A. Arenas

Bluetongue epidemiology in wild ruminants from Southern Spain

Serum samples from 210 wild ruminants collected between 2006 and 2007 in southern Spain were tested for antibodies against bluetongue virus (BTV) by means of a competitive ELISA assay. Eighty-seven of the 210 wild ruminants analysed (41%) showed antibodies against BTV. Statistically significant differences were found in the seroprevalence among species: 66% (65 of 98) for red deer (Cervus elaphus), 50% (ten of 20) for fallow deer (Dama dama), 33% (three of nine) for mouflon (Ovis aries musimon) and 11% (nine of 83) for Spanish ibex (Capra pyrenaica). Overall, the sites where seropositive wild ruminants were found coincide with the areas where BTV had been detected in livestock, but in eastern Sierra Morena, the virus circulated in wild ruminants, although it had not been detected in domestic ruminants in the same areas. Wild ruminants over 1-year of age (sub-adults and adults) had significantly higher seroprevalences than juvenile animals. Statistically significant differences were also observed between BTV seroprevalence and management (free-ranging vs. captivity) with higher prevalence in free-ranging animals. The high seroprevalences obtained suggest that BTV is widespread in wild ruminants in southern Spain. This factor could have an important influence on the evolution of the infection in domestic livestock and indicates the need to include wild ruminant species in BTV surveillance or control programs.

West Nile fever outbreak in horses and humans, Spain, 2010.

TO THE EDITOR: West Nile virus (WNV) is a member of the genus Flavivirus within the Japanese encephalitis antigenic complex. The enzootic virus cycle involves transmission between avian hosts and ornithophilic mosquitoes, whereas humans and horses are considered dead-end hosts. Given the recent increase of WNV infection in humans and horses in Europe, concern has been raised regarding public and animal health.

Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids.

A cross-sectional study was carried out on equids (horses, mules and donkeys) in Andalusia, Southern Spain, to assess the level of exposure to equine piroplasmosis and to investigate risk factors associated with these infections. At least one animal seropositive for Theileria equi and/or Babesia caballi was detected in 222/380 (58.4%) herds sampled by competitive inhibition ELISAs. The seroprevalences for B. caballi and T. equi were 13.2% and 56.1%, respectively; there was serological evidence of co-circulation of both piroplasms in 10.8% of herds. Antibodies against equine piroplasms were detected in 286/537 (53.3%) animals; 61 (11.4%) were seropositive for B. caballi, 270 (50.3%) were seropositive for T. equi and 24 (8.4%) were seropositive for both T. equi and B. caballi. There was a significantly higher seroprevalence of B. caballi in mules (32.1%) compared with donkeys (17.0%) and horses (7.9%), and a significantly higher seroprevalence of T. equi in mules (66.1%) in comparison with horses (48.6%), but not donkeys (47.2%). There were significant differences in prevalence of both piroplasms among locations; the seroprevalence of B. caballi ranged from 0 to 22.5%, while the seropositivity to T. equi ranged from 26.7 to 63.3%. A multiple logistic regression model indicated that the risk factors associated with a higher T. equi seroprevalence were increased age, presence of ticks and vaccination against other diseases. Risk factors associated with a higher seroprevalence of B. caballi were species (mules compared to horses), entry of horses in the last 6months, presence of ticks and presence of shelter. The findings indicate widespread exposure to equine piroplasmosis in Southern Spain.

Role of wild ruminants in the epidemiology of bluetongue virus serotypes 1, 4 and 8 in Spain

Although the importance of wild ruminants as potential reservoirs of bluetongue virus (BTV) has been suggested, the role played by these species in the epidemiology of BT in Europe is still unclear. We carried out a serologic and virologic survey to assess the role of wild ruminants in the transmission and maintenance of BTV in Andalusia (southern Spain) between 2006 and 2010.A total of 473 out of 1339 (35.3%) wild ruminants analyzed showed antibodies against BTV by both ELISA and serum neutralization test (SNT). The presence of neutralizing antibodies to BTV-1 and BTV-4 were detected in the four species analyzed (red deer, roe deer, fallow deer and mouflon), while seropositivity against BTV-8 was found in red deer, fallow deer and mouflon but not in roe deer. Statistically significant differences were found among species, ages and sampling regions. BTV RNA was detected in twenty-one out of 1013 wild ruminants (2.1%) tested. BTV-1 and BTV-4 RNA were confirmed in red deer and mouflon by specific rRT-PCR.BTV-1 and BTV-4 seropositive and RNA positive wild ruminants, including juveniles and sub-adults, were detected years after the last outbreak was reported in livestock. In addition, between the 2008/2009 and the 2010/2011 hunting seasons, the seroprevalence against BTV-1, BTV-4 and BTV-8 increased in the majority of provinces, and these serotypes were detected in many areas where BTV outbreaks were not reported in domestic ruminants. The results indicate that wild ruminants seem to be implicated in the dissemination and persistence of BTV in Spain.

Seroprevalence and risk factors associated to Mycobacterium bovis in wild artiodactyl species from southern Spain, 2006-2010.

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries. A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1–8.9) animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for animal and public health.

Monitoring of the West Nile Virus epidemic in Spain between 2010 and 2011

West Nile virus (WNV) is a mosquito-transmitted flavivirus recognized as an emerging and re-emerging pathogen in different countries. This study describes the monitoring of the first WNV epidemic in Spain between 2010 and 2011. Between September and December 2010, 36 outbreaks of WNV in horses were reported in three different provinces of Andalusia (southern Spain), with no apparent spread outside this area. The temporal distribution and the clinical signs observed during the WNV epidemic in Spain were, in general, similar to those reported in Europe and in the Mediterranean Basin. Morbidity, mortality and fatality rate in the affected herds were 4.6, 1.4 and 35.3%, respectively. Thirty-six of 75 (47.4%) suspected herds investigated presented at least one IgM seropositive animal. The individual seroprevalence in unvaccinated animals from the infected holdings was 51.7%. RNA WNV lineage 1 virus was confirmed from blood and cerebrospinal fluid samples in a lethally infected horse. The entomological survey showed that the most abundant mosquito species detected in the affected area was Culex pipiens. A cross-sectional study was carried out in non-suspected herds between April 2010 and February 2011 in the affected area. The individual seroprevalence was 11.0%, and six of the 38 herds sampled (15.8%) presented at least one seropositive animal. The results showed active WNV circulation several months before the first outbreak was reported in horses. The seropositivity found in municipalities where clinical cases were not reported indicates a higher geographical dissemination of the virus. Significantly higher seroprevalences were detected in areas close to Morocco. Furthermore, 90 wild ruminants were tested for the presence of antibodies against WNV, but the results were all negative.

Factors affecting seroprevalence of Toxoplasma gondii in the endangered Iberian lynx (Lynx pardinus)

Wild felids are considered important in maintaining the sylvatic cycle of Toxoplasma gondii. Although, T. gondii antibodies have been reported in several species of wild felids, little is known of the epidemiology and risk factors associated with T. gondii infection in wild cats. The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. In the present study, seroprevalence and associated risk factors for T. gondii infection in a large population of Iberian lynx in Spain were determined. Serum samples from 129 Iberian lynx collected from 2005 to 2009 and 85 wild rabbits (Oryctolagus cuniculus), sharing the habitat with the Iberian lynx, were tested for antibodies to T. gondii by the modified agglutination test (MAT) using a cut-off value of 1:25. Antibodies to T. gondii were found in 81 of 129 (62.8%) Iberian lynx. Seroprevalence to T. gondii in Iberian lynx significantly increased with age (P<0.001). T. gondii seroprevalences were similar in free-ranging (66.7% of 93) and wild-caught captive lynx (69% of 84) but significantly lower in captive-born lynx (22.5% of 40). Seroprevalence was higher in lynx with concurrent Cytauxzoonfelis (88% of 25) but not with concurrent Feline Leukemia Virus (FeLV) infection (53.8% of 13). There were no significant differences in seroprevalence between sexes, geographic region and year of sample collection (2005-2009). Oocysts of T. gondii were not detected microscopically in fecal samples from 58 lynx. Wild rabbits are considered the most important food for the lynx. Antibodies to T. gondii were found in 14 (11.9%) of 85 rabbits tested. The present results indicate that T. gondii infection is widespread in the two areas where Iberian lynx survive in Spain. The fact that four captive-born lynx seroconverted was indication of contact with T. gondii also in the Captive Breeding Centers, hence, control measures to prevent T. gondii infection would be necessary in these centers.

An evaluation of the application of infrared thermal imaging to the tele-diagnosis of sarcoptic mange in the Spanish ibex (Capra pyrenaica).

The application of infrared thermal imaging to the diagnosis of sarcoptic mange in the wild Spanish ibex (Capra pyrenaica) was evaluated. Seventy-three ibexes (35 males, 38 females) of varying ages were studied. Each animal was observed using conventional binoculars (OT) to detect lesions characteristic of mange. Infrared thermography (IR) was then performed and the resultant image judged negative or positive. The distance from the thermograph to the animal was measured, and the animal killed. Skin samples were taken for mite detection by routine laboratory diagnosis (LAB). The most sensitive and specific technique for the tele-diagnosis of sarcoptic mange in the Spanish ibex is OT, as it permits diagnosis over greater distances than IR, which sensitivity is impaired at distances >100m. When disease prevalence is low, such as in initial and final phases of an epidemic, a more sensitive technique would be valuable in detecting all affected animals.

Epidemiological surveillance of bluetongue virus serotypes 1, 4 and 8 in Spanish ibex (Capra pyrenaica hispanica) in southern Spain.

A cross-sectional study was carried out to assess the prevalence and circulation of bluetongue virus (BTV) in Spanish ibexes (Capra pyrenaica hispanica). A total of 770 sera samples, 380 blood samples and 34 spleen samples were collected between 2006 and 2009 in Andalusia (southern Spain), a region and time period with a wide circulation of BTV in livestock. Thirty-one out of 770 (4.0%; CI(95%): 2.6-5.4) sera samples analyzed by ELISA showed antibodies against BTV. Twenty-four out of 31 seropositive samples were tested against BTV serotypes 1, 4 and 8 by serum neutralization test (SNT). Neutralizing antibodies against BTV-1 and BTV-4 were detected in seven and ten animals, respectively, four of them showed neutralizing antibodies to both serotypes. The animals seropositive to BTV-4 were sampled between 2006 and 2008, while BTV-1 circulation was confirmed in ibexes sampled between 2007 and 2009. None of the ibexes presented neutralizing antibodies against BTV-8. Statistically significant differences were found among regions and years, which is in coincidence with what occurred in domestic ruminants. There were no statistically significant differences between sexes, age classes and habitats (captivity vs. free-living). BTV RNA was not found in any of the 380 blood samples analyzed. However, BTV-1 RNA was detected from spleen in one Spanish ibex from Málaga province in August 2008. This finding evidences the presence of BTV-1 in Spanish ibex in a municipality where BT outbreaks were not detected in domestic ruminants during that period. Results of the present study show that Spanish ibexes were exposed and responded serologically to both BTV-1 and BTV-4. The low seroprevalence obtained suggests that Spanish ibex is not a relevant species in the dissemination of BT. However, the detection of BTV-1 RNA and the presence of seropositive ibexes in areas where BT outbreaks were not detected in livestock, could not exclude a significant role in the epidemiology of BTV in certain areas.

Avian influenza in wild waterfowl and shorebirds in the Donana National Park: Serological survey using the enzyme-linked immunosorbent assay.

The indirect ELISA was used to detect antibodies to influenzavirus A in the sera of wildfowl from the Donana National Park. Of the 712 birds examined, 44 (6.2%) were seropositive. Positive birds belonged to 10 of the 13 species studied. Infection rates varied widely: spoonbill (Platalea leucorodia, 32.2%), mallard (Anas platyrhynchos, 9.9%), gadwall (Anas strepera, 8.6%), red-crested pochard (Netta rufina, 8.1%), pochard (Aythya ferina, 6.4%), shoveler (Anas clypeata, 5%), great crested grebe (Podiceps cristatus, 4.3%), avocet (Recurvirostra avosetta, 3.1%), grey heron (Ardea cinerea, 3.1%) and coot (Fulica atra, 0.8%). Although infection rates were not high, the wide range of avian species susceptible to influenzavirus A suggests circulation of the virus amongst wildfowl at Donana.