Sequence Analysis of the msp4 Gene of Anaplasma phagocytophilum Strains

Abstract

The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.

Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium

“…This high level of infection and associated immunity could contribute to the relative lack of acute clinical cases of BGA in monitored herds. However, this may also be linked to A. phagocytophilum strain heterogeneity and vertebrate diversity (de la Fuente et al 2005a;Granquist et al 2010). Infection rates estimated for Babesia spp.…”

Toxoplasma gondii, Neospora caninum and tick-transmitted bacterium Anaplasma phagocytophilum infections in one selected goat farm in Slovakia

“…phagocytophilum was detectected by real time PCR amplification protocol of the msp2 gene as described by Courtney et al (2004). In selected positive samples msp4 gene was amplified and sequenced using pair of MAP4AP5 and MSP4AP3 (De la Fuente et al 2005).…”

Experimental Infection of C3H/HeJ Mice with the NY18 Isolate of Anaplasma phagocytophilum

“…A fragment of 468 nucleotides comprising 16S rDNA positions -1 -467 of the Anaplasma marginale Florida strain reference sequence (Genbank accession number AF309867) was amplified by PCR using oligonucleotide primers 16SANA-F (59-CAG AGT TTG ATC CTG GCT CAG AAC G-39) and 16SANA-R (59-GAG TTT GCC GGG ACT TCT TCT GTA-39) as described previously. 18,45 The A phagocytophilum species-specific major surface protein (msp)4 PCR was used to corroborate pathogen identification as reported previously. 18 The msp4 and 16S rDNA gene sequences were amplified from 1 ml (0.1-10 ng) of DNA by PCR using 10 pmol of each primer in a 50-ml volume (1.5 mM MgSO 4 , 0.2 mM dNTP, 1X AMV/Tfl 5X reaction buffer, 5 u Tfl DNA polymerase) using the Access RT-PCR system (Promega, Madison, WI).…”

“…18,45 The A phagocytophilum species-specific major surface protein (msp)4 PCR was used to corroborate pathogen identification as reported previously. 18 The msp4 and 16S rDNA gene sequences were amplified from 1 ml (0.1-10 ng) of DNA by PCR using 10 pmol of each primer in a 50-ml volume (1.5 mM MgSO 4 , 0.2 mM dNTP, 1X AMV/Tfl 5X reaction buffer, 5 u Tfl DNA polymerase) using the Access RT-PCR system (Promega, Madison, WI). Reactions were performed in an automated DNA thermal cycler (Eppendorf Mastercycler personal, Westbury, NY) for 35 cycles.…”