1992

Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis.

In North America, the role of wild ruminants in the epidemiology of anaplasmosis has not been clearly defined. Such information is particularly meager in regard to bighorn sheep. We report the susceptibility of two Rocky Mountain bighorn sheep (Ovis canadensis canadensis) to experimental infection with a well characterized field isolate of Anaplasma ovis obtained from domestic sheep in Idaho. Both bighorn sheep developed infection resulting in severe clinical disease, with relatively high parasitemias, icterus and anemia. One animal required tetracycline therapy and responded well to treatment, while the other recovered uneventfully without treatment. Both bighorn sheep were spleen-intact, a condition that in A. ovis-exposed domestic sheep typically is associated with mild infection. The results indicate that bighorn sheep may be adversely affected if exposed to the organism in nature.

Anaplasma↗

PubMed1993

Characterization of an Anaplasma ovis isolate from desert bighorn sheep in southern California.

To understand the role of bighorn sheep (Ovis canadensis) in the epidemiology of anaplasmosis, we recovered a field isolate from a suspected enzootic area in southern California (USA). Whole blood was collected from three desert bighorn sheep (Ovis canadensis nelsoni) and inoculated into a susceptible splenectomized domestic sheep, calf and a susceptible spleen-intact bighorn sheep. No infection occurred in the calf, but a detectable infection developed in both the domestic sheep and bighorn sheep 24 days after inoculation. The infection in both domestic and bighorn sheep resulted in severe clinical disease but was resolved with the use of tetracycline. Using monoclonal antibodies and DNA probes, we confirmed that the isolate was Anaplasma ovis.

Anaplasma↗

PubMed2004

Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer.

Serum samples obtained from white-tailed deer (Odocoileus virginianus) in Connecticut (n=218) and South Carolina (n=20) (USA) during the period 1992-2002 were analyzed for antibodies to whole-cell or recombinant antigens (i.e., fusion proteins) of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, etiologic agents of Lyme borreliosis and granulocytic ehrlichiosis, respectively. In enzyme-linked immunosorbent assays (ELISAs) with whole-cell B. burgdorferi, the overall seropositivity rate for Connecticut (53%) exceeded that for South Carolina (30%). In separate tests of seven recombinant antigens of B. burgdorferi by an ELISA, seroprevalence for the VlsE antigen was highest (48%) in Connecticut followed by outer surface protein (OspF) (21%), whereas serum reactivities to the protein (p) 41-G antigen (55%) and VlsE (25%) were most frequent for South Carolina sera. In analyses for antibodies to the recombinant protein (p) 44 antigen of A. phagocytophilum, seroprevalences of 52% and 25% were recorded for Connecticut and South Carolina samples, respectively. These findings paralleled those determined by indirect fluorescent antibody staining methods with whole cells (43% and 30%). Moreover, there was good agreement (74%) in results of Western blot analyses and an ELISA when a subset of 39 sera was screened with whole-cell or recombinant p44 antigens of A. phagocytophilum. An ELISA with highly specific recombinant VlsE or p44 antigens can be used in conjunction with other antibody tests to determine whether deer living in different regions of eastern United States were exposed to B. burgdorferi or A. phagocytophilum.

Anaplasma phagocytophilum↗

PubMed1975

Propagation of Anaplasma marginale in bovine lymph node cell culture.

Anaplasma marginale was propagated in cell cultures derived from bovine lymph node (LN). Treatment of host cells with diethylaminoethyl dextran (DEAE-D) before inoculation and centrifugation of inoculum onto the monolayers resulted in significant numerical increases of A marginale. The direct fluorescent antibody technique (FAT) was used for detection of the organism in culture. The rat was combined with the standard microscopic count procedure to obtain numerical estimates of the organism in cell culture. Infection of LN cells was irregular, with some cells containing many organisms and others containing none. The organisms were dispersed or in inclusions in the cytoplasm of LN cells. Numerical increases of organisms occurred within 6 hours and these were greatest at 12 to 24 hours. After 24 hours, the organisms decreased rapidly, but small numbers of them were observed for at least 7 days. The average generation time in culture was approximately 17.1 hours.

Anaplasma↗

PubMed1975

Study of an attenuated Anaplasma marginale vaccine in Mexico natural challenge of immunity in an enzootic area.

An evaluation was made of the protection induced by an attenuated Anaplasma marginale vaccine in young purebred cattle against the challenge exposure of naturally transmitted anaplasmosis in enzootic areas of Mexico. The cattle, which were raised in isolation units free of arthropods, consisted of 10 Brown Swiss calves (1 to 13 months of age) and 8 Holstein calves (5 to 7 months of age). They were paired by breed, age, and body weight, and allotted to 2 equal groups. Calves in 1 group were vaccinated, and at 6 weeks after vaccinations were done, calves in both groups were placed in the field where they were raised for approximately 1 year. Two Holstein and 3 Brown Swiss calves of the nonvaccinated group (group 2) developed clinical anaplasmosis, and the remaining calves of this group had hematologic evidence of the disease during the 2 to 4 months after introduction to the field. The vaccinated group, which remained free of anaplasmosis, showed consistently greater weight gain than did the controls. Among the Holstein calves, the maximum weight difference in favor of the vaccinated group was 50 kg/head at 5.5 months after field exposure, and among the Brown Swiss calves, the differences in weight gain in favor of vaccinated calves at the end of the 12-month period was between 11 and 30 percent. It is concluded that the vaccine provided a means for safe adaptation of high-quality young cattle to the tropics by protecting them against what appears to be the major obstacle to this practice, anaplasmosis.

Agglutination Tests↗