ankA Gene Research Articles

Detection ofAnaplasma phagocytophilumin Ixodid Ticks from Equine-Inhabited Sites in the Southeastern United States

Anaplasma phagocytophilum is a vector-borne, obligate intracellular bacterium that invades the neutrophils and eosinophils of infected individuals, causing granulocytic anaplasmosis. Equine cases have previously been reported in the United States from California, Florida, and Connecticut, but limited surveillance studies in the Southeast have been conducted. The objective of this study was to determine A. phagocytophilum prevalence in Ixodes scapularis ticks at southeastern U.S. horse-inhabited sites to evaluate the potential risk for equine exposure to A. phagocytophilum-infected ticks in these areas. Samples of I. scapularis were collected from selected barrier islands and Georgia mainland sites where feral and domestic equine populations are present, respectively. Ticks were individually tested for infection by amplification of the A. phagocytophilum ankA gene. The collective prevalence of A. phagocytophilum in I. scapularis ticks was 20% (n=808).

Identification of Anaplasma phagocytophilum in tick populations in Estonia, the European part of Russia and Belarus

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.

PCR for detection of tick-borne Anaplasma phagocytophilum pathogens: a review

Tick-borne infections such as granulocytic anaplasmosis number among emerging infectious diseases. Anaplasma phagocytophilum is an obligate intracellular bacterial parasite infecting the granulocytes of vertebrates. This bacterium is the aetiologic agent of HGA (human granulocytic anaplasmosis). Molecular methods allow quick and accurate detection of pathogens in ticks, humans, or animals. Monitoring of the environment for A. phagocytophilum involves both classical and nested PCR, since these simple methods are most efficient. As markers, parts of the 16S rRNA, ankA, groESL, msp2, or msp4 genes are used for analyses. Molecular methods have enabled analysis of the genomes of pathogens, differentiation between strains and thus, in association with ecological studies, they facilitate an understanding of their biology, pathogenicity and mode of diffusion in the natural environment.  

Anaplasmosis in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum

Two dogs in France were diagnosed with Anaplasma phagocytophilum infection by real-time PCR. The most remarkable hematologic and biochemical findings were severe thrombocytopenia, mild neutrophilia, morulae in neutrophils, and increased serum concentration of the α2-globulin fraction detected by agarose gel electrophoresis of serum proteins. Using sequencing of the partial 16S rRNA and ankA genes, molecular characterization of the A. phagocytophilum strains showed that the organisms from both dogs were identical to the European strains isolated from horses and people. Based on phylogenetic analysis, the ankA gene was more discriminating than the 16S rRNA gene in distinguishing the majority of European and American strains of A. phagocytophilum infecting people and animals. Three isolates of A. phagocytophilum, 1 from Spain (cow) and 2 from Norway (sheep and deer), were external to the European and American clades.

New Genetic Variants ofAnaplasma phagocytophilumandAnaplasma bovisfrom Korean Water Deer (Hydropotes inermis argyropus)

Wild deer are one of the important natural reservoir hosts of Anaplasma species, which cause granulocytic anaplasmosis in equines, canines, and humans. The objective of the present study was to determine whether and what species of Anaplasma naturally infect Korean water deer (KWD) in the Republic of Korea. A total of 66 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife in Korea between March 2008 and May 2009. Polymerase chain reaction (PCR) was performed using 16S ribosomal (r)RNA, with ankA, groEL, and msp2 gene primers to amplify the genes of Anaplasma and Ehrlichia. Using 16S rRNA-based nested PCR, Anaplasma phagocytophilum and Anaplasma bovis were detected in 42 (63.6%) and 23 (34.8%) of 66 KWD spleens, respectively. The 42 A. phagocytophilum were classified into five genotypes and the 23 A. bovis were classified into two genotypes by sequence analysis. By ankA-, groEL-, and msp2-based nested PCR, A. phagocytophilum was detected in 1 (1.5%), 7 (10.6%), and 3 (4.6%) of 66 samples, respectively. These gene sequences had only one genotype. Five of seven obtained 16S rRNA gene sequences have never been identified. The ankA, groEL, and msp2 obtained gene sequences represented new genotypes. This is the first report of A. phagocytophilum and A. bovis in KWD, suggesting that they may act as reservoirs for anaplasmosis zoonotic pathogens.

