Detection of Anaplasma phagocytophilum in Ixodes ricinus ticks from Norway using a realtime PCR assay targeting the Anaplasma citrate synthase gene gltA
© Henningsson et al. 2015
Received: 3 March 2015
Accepted: 16 July 2015
Published: 1 August 2015
A TaqMan real-time PCR assay targeting the Anaplasma citrate synthase gene, gltA, was developed and used for detection of Anaplasma phagocytophilum in 765 Ixodes ricinus ticks collected from dogs and cats in northern Norway (n = 669) and Telemark county in southern Norway (n = 96).
Among the ticks from northern Norway the prevalence of A. phagocytophilum was 3.0 %, while the prevalence in southern Norway was 2.1 % (p = 0.63). The gltA PCR assay showed a high analytical sensitivity (30 genomic units) and efficiency (98.5 %), and its utility in clinical diagnostics should be evaluated in future studies.
This is the first report of A. phagocytophilum occurrence in ticks collected north of the Arctic Circle in Norway. The prevalence is comparable to that found in Telemark county in southern Norway.
KeywordsAnaplasma phagocytophilum Ixodes ricinus TaqMan realtime PCR Norway gltA Prevalence
Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen transmitted by Ixodes ticks . The bacterium is a well-documented pathogen in veterinary medicine, causing tick-borne fever (TBF) in domestic ruminants and horses [2, 3]. The first verified cases of human granulocytic anaplasmosis (HGA) were reported from the USA in 1994 [4, 5], and the first verified European cases were reported from Slovenia in 1997 . HGA cases have been reported from southern Norway and there is serological evidence of Anaplasma endemicity [7–10]. The pathogen is also prevalent in Norwegian livestock , and serological findings in cattle have indicated the presence of A. phagocytophilum in Nordland county in northern Norway . Anaplasma phagocytophilum is a possible emerging tick-borne pathogen in northern Norway, but reports on human disease are lacking.
The prevalence of A. phagocytophilum infection in the European tick Ixodes ricinus varies in different areas and between developmental stages of the tick . In Norway, recent studies indicate a prevalence in host-seeking I. ricinus of 1.4 % to 19.4 % with great regional differences [9, 14, 15].
In vitro cultivation and blood smear microscopy are difficult and cumbersome methods for detection of A. phagocytophilum in clinical samples, and therefore clinical diagnostics is mainly based on serology and PCR methods . Major advantages of PCR methods are rapid test results, high sensitivity and the possibility of quantifying pathogen load. Several PCR assays have been described for the detection of A. phagocytophilum in clinical samples and in ticks [17–19]. Real time PCR offers gains of rapidity and quantitativity and runs in a closed-system format, which eliminates risks of carry-over contamination. The A. phagocytophilum gene gltA codes for an essential housekeeping enzyme, citrate synthase. Mutations in such genes are predominantly point mutations that conserve enzyme function and they are not subject to the effects of diversifying selection that may affect surface proteins targeted by the immune system. This makes them attractive targets for phylogenetic studies, making extensive high-quality sequence information available. They are less conserved than 16 s rRNA genes and generally lack secondary structure, which simplifies the design of species-specific PCR.
In this study, we developed a TaqMan real-time PCR assay targeting gltA for the detection of A. phagocytophilum. Using this test, we aimed at extending previous studies at the ticks’ northern distribution limit by investigating the prevalence of A. phagocytophilum in I. ricinus ticks collected during a three-year period from different locations in northern and southern Norway.
Study area and design
Nucleic acid extraction and design of A. phagocytophilum PCR
Nucleic acid extraction was done as previously described , individually from each tick and stored at – 20 °C until use.
Primers, probes and cycling parameters
5’ hydrolysis probe
A synthetic plasmid, pAP-GltA, containing the amplicon sequence cloned in pUC57 was obtained from Genscript Corporation (Scotch Plains, NJ). This plasmid was used as a positive control. Serial dilutions of the plasmid were used to determine analytical sensitivity and as a quantitation standard.
Differences in A. phagocytophilum prevalence were analysed by Chi-Square test, p < 0.05 was regarded as significant.
No ethical approval was required for this study since removal of ticks was part of routine care of the pets.
Collected ticks, their origin and prevalence of Anaplasma phagocytophilum
No. of A. phagocytophilum-infected ticks (%)
No. of A. phagocytophilum-infected ticks from dogs (%)
No. of A. phagocytophilum-infected ticks from cats (%)
Total North Norway
In silico evaluation of the PCR test
Analytical sensitivity and efficiency of the gltA PCR assay
Analysis of a 10x serial dilution of pAP-GltA from 3.106 to 30 genomic units (GU)/5 μL (in duplicate) using the analysis software of the Applied Biosystems StepOne genetic analyzer gave a PCR efficiency of 98.5 % based on the slope of the standard curve of the Cq values (coefficient of determination, R2 = 0.998). Testing of 10 replicates of 30 GU (9/10 positive) and 3 GU (5/10 positive) gave a cut-off of 30 GU (Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines) .
A. phagocytophilum prevalence and geographical distribution
The overall prevalence of A. phagocytophilum in the collected ticks was 2.9 % (22/765) (Table 2). Of the ticks collected from dogs and cats 3.2 % (15/463) and 2.3 % (7/302), were positive for A. phagocytophilum, respectively (p = 0.46).
Among the adult female ticks, 2.8 % (19/690) were positive for A. phagocytophilum, while 4.3 % (3/69) of the adult male ticks were positive (p = 0.45). None of the four collected nymphs were positive for A. phagocytophilum.
