Jordi Casal

Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from post-weaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark.

Longitudinal case-control studies were performed in post-weaning multisystemic wasting syndrome (PMWS) affected farms from Denmark and Spain using similar designs. Fourteen independent batches of 100-154 pigs per batch were monitored from birth to PMWS outbreak occurrence. Pigs displaying PMWS-like signs and matched healthy cohorts were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. Porcine circovirus type 2 (PCV2) quantitative PCR (qPCR) and serology techniques were applied to analyse longitudinally collected sera and/or nasal and rectal swabs. Results showed that PCV2 load increased in parallel to waning maternal antibody levels, reaching the maximum viral load concurrent with development of clinical signs. PMWS affected pigs had higher PCV2 prevalence and/or viral load than healthy pigs in all collected samples at necropsy (p<0.0001-0.05) and even in sera and nasal swabs at the sampling prior to PMWS outbreak (p<0.01-0.05). Danish farms had a higher PCV2 prevalence in young piglets as well as an earlier PMWS presentation compared to Spanish farms. PMWS diagnoses were confirmed by laboratory tests in only half of pigs clinically suspected to suffer from PMWS. Positive and significant correlations were found among PCV2 viral loads present in sera, nasal swabs, rectal swabs and lymphoid tissues (R=0.289-0.827, p<0.0001-0.01), which indicates that nasal and rectal swabs were suitable indicators of PCV2 excretion. Sensitivity and/or specificity values observed from both tests used separately or combined suggested that qPCR and/or serology tests are not apparently able to substitute histopathology plus detection of PCV2 in tissues for the individual PMWS diagnosis within PMWS affected farms. However, qPCR appears to be a potential reliable technique to diagnose PMWS on a population basis.

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005

Abstract ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991–1995, 18 strains) and a second set (n =46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991–1995 to 89.5% in 2000–2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000–2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991–1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000–2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.

Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swine herds

Hepatitis E is a zoonotic disease and is highly prevalent in European swine livestock. There is a need to compare the infection dynamics of hepatitis E virus (HEV) between herds with the same production system and determine the percentage of animals that could arrive infected at slaughter age. Therefore, a longitudinal study was performed in six Spanish farrow-to-finish affected farms. Twenty piglets per farm were monitored from nursery to slaughter. RT-PCR and serology techniques were applied to analyze longitudinally collected sera and/or faecal samples. Liver and bile samples were also taken at the abattoir. Anti-HEV IgM were firstly detected at 7 weeks of age in 5 farms whereas at 13 weeks of age in 1 farm (farm 2). At slaughter age 50-100% of pigs had seroconverted to anti-HEV IgG in the former 5 farms whereas in the other herd only 5% of pigs were IgG seropositive (farm 2). Six out of 96 livers and 5 out of 80 biles analyzed were HEV positive at the abattoir (total percentage of infected animals: 11.5%). All these positive animals had already seroconverted except 2 pigs of farm 2. Hence, pigs can be seronegative at slaughter age being infected during the latest fattening period. Manipulation of HEV-infected livers or other organs from pigs could be considered a possible route of transmission in Spanish abattoirs. This study represents the first longitudinal survey on swine HEV infection dynamics conducted in different herds.

Possible introduction of bluetongue into the Balearic Islands, Spain, in 2000, via air streams

BLUETONGUE is included in the Office International des Epizooties List A of diseases. It is caused by an orbivirus that affects wild and domestic ruminants and usually only causes clinical disease in sheep flocks and some species of deer. Its main form of transmission is through blood-sucking Diptera (midges) of the genus Culicoides, belonging to the Ceratopogonidae family. Bluetongue reappeared in the western Mediterranean in 1998, 40 years after it had been eradicated. In 1998 and 1999, the virus was detected in eastern Greece (mainly serotypes 4 and 9), and an epidemic caused by serotype 4 occurred in 1999 in Bulgaria and Turkey. In December 1999, serotype 2 was detected in Tunisia and, the following summer, disease caused by the same serotype spread to north-east Algeria, Sardinia, Sicily, the Italian region of Calabria, Corsica (France) and the Balearic Islands (Spain). The spread of the disease in the Mediterranean basin has been reviewed by Mellor and Wittmann (2002). In the Balearic Islands, between October and November 2000, a total of 505 outbreaks of infection were declared in sheep flocks, with 391 outbreaks on Mallorca and 114 on Minorca; 4106 animals were affected clinically. After an outbreak had been detected, control measures were implemented. These included banning the local movement of susceptible animals, banning shipments to the Spanish mainland and other countries, the emergency vaccination of all small ruminants older than 30 days with attenuated vaccines, establishing protection and surveillance zones, and vector control measures. The epidemic was stopped and no more outbreaks appeared during 2001 and 2002. Miranda and others (2003) reported the presence of Culicoides imicola, the main vector of bluetongue, on both Mallorca and Minorca. The introduction of bluetongue to