Pedicle myiasis by Lucilia caesar (Diptera, Calliphoridae): An emerging disease in roe deer from north-western Spain.

A total of 35 male roe deer (Capreolus capreolus) from Lugo province (north-western Spain) were examined for pedicle myiasis between 2020 and 2022. All these animals had died by different causes and were examined or preserved just after their death to avoid post-mortem infestations. After external and internal head inspection, five animals were diagnosed with severe myiasis at the basis of the antlers (14.29%; 95% confidence interval = 5.38-31.04). All the affected bucks presented a cutaneous wound (1.5-15 cm in diameter) around the pedicles with extensive tissue destruction, exposition of frontal and parietal bones, and massive infestation by dipteran larvae (2-12 mm long). Four of five roe deer showed whitish egg clusters adhered to the basis of the antlers. Moreover, in one animal, six larvae had penetrated the cranial cavity, and in two of them larvae were also found in the nasopharyngeal cavity. All the cases were recorded in summer (May-August). Morphological identification and subsequent molecular confirmation revealed that all animals were infested by different larval stages of Lucilia caesar (Diptera: Calliphoridae). This study represents the first report of pedicle myiasis in Spain. Since this disease is reported sporadically, the detection of five cases in a short period of time suggests an increase in the incidence of this myiasis.

A case report of fatal feline babesiosis caused by Babesia canis in north western Spain.

BACKGROUND: In Europe, Babesia infections in cats are sporadic and only partial knowledge is currently available since the number of described cases including both the clinical presentation and the molecular identification of the Babesia species involved is limited. In the present case report, the clinical signs, the epidemiological data and the molecular results suggest that this is the first reported fatal case of feline babesiosis caused by Babesia canis. CASE PRESENTATION: A six month old female European shorthair cat from north-western Spain died after being hospitalized for two days. This animal was pregnant and showed anorexia, lethargy, weakness, jaundice and fever with increased respiratory and heart rates. Haematological analysis revealed haemolytic regenerative anaemia, thrombocytopenia and leukocytosis. The presence of piroplasms was assessed using a PCR targeting the 18S rRNA gene of Babesia spp. and Theileria spp.; the sample resulted positive and B. canis was identified by DNA sequence analysis. The possible existence of co-infections with other vector-borne pathogens such as Anaplasma spp., Bartonella spp., Borrelia burgdorferi s.l., Cytauxzoon spp., Ehrlichia spp., Hepatozoon canis, Mycoplasma spp. or Rickettsia spp. was excluded by qPCR. CONCLUSIONS: Our results together with previous reports on Babesia infections in cats from Europe suggest that feline babesiosis should be included in the differential diagnosis of animals with anaemia, thrombocytopenia, anorexia and lethargy, especially in young or immunocompromised animals from endemic areas for canine babesiosis.

Prevalence and molecular characterization of Rickettsia spp. in questing ticks from north-western Spain.

Tick-borne rickettsioses, most of them belonging to the spotted fever group (SFG), have been recognized as important emerging vector-borne zoonotic diseases. In order to determine the presence of Rickettsia spp. in questing ticks from north-western Spain, 1056 Ixodes ricinus, 19 Dermacentor marginatus, 17 Dermacentor reticulatus and one Ixodes acuminatus were processed. Rickettsia DNA was detected by PCR targeting rOmpA and rOmpB genes. A total of 219 (20.7%) I. ricinus, 19 (100%) D. marginatus and four D. reticulatus (23.5%) were positive. The prevalence was significantly higher in I. ricinus from coastal areas and in winter. Five species were identified: Rickettsia felis, Rickettsia monacensis, Rickettsia raoultii, Rickettsia slovaca and "Candidatus Rickettsia rioja". Our results reveal a significant presence of some pathogenic Rickettsia species in questing tick populations from this area which involves a noticeable risk of rickettsiosis. As R. raoultii, R. slovaca and "Ca. R. rioja" DNA were identified in I. ricinus, considered an unusual vector for these Rickettsia species, further studies are needed to unravel the role of that tick species in the maintenance and transmission of these three Rickettsia species in north-western Spain.

