Preliminary study of natural reservoirs as sentinels of Anaplasma phagocytophilum and Ehrlichia chaffeensis in Soria , northern Spain

The serum of foxes and red deer from the Province of Soria (northern Spain) was screened in indirect immunofluorescence assays to determine whether these animals could be used as sentinels of the tick-borne pathogens Anaplasma phagocytophilum and Ehrlichia chaffeensis. The results suggest that foxes and red deer would not make good sentinels in epidemiological studies on E. chaffeensis in this region, although red deer could be used as such for the study of A. phagocytophilum transmission.


INTRODUCTION
Ehrlichia chaffeensis and Anaplasma phagocytophilum are the causal agents of severe emerging and reemerging human diseases [1].Both are transmitted through the bite of an infected tick.In Europe, the primary vector is Ixodes ricinus, for which red deer (Cervus elaphus), foxes (Vulpes vulpes), cattle, sheep, goats and horses act as reservoirs [2].In Spain, little is known about the epidemiology of these diseases.The present work reports a preliminary study, undertaken in the Province of Soria, to record the prevalence of the above pathogens in foxes and red deer, and to determine the value of these animals as sentinel species.

Study Area
The Province of Soria is located in northern Spain (central point 41˚25'0"N, 2˚28'0"W; altitude range 1100 -1650 m).It has a continental climate with cold winters and mild summers.The region has extensive forested areas and high shrubland [3].

Collection of Serum Samples
All the serum samples used in this work-30 from wild red deer and 30 from wild foxes-came from our group's frozen (−20˚C) serum collection.

Serological Assays
Indirect immunofluorescence assays (IFA) were performed to detect antibodies to A. phagocytophilum and E. chaffeensis in the tested serum samples according to Santos et al. [4] and Brouqui et al. [5].Samples were diluted 1:40 in PBS and incubated on slides prepared with HL-60 cells infected with A. phagocytophilum (prepared with Arkansas strain) or DH82 cells infected with E. chaffeensis (prepared with Webster strain) (both types of slides were kindly supplied by R. Sousa and A.S. Santos of the Centro de Estudos de Vectores e Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge, Portugal).The fluorescein-labelled conjugate was adapted according to the animal species studied (Sigma, St Louis, MO).Results were interpreted as positive when IgG titres of ≥40 were recorded.All positive samples were serially diluted to determine the endpoint titre, which was expressed as the reciprocal of the serum dilution.

Tick Counts
The number of different ticks on each animal from which serum was extracted was recorded in a database at the time of serum preparation.Ticks were enumerated following the method of Dominguez-Peñafiel et al. [6].

RESULTS AND DISCUSSION
None of the fox serum samples examined was positive for E. chaffeensis, while three were positive for A. phagocytophilum.All the red deer serum samples were positive for A. phagocytophilum but negative for E. chaffeensis.
A total of 109 ticks were obtained from the red deer: 37 males (33.94%), 56 females (51.37%) and 11 nymphs (10.09%).The most common species identified was Ixodes ricinus, with 35 males (41.17%),50 females (58.82%), followed by Haemaphysalis punctata with 1 male (20%) and 4 females (80%), and Rhipicephalus bursa with 1 male (33.33%) and 2 females (66.66%).As expected, most of the ticks found were adults (93 [85.32%]).The other work in the study area reported the same tick species on the same hosts [3].Some 20% of the foxes were parasitised by ticks.The most common species identified (always as adults) were Rhipicephalus sanguineus (50%), Ixodes canisuga (16.6%),Ixodes ricinus (16.6%) and Ixodes hexagonus (16.6%).These species are similar to those recorded on foxes from Thuringia (Germany) [7], and the same as those recorded in foxes in eastern Spain [8] and the Spanish south [9].Dermacentor variabilis and species of Amblyoma are known to act as vectors of A. phagocytophilum and Ehrlichia spp., but whether other ticks do so is unknown.The present results for the red deer suggest studies are needed to identify the role of other tick species in A. phagocytophilum transmission.
E. chaffeensis is the causal agent of human monocytotropic ehrlichiosis, and foxes are potential reservoirs for this bacterium [10].A. phagocytophilum is the causal agent of monocytic anaplasmosis, and this pathogen has been confirmed in red deer in Slovakia [11], as well as in sheep, ticks and foxes in Hungary [12].In Spain, it has been detected serologically and by PCR (in both the north and south) in ticks, roe deer, and cattle [13,14].
Since Ehrlichia and Anaplasma species can cause human disease [15][16][17][18], the prevalence of these pathogens in the study area, where contact between wild animals and people through outdoor activities and work, should be monitored.The present results suggest that foxes and red deer would not make good sentinels in epidemiological studies on E. chaffeensis in the study region, although red deer could be used as such for the study of A. phagocytophilum transmission.Red deer would seem to be involved in the natural cycle of this pathogen in this province.