A Novel High-Resolution Melt PCR Assay Discriminates Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis”

Abstract

f "Candidatus Neoehrlichia mikurensis" (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, "Candidatus N. mikurensis" was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found. Pathogenic Anaplasmataceae of the genera Anaplasma, Ehrlichia, and Neoehrlichia are transmitted by ixodidae. Anaplasma phagocytophilum, the agent of human, equine, canine, and feline granulocytic anaplasmosis (GA) and tick-borne fever of ruminants, occurs throughout central and northern Europe (1). Anaplasma phagocytophilum genotypes in North America and Europe apparently differ in host tropisms and clinical symptoms, with ruminant tick-borne fever and equine GA found predominantly in Europe and human GA predominantly in North America (1). Although human GA is often asymptomatic, 7% of diagnosed clinical cases require intensive medical care and 0.6% of the cases are fatal (2). Anaplasma phagocytophilum is transmitted by Ixodes species. In Eurasia, vector competence has not been experimentally established for any tick species, but the bacterium was detected in many different Ixodes species (1), including Ixodes ricinus (3, 4) and Ixodes hexagonus (1, 5).Recently, "Candidatus Neoehrlichia mikurensis" was identified as a new member of the Anaplasmataceae. This emerging pathogen is also transmitted by Ixodes ticks and can cause severe disease in humans (6-8) and dogs (9). It was first identified in Ixodes ovatus and Rattus norvegicus in Japan (10). Bacterial DNA was detected in liver, heart, and spleen tissue, and intracellular bacteria were found in splenic endothelial cells. In two human cases, the pathogen occurred in granulocytes (11). Previously, very closely related 16S rRNA gene sequences were reported as the Schotti variant of Ehrlichia-like bacteria in the Netherlands (12) and Ehrlichia sp. "Rattus Strain" in China (13). "Candidatus Neoehrlichia mikurensis" appears to be one of the most prevalent pathogens in I. ricinus in Europe (14-17), suggesting the possibility that many subclinical human infections occur. Therefore, the aim was to develop a real-time high-resolution melt PCR (HRM-PCR) assay discriminating A. phagocytophilum and "Candidatus Neoehrlichia mikurensis."Six rodent species were trapped in 2010 and 2011 at four different sites in Berlin (Table 1). Spleen samples were collected during necropsy from cats from animal shelters in Berlin (2006Berlin ( to 2008. Host-associated, nonengorged, or partially engorged I. ricinus and I. hexagonus ticks were collected from dogs in Berlin and Brandenburg by their owners and sent to the Small Animal Clinic, Freie Universität Berlin, Berlin, Germany. Questing Dermacentor reticulatus ticks were collected ...

Infections with the tick-borne bacterium Candidatus Neoehrlichia mikurensis

“…Studies on the prevalence of C. Neoehrlichia mikurensis in ticks removed from people in The Netherlands [59] and Germany [28] have estimated that every ninth to 12th tick bite represents a risk of transmission of C. Neoehrlichia mikurensis, although the risk of becoming infected is likely to be considerably lower [59]. Infected ticks and rodents have been found in the vicinity of large cities, such as Berlin, Budapest, Guangzhou, Leipzig, and Zurich, indicating that large populations may be exposed to this new infectious agent [6,24,30,31,36,60].…”

“…Until 2014, C. Neoehrlichia mikurensis had not been found in the larval stage of ticks [43], and it had been inferred that it is not transmitted transovarially [30]. However, it was recently reported that four of ten larvae sampled in Austria were …”

“…Seven species of ticks have been reported to be infected by C. Neoehrlichia mikurensis: I. ricinus [48]; I. persulcatus [5,15]; I. ovatus [1]; Ixodes frontalis [57]; Ixodes hexagonus [30]; Dermacentor reticulatus [29,30]; and Haemaphysalis concinna [13]. It should be emphasized that the infection rates of C. Neoehrlichia mikurensis are highest among the Ixodes species, indicating that these are the primary vectors; the importance of the other tick genera as vectors is disputable, as they rarely carry C. Neoehrlichia mikurensis ( Table 1).…”

“…It has been detected in Mongolia [12], China [6,12,13], Japan [1,14], Russia [4,5,15,16], the Czech Republic [9,17,18], Slovakia [19][20][21], Moldova [22], Hungary [23][24][25], Austria [18,26], Germany [11,[27][28][29][30][31][32], Switzerland [33][34][35][36][37], Poland [38,39], Romania [40], France [28,41,42], Belgium [43], Italy [44][45][46], Spain [47], The Netherlands [3,42,43,48], and the Scandinavian countries [2,…”

Vector-borne pathogens in dogs and red foxes from the federal state of Brandenburg, Germany

“…The protocol used for this PCR was previously described by Krücken et al (2013). Briefly, reaction tubes contained 0.3 mM dNTPs, 0.3 µM of each primer, 0.1 U/µl Phusion Hot Start II High-Fidelity DNA Polymerase and 2 µl template DNA (20 -100 ng/µl) in 25 µl 1 × EvaGreen® Fluorescent DNA Stain (Jena Bioscience, Germany), 1 × Phusion HF buffer.…”