Cryptosporidium species and subtype analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain.

Faecal specimens from diarrhoeic pre-weaned lambs (n = 171) and goat kids (n = 118) were collected in 37 sheep and 23 goat flocks, respectively, from NW Spain and microscopically examined for the presence of Cryptosporidium oocysts. Positive specimens were selected for molecular characterization. Presence of Cryptosporidium oocysts were significantly higher in specimens from goat kids (62.7%) than from lambs (31.6%). PCR products of the SSU rRNA locus were obtained for 108 isolates, and three Cryptosporidium species were identified. Cryptosporidium parvum was the most common species identified from both lambs (74.4%) and goat kids (93.8%). The remaining PCR products from lambs (25.6%) and goat kids (7.7%) were identified as Cryptosporidium Ubiquitum and Cryptosporidium xiaoi, respectively. Five C. parvum subtypes were identified; IIaA13G1R1, IIaA14G2R1, IIaA15G2R1 and IIaA16G3R1 were found in both host species, and IIdA17G1 was only detected in goat kids. Subtype IIaA15G2R1 was the most common and widely distributed. The present study provides the first description of subtypes IIaA13G1R1 in both small ruminant species, IIaA14G2R1 in sheep and IIaA16G3R1 in goats. Our results also reveal that diarrhoeic pre-weaned lambs and goat kids must be considered important reservoirs of Cryptosporidium species with zoonotic potential, such as C. parvum and C. ubiquitum.

Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain).

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1-80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March-May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.

Seroprevalence of Borrelia burgdorferi sensu lato in roe deer (Capreolus capreolus) from northwestern Spain.

In the present study, the seroprevalence of Borrelia burgdorferi sensu lato in roe deer in relation to different parameters in northwestern Spain was investigated. A total of 154 roe deer hunted between April 2007 and October 2008 from different localities of Galicia (northwest Spain) were examined. From each animal, a blood sample and all attached ticks found were collected. All the specimens for tick stages (larva, nymph, and adult) were speciated based on reference keys. Antibodies against B. burgdorferi were detected by indirect immunofluorescence (titer > or = 1:64). The percentage of roe deer seropositive for B. burgdorferi was 68.8% (106/ 154), of which 88.7% (94/106) were parasitized by ticks. Ixodes ricinus was the only species identified and was detected in 83.1% of roe deer with a mean (standard deviation [SD]) intensity of 46 +/- 47 ticks. Individual host characteristics such as age or sex did not have any effect on the prevalence of B. burgdorferi, but significant seasonal variation was observed, with higher prevalences in April-July than in August-October. Antibodies against B. burgdorferi were related to the presence of ticks. When analyzing all the factors together, the total number of ticks parasitizing roe deer was found as the most influential factor on B. burgdorferi prevalence. The results of this study have shown that roe deer in the northwest of Spain are highly exposed to B. burgdorferi and that exposure is related to the presence of I. ricinus.

Cephenemyia stimulator (Diptera: Oestridae) myiasis in a roe deer (Capreolus capreolus) from Portugal.

Herein we present a nasopharyngeal myiasis case by Cephenemyia stimulator in a roe deer hunted in Trás-os-Montes (NE Portugal). Preliminary inspection showed one larva sorting through the nostrils and the examination of the nasopharyngeal cavity showed more than 15 larvae in the glottis and retropharyngeal recesses. Four larvae were collected and stored into 70% ethanol for morphological and molecular identification. Three of the larvae were identified as third instars and the other one as a prepupa of Cephenemyia stimulator, being the first confirmation of this species in roe deer from Portugal. C. stimulator is currently widely distributed in roe deer from central and northern Spain so, the transboundary natural dispersal of these cervids would explain the introduction of this myiasis in Portugal. Further studies are needed to monitor the spreading of this infection in the westernmost populations of European roe deer.

