Genetic Variants of Anaplasma phagocytophilum in Wild Caprine and Cervid Ungulates from the Alps in Tyrol,Austria

Abstract

The occurrence of genetic variants of Anaplasma phagocytophilum was studied in wild ungulates from the northern and central eastern Alps in Tyrol, Austria. For this purpose, spleen samples collected from 53 game animals during the hunting season 2008/2009 (16 roe deer [Capreolus capreolus], 10 red deer [Cervus elaphus], 16 Alpine chamois [Rupicapra r. rupicapra], 7 Alpine ibex [Capra i. ibex], and 4 European mouflons [Ovis orientalis musimon]) were analyzed. Thirty-five animals originated from the Karwendel mountains, 12 from the Kaunertal area (Ötztal Alps), and the remaining from other mountainous areas in Tyrol. DNA extracts were screened with a real-time polymerase chain reaction targeting the msp2 gene of A. phagocytophilum. A total of 23 (43.4%) samples, from all ungulate species studied, were A. phagocytophilum positive. As of the date of this article, A. phagocytophilum has not been reported in the Alpine ibex. The positive samples were investigated further with polymerase chain reactions for amplification of the partial 16S rRNA, groEL, and msp4 genes. Sequence analysis using forward and reverse primers revealed seven different 16S rRNA gene variants. No variant could be attributed to any particular ungulate species. The groEL gene revealed 11 different variants, which grouped in the phylogenetic analysis into two distinct clusters: one cluster contained the sequences from roe deer, whereas the sequences of the other species formed the second cluster. The msp4 gene showed a high degree of variability in the amplified part with a total of 10 different sequence types. The results show that the wild mountain ungulates were infected to a considerable extent with various variants of A. phagocytophilum. The pathogenicity of the variants and the reservoir competence of the species investigated in this study deserve further attention in future studies.

Introduction

Anaplasma phagocytophilum, an obligate intracellular bacterium, causes tick-borne fever in ruminants and granulocytic anaplasmosis in dogs, cats, horses, and humans. The pathogen is transmitted by the tick Ixodes ricinus, which is an abundant tick throughout Europe and is not host specific (Woldehiwet 2010). The prevalence of A. phagocytophilum in the vector varies greatly in different regions in Europe from 0.25% to 57.14% (e.g., Alberdi et al. 1998, Mantelli et al. 2006). In Austria, it ranges on average between 5.1% and 8.7% in I. ricinus, but point prevalences have been found to be higher (Sixl et al. 2003, Hartl 2004, Polin et al. 2004, Huber 2006). Recently, experimental transovarial transmission of A. phagocytophilum with a maximum efficiency of 40% has been shown in the North-American one-host tick Dermacentor albipictus (Baldridge et al. 2009). Transovarial transmission in I. ricinus has not been shown yet; therefore, a vertebrate reservoir host is necessary for the maintenance of the ecological cycle in nature.

In Europe, roe deer (Capreolus capreolus) and red deer (Cervus elaphus) are in discussion as reservoir hosts, but A. phagocytophilum (DNA and/or antibodies) also has been detected in sika deer (Cervus nippon), chamois (Rupicapra r. rupicapra), European mouflon (Ovis orientalis musimon), wild boar (Sus scrofa), hares (Lepus europaeus), red foxes (Vulpes vulpes), the European brown bear (Ursus arctos), and small rodents (Alberdi et al. 2000, Liz et al. 2002, Petrovec et al. 2002, 2003, Oporto et al. 2003, Hulínská et al. 2004, Polin et al. 2004, Skarphédinsson et al. 2005, Beninati et al. 2006, Smetanová et al. 2006, Adamska and Skotarczak 2007, De la Fuente et al. 2008, Zeman and Pecha 2008, Bown et al. 2009, Karbowiak et al. 2009, Robinson et al. 2009, Stefanidesova et al. 2008, Vichová et al. 2010).

