Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain.

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n=53), 29% of foxes (n=48), 29% of stone martens (n=21) and in 25-50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.

Molecular investigation of the occurrence of Coxiella burnetii in wildlife and ticks in an endemic area.

At present few studies have been carried out on the distribution and incidence of Coxiella burnetii infection in wildlife. Therefore, the aim of this study was to investigate the distribution of C. burnetii in the main wild species in the Basque Country (Northern Spain), such as carnivores, cervids, wild boar, lagomorphs and several species of birds. Tissues from a total of 601 animals and 340 adult ticks collected from them were analyzed by PCR. DNA of C. burnetii was detected in 5.1% of roe deer (Capreolus capreolus), 4.3% of wild boar (Sus scrofa), 9.1% of European hare (Lepus europaeus), and among wild birds, in 11% of vultures (Gyps fulvus) and 14% of black kites (Milvus migrans). These results showed that C. burnetii circulates in wildlife in Spain participating in the cycle of Q fever in nature. All of the adult ticks analyzed were negative for C. burnetii, suggesting that ticks do not play an important role in the transmission of C. burnetii in this area.

Lack of evidence of paratuberculosis in wild canids from Southwestern Europe.

Wild carnivores are at the top of the trophic chain. They are predators and carrion consumers, and thus, prone to come in contact with disease agents contaminating the environment or infecting live or dead animals. We hypothesized that wild canids could be used as sentinels for the detection of regions with higher Mycobacterium avium paratuberculosis (MAP) prevalence in wild and domestic animals. To test this hypothesis, we set up an ELISA to test 262 wolf (Canis lupus) and fox (Vulpes vulpes) sera for MAP-specific antibodies and processed a subset of samples for culture (n = 61), MAP-specific PCR (15) and histopathology (14). In wolves, the optical density (OD) values in the ELISA were continuously distributed. Ten fox sera (4%) had OD readings of over twice the mean, suggesting contact with mycobacteria. However, all samples tested by PCR were negative for both IS900 and ISMAP02 sequences, and samples cultured for MAP yielded no growth. No visible paratuberculosis or tuberculosis-compatible lesions were recorded. On histopathological examination, no lesions compatible with mycobacterial diseases were observed. These results suggest that wild canids show little or no evidence of paratuberculosis and are unlikely to be useful sentinels for the detection of MAP in Southwestern Europe.

Angiostrongylus species in wild carnivores in the Iberian Peninsula.

A survey of Angiostrongylus parasites was carried out between 2003 and 2006 in wild carnivore species in the Basque Country (Northern Spain). Parasitological examination consisted in the dissection of heart and lungs for the extraction of adult worms. Nematodes were identified using morphometrical features and also PCR amplification and sequencing analysis. The animal species included in this study were Eurasian badger (Meles meles), Weasel (Mustela nivalis), Beech marten (Martes foina), Pine marten (Martes martes), Polecat (Mustela putorius), American mink (Mustela vison), Red fox (Vulpes vulpes), Wolf (Canis lupus), Wild cat (Felis silvestris), and Small-spotted genet (Genetta genetta). Angiostrongylus parasites were only found in foxes and badgers at prevalences of 33.3% and 24%, respectively. Identification of the nematodes by morphometrical features revealed that foxes were infected with A. vasorum while badgers were infected by a different species of Angiostrongylus most likely A. daskalovi. Sequencing data of the second internal transcribed spacer region of ribosomal DNA (ITS2) of isolates from each species confirmed the species difference. The high prevalence of Angiostrongylus found in the present survey, indicates that the wild cycle of two different species of Angiostrongylus is present in the Basque Country. To our knowledge this is the first report of A. daskalovi in the Iberian Peninsula.