Molecular evidence of Anaplasma phagocytophilum infection in wild cervids and feeding Ixodes ricinus ticks from west-central Poland

seasons. Roe deer were sampled during the summer and the fall in 10 districts (Czarnkow, Krzykosy, Oborniki, Margonin, Miedzychod, Murowana-Goslina, Poniec, Sierakow, Trzcian- ka, Wielen ´ ), whereas samples from red and fallow deer were obtained only during the fall in nine (listed above with the exception of Krykosy) and two districts (Margonin, Murowa- na-Goslina), respectively. In total, 238 partially or fully engorged female and 63 non-engorged male I. ricinus ticks were obtained from 51 animals (20 fallow deer, 18 roe deer and 13 red deer). Total DNA was extracted from blood and I. ricinus ticks using the genomic Mini AX Blood (A&A Biotechnology, Gdynia, Poland) and Sherlock AX kit (A&A Biotechnology), respec- tively. Blood and tick samples were analysed for the presence of A. phagocytophilum DNA by PCR using three different primer sets: EHR521-EHR747, LA6-LA1 and HGE396-HGE921, based on the sequences of the 16S rDNA, ankA and hge-44 genes, respectively (4,5). PCR products were considered positive when A. phagocytophilum DNA was detected by at least two primer sets. Additionally, selected PCR-positive samples tested with the primers LA6-LA1 specific for a 444-bp region of the ankA gene were sequenced with the ABI PRISM 3100 Genetic Analyser (Applied Biosystem 850, Foster City, CA, USA) according to the manufacturer's protocol. Sequences were edited and compared with gene sequences deposited in the GenBank database using the NCBI BLAST program (U.S. National Institute of Health, Bethesda, Maryland).

Distinct Ecologically Relevant Strains ofAnaplasma phagocytophilum

Distinct Ecologically Relevant Strains of<i>Anaplasma phagocytophilum</i>

Co-phylogenetic analysis of Anaplasma phagocytophilum and its vectors, Ixodes spp. ticks

The coevolutionary history of Ixodes spp. ticks, the obligately tick-transmitted bacterial pathogen Anaplasma phagocytophilum, and its various rodent reservoir hosts world-wide is not known. According to coevolution theory, the most recently evolved of tick-bacterial complexes could have difficulty maintaining A. phagocytophilum in nature, because transmissibility has not been efficiently maximized. This study was intended to examine the phylogeographic history of I. ricinus-subgroup ticks and A. phagocytophilum, provide an estimate for the date of the divergence of A. marginale and A. phagocytophilum, and evaluate whether there is correspondence between tick and Anaplasma spp. trees. Analysis of Ixodes spp. ticks showed a New World clade consisting of I. scapularis and I. pacificus, European I. ricinus as a sister group to this clade, and Asian I. persulcatus as basal. Of the three A. phagocytophilum genes evaluated, the most resolution was provided by the ankA gene. ankA sequences formed an Old World clade with eastern North America strains as a sister clade. California strains were highly diverse and did not form a clade. Base substitution rates were very comparable along both A. marginale and A. phagocytophilum lineages. Based on 16S rDNA analysis, maximum and minimum divergence times of A. phagocytophilum and A. marginale were calculated to be 78,296,703 and 43,415,708 years, respectively. If A. phagocytophilum did closely coevolve with specific I. ricinus-subgroup tick species, then A. phagocytophilum strains could have specialized on local tick species and optimized local infectivity in the Old World and eastern US. However, lack of absolute resolution of tick trees and conflicting prevalence data (with low prevalence in Asia and western North America) preclude us from inferring a tight coevolutionary relationship of tick species from this phylogeographic analysis.

The Aspergillus nidulans snt Genes Are Required for the Regulation of Septum Formation and Cell Cycle Checkpoints

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before forming a septum. Previous genetic results suggest that the ability to separate nuclear division and septum formation depends upon a threshold level of activity of the cyclin-dependent kinase NIMX(cdk1). Mutations in nimX and nimT, the gene encoding the NIMX(cdk1)-activating phosphatase, have revealed that Tyr-15 phosphorylation is important for determining the timing of the formation of the first septum. Here, we describe a screen for suppressors of nimT23 (snt), designed to identify additional components of the pathway regulating septum formation. We show that a subset of the snt mutants are defective in the temporal regulation of septum formation and in cell cycle checkpoint responses. Molecular characterization of sntA shows that it is allelic to the previously described ankA gene, which encodes the NIMX(cdk1) Tyr-15 kinase. Additional experiments described in this study show that nutritional conditions modulate the timing of septum formation and alter the phenotypes displayed by the snt mutants. A model that suggests that the timing of septum formation is influenced by DNA damage and glucose availability via the sntA and sntB gene products is proposed.