Genetic heterogeneity exists within the species A. phagocytophilum, and variants show differences in vectors, host tropism, pathogenicity, and geographical distribution (Massung et al. 2002, Stuen et al. 2002, Carpi et al. 2009). For example, in Slovenia, genetic diversity of the 16S rRNA and groESL genes was detected in sequences derived from roe deer and red deer. All red deer sequences clustered with those derived from humans cases, whereas the roe deer sequences clustered separately (Petrovec et al. 2002). Two distinct European lineages were discovered in a phylogenetic analysis of the groEL gene (Alberti et al. 2005). On the basis of the 16S rRNA gene, a host species segregation was detected in the Czech Republic (Zeman and Pecha 2008).

The purpose of this study was to identify A. phagocytophilum infections in typical mountain ungulates and other wild ruminants adapted to the mountain ecosystem of the Alps in Tyrol, Austria, and to analyze potential genetic variants and species segregation in this host population.

Materials and Methods

Spleen samples were collected from 53 wild ungulates (16 roe deer, 10 red deer, 16 Alpine chamois, 7 Alpine ibex [Capra i. ibex], and 4 European mouflons) during the hunting season from August 2008 to January 2009 in Tyrol, Austria.

Thirty-five animals (14 chamois, 2 mouflons, 10 roe deer, 8 red deer, and 1 ibex) were from the Karwendel mountains, 12 animals (5 ibex, 4 roe deer, 2 mouflons, and 1 chamois) from the Kaunertal area of the Ötztal Alps, and the rest (6 animals) originated from two other mountain hunting grounds of the Unterland part of Tyrol, Austria (Fig. 1).

FIG. 1.

Map of Austria showing the study areas, main rivers and cities, and neighboring countries. The Federal State of Tyrol is shaded in gray.

The Karwendel mountains are located between the Isar and Inn rivers and belong to the range of the northern Calcareous Alps and cover an area of >750 sq km with 125 peaks above 2000 m (highest: Birkkarspitze, 2749 m). In contrast, the Ötztal Alps are located south to the River Inn and belong to the Central Eastern Alps and include the largest and highest ranges in Austria. There are several peaks higher than 3000 m (highest: Wildspitze, 3768 m). The Kaunertal is one of three valleys that open north into the Inn Valley.

Red deer, roe deer, and chamois are abundant native ungulate species throughout Tyrol, and reintroduction of the ibex, following its extermination in Austria in the 18th century, started in the Ötztal Alps and the Karwendel in the 1950s. The introduced mouflon is restricted to small areas in Tyrol. The habitat use of all species is characterized by seasonal differences related to availability of suitable foraging sites. The range of roe deer comprises usually altitudes from 600 to 900 m but may reach 1200 m in summer. Red deer, however, colonize winter habitats at 600–900 m but can be found during the summer season in subalpine areas of up to 1800 m. The typical mountain ungulates chamois and ibex reside usually at higher altitudes; chamois is a characteristic inhabitant of the timberline range (subalpine zone), whereas ibex prefers to live in the upper subalpine (1800–2300 m) and mainly alpine zones. The habitat range of the introduced mouflons is comparable to that of the chamois.

The sex, age, and body condition of the animals used in this study were provided by the hunters. There was no information as to the infestation of the ungulates with ectoparasites.

Approximately 10 g of spleen tissue was preserved in 70% ethanol. From each sample, 25–50 mg subsample was prepared using a sterile mortar and scalpel blade, and DNA was extracted individually using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany). Samples were incubated overnight at 55°C and DNA was extracted in a 200 μL volume. Quality and quantity of extracted DNA were tested with a spectrophotometer (NanoDrop^® ND-1000; Peqlab, Erlangen, Germany). As the extracted total DNA content was very high, averaging 130 ng/μL, the samples were diluted again with 100 μL elution buffer.