Evaluation of the efficacy of two postweaning colibacillosis vaccines in a field herd with PRRS circulation during postweaning stage.

Postweaning diarrhea (PWD) and PRRS are two major concerns in swine production, which association has not been consistently explored. In the current scenario of restrictions in the use of antibiotics and ZnO, vaccination is more relevant to control PWD, but PRRS virus circulation may compromise the immune protection conferred by postweaning colibacillosis vaccines. We evaluated the efficacy of two postweaning colibacillosis vaccines (parenteral and oral) in a commercial herd affected by an outbreak of PWD and with PRRS circulation in postweaning. Five groups were studied during the postweaning period: one control (Group 1) and four vaccinated: two with each postweaning colibacillosis vaccine administered alone (Groups 2 and 3) or with sow vaccination against PRRS (Groups 4 and 5). We evaluated the effects on piglet weight, average daily weight gain and in the percentage of piglets with diarrhea, its duration, lethality and mortality. PRRS viremia and anti-PRRS antibodies were evaluated by qPCR and ELISA. Regarding control group, colibacillosis vaccination generally improved most of the measured parameters; but significant improvements were only observed in Groups 4 and 5 (p < 0.05). Moreover, cases of diarrhea occurred at different ages: in Groups 2 and 3 the peak of cases occurred just after ZnO was removed from the feed compared to Group 1, while in Groups 4 and 5 no peak was observed. This suggests that postweaning colibacillosis vaccination may be compromised by the PRRS circulation. In PRRS endemic herds an effective protection against PWD through vaccination may require PRRS vaccination to obtain a better performance.

Monitoring of porcine circovirus type 2 infection through air and surface samples in vaccinated and unvaccinated fattening farms.

Air and surfaces of swine farms are the two alternative samples to obtain information about the health status of the herd. The aim of this study was to assess air and surface sampling for the detection of porcine circovirus type 2 (PCV2) in vaccinated and unvaccinated fattening farms, studying the relationship between the viral load in these samples with the viremia at herd level. Three swine fattening batches (one unvaccinated; two vaccinated) were monitored at 10, 12, 14, 16 and 18 weeks old; at each stage, blood, air and different surfaces were sampled and analysed by qPCR. In all herds, PCV2 was detected in all types of samples. Whenever viremia was detected, PCV2 was also detected in air and surface samples, even in those cases with a low estimated prevalence (1.6%); moreover, in two out of the three herds, PCV2 was detected in air and surface samples earlier than in the blood of the sampled population. In addition, a good correlation between the viremia of pig population and the PCV2 load in air and surface samples was found in both cases (τ = 0.672 and 0.746, respectively; p <0.05). These results show that air and surface samples could be useful tools to monitor PCV2 infection, being suitable for detecting the virus in cases of low prevalence and even before pigs develop viremia; therefore, these sampling techniques would speed up the implementation of the required measures to prevent productive and economic losses due to PCV2 infection.

Antimicrobial Susceptibility of Enterotoxigenic Escherichia coli from Diarrhoeic Neonatal Calves in Spain.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens involved in neonatal calf diarrhoea (NCD) causing high economic losses in dairy farms. Antibiotic treatment is common in cases of systemic illness caused by NCD, but antimicrobial susceptibility tests (AST) are usually not performed. Thus, the aim of this study was to characterize the antimicrobial susceptibility of ETEC strains obtained from calves with diarrhoea between 2018-2020. Faecal samples (n = 420) were analyzed to detect the typical ETEC virulence factors F5 and STa. Positive samples were cultured to identify and isolate ETEC strains (n = 41) and ASTs were performed. Our results are alarming since ETEC strains resistant to three or more families of antimicrobials were detected in all isolates. Only four antibiotics (ceftiofur, cefoperazone, cefquinome and gentamicin) presented efficacy against more than 90% of the ETEC strains, while the other ten antibiotics were effective against less than 40% of the strains. In addition, a high number of strains were resistant to most first-line antimicrobials used in veterinary practice. For this reason, when ETEC infection is suspected, an AST must always be performed to select the most appropriate antimicrobial in each case and to avoid the emergence of new resistance mechanisms.

Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain.

Metastrongyloid and trichuroid cardiorespiratory nematodes of dogs and cats are considered emergent in several European countries, and an increase in the number and extent of endemic foci has been described. Since data on their prevalence are limited in this continent, faecal samples from new arrivals (365 dogs and 65 cats) at two animal shelters in North-western Spain were analysed using both floatation and Baermann techniques. In order to confirm the microscopic identification of Metastrongylidae first stage larvae, molecular characterization based on the sequence of the ITS-2 was performed. The possible influence of some variables such as the species, sex and age of the animals and the co-infection with other gastrointestinal parasites on the prevalence of cardiorespiratory nematodes was analysed. The most prevalent metastrongylid was Aelurostrongylus abstrusus (15.4%) followed by Angiostrongylus vasorum (4.1%) and Crenosoma vulpis (1.1%). Regarding trichuroids, Eucoleus aerophilus and/or Eucoleus boehmi eggs were detected in 28 dogs (7.7%) and four cats (6.2%). Almost all animals positive to cardiorespiratory nematodes (86.8%) were co-infected with gastrointestinal parasites. The prevalence of Metastrongylidae and respiratory trichuroids was significantly higher in dogs co-infected with Taenia spp. and Toxocara canis or Giardia duodenalis and Sarcocystis spp., respectively. In cats, a significant higher prevalence of Metastrongylidae nematodes was found in animals co-infected with Toxocara cati. Our results reveal that cardiorespiratory nematodes are common in companion animals from north-western Spain, showing higher prevalences than those previously reported from this country. This investigation represents the first report of C. vulpis, E. aerophilus and E. boehmi in dogs from Spain. The identification of a number of zoonotic parasites is of public health concern. Our results indicate that these nematodes should be included in the differential diagnosis of dogs and cats from north-western Spain showing respiratory or cardiac clinical signs.

Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

Mixed Nasopharyngeal Myiasis by Bots and Blowflies in a Roe Deer (Capreolus capreolus).

A mixed nasopharyngeal infestation by larvae of Cephenemyia stimulator (Diptera: Oestridae) and Lucilia caesar (Diptera: Calliphoridae) was detected in a roe deer (Capreolus capreolus) killed in a car collision in Lugo, Spain, in July 2020. This article stresses the importance of correctly identifying larvae in myiasis cases.

Tick infestation (Acari: Ixodidae) in roe deer (Capreolus capreolus) from northwestern Spain: population dynamics and risk stratification.

During the 2007 and 2008 hunting seasons (April-October) the skin of 367 roe deer (Capreolus capreolus L.), hunted in different preserves from Galicia (Northwestern Spain), were examined for ticks (Acari: Ixodidae). The overall prevalence of infestation by ticks was 83.1%. The predominant species was Ixodes ricinus (83.1%), whereas a single Dermacentor marginatus specimen appeared in one roe deer. All developmental stages of I. ricinus were found parasitizing roe deer, the adults being the most frequent (82.2%), followed by nymphs (45.6%) and larvae (27.2%). The mean intensity of infestation by I. ricinus was 43.2 ± 49.85; most of them were adults (30.7 ± 31.64) and in a lesser extend nymphs (16.9 ± 24.74) and larvae (10.7 ± 29.90). Ixodes ricinus was present all over the study with percentages that oscillated between 100% in spring and 57.4% in autumn. CHAID algorithm showed the sex of roe deer as the most influential factor in tick prevalence, followed by the climatic area. The different developmental stages of I. ricinus were more frequent in males than in females, and the prevalence of adults and larvae were higher in roe deer from coastal areas than in those from mountainous and central areas, whereas nymphs were more frequent in mountainous areas. Host age and density were not determinants for tick infestation. Our results confirm that roe deer are important hosts for I. ricinus in northwestern Spain, serving as a vehicle for the geographic distribution of these ticks.

