Development and Use of Specific Polymerase Reaction for the Detection of an Organism Resembling Ehrlichia Sp. In White-Tailed Deer

Little, Susan E.; Dawson, Jacqueline E.; Lockhart, J. Mitchell; Stallknecht, David E.; Warner, Cynthia K.; Davidson, William R.

doi:10.7589/0090-3558-33.2.246

Cited by 40 publications

(50 citation statements)

References 2 publications

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“…Schotti variant with primers thought to be specific for A. phagocytophilum underlines the necessity to confirm the specificity of the PCR products by sequence analysis. Amplification of related species has also been reported for other primer combinations (24,27).…”

Section: Discussionmentioning

confidence: 92%

High Diversity ofankASequences ofAnaplasma phagocytophilumamongIxodes ricinusTicks in Germany

Loewenich¹,

Baumgarten²,

Schröppel³

et al. 2003

J Clin Microbiol

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In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

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Section: Discussionmentioning

confidence: 92%

High Diversity ofankASequences ofAnaplasma phagocytophilumamongIxodes ricinusTicks in Germany

Loewenich¹,

Baumgarten²,

Schröppel³

et al. 2003

J Clin Microbiol

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“…We were aware of the existence of an undescribed Anaplasma sp. of WTD that is amplified in 16S PCR assays by using primers GE9F and GA1UR (34). Additionally, it has been reported that in blood samples containing a high concentration of Anaplasma (Ehrlichia) platys DNA, E. equi primers have induced false priming (52).…”

Section: Discussionmentioning

confidence: 99%

“…RT-nPCR was performed on RNA extracted from deer blood, cell culture, and tissues in the following manner. Reverse transcription of 16S r-RNA to cDNA and subsequent primary amplification using primers ECC and ECB were carried out in a single-tube reaction, followed by secondary amplification as described previously (44), except that secondary primers GE9F and GA1UR were used to generate an internal 411-bp fragment (13,34).…”

Section: Methodsmentioning

confidence: 99%

Experimental Infection of White-Tailed Deer with Anaplasma phagocytophilum , Etiologic Agent of Human Granulocytic Anaplasmosis

et al. 2005

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Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, >64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of >64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.

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“…WTD also are important hosts for Lone Star ticks, Amblyomma americanum, and the black-legged tick, I. scapularis, which are proven vectors of these zoonotic disease agents (2,18,36,54). Examination of wild WTD also has disclosed molecular evidence of another rickettsial agent, termed WTD agent, which is closely related to, but not identical with, Anaplasma platys (7,14,38). Thus far, the only known vertebrate hosts of the WTD agent are wild WTD, as well as possibly mule deer and black-tailed deer (21).…”

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confidence: 99%

“…strain based on PCR and nucleotide sequencing, describe its morphological characteristics by light and electron microscopy, and demonstrate its ability to infect a naive WTD fawn. Dumler et al (17), specifically the WTD agent, E. chaffeensis, and E. ewingii (38,41,60). Two captive, 4-month-old WTD fawns, WTD76 and WTD81, were injected intradermally, subcutaneously, intravenously, and intraperitoneally with 2 ml of blood by each route (8 ml total) from blood pooled from three of the wild deer.…”

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confidence: 99%

Isolation of anAnaplasmasp. Organism from White-Tailed Deer by Tick Cell Culture

et al. 2003

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We used tick cell culture to isolate a bacterium previously referred to as the "white-tailed deer (WTD) agent" from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34°C, and 8 days later, Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first to Anaplasma platys and second to Anaplasma phagocytophilum, supporting its placement in the genus Anaplasma. Transmission electron microscopy of this Anaplasma sp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with the Anaplasma sp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.The genus Anaplasma comprises obligate intracellular rickettsial pathogens that are biologically transmitted by ixodid ticks (15,20,32,36,54). They target circulating blood cells of wild and domestic animals, as well as of humans, and are of global veterinary and human health importance. Although the type species, Anaplasma marginale, was described early last century (56) and has been intensively studied (33), it was isolated in continuous culture only recently (46). A continuous culture system for this obligate intraerythrocytic parasite became available with the development of cell lines from the black-legged tick, Ixodes scapularis (45), the vector for a range of different pathogens from viruses to protozoa (52,54,55 , abstr. 165, 1995) and Anaplasma phagocytophilum (47, 48), formerly known as Ehrlichia equi, Ehrlichia phagocytophila, Cytoecetes phagocytophila, or the human granulocytic ehrlichiosis agent (17). Tick cell culture has allowed not only primary isolation and continuous in vitro maintenance of these pathogens but also analysis of differential expression of specific antigens in the vector and vertebrate host (28). Other, as yet uncultivable, tick-borne pathogens might be candidates for isolation in ixodid tick cell lines.White-tailed deer (WTD; Odocoileus virginianus) are proven vertebrate reservoirs for Ehrlichia chaffeensis (40,41) and are known to be naturally infected with at least two related zoonotic rickettsiae, Ehrlichia ewingii (60) and A. phagocytophilum (6, 39). Although some wild WTD have been shown to carry antibodies that recognize A. marginale, their role in the epizootiology of bovine anaplasmosis is debated (31,42,44). WTD also are important hosts for Lone Star ticks, Amblyomma americanum, and the black-legged tick, I. scapularis, which are proven vectors of these zoonotic disease agents (2,18,36,54). Examination of wild WTD also has disclosed molecular evidence of another rickettsia...

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Development and Use of Specific Polymerase Reaction for the Detection of an Organism Resembling Ehrlichia Sp. In White-Tailed Deer

Cited by 40 publications

References 2 publications

High Diversity ofankASequences ofAnaplasma phagocytophilumamongIxodes ricinusTicks in Germany

High Diversity ofankASequences ofAnaplasma phagocytophilumamongIxodes ricinusTicks in Germany

Experimental Infection of White-Tailed Deer with Anaplasma phagocytophilum , Etiologic Agent of Human Granulocytic Anaplasmosis

Isolation of anAnaplasmasp. Organism from White-Tailed Deer by Tick Cell Culture

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