Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi.
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A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States. (+info)
Reinfection with Anaplasma phagocytophilum in BALB/c mice and cross-protection between two sympatric isolates.
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Infection with Anaplasma phagocytophilum in white-footed mice results in partial protection against reinfection with the same agent. However, humans and domestic animals may be sequentially exposed to different isolates of the agent circulating in the same or adjacent foci. We investigated whether immune response to a tick-borne infection with A. phagocytophilum provides protection against homologous and heterologous challenges. BALB/c mice were infected with one of the two sympatric isolates of A. phagocytophilum via tick bite and challenged 16 weeks later by Ixodes scapularis nymphs infected with either the same or the alternative isolate. As controls, groups of infected mice were challenged by uninfected ticks to confirm an absence of reactivation of the original infection or groups of naive mice were fed upon by ticks from cohorts used for an infectious challenge. Xenodiagnostic I. scapularis larvae were fed upon each mouse at 14 and 21 days postchallenge (PCH) and tested for the presence of A. phagocytophilum as freshly molted nymphs. Blood samples for quantitative PCR were collected at 7, 14, 21, and 70 days PCH. Serum samples were collected weekly to monitor development of immune response. The proportion of infected animals, levels of bacteremia, and the prevalence of infection in xenodiagnostic ticks were higher in groups of control mice exposed to A. phagocytophilum for the first time than in mice reinfected with either homologous or heterologous isolates. The presence of antibodies against A. phagocytophilum did not protect mice from a challenge with either homologous or heterologous isolates, however the ensuing reinfection was significantly milder and of a shorter duration than the first infection with either isolate. (+info)
Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection.
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Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O(2)(-)) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b(558) mobilization to the membrane, as well as colocalization of Rac and p22(phox). This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O(2)(-). Indeed, A. phagocytophilum rapidly detoxifies O(2)(-) in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22(phox) and gp91(phox). Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing. (+info)
Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome.
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The intracellular organism Anaplasma phagocytophilum causes human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes. Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst. To investigate the mechanism of survival, we first assessed the kinetics of A. phagocytophilum entry into neutrophils by using double-labeling confocal microscopy. At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively. The neutrophil respiratory burst in the presence of A. phagocytophilum was assessed by a kinetic cytochrome c assay and by measurement of oxygen consumption. Neutrophils in the presence of A. phagocytophilum did not produce a significant respiratory burst, but A. phagocytophilum did not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added. Immunoelectron microscopy of neutrophils infected with A. phagocytophilum or Escherichia coli revealed that NADPH oxidase subunits gp91(phox) and p22(phox) were significantly reduced at the A. phagocytophilum phagosome after 1 and 4 h of incubation. In neutrophils incubated simultaneously with A. phagocytophilum and E. coli for 30, 60, and 90 min, gp91(phox) was present on 20, 14, and 10% of the A. phagocytophilum phagosomes, whereas p22(phox) was present in 11, 5, and 4% of the phagosomes, respectively. Similarly, on E. coli phagosomes, gp91(phox) was present in 62, 64, and 65%, whereas p22(phox) was detected in 54, 48, and 48%. We conclude that A. phagocytophilum does not suppress a global respiratory burst and that, under identical conditions in the same cells, A. phagocytophilum, but not E. coli, significantly reduces gp91(phox) and p22(phox) from its phagosome membrane. (+info)
CXCR2 blockade influences Anaplasma phagocytophilum propagation but not histopathology in the mouse model of human granulocytic anaplasmosis.
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Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils and causes human granulocytic anaplasmosis. Infection induces neutrophil secretion of interleukin-8 or murine homologs and perpetuates infection by recruiting susceptible neutrophils. We hypothesized that antibody blockade of CXCR2 would decrease A. phagocytophilum tissue load by interrupting neutrophil recruitment but would not influence murine hepatic pathology. C3H-scid mice were treated with CXCR2 antiserum or control prior to or on day 14 after infection. Quantitative PCR and immunohistochemistry for A. phagocytophilum were performed and severity of liver histopathology was ranked. Control mice had more infected cells in tissues than the anti-CXCR2-treated group. The histopathological rank was not different between treated and control animals. Infected cells of control mice clustered in tissue more than in treated mice. The results support the hypothesis of bacterial propagation through chemokine induction and confirm that tissue injury is unrelated to A. phagocytophilum tissue load. (+info)
A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle.
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Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi. (+info)
Polymorphism and transcription at the p44-1/p44-18 genomic locus in Anaplasma phagocytophilum strains from diverse geographic regions.
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A polymorphic multigene family (p44) of Anaplasma phagocytophilum encodes the immunodominant 44-kDa major outer membrane proteins. With p44-specific PCR and gene-specific probes, p44-1 was found in all human isolates from New York State but not in isolates from Minnesota, whereas p44-18 and two other p44 species were found in isolates from both regions. We therefore sequenced the genomic locus corresponding to the p44-1/p44-18 tandem locus of A. phagocytophilum HZ in 14 other geographically divergent strains from various hosts. The locus was found in all 14 strains, and p44-18 was conserved among all 13 United States isolates studied. In all nine northeastern strains, p44-1 was conserved. However, in three of the Minnesota strains and in one California strain, p44-1 was replaced at this genomic locus by the novel gene p44-61 (p44-61/18), whose hypervariable region (hv) was a chimera of p44-20hv and p44-23hv. The conserved base sequence within the hv region linked the two segments. In contrast, in the Old Sourhope strain isolated from sheep in the United Kingdom, only a single and distinct p44, p44-OS, was found in this locus. This suggests different rates of evolution of p44-1 and p44-18 at this locus and conservation of the locus within strains isolated from the same geographic region. Locus-specific reverse transcription-PCR revealed expression of p44-1 by New York and p44-61 by Minnesota strains at this locus. These p44 loci provide insight into the molecular evolution and functional divergence of p44 paralogs and may serve as markers for typing strains from different geographic regions. (+info)
Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal.
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A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus. (+info)