The Goat as a Risk Factor for Parasitic Infections in Ovine Flocks.

Small ruminants in northwestern Spain are frequently managed in mixed flocks. Sheep-goat joint management has not been considered a risk factor for parasite infection, so the main objective of this study was to establish if mixed management with goats supposes a risk factor for parasitic infections in ovine flocks. Two thousand and ninety-three sheep were sampled from 74 commercial meat ovine flocks for diagnostic of the main parasites. Goat contact was a risk factor for sheep to be infected by protostrongylids, Dictyocaulus filaria, gastrointestinal nematodes and Eimeria spp. In contrast, Moniezia, Fasciola hepatica, Dicrocoelium dendriticum and Paramphistomidae prevalences were not influenced. Sheep-goat mixed management can be considered as a risk factor, since goats would act as a source of pasture contamination for interspecific parasites (protostrongylids, Dictyocaulus filaria and gastrointestinal nematodes). In relation to host-specific parasites, such as Eimeria spp., goats cannot be considered as a source for sheep, but competition for food and spaces between both small ungulates can suppose a reduced grazing area to sheep, provoking high environmental contamination and stress that facilitate their infection. Future epidemiological studies for parasitic infections in small ruminants should consider sheep-goat mixed management as a possible risk factor to be included in multivariate analyses.

An Update on Cephenemyiosis in the European Roe Deer: Emergent Myiasis in Spain.

Cephenemyia stimulator is a Palearctic species developing in the nasal cavity and pharynx of roe deer (Capreolus capreolus). It is widely spread in the range of distribution of this ungulate in Europe. Since the first report of C. stimulator in Spain in 2001, a rapid geographic expansion has been observed, first in the north of the country, with high prevalence and intensities of infestation that caused some mortal cases, and, lately, also in Extremadura and Andalucía, the southernmost populations of European roe deer. These observations suggest an adaptation of this parasite to different ecosystems of the Iberian Peninsula. Almost simultaneously, C. stimulator is also expanding its range to northern Europe, with the first cases being reported in Sweden. Thus, Cephenemyia stimulator may be an example of a parasite currently displaying distributional changes along its southernmost and northernmost range margins. Thus, it is of the utmost importance to unravel all the epidemiological and clinical aspects of this myiasis, as well as implementing surveillance measures including reliable and non-invasive diagnostic techniques to monitor its expansion and adaptation to different ecosystems and/or hosts and to reduce the negative impact on roe deer populations.

The Age-Related Cryptosporidium Species Distribution in Asymptomatic Cattle from North-Western SPAIN.

An age-related distribution of Cryptosporidium species has been reported in cattle, with C. parvum being predominant in suckling calves, C. bovis and C. ryanae being predominant in post-weaned calves and C. andersoni being predominant in adults. However, variants to this pattern have recently been reported. Thus, fecal samples (n = 594) from asymptomatic cattle were collected in north-western Spain. Animals were classified as <1 month (G1), 1-2 months (G2), 2-12 months (G3), 12-24 months (G4) and >2 years (G5). Cryptosporidium detection and species identification were performed by SSU rRNA PCR. Individual Cryptosporidium prevalence was 16.7%; it significantly decreased with age. Cryptosporidium parvum was predominant in G1 and C. bovis was predominant in the rest of the age classes; C. bovis and C. ryanae were especially prevalent in G2 and G3. Cryptosporidium occultus was not found in suckling calves. Finally, C. andersoni and C. xiaoi were occasionally detected in G5. The presence of C. parvum in all age classes implies significant animal and public health concerns. The predominance of C. bovis in cattle older than 1 month supports the idea that the age-related pattern of Cryptosporidium species described in cattle is not fully consistent, and thus further studies are still needed to identify those factors determining the species distribution.

Molecular detection of myxoma virus in the environment of vaccinated rabbitries.