All samples were screened for the presence of A. phagocytophilum with a real-time polymerase chain reaction (PCR) targeting a 77-bp portion of the msp2 gene, using primers ApMSP2f and ApMSP2r and the probe ApMSP2p labeled with HEX-TAMRA (Courtney et al. 2004). The real-time PCR was carried out in a BioRad iCycler IQ (Bio-Rad, Munich, Germany) using the Hot StarTaq Polymerase Kit (Qiagen, Hilden, Germany) in a total volume of 25 μL including 5 μL DNA sample. Cycling conditions were initial activation of the enzyme at 95°C for 15 min and then 40 cycles of denaturation at 94°C for 15 s and annealing–extension at 60°C for 60 s. Positive samples were analyzed further with nested PCRs targeting a variable part of the 16S rRNA of A. phagocytophilum, using primer pairs ge3a/ge10r in the first reaction and ge9f/ge2 in the second reaction (Massung et al. 1998). The groEL gene was amplified with a heminested PCR using primer pair EphplgroEL-F/EphplgroEL-R in the first reaction and EphplgroEL-F/EphgroEL-R in a second amplification specific for A. phagocytophilum (Alberti et al. 2005). The partial msp4 gene nested PCR was carried out following the approach developed by Bown et al. (2007), using primers MSP4AP5/MSP4AP3 (de la Fuente et al. 2005), followed by a second nested PCR amplification using primers msp4f/msp4r (Bown et al. 2007). The 16S rRNA PCR was carried out on an Gene Amp PCR System 2700 (Applied Biosystems, Weiterstadt, Germany), the groEL PCR and msp4 PCR were carried out on an Mastercycler^® gradient (Eppendorf, Hamburg, Germany) with an initial activation step (15 min at 95°C), then 40 cycles (30 s at 94°C, 30 s at 55°C, 1 min at 72°C—16S rRNA gene; 30 s at 94°C, 30 s at 55°C, 45 s at 72°C—groEL; 30 s at 94°C, 45 s at 54°C, 1 min at 72°C—msp4), and a final extension (10 min at 72°C). All reactions were carried out in a 50 μL volume including 5 μL DNA sample. The nested reactions were carried out at the same temperature and cycle conditions as the first reactions; however, for 16S rRNA, only 25 cycles were carried out with 1 μL of the first amplification product. The HotStarTaq Polymerase Kit (Qiagen) was used for all PCR experiments. PCR-clean water was used as negative and DNA from A. phagocytophilum-positive I. ricinus ticks as positive controls. Successful amplification was verified with 2.0% agarose gel electrophoresis after staining with GelRed™ (Biotium, Hayward, CA) and visualization under UV light.

PCR products were purified with the QIAquick PCR Purification Kit (Qiagen) and sequenced with forward and reverse primers of the nested reactions by Eurofins MWG Operon (Martinsried, Germany).

Sequence analysis was carried out using the following programs: Chromas©Lite (www.technelysium.com.au/chromas_lite.html), BLASTn (www.ncbi.nlm.nih.gov.library.vu.edu.au/BLAST/), Transeq (www.ebi.ac.uk/emboss/transeq/), and ClustalW (www.ebi.ac.uk/clustalw/index.html).

Phylogenetic analysis was carried out with the PHYLIP 3.67 software package (Felsenstein 1989) using the neighbor-joining method. Distance matrices were calculated with the Kimura two-parameter method using DNADIST. A bootstrap analysis was performed to test the stability of the trees with 1000 resamplings using SEQBOOT. Consensus trees were derived with CONSENSUS.

Sex-specific, age-class, and body condition-related prevalences of A. phagocytophilum infection were calculated. Chi-squared tests or chi-squared test for 3 × 2 contingency table were used to compare the rate of infection between the sexes, age-classes (<1, 1–3, > 3 years), and body conditions (good vs. bad). In all statistics, p-values of <0.05 were considered significant.

Results

A total of 23 of the 53 (43.4%) animals were tested positive for A. phagocytophilum DNA by real-time PCR (Table 1). DNA of A. phagocytophilum was detected in all ungulate species studied.

Table 1.

Detection of Anaplasma phagocytophilum DNA by Real-Time Polymerase Chain Reaction Targeting the msp 2 Gene in the Spleen of Wild Ungulates from the Alps in Tyrol, Austria, Collected During the Hunting Season 2008/2009

	Total no. of animals (no. of positive animals)
Species	August 2008	September 2008	October 2008	November 2008	December 2008	January 2009	NR
Roe deer	6 (5)	1 (1)	0	2 (1)	4 (1)	2 (0)	1 (0)
Red deer	1 (1)	1 (1)	1 (1)	2 (1)	2 (1)	1 (0)	2 (2)
Chamois	3 (0)	3 (0)	3 (2)	1 (0)	6 (2)	0
Ibex	0	0	0	2 (2)	5 (0)	0
Mouflon	0	0	0	1 (0)	3 (2)	0
Total	10 (6)	5 (2)	4 (3)	8 (4)	20 (6)	3 (0)	3 (2)

NR, not recorded.

A total of 19 of 35 animals from the Karwendel mountains were positive, but none of the 12 animals from the Kaunertal area. The remaining four positive animals were from the other mountainous hunting grounds in Tyrol.

Rates of infection with A. phagocytophilum were not statistically different (p > 0.05) between samples from autumn (September to November) or winter (December and January) (50% and 26.1%, respectively), between male and female animals (42.3% or 40.0%, respectively), or between animals in good and bad body condition (44.1% and 33.3%, respectively; Tables 1 and 2). However, the infection rate for A. phagocytophilum showed a significant variation among the age-classes (p < 0.05): animals >3 years of age tested positive significantly less frequently for A. phagocytophilum than the younger animals, whereas rates of infection did not differ (p > 0.05) between <1- and 1–3-year-old animals (Table 2).

Table 2.

Details of the Ungulates Studied for Anaplasma phagocytophilum DNA During the Hunting Season 2008/2009 in Austria

	Total no. of animals/no. of positive animals (%)
		Sex			Age groups				Body condition ^a
Species	All	Male	Female	NR	Group A (<1)	Group B (>1–3)	Group C (>3)	NR	Good	Bad	NR
Roe deer	16/8 (47.1)	6/2 (33.3)	10/6 (60.0)		2/2 (100.0)	4/4 (100.0)	9/1 (11.1)	1/1 (100.0)	13/7 (53.8)	3/1 (33.3)
Red deer	10/7 (70.0)	5/3 (60.0)	3/2 (66.7)	2/2 (100.0)	2/1 (50.0)	3/3 (100.0)	3/1 (33.3)	2/2 (100.0)	6/4 (66.7)		4/3 (75.0)
Chamois	16/4 (25.0)	12/3 (25.0)	4/1 (25.0)		1/1 (100.0)	4/2 (50.0)	11/1 (9.1)		5/0 (0)	11/4 (36.4)
Ibex	7/2 (28.6)	1/1 (100.0)	6/1 (16.7)			2/0 (0)	5/2 (40.0)		7/2 (28.6)
Mouflon	4/2 (50.0)	2/2 (100.0)	2/0 (0)		2/1 (50.0)		2/1 (50.0)		3/2 (66.7)	1/0 (0)
Total	53/23 (43.4)	26/11 (42.3)	25/10 (40.0)	2/2 (100.0)	7/5 (71.4)	13/9 (69.2)	30/6 (20.0)	3/3 (100.0)	34/15 (44.1)	15/5 (33.3)	4/3 (29,4)

Judged by the hunter.

Amplicons of the partial 16S rRNA gene were obtained in all 23 cases. Multiple alignment revealed seven different sequence types. A single sequence type was found for both chamois and Alpine ibex, whereas among the mouflon, red deer, and roe deer, individual animals carried sequences varying from other individual animals of the same species. The variations were found in four nucleotide positions within the first 52 bases (Table 3, including GenBank accession numbers).

Table 3.

Number of Occurrence of the Sequence Types of the 497-bp Partial 16S rRNA Gene Detected in the Ungulate Species Studied

	Sequence type 16S rRNA gene ^a (nucleotide differences) ^b
	B^c	J^d	S^e	T^f	W^g	X^h	Yⁱ
Ungulate species	(AAGG)	(GAAA)	(AGGG)	(GGGG)	(AAAG)	(GAAG)	(GAGG)
Roe deer	1	n.a.	1	n.a.	n.a.	3	3
Red deer	1	1	4	1	n.a.	n.a.	n.a.
Chamois	n.a.	n.a.	n.a.	n.a.	4	n.a.	n.a.
Ibex	n.a.	n.a.	2	n.a.	n.a.	n.a.	n.a.
Mouflon	n.a.	n.a.	n.a.	n.a.	1	n.a.	1
Total	2	1	7	1	5	3	4

Nomenclature is for ease of understanding only.

Nucleotide positions 3, 4, 11, and 52 in relation to the 497 bp obtained from the sequences from this study.

GenBank accession numbers FJ812388 and FJ812389.

GenBank accession number FJ812390.

GenBank accession numbers FJ812391–FJ812397.

GenBank accession number FJ812398.

GenBank accession numbers FJ812399–FJ812402, GU265827.

GenBank accession numbers FJ812403–FJ812405.

GenBank accession numbers FJ812406–FJ812409.

n.a., not amplified.

The amplification of the groEL gene was successful in 22 cases. Eleven sequence variants were obtained from the amplified region, with differences in a total of 25 nucleotide positions. The sequences were submitted to GenBank (accession numbers GQ988753–GQ988774). The construction of a phylogenetic tree using 10 consensus strains with 530 bp from this study (for one sequence only 508 bp could be evaluated) and a selection of sequences available in GenBank revealed two main clusters, one contained A. phagocytophilum sequences from roe deer and goats, and the other one from all the other species, including human patients (Fig. 2). Translation of the nucleotide sequences into amino acids revealed that only two nucleotide differences resulted in amino acid exchanges (position 10: asparagine → serine; position 46: serine → alanine).

FIG. 2.

Phylogenetic clustering of the partial groEL gene of Anaplasma phagocytophilum derived from wild ungulates from the Alps in Tyrol, Austria. The tree was obtained using the neighbor-joining method with the software package PHYLIP 3.67 after alignment with ClustalW of consensus sequences of the 530-bp partial sequence of the groEL genes obtained in this study (this study: bold print) and A. phagocytophilum sequences available from GenBank from various sources. Distance matrices were calculated using the Kimura two-parameter method. Selected bootstrap values in % (1000 repeats) are presented at the respective nodes. GenBank accession numbers are indicated in parentheses; for sequences from this study also the lettering found in Table 4 is indicated. *Also found in Alpine ibex; **also found in mouflon and chamois.

Great variability was also found in the 340-bp sequences of the partial msp4 gene, which was amplified in 21 cases. Ten different sequence types were identified, with differences in altogether 20 nucleotide positions. These have been deposited in GenBank under accession numbers GU265828–GU265848; eight of these sequence types have not been described before and are new in comparison to sequences deposited in the GenBank. Translation revealed that these nucleotide differences resulted in a diverse amino acid composition.

However, 16S rRNA, groEL, and msp4 gene variants did not match into distinct type strains. In only two cases appeared a combination of gene variants in more than one animal (Table 4).

Table 4.

Details of the Sequence Type Combinations Detected in the Samples in This Study for the Partial 16S rRNA, groEL, and msp4 Genes

Sequence type
16S rRNA^a	groEL^a	msp4^a	Host species
B	m	e	Red deer
B	f	n	Roe deer
J	h	j	Red deer
S	g	n	Roe deer
S	n.a.	n.a.	Red deer
S	n	f	Red deer
S	p	h	Red deer
S	q	i	Red deer
S	n	f	Ibex
S	n	f	Ibex
T	h	g	Red deer
W	j	k	Chamois
W	i	k	Chamois
W	h	n.a.	Chamois
W	o	i	Chamois
W	h	l	Mouflon
X	f	n	Roe deer
X	k	m	Roe deer
X	g	n	Roe deer
Y	g	n	Roe deer
Y	g	n	Roe deer
Y	f	n	Roe deer
Y	h	i	Mouflon

Upper case letters indicate 16S rRNA sequence types; lower case letters indicate groEL sequence types; lower case italicized letters indicate msp ⁴ sequence type. Bold text indicates combinations appearing more than once. Sequence type nomenclature is for ease of understanding only (no official type or strain name).

Discussion

DNA of A. phagocytophilum has been detected in a variety of wild ruminant species across their distribution range in the northern hemisphere (e.g., Massung et al. 2005, Beninati et al. 2006, Kawahara et al. 2006). In the present study, DNA of A. phagocytophilum was detected in all ungulate species studied, suggesting this group of animals play a role in the maintenance of A. phagocytophilum in nature in Central Europe. The highest infection rate was found in red deer and roe deer (70% and 47.1%, respectively). Two of four mouflons were positive. Chamois and ibex were least frequently infected (25% and 28.6%, respectively). Previously identified infection rates in roe deer ranged on average between 20% and 50% (Alberdi et al. 2000, Oporto et al. 2003, Petrovec et al. 2003, Beninati et al. 2006, Adamska and Skotarczak 2007, Zeman and Pecha 2008, Bown et al. 2009). However, individual results varied, for example, from 13% to 86% or from 43% to 74% (Petrovec et al. 2002, 2003, Hulínská et al. 2004, Polin et al. 2004). Therefore, our results in roe deer fall in the upper range of infection rates from studies in Europe. In a study including chamois from Switzerland, 28.2% of the animals had antibodies against A. phagocytophilum, but A. phagocytophilum DNA was not detected in any of them (Liz et al. 2002). Conversely, A. phagocytophilum DNA was detected in 25% of the chamois from Tyrol in the present study. A total of 11 chamois in the present study had bad body conditions (69%), of which 4 were positive for A. phagocytophilum DNA. The question arises whether poor body condition facilitates infection with A. phagocytophilum in chamois or whether A. phagocytophilum infection promotes factors leading to poor body condition.

Interestingly, differences were found in the infection rates in animals from the different hunting grounds. None of the animals from the Kaunertal area were positive, which was statistically significantly less when compared with the animals from the Karwendel mountains, which are on average lower mountains. However, because of the relatively low number of samples, it is not possible to draw a final conclusion from these data. Further, information on the altitude where the animals were hunted was not available.

Animals were tested only once postmortem during the hunting; therefore, no conclusion can be drawn as to persistence of the infection in these animals. A large part was positive at the end of the tick activity season. However, in general, variations in infection rates between mammalian species may be explained by different biological behavior of A. phagocytophilum strains or different susceptibility of the animals. Recently, it was shown in the United Kingdom that different genotypes of A. phagocytophilum seem to circulate in coexisting ecological niches (Bown et al. 2009). Also, variation of the prevalence in the vector tick or higher infection pressure in periods of peak tick activity may influence the occurrence of A. phagocytophilum in the mammals.

A. phagocytophilum has so far not been described in the Alpine ibex, and to the knowledge of the authors, there is just one report of an I. ricinus infestation of an Alpine ibex (Couturier 1962). Usually, Alpine ibex concentrate in high mountainous areas above the timberline; however, with declining availability of food near the end of winter, they migrate to lower altitudes reaching lowest levels near the valley beds in May (Meile et al. 2003). In the past, several studies conducted in the Alps, including Slovenia, Austria, and Switzerland, have shown that I. ricinus ticks rarely occur at altitudes above 1200 m (Rosický et al. 1961, Mahnert 1971, Aeschlimann 1972, Kaaserer 1974, Cotty et al. 1986). However, I. ricinus occasionally were recorded at 1800 m above sea level (a.s.l.) in Slovenia (Rosický et al. 1961) and at 2500 m a.s.l. in Tyrol, Austria (Mahnert 1971). As I. ricinus are active when average day time temperatures rise above 7°C (Korenberg 2000), ibex may encounter ticks, especially when occupying the areas of the valley beds in spring. In addition, a recently published paper reported an expansion of I. ricinus to higher altitudes, possible related to the climate changes in the Czech Republic (Materna et al. 2008).

The results of this study did not support the hypothesis of strict mammalian host species segregation of different 16S rRNA gene variants of A. phagocytophilum, as suggested by other authors (Petrovec et al. 2002, Zeman and Pecha 2008). However, in accordance with Zeman and Pecha (2008), a 16S rRNA gene sequence variant was detected, which seems to be present in several different mammalian species, including roe and red deers (strain “B” in the present study; “HV” in Zeman and Pecha 2008) (Table 3). We also detected the Czech variants “DV” (“S” in the present study; in roe deer, red deer, and ibex), “RV” (“X” in the present study; only in roe deer), and “SV” (“W” in the present study; in chamois and mouflon). Therefore, on the 16S rRNA gene level, the same variants were found among wild ruminants in Austria and in the Czech Republic.

The groEL gene sequences in this study formed two distinct clusters: one contained all the sequences derived from roe deer and the other one from the other species. Other researchers previously found a similar phenomenon in Germany, Sardinia/Italy, the Czech Republic, and Poland (Petrovec et al. 2002, von Loewenich et al. 2003, Alberti et al. 2005, Rymaszewska 2008). Therefore, the question arises whether there is an A. phagocytophilum variant specific for roe deer, which may not have appeared in human infections (Petrovec et al. 2002).

Great heterogeneity has been also found in the partial msp4 gene in this study. Of the 10 different variants, 8 have not been described before. The detected sequences were also different from the sequences analyzed by de la Fuente et al. (2005). The polymorphic multigene family p44 encoding the immunodominant major surface proteins (msp) is likely to be important in the pathogenesis in the mammalian host, and therefore, variation in the coding region could have an effect on host tropism, pathogenicity, and persistence in animals (Brayton et al. 2001, Lin et al. 2004, Stuen 2007).

The differences in the msp4 gene also resulted in diverse amino acid exchanges, whereas on the basis of the protein level of the heat shock protein, the nucleotide differences in the partial groEL gene resulted in only two amino acid exchanges.

Previous studies showed that a wide variety of A. phagocytophilum variants on the basis of several genes seem to circulate in wild mammals and ticks in different geographic regions.

In two cases (one red deer and one chamois), no amplicons of the msp4 gene could be obtained. For the red deer in question, it was also not possible to amplify the partial groEL gene. This may be explained by the variability of the gene or by reaching the limit of sensitivity of the analytical method.

No clear sequence typing could be achieved, even though A. phagocytophilum from roe deer seem to share more genetic similarities than those of the other ungulates on the basis of the genes studied. This supports the suggestion that A. phagocytophilum may have distinct ecotypes in nature (Bown et al. 2009).

Our results clearly showed that the wild ungulates from Tyrol, Austria, were infected to a high extent by A. phagocytophilum and that several genetic variants of A. phagocytophilum appear to circulate in nature, even in one geographical region. The pathogenicity of the variants and the reservoir competency of the ungulate species studied deserve further attention in future studies.

Footnotes

Disclosure Statement

The authors declare that no competing financial interests exist.

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