The sequences of groESL operon of Anaplasma phagocytophilum among human patients in Slovenia Free

Abstract

Anaplasmosis comprises a group of emerging tick-borne diseases. It is mostly mild and self-limiting disease. The causative agent Anaplasma phagocytophilum is a pathogen known to cause disease not only in humans but also in ruminants, horses, and dogs. Anaplasma phagocytophilum shows differences in clinical severity, disease manifestation, reservoir competency, and antigenic diversity. Deer have been suggested as a reservoir animal. The heterogeneity of the groESL operon, as well as other genes of A. phagocytophilum, in animals and in a tick vector Ixodes ricinus has been described elsewhere. Only few studies report PCR-confirmed human cases of anaplasmosis. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia.

Between the years 1996 and 2008, blood samples of human patients with clinical signs of anaplasmosis were tested for the presence of anaplasmal DNA. DNA was extracted from acute blood samples of patients that seroconverted or had at least a fourfold rise in antibody titer against A. phagocytophilum antigen. For initial screening of all samples, PCR for a small part of the 16S rRNA gene of A. phagocytophilum was used (Kolbert, 1996). Positive samples were additionally tested with a nested PCR targeting a 1256-bp segment of the groESL operon (Sumner et al., 1997) and some of them for a larger fragment of the 16S rRNA gene. To detect A. phagocytophilum variants, all amplicons of the groESL operon and of a larger fragment of the 16S rRNA gene were further sequenced on both strands (Sumner et al., 1997; Massung et al., 1998). The sequences were analyzed by using treecon software (Van der Peer & de Wachter, 1994), and a phylogenetic tree was constructed with the neighbor-joining method. Support for the tree nodes was calculated with 1000 bootstrap replicates. The blood samples were collected, processed, and analyzed in separate years. This way the possibility of contamination was minimized.

In the ESCAR guidelines, one of the definitions of a confirmed case of human anaplasmosis is a febrile illness with a history of a tick bite or tick exposure and demonstration of A. phagocytophilum infection by seroconversion or at least a fourfold rise in antibody titer and/or positive PCR result with subsequent sequencing of amplicons (Brouqui et al., 2004). During 1996–2008, there were 66 serologically confirmed cases of human anaplasmosis in Slovenia according to the guidelines of ESCAR (Table 1). Of 66 confirmed cases, 46 were tested with a screening PCR and 28 (60.9%) of them were positive for the presence of A. phagocytophilum DNA (Table 1). Of 28 samples, 27 had amplified and sequenced the groESL operon and eight of them a larger fragment of 16S rRNA gene (Table 1). The homology search and the alignment of the groESL sequences showed only one genetic variant, 100% identical to the published sequence from a human patient (GenBank accession no. AF033101) and from a tick I. ricinus (GenBank accession no. EU246961) from Slovenia, as well as from a German (GenBank accession no. AF482760) and Swedish (GenBank accession no. AY529490) horse. Sequencing analysis of a larger fragment of the 16S rRNA gene from human patients revealed 100% identity among each other and to a reference sequence from a Swedish horse (GenBank accession no. AY527214).

Slovenia is a small country with diverse climate, vegetation, and animal representatives. Anaplasmosis in dogs in Slovenia is an emerging disease, causing from mild to a very serious illness, and even death (Tozon et al., 2003). On the other hand, human anaplasmosis is a rare and mild disease (Lotrič-Furlan et al., 2001). Studies from elsewhere report of different variants of groESL operon of A. phagocytophilum from animal samples (horses and dogs from Italy, sheep from Norway, deer from Austria and Slovenia) (Alberti, 2005; Stuen, 2006; Petrovec et al., 2003, 2002) and from ticks (Germany, Austria, Slovenia) (von Loewenich, 2003; Sixl et al., 2003; Strašek Smrdel et al., 2010). In Slovenia, in roe and red deer (Capreolus capreolus and Cervus elaphus, respectively) (Petrovec et al., 2002) and in ticks I. ricinus (Strašek Smrdel et al., 2010), different sequences of the groESL operon were found in two genetic lineages. A much lower diversity of sequences of groESL operon has been detected in samples from dogs and wild boar (Sus scrofa) from Slovenia. In dogs, two genetic variants and in wild boar one genetic variant, genetic variant of A. phagocytophilum have been established (Strašek Smrdel et al., 2008a, b). These sequences clustered in one genetic lineage, together with red deer sequences. Despite the fact that great diversity of groESL operon sequences in ticks and deer in Slovenia has been detected (Petrovec et al., 2002; Strašek Smrdel et al., 2010), only one genetic variant was present among all tested (27) human patients from this study, as well as in wild boar samples from previous study (Strašek Smrdel et al., 2008b). An identical variant of the groESL operon has previously been found also in ticks I. ricinus (Petrovec et al., 1999; Strašek Smrdel et al., 2010), dog samples (Strašek Smrdel et al., 2008a), and a human patient (Petrovec et al., 1999) in Slovenia, but not in roe deer and red deer samples (Petrovec et al., 2002). Results from this and previous studies of wild boar, deer, tick, human, and dog samples from Slovenia might suggest that wild boar could represent a reservoir for a variant of the groESL operon of A. phagocytophilum that causes human anaplasmosis in human patients and dogs from Slovenia. On the other hand, only this variant might be competent enough to replicate in wild boar, dogs, and humans, but not in deer. In contrast to our results, in the neighboring country Austria, two genotypes of groEL gene in two human patients have been found recently. They differ in a single A/G polymorphism (Haschke-Becher et al., 2010). After all, although Slovenia has the largest number of PCR detected and sequenced human samples of A. phagocytophilum so far, it might also be a country too small to detect greater genetic diversity among human samples of anaplasmosis.

This is the first report of the PCR-confirmed human cases of anaplasmosis in Slovenia. No variability in the groESL operon among human patients in Slovenia has been found. The same genotype of the groESL operon was found in human and wild boar samples. Is it possible that wild boar might serve as a reservoir for this variant of A. phagocytophilum in Slovenia? Or is this variant competent enough to replicate only in boar and humans? Other genetic markers need to be analyzed from multiple strains to draw a final conclusion.

References