Myxoma virus (MYXV) is the aetiological agent of myxomatosis, a systemic, mostly lethal disease that affects European rabbits. Vaccination against it, although widespread, has not been completely effective and disease outbreaks still take place on farms which carry out vaccination programmes. Since some of these cases have been attributed to airborne transmission or the spread of the virus via inanimate vectors, the aims of this study were to determine MYXV contamination levels and distribution in the environment of vaccinated farms and to ascertain whether the detected virus corresponded to field strains. For that, environmental samples from several areas, tools and employees from four (three infected and one uninfected) rabbitries were taken and analysed by qPCR. MYXV was detected in the environment of all the infected farms, whereas all the samples from the non-infected farm were negative. Furthermore, all the positive samples contained viral DNA compatible with field strains of the virus. These results lead us to believe that the administration of currently available commercial vaccines does not prevent infected animals from shedding the field virus. Moreover, viral DNA was also found in items that are not in direct contact with the animals, which could play a role in the transmission of the infection throughout the farm and to other farms. Therefore, this study proves that current vaccination schemes on their own are not sufficient to prevent this disease and should be accompanied by adequate biosecurity measures.

Genotype and subtype analysis of Cryptosporidium isolates from calves and lambs in Galicia (NW Spain).

Faecal specimens from diarrhoeic pre-weaned calves (n=61) and lambs (n=127) collected over a 1-year period (2008-2009) at 27 cattle and 28 sheep farms in Galicia (NW Spain) were examined for the presence of Cryptosporidium oocysts and positive specimens were selected for molecular examination. Overall, 30 calves (49.2%) and 39 lambs (30.7%) tested positive for Cryptosporidium by microscopy. PCR products of the SSU rRNA locus were obtained for 27 Cryptosporidium positive calf isolates and 23 lamb isolates. Restriction analyses generated profiles of C. parvum in all isolates except for 9 lamb specimens from 5 farms that yielded banding patterns and sequences indicative of the Cryptosporidium cervine genotype. Sequence analyses of the glycoprotein (GP60) gene revealed that all but 1 C. parvum isolate from calves belonged to the subtype IIaA15G2R1 and 1 isolate was identified as the novel subtype IIaA13G1R1. Two different subtypes were identified in sheep flocks including IIaA16G3R1, which was seen in 7 lamb isolates from a single farm and subtype IIaA15G2R1, identified in 3 isolates from 2 farms. These findings suggest a limited genetic diversity within C. parvum in ruminant livestock from this geographical area, although both calves and lambs should be considered as a reservoir for zoonotic subtypes.

Prevalence and molecular characterization of Anaplasma phagocytophilum in roe deer (Capreolus capreolus) from Spain.

Anaplasma phagocytophilum can infect a wide range of vertebrates; nevertheless, some genetic variants are associated with particular species of tick vectors and animal hosts. It has been suggested that roe deer (Capreolus capreolus) mainly acts as a reservoir of several A. phagocytophilum non-pathogenic variants for other animal species. The aim of this study was to determine the prevalence and identify the genetic variants of A. phagocytophilum in roe deer from Spain in order to assess host-pathogen associations and their pathogenic potential. The spleens of 212 roe deer hunted in Spain were individually collected and analysed by a commercial qPCR kit in order to detect the presence of A. phagocytophilum DNA. Positive samples were further characterized at groESL, 16S rRNA and msp2 partial genes. The possible influence of several intrinsic (age and sex) and extrinsic factors (ecological area) on A. phagocytophilum prevalence was analysed using a logistic regression. Overall, 41.5 % of the samples resulted positive to A. phagocytophilum. The percentage of infected roe deer was significantly higher in the Mediterranean and Oceanic areas than in the Continental and Mountain regions; nevertheless, prevalence was not related to age or sex. Sequence analysis at groESL and 16S rRNA genes allowed the identification of three ecotypes (I to III) and four variants ("Y", "X", "W", "I"), respectively. A high percentage of roe deer from Spain is infected with different variants of A. phagocytophilum; these results have implications for public and animal health since some of these ecotypes and variants have been previously identified in both human and animal clinical cases.