Of the ticks collected in the three northernmost counties in Norway, 3.0 % (20/669) were positive for A. phagocytophilum (Nordland 2.8 % (18/647); Troms 9.1 % (2/22); the tick collected in Finnmark could not be included due to the pet’s recent stay outside the study area. Of the ticks collected in Telemark county, 2.1 % (2/96) were positive for A. phagocytophilum (Fig. 1). There was no significant difference in A. phagocytophilum prevalence in ticks collected in northern Norway compared to ticks collected in Telemark county (p = 0.63).
In this study we developed a real-time PCR assay targeting gltA with the purpose of direct detection of A. phagocytophilum in ticks. By using a large collection of ticks we give new insights into the epidemiology of A. phagocytophilum in ticks in northern Norway in relation to a southern Norwegian county (Telemark). We report here, for the first time, the presence of A. phagocytophilum in feeding ticks collected north of the Arctic Circle in Norway.
Previous studies have demonstrated the presence of I. ricinus in northern Norway as well as a high prevalence of Borrelia burgdorferi sensu lato in ticks in the region around the Arctic Circle [15, 20, 21]. Serological studies in two sheep flocks in Brønnøy found a high seroprevalence against A. phagocytophilum; 97 % and 100 %, respectively (Stuen et al., unpublished data). In addition, a serological study in dogs from northern Norway found a seropositivity of 3 % (4/120); three of the dogs were from Brønnøy . The presence of A. phagocytophilum has recently been reported in a smaller number of I. ricinus ticks collected by flagging vegetation in Brønnøy in the county of Nordland . We found an overall prevalence of A. phagocytophilum in feeding ticks of 3.0 % in northern Norway, which is about the same prevalence as we found in Telemark county in southern Norway, and also comparable to the prevalence in I. ricinus in more southern parts of Europe . The majority of the ticks in this study were collected in Nordland county (85 %), which includes the archipelago of Brønnøy, recognised for its high tick abundance . Climate, day length, habitat, mixture of hosts, and host abundance seem to be favourable for I. ricinus in this area, and apparently also for maintenance of A. phagocytophilum in the tick population.
Anaplasma phagocytophilum is internationally regarded as an emerging tick-borne pathogen, and areas of endemicity include parts of North America, Europe and Asia . These regions correspond to areas where the appropriate tick vectors are found (I. ricinus in Western Europe, I. persulcatus in Eastern Europe and Asia, I. scapularis and I. pacificus in Eastern and Western USA respectively) . Tick population density and geographical distribution are affected by changes in climate, vegetation and host abundance , and continuous surveillance of ticks and tick-borne pathogens at their distribution limits are important for early detection of altered risk scenarios and threats to public health. Although increasingly detected, symptomatic HGA still appears to be rather rare in Europe [17, 29], as opposed to the situation in the USA where HGA is a notifiable disease with increasing incidence . However, human seroprevalence in Europe varies between 2–28 % . It is unclear whether the discrepancy between the seroprevalence and the low number of symptomatic cases results from underdiagnosis of HGA, asymptomatic serologic reactions, reduced virulence of circulating A. phagocytophilum strains in Europe or even infections that cause cross-reactive serologic responses . In any case, A. phagocytophilum is endemic in Europe where it is the most widespread tick-borne pathogen in animals , and should therfore also be regarded as a potentially emerging human pathogen. Tick-exposed patients presenting with fever, leukopenia and elevated serum transaminases should have HGA included in the differential diagnoses .
The PCR method developed showed a high analytical sensitivity (30 GU) and efficiency (98.5 %). Based on the in silico analysis, the test is expected to be highly specific. It is rapid, and the results were found to be unambiguous and straightforward to interpret. We found this PCR assay to be useful for detection of A. phagocytophilum in ticks, and it may also be useful in clinical diagnostics in both human and veterinary medicine, but further evaluation of the method using clinical specimens will be needed.
In silico testing showed that 79/85 (93 %) of Anaplasma phagocytophilum gltA sequences are highly homologous to the primers and probe and will be efficiently detected. The same applies to a slightly more divergent sequence represented by isolate R33 (JX841254)  which, according to information presented in the sequence file, was detected in reindeer (Rangifer tarandus) imported to France. However, some far-eastern isolates are expected to be detected at low sensitivity, if at all. These include a group represented by strain KC478600, isolated from a rat (Rattus norvegicus) in southeastern China , and a single isolate, Khablx (AY339603), from I. persulcatus in the Russian far east .
The present study implies that both humans and pets may contract anaplasmosis also in northern parts of Norway, and that physicians as well as veterinarians need to be aware of the disease. The PCR assay targeting gltA performed well, and may be useful in clinical diagnostics in the future, but may fail to detect certain far-eastern isolates.
The authors would like to thank Malin Lager and Olaf Dienus, Department of Clinical Microbiology, County Hospital Ryhov, Jönköping, for performing the laboratory work, and Sture Löfgren for valuable help with the manuscript. The primers and probe were designed during a class exercise in PCR primer design for MSc students directed by AJ at Telemark University College. We would like to thank all the students who participated, and particularly Nina Narmoe Egerberg whose primer selection was the basis for the present test. We are in debt to the personnel at the veterinary clinics in northern Norway and Telemark that examined the pets and collected the ticks, and to the pet owners in these regions; without their support this study would not have been realisable. This work was supported by grant SFP912-10 from the Northern Norway Regional Health Authority (Helse Nord RHF), and grant SLS-100221 from the Scandinavian Society of Antimicrobial Chemotherapy, as well as grants from the Division of Medical Services, County Hospital Ryhov, Jönköping.
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