Antibodies to Rickettsia spp. and Borrelia burgdorferi in Spanish Wild Red Foxes (Vulpes vulpes)

Interest in emerging and re-emerging rickettsial infections (such as those caused by Rickettsia typhi and Rickettsia slovaca) and in Lyme disease (caused by Borrelia burgdorferi) has grown in Spain over recent years. Epidemiologic studies have been conducted to determine the prevalence of these infections among humans and to identify the reservoirs and vectors of the pathogens (Bernabeu-Wittel et al. 1999; Antón et al. 2008; Lledó et al. 2014).

Contact between wildlife and domestic animals indirectly increases contact between wildlife and humans and therefore the likelihood of pathogen transmission. Foxes often live in periurban and agricultural areas and adapt well to domestic animals and humans (Fishman et al. 2004). Direct or indirect human contact with these animals is therefore possible. The literature contains no information on whether the foxes of central Spain are affected by R. typhi, R. slovaca, or B. burgdorferi. We investigated the prevalence of these infections in foxes living in the province of Soria (central point 41°25′0′′N, 2°28′0′′W; 1,100–1,650 m in elevation) and examined the value of these mammals as a sentinel species.

We examined serum samples from 314 wild red foxes (Vulpes vulpes). The samples were from the frozen (−20 C) serum collection of Anthropozoonosis research group of Alcala University, Alcala de Henares, Spain. The foxes were captured alive or found dead (usually roadkills) by forestry agents. Numbers by sex and age category were as follows: 153 males (48.7%) and 161 females (51.3%); 49 (15.6%) juveniles (i.e., with milk teeth; <1 yr old), 182 (58.0%) adults (i.e., with permanent, unworn teeth; 1–5 yr old), and 83 (26.4%) “old” (i.e., with markedly worn teeth; >5 yr old). This research was approved by the Research Bioethics and Animal Welfare Committee of the Universidad de Alcalá.

Serum samples were examined for antibodies to Rickettsiae by using an in-house indirect immunofluorescence system (Phillip et al. 1976). Vero E6 cells (ATCC 1586, Sigma-Aldrich, St. Louis, Missouri, USA) infected with R. typhi (Wilmington strain) or R. slovaca (strain 246 CDC) were used to provide antigens. Antibodies to B. burgdorferi were detected using the same system, but following the method of Russell et al. (1984) and using B. burgdorferi sensu stricto (strain B31 ATCC 35210) as an antigen source; these bacteria were previously propagated in Barbour Kelly medium (Sigma-Aldrich) and fixed on spot slides. Fluorescein-labeled rabbit anti-dog immunoglobulin G (IgG) antibodies (Sigma-Aldrich) were used to reveal the binding antibodies. Positive and negative control sera were also examined. Sera showing a typical pattern of fluorescence and an IgG antibody titer of ≥1/64 were considered positive. All positive samples were serially diluted to determine the endpoint titer, expressed as the reciprocal of the maximum serum dilution giving a positive result.

The number of species of arthropods on each animal from which serum was extracted was recorded in a database at the time of serum preparation. Arthropods were enumerated following the method of Dominguez (2004). Ticks were detected on 57 foxes (18.2%) and fleas on 163 (51.9%). The χ² or Fisher’s exact test was used to compare differences in proportions, using two-way tables. Significance was set at P<0.05.

Six of 314 foxes (1.9%) had antibodies against R. typhi. The prevalence was 1.9% (3/161) in female foxes and 2.0% (3/153) in males. Four adults (2.19% [4/182]) and two old animals (2.40% [2/83]) were positive. Two of six antibody-positive foxes had fleas (Table 1). Positive serum antibody titers ranged from 64 to 256.

Twenty-one of 314 foxes (6.7%) had antibodies to R. slovaca. Ten of 153 males (6.53%) and 11 (6.8%) of 151 females were positive. Two of 49 juveniles were positive (4%), as were 15 of 182 adults (8.2%) and four of 83 old foxes (4.8%). Five of 21 antibody-positive foxes (23%) had ticks (Table 2). Positive antibody titers ranged from 40 to 512.

Twenty-six of 314 foxes had antibodies to B. burgdorferi (8.3%). Eight of 153 (5.2%) males and 18 of 151 (11.1%) females were positive. Six of 49 (12%) young, 13 of 182 (7.1%) adults, and seven of 83 (8%) old foxes were positive. Six of 26 antibody-positive foxes (23%) had ticks at the time of capture (Table 2). Positive antibody titers ranged from 64 to 1,024. No significant differences were seen in the prevalence of any infection with respect to age or sex.

We believe this to be the first detection of antibodies to R. typhi and R. slovaca in foxes, indicating infection of Spanish red foxes with the agents of spotted fever and typhus. Given the numerous cross-reactions that occur among the species of Rickettsia (Teysseire and Raoult 1992), more specific serologic tests and molecular studies are required to confirm these results.

The prevalence of R. typhi antibodies we recorded is lower than that reported for domestic dogs (Canis lupus familiaris) in the study area (12.3%; Lledó et al. 2003) and in Catalonia (northeastern Spain) (4.5%; Nogueras et al. 2013). It is also lower than that reported for dogs in Portugal (26.9%; Bacellar et al. 1995), but higher than that reported for rodents and dogs in Egypt (0.4%; Soliman et al. 1989).

The flea Xenopsylla cheopis has traditionally been regarded the vector of R. typhi, but in this study no foxes were found with X. cheopis. Two other species of flea were detected, however, that in recent years have been designated possible R. typhi vectors: Ctenocephalides canis (Parola et al. 2003) and especially Ctenocephalides felis (Farhang-Azad et al. 1984). Both are primarily found on cats (Felis catus) or domestic dogs, but an association with wild animals has also been reported (Torina et al. 2013). Ctenocephalides canis, the second most commonly detected flea, was found on 8.3% of the foxes, whereas C. felis, which shows greater affinity for feeding on humans, was detected on 1.3% (Farhang-Azad et al. 1984). Both species were detected on foxes with antibodies to R. typhi.

The detected prevalence of R. slovaca antibodies was 6.7%. The literature contains no information on the prevalence of this pathogen in foxes, although prevalence studies on other members of the spotted fever group have been performed in dogs. The results, however, vary widely. For example, figures of 24–50% are reported for northwestern Spain (Amusategui et al. 2008), 85.6% for Portugal (Bacellar et al. 1995), and 11.6% and 3.9% for Brazilian dogs from rural and urban areas, respectively (Labruna et al. 2007).

The presence of the tick Dermacentor marginatus on one fox should be noted. This tick is the main vector of R. slovaca (Sekeyová et al. 1998); in the region of Castilla y León, it commonly bites humans, and it is the most common tick in the province of Soria (Fernández-Soto et al. 2003).

The highest antibody prevalence we found was for B. burgdorferi (8.3%). Sobrino and Cortazar (2008) also reported 8.3% prevalence in foxes from the northern third of Spain. Other studies have indicated 7–15% in Germany (Schoffel et al. 1991; Heidrich et al. 1999). A prevalence of 21.9% was reported for domestic canids in the province of Soria (Merino et al. 2000). In Spain, the main vector of B. burgdorferi is Ixodes ricinus (Estrada-Peña et al. 1995), the tick most commonly found in our study.

We have shown that Rickettsia spp. and B. burgdorferi, and their vectors, are present in foxes in the province of Soria. Further studies are needed to determine the role of red foxes in the natural endemic cycles of these pathogens.