Fox on the run--molecular surveillance of fox blood and tissue for the occurrence of tick-borne pathogens in Austria.

BACKGROUND: The red fox (Vulpes vulpes) is a widespread species, harbouring many pathogens relevant for humans and pets. Indeed, Anaplasma spp., Ehrlichia canis and Rickettsia spp. among the bacteria and Hepatozoon canis as well as Babesia sp. among the parasites have been the focus of several studies.
FINDINGS: In a cohort of 36 foxes shot on one day in the north-eastern part of Austria, Babesia microti-like pathogens were found in 50%, while H. canis was detected in 58.3% of the samples. The spleen was more useful for detection of H. canis, whereas B. microti-like parasites were more frequently found in the blood. Bacteria could not be confirmed in any of the cases to demonstrate the occurrence of such tick-borne pathogens using PCR and sequencing on blood and spleen samples.
CONCLUSIONS: The occurrence of B. microti-like and H. canis parasites raised many questions, because these infections have never been found autochthonously in dogs. Furthermore in the case of H. canis the main vector tick, Rhipicephalus sanguineus, is absent in the sampling area, leaving space for further hypotheses for transmission such as vertical transmission, transmission via ingestion of prey animals or other vector ticks. Further studies are needed to evaluate the risks for pets in this area. PCRs delivered differing results with the different tissues, suggesting the use of both spleen and blood to obtain an integral result.

Findings

Background

Red foxes (Vulpes vulpes) are among the most widely distributed mammals in the world and are invading many urban areas due to a good adaptation to human environments, and to rabies vaccination [1]. As a result foxes might play a big role in spreading pet-relevant pathogens and parasites such as mites and ticks [2]. Recently they have been discussed as a potential reservoir for blood parasites like Anaplasma phagocytophilum [3], Hepatozoon canis [4], Babesia sp. [5], Ehrlichia canis [6] and Rickettsia spp. [2]. Due to their close vicinity to domestic habitats they may act as a transmission interface for some of these pathogens to pets and humans [5].

Babesia microti-like parasites – also known as Babesia sp., Babesia annae or Theileria annae – are frequently found in foxes in countries such as Croatia [7], Portugal [5] and Spain [8]. The common assumption is that Ixodes hexagonus is involved in the transmission cycle [9], and a recent study identified I. ricinus and I. canisuga as carriers and therefore as potential vectors [10]. These ticks could also serve as a transmission interface to dogs, where Babesia may cause azotaemia, haemolytic anaemia, renal failure and mortality [11].

Hepatozoon canis affects canids and its occurrence is mostly linked to the distribution of the main vector tick Rhipicephalus sanguineus [12], already displaying exceptions in countries such as Austria, Germany or Hungary [12-14].

The aim of this study is to evaluate the role of foxes in terms of their blood pathogens and to discover potential reservoirs for tick-borne diseases in northern latitudes.

Method

Foxes shot on 18 January 2014 in the district of Gänserndorf (in the northeast of Lower Austria) were further processed on the same day. From the 36 foxes, 35 spleen samples and 17 blood samples were obtained. Extraction of DNA from blood and tissue was performed as previously described [14]. Primers detecting Anaplasma sp., Babesia sp. (piroplasms), Ehrlichia canis, Hepatozoon canis and Rickettsia sp. were used (Table 1). The PCRs were conducted on the Eppendorf Mastercycler pro S (Eppendorf AG, Hamburg, Germany) using protocols published elsewhere [14]. To confirm the sequence, positive samples were randomly chosen and the amplifications were purified by Fast-kit (Bio-Rad Laboratories, Vienna, Austria) according to the manufacturer’s recommendations and sent for sequencing (Microsynth AG, Balgach, Switzerland; LGC, Teddington, UK). Sequences obtained were further processed by GeneDoc (http://genedoc.software.informer.com/2.7/) and blasted on GenBank® (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Table 1

PCR parameters for amplification of DNA of target organisms

Target organism	Forward primer (5’-3’)	No. of cycles	Annealing temperature (°C)	Primer concentration (pmol)	Product size (bp)	Reference
	Reverse primer (5’-3’)
Anaplasma sp.	Ehr.u.for: GTT TGA TCC TGG CTC AGG AYD AAC	30	66.8	12.5	619	[15]
	ERB2rev: CTC TTT CGA CCT CTA GTC TAG C
Piroplasms (nested)	1st	40	68	25	561	[16]
	BTH-1 F: CCT GAG AAA CGG CTA CCA CAT CT
	BTH-1R: TTG CGA CCA TAC TCC CCC CA
	2nd
	GF2: GTC TTG TAA TTG GAA TGA TGG	40	60	50
	GR2: CCA AAG ACT TTG ATT TCT CTC
Ehrlichia canis	Ehr.u.for: GTT TGA TCC TGG CTC AGG AYD AAC	30	65.0	20	619	[15]
	Ehr.CCE.rev: CTC TTT CGA CCT CTA GTC TAG C
Hepatozoon canis	HEPF: ATA CAT GAG CAA AAT CTC AAC	35	57.0	10	660	[17]
	HEPR: CTT ATT ATT CCA TGC TGC AG
Rickettsia sp.	ITS-F: GAT AGG TCG GGT GTG GAA G	35	52	1	342 – 533	[18]
	ITS-R: TCG GGA TGG GAT CGT GTG

Ethical statement

Fox were shot during routine hunting events under the restrictions of the game laws of the province of Lower Austria.

Results

The investigation of the blood and spleen samples identified 18 B. microti-like pathogen-positive foxes, 21 foxes harbouring H. canis and four foxes with double infections (Table 2), leading to prevalences of 50%, 58.3% and 11.1%, respectively. PCRs for detecting piroplasms (Babesia sp. nested) in blood and spleen detected 13 (76.5% of the blood samples) and 11 (31.4% of the spleens) B. microti-like pathogens, respectively. Sequences of these pathogens showed 98–100% similarity to B. sp. “Spanish dog” (e.g. GenBank® accession no. AF188001.1 or EU583387.1). Using the Hepatozoon-specific primers, 21 foxes tested positive for H. canis. The investigation of the spleen samples identified 18 positive results (51.4%), whereas in the blood samples only six positive results (35.3%) were found. Seven more PCR products, positive on the gel, provided no conclusive sequence data, and therefore were noted as false positives. All conclusive sequences delivered 99–100% similarity to H. canis found in GenBank® (e.g. accession no. AY150067.2, DQ111754.1, JN584477.1 or KC509526.1).

Table 2

PCR results of spleen and blood compared to sequencing results of the investigated foxes (pos = representing a positive PCR product on the gel, neg = delivering no band on the gel, or -like = confirmed sequence of this pathogen in the substrate, “f” indicates false positive samples showing a gel band, but not confirmed during sequencing)

	PCR
Fox	Piroplasms nested	H. canis	Pathogens detected	GenBank® accession no
1	B. microti-like	pos. f	B. microti-like	KM115968
2	H. canis	H. canis	H. canis	KM115969
3	H. canis	pos.	H. canis	KM115970
4	H. canis	H. canis	H. canis	KM115971
5	B. microti-like	neg.	B. microti-like	KM115972
6	B. microti-like	pos. f	B. microti-like	KM115973
7	H. canis	H. canis	H. canis	KM115974
8	B. microti-like	neg.	B. microti-like	KM115975
9	B. microti-like	neg.	B. microti-like	KM115976
10	pos	pos. f	unclear
11	B. microti-like	pos. f	B. microti-like	KM115977
12	B. microti-like	neg.	B. microti-like	KM115978
13	H. canis	H. canis	H. canis	KM115979
14	B. microti-like	H. canis	B. microti-like/H. canis	KM115980/KM115981
15	B. microti-like/ H. canis	pos.	B. microti-like/H. canis	KM115982/KM115983
16	H. canis	H. canis	H. canis	KM115984
17	B. microti-like	pos. f	B. microti-like	KM115985
18	H. canis	H. canis	H. canis	KM115986
19	H. canis	H. canis	H. canis	KM115987
20	B. microti-like	H. canis	B. microti-like/H. canis	KM115988/KM115989
21	B. microti-like	neg.	B. microti-like	KM115990
22	H. canis	H. canis	H. canis	KM115991
23	B. microti-like	neg.	B. microti-like	KM115992
24	H. canis	H. canis	H. canis	KM115993
25	B. microti-like	neg.	B. microti-like	KM115994
26	H. canis	H. canis	H. canis	KM115995
27	H. canis	H. canis	H .canis	KM115996
28	B. microti-like	pos. f	B. microti-like	KM115997
29	H. canis	H. canis	H. canis	KM115998
30	B. microti-like	H. canis	B. microti-like/H. canis	KM115999/KM116000
31	H. canis	H. canis	H. canis	KM116001
32	pos.	H. canis	H. canis	KM116002
33	H. canis	H. canis	H. canis	KM116003
34	B. microti-like	neg.	B. microti-like	KM116004
35	H. canis	H. canis	H. canis	KM116005
36	B. microti-like	pos. f	B. microti-like	KM116006

In none of the blood or spleen samples could Anaplasma sp., E. canis or Rickettsia spp. be detected.

Discussion

Foxes are known to be major reservoirs for Babesia microti-like parasites [5]. The high prevalence of 50% found in this study and in this population is therefore not surprising and reflects a similar situation in Germany with 46.4% [10], Portugal with 69.2% [5] and Spain with 14% to 50% [8].

The 58.3% positive H. canis foxes in Austria are in concordance with four positive foxes out of nine found in Slovakia [19], 45.2% in Germany [20], 16 out of 111 investigated foxes (11.6%) in Poland [21] or 8% in Hungary [22]. To date H. canis is not found endemically in dogs in these areas, nor is R. sanguineus known to occur autochthonously [12,19,21,23], although H. canis has already been found in dogs in areas lacking the main vector tick in Germany [13,20] and Hungary [12,22].

Conclusion

Foxes represent a good reservoir for several zoonotic and pet-relevant diseases. In terms of blood parasites this seems more the rule than the exception. Human- and pet-relevant agents such as Babesia microti-like pathogens and H. canis could be found in a relatively small set of fox samples originating from north-eastern Austria. Especially, the occurrence of H. canis in considerable numbers in this population so far north raises many questions such as the potential impact on domestic animals, reservoirs and infection pathways. Moreover, the main vector tick, Rhipicephalus sanguineus, is absent in the sampling area. Therefore other transmission pathways such as vertical transmission, transmission via ingestion of preyed animals or other vector ticks need to be evaluated.

Thus foxes have to be considered during treatment strategies and B. microti-like as well as H. canis pathogens have to be recognized as an unnoticed threat in northern areas. The use of piroplasm PCRs could help to identify both B. microti-like and H. canis pathogens prior to screening, followed by PCRs with species-specific primers.