Detection of Borrelia burgdorferi Sensu Lato and Anaplasma phagocytophilum in Small Mammals and Ectoparasites in Hungary

Introduction

S mall mammals are important hosts for the larval and nymphal stages of numerous hard tick species and, as a consequence, they may serve as reservoirs for many tick-borne pathogens, including Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum.

Borrelia burgdorferi s.l. is the causative agent of Lyme borreliosis, which is the most prevalent vector-borne human disease in the temperate zone of the Northern Hemisphere. Borrelia afzelii, Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia spielmanii are the most important pathogenic Borrelia species in Europe and each is associated with different vertebrate reservoirs (Bowman and Nuttall 2008).

Granulocytic anaplasmosis is caused by A. phagocytophilum. This pathogen has also been found in several vertebrate species (rodents, carnivores, equids, and ruminants), but the main reservoir in Europe is as yet unknown (Woldehiwet 2010).

The most important vector for both B. burgdorferi s.l. and A. phagocytophilum in Europe is Ixodes ricinus, which is also one of the most prevalent tick species in Hungary (Sréter et al. 2005, Földvári et al. 2007).

The aim of this preliminary study was to investigate the presence of Lyme disease spirochetes and A. phagocytophilum in wild-living small mammals, in their ectoparasites, and in field-collected ticks in Hungary and to gain information about the occurrence and possible coexistence of these pathogens at a selected region.

Materials and Methods

The study area is a mosaic of grasslands and oak woods situated in southeastern Hungary. Small mammals were live-trapped in April, July, October, and November 2009 and common hamsters were obtained from trappers in May and October 2009. Ectoparasites were combed off and lung, liver, spleen, and kidney samples were collected. After identification, ticks were pooled according to species (30 larvae, 15 nymphs, 10 or fewer males or females per pool). DNA was extracted by an X-tractor Gene nucleic acid extraction robot (Corbett Robotics Pty. Ltd., Queensland, Australia) with the Total RNA Isolation Kit, Nucleospin 96 RNA (Macherey-Nagel GmbH & Co. KG, Düren, Germany), except for the DNase incubation step as described in Gyuranecz et al. (2010a, 2010b) A. phagocytophilum and B. burgdorferi s.l. were detected by polymerase chain reaction. Primers SL forward and reverse were used to amplify a portion of the Borrelia osp A gene as described in Demaerschalck et al. (1995). Borrelia species were determined by sequencing and analyzing the amplicons. A. phagocytophilum DNA was detected and identified by species-specific primers MSP4AP3 and MSP4AP5 (de la Fuente et al. 2005).

Results

Overall, 277 small mammals (100 common hamsters [Cricetus cricetus], 38 common voles [Microtus arvalis], 110 yellow-necked mice [Apodemus flavicollis], and 15 striped field mice [Apodemus agrarius] were trapped, and 8 Eurasian pygmy shrews [Sorex minutus] and 6 common shrews [Sorex araneus]) were by-catched. Neither A. phagocytophilum nor B. burgdorferi s.l. was detected in any of the tissue samples.

A total of 806 ticks (404 I. ricinus, 374 Ixodes acuminatus, and 28 Haemaphysalis concinna) and 25 fleas (15 Ctenophtalmus assimilis and 10 Nosopsyllus fasciatus) were removed from small mammals. A. phagocytophilum was not found in any of the feeding ticks. Three pools containing Ixodes larvae (collected from one A. flavicollis and two A. agrarius), one pool of I. ricinus nymphs (collected from M. arvalis), and one pool of I. acuminatus females (collected from C. cricetus) contained B. afzelii (Table 1). None of the flea samples were positive for any of the examined pathogens.

A total of 1106 I. ricinus and 476 H. concinna were collected from the vegetation. Six I. ricinus pools and three H. concinna pools contained B. afzelii. B. garinii was found in one I. ricinus pool. We detected A. phagocytophilum in three I. ricinus pools (two female and one nymph pools).

Discussion

A large number of I. acuminatus ticks were found, but only on common hamsters (C. cricetus), not on other rodents. Only a few I. acuminatus were collected previously in Hungary: three females from hedgehogs (Babos 1965) and four females from dogs (Földvári et al. 2007). According to our results, I. acuminatus might be more prevalent in Hungary than we previously thought. An explanation for the low number of previously collected specimens of this species is that these nidiculous ticks are usually found on rodents and small insectivores and in their burrows (Bowman and Nuttall 2008), so it is unlikely that they are collected by flagging or dragging.

Four pools of Ixodes larvae were found positive for B. afzelii. Larvae in three out of the four pools were removed from small mammals. The infection of these larvae with borreliae could be the result of either cofeeding or that the small mammals were infected with Borrelia, but the infection was not detected. The ticks in the fourth positive larva pool were collected from the vegetation. These infected larvae may be evidence for transovarial transmission, although it was reported that B. afzelii is rarely, if ever, inherited by ticks (Bowman and Nuttall 2008).

The present study is the first report of borreliae from I. acuminatus and H. concinna from Hungary.

A. phagocytophilum was previously detected in I. ricinus collected from foxes (Sréter et al. 2004) and in field-collected ticks (this study) in Hungary, but we have not been able to find it in small mammals yet, despite the fact that recent studies suggest that rodents can be important reservoirs of this pathogen (Woldehiwet 2010). The small number of positive samples in field-collected ticks suggests a low prevalence of this pathogen at the study site. This provides a possible explanation for our lack of success in detecting these bacteria in small mammal tissue samples.

The prevalence of pathogens in field-collected ticks and in ticks collected from small mammals was very low and, despite the fairly high number of captured rodents, we were not able to detect them in any tissue samples. This may reflect the fact that prevalence varies greatly between different regions. There is also a possibility that the incompetent or less competent host species present at the study site outnumber the reservoir animals, thus diluting the prevalence of these pathogens. However, to prove the presence and determine the characteristics of this dilution effect, further information about the host community of the area and the analysis of tick blood-meals is needed.