Prevalence of Anaplasma phagocytophilum and Coinfection with Borrelia burgdorferi Sensu Lato in the Hard Tick Ixodes ricinus in the City of Hanover (Germany)

Abstract

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.

Introduction

A naplasma phagocytophilum is a Gram-negative, obligate intracellular α-proteobacterium affecting neutrophil granulocytes and causing human granulocytic anaplasmosis (HGA). HGA leads to a variety of clinical signs, for example, malaise, fever, myalgia, and headaches. In ruminants like sheep and cattle, the agent causes tick-borne fever and leads to economical losses due to reduced milk yield and abortion (Hudson 1950, Stamp et al. 1950). The pathogen is also known to cause anaplasmosis in horses and dogs (Gribble 1969, Pusterla et al. 1998). In Europe, A. phagocytophilum as well as the Lyme disease agent Borrelia burgdorferi sensu lato (sl) are mainly transmitted by I. ricinus ticks (Strle 2004, Gray et al. 2009). In the present study the infection rate of A. phagocytophilum and coinfections with B. burgdorferi sl in I. ricinus ticks were investigated to close the gap of missing data from Northern Germany.

Materials and Methods

Tick material

One thousand six hundred forty-six ticks (389 males, 388 females, and 869 nymphs) were collected as eight cohorts from March to October 2005 from 12 different recreation areas in the city of Hanover, the capital of the northern German federal state Lower Saxony.

A. phagocytophilum real-time PCR

Isolation of tick genomic DNA was performed as described by Strube et al. (2010) followed by quantitative real-time PCR (qPCR) according to Courtney et al. (2004). The reaction was set up using the Brilliant^® QPCR Master Mix (Stratagene): 7.72 μL double-distilled H₂O, 12.5 μL Brilliant buffer (containing SureStart^® Taq Polymerase), 0.15 μL forward and reverse primer (50 μM each) 0.1 μL probe (50 μM), 0.38 μL diluted ROX (reference dye, 1:500 dilution), and 4 μL tick DNA template. QPCR cycling conditions were set according to Courtney et al. (2004), but the number of cycles was increased to 45. A plasmid dilution series comprising the target sequence served as positive control and standard series. Experiments and data analysis were performed using the Mx3005 Multiplex Quantitative PCR System (Stratagene).

Additionally, ticks were evaluated for coinfection with B. burgdorferi sl based on preinvestigation by Montenegro (2008).

Statistical analysis

Results were statistically analyzed using the chi-square test (SigmaStat^® software, version 3.11) followed by Bonferroni-Holm correction in case of seasonal variations.

Results

A. phagocytophilum prevalence in Ixodes ricinus ticks

Infection with A. phagocytophilum was detected in 3.2% (52) of the 1646 examined I. ricinus ticks. Adult ticks showed a significantly higher (p=0.05) infection rate of 4.1% (32/777) than nymphs with 2.3% (20/869). Among the adult ticks an equal number of male (16/389) and female (16/388) ticks was found to be positive. Detailed prevalence data and significant differences are shown in Figure 1.

FIG. 1.

Seasonal variation of Anaplasma phagocytophilum infection rates in Ixodes ricinus ticks of different developmental stages. Connection lines indicate significant difference between prevalence rates.

Coinfection with B. burgdorferi sl

In 15 of 1646 ticks (0.9%), which were coinfected with Borrelia spirochetes, four genospecies were detected: B. valaisiana, B. afzelii, B. burgdorferi sensu stricto (ss), and B. garinii. Eighty percent (12/15) of coinfected ticks were adults. From these, two-thirds (8/12) represented females and one-third (4/12) males. The remaining 20% (3/15) were nymphs. Seven ticks were monoinfected and 5 ticks were infected with two different Borrelia genospecies. Detailed results are shown in Table 1.

Table 1.

Coinfection of Anaplasma phagocytophilum–Infected Ticks with Different Genospecies of the Borrelia burgdorferi sl-Complex

	Anaplasma phagocytophilum+Borrelia burgdorferi sl coinfection
Tick cohort	No. of ticks	%	Tick stage	Borrelia genospecies
March/April	1	0.5	Female	B. garinii+B. burgdorferi ss
April/May	5	2.2	Male	B. garinii+B. burgdorferi ss
			Male	not differentiable
			Female	B. burgdorferi ss
			Female	B. valaisiana
			Nymph	B. garinii+B. burgdorferi ss
May/June	0	0	—	—
June/July	3	0.9	Female	B. valaisiana
			Female	B. garinii
			Female	not differentiable
July/August	2	0.9	Male	B. garinii+B. valaisiana
			Nymph	B. afzelii
August/September	1	0.5	Nymph	not differentiable
September	1	0.5	Female	B. afzelii+B. garinii
September/October	2	1.4	Male	B. valaisiana
			Female	B. valaisiana
total	15	0.9	4 males, 8 females, 3 nymphs

Discussion

A. phagocytophilum has been detected in tick vectors in nearly all European countries. Prevalences range from 0.3% in France to 13.4% in the European part of Russia (Cotte et al. 2010, Katargina et al. 2011). In Germany, A. phagocytophilum infection rates in I. ricinus range between 1% and 4.5% in southern Germany, 2.3% in central Germany, and 1% in northeastern Germany (Hildebrandt et al. 2002, 2003, Hartelt et al. 2004, Silaghi et al. 2008, Franke et al. 2011). The present study revealed a total A. phagocytophilum infection rate of 3.2% for Hanover, a city located in northern Germany. Prevalence differences to the studies mentioned above could result from prevalence variability between years, collecting time points, and ecological factors (Pichon et al. 2006, Hildebrandt et al. 2010). In the present study adult ticks showed significantly higher infection rates in contrast to nymphs, which may be explained by them simply having taken an additional blood meal. Further, fluctuations in the A. phagocytophilum infection rate (0.5–7.4%) were observed among tick cohorts collected between March and October. These findings may result from ecological factors, such as microclimate conditions, vegetation, tick density, and the availability of suitable reservoirs or hosts, which affect the occurrence of tick-borne pathogens in nature (Hildebrandt et al. 2003).

Coinfection with spirochetes from the B. burgdorferi sl-complex was found in 0.9% of the investigated Hanoverian ticks und was thus comparable to the results of Pichon et al. (2006) and Hildebrandt et al. (2003), who found a B. burgdorferi sl. coinfection rate of 0.8% and 0.7%, respectively. In the present study, B. garinii was found to be the most frequent Borrelia genospecies in coinfected ticks followed by B. valaisiana, B. burgdorferi ss, and B. afzelii. For B. burgdorferi ss both birds and rodents are competent reservoirs, whereas the genospecies B. garinii and B. valaisiana are bird associated and B. afzelii is rodent associated, respectively (Humair et al. 1995, Kipp et al. 2006). Genospecies distribution of the present study may be explained by the hypothesis that the majority of collected ticks fed on birds during their last blood meal. Since A. phagocytophilum prevalence data can vary between years, a further study is in progress to track future development of the prevalence situation.

Footnotes

Disclosure Statement

The authors declare that no competing financial interests exist.

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