Cristina Lorca-Oró

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.

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.

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.

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.

Long-Term Dynamics of Bluetongue Virus in Wild Ruminants: Relationship with Outbreaks in Livestock in Spain, 2006-2011

Wild and domestic ruminants are susceptible to Bluetongue virus (BTV) infection. Three BTV serotypes (BTV-4, BTV-1 and BTV-8) have been detected in Spain in the last decade. Even though control strategies have been applied to livestock, BTV circulation has been frequently detected in wild ruminant populations in Spain. The aim of the present study is to assess the role for wild ruminants in maintaining BTV after the vaccination programs in livestock in mainland Spain. A total of 931 out 1,914 (48.6%) serum samples, collected from eight different wild ruminant species between 2006 and 2011, were BTV positive by ELISA. In order to detect specific antibodies against BTV-1, BTV-4 and BTV-8, positive sera were also tested by serumneutralisation test (SNT). From the ELISA positive samples that could be tested by SNT (687 out of 931), 292 (42.5%) showed neutralising antibodies against one or two BTV serotypes. For each BTV seroptype, the number of outbreaks in livestock (11,857 outbreaks in total) was modelled with pure autoregressive models and the resulting smoothed values, representing the predicted number of BTV outbreaks in livestock at municipality level, were positively correlated with BTV persistence in wild species. The strength of this relationship significantly decreased as red deer (Cervus elaphus) population abundance increased. In addition, BTV RNA was detected by real time RT-PCR in 32 out of 311 (10.3%) spleen samples from seropositive animals. Although BT outbreaks in livestock have decreased substantially after vaccination campaigns, our results indicated that wild ruminants have been exposed to BTV in territories where outbreaks in domestic animals occurred. The detection of BTV RNA and spatial association between BT outbreaks in livestock and BTV rates in red deer are consistent with the hypothesis of virus circulation and BTV maintenance within Iberian wild ruminant populations.

Immunization with DNA vaccines containing porcine reproductive and respiratory syndrome virus open reading frames 5, 6, and 7 may be related to the exacerbation of clinical disease after an experimental challenge.

Pigs were immunized with DNA plasmids containing different open reading frames (ORFs) of a porcine reproductive and respiratory syndrome virus (PRRSV) genotype I strain. One group was injected with three inoculations of ORF7, a second group was immunized with three inoculations of plasmids containing ORF5 and ORF6, and a third group was kept as controls. Later, +21 days after the last inoculation, animals were challenged with the homologous strain. After the challenge, PRRSV-specific interferon (IFN)-γ-secreting cells and anti-PRRSV IgG antibodies developed faster in DNA vaccinated pigs (p<0.05). However, DNA-immunized pigs showed an exacerbation of the disease compared to the unvaccinated challenged pigs. The data suggest that previous immunization with DNA vaccines against glycoprotein 5 and/or matrix protein of PRRSV, as well as nucleoprotein but to a lesser degree, could result in an exacerbation of the clinical course in terms of fever upon challenge.

Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection

Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication.

Toxoplasma gondii Infection in Seagull Chicks Is Related to the Consumption of Freshwater Food Resources.

Understanding the spread of Toxoplasma gondii (T. gondii) in wild birds, particularly in those with opportunistic feeding behavior, is of interest for elucidating the epidemiological involvement of these birds in the maintenance and dissemination of the parasite. Overall, from 2009 to 2011, we collected sera from 525 seagull chicks (Yellow-legged gull (Larus michahellis) and Audouin’s gull (L. audouinii)) from 6 breeding colonies in Spain and tested them using the modified agglutination test (MAT) for the presence of antibodies against T. gondii. Chick age was estimated from bill length. Main food source of seagull chicks was evaluated using stable isotope analyses from growing scapular feathers. Overall T. gondii seroprevalence was 21.0% (IC95% 17.5–24.4). A generalized linear mixed-effects model indicated that year (2009) and food source (freshwater) were risk factors associated to the individual risk of infection by T. gondii, while age (days) was close to significance. Freshwater food origin was related to the highest seroprevalence levels, followed by marine origin, supporting freshwater and sewages as important routes of dispersion of T. gondii. Year differences could indicate fluctuating rates of exposure of seagull chicks to T. gondii. Age ranged from 4 to 30 days and seropositivity tended to increase with age (P = 0.07), supporting that seropositivity is related to T. gondii infection rather than to maternal transfer of antibodies, which in gulls is known to sharply decrease with chick age. This study is the first to report T. gondii antibodies in Yellow-legged and Audouin’s gulls, thereby extending the range of intermediate hosts for this parasite and underscoring the complexity of its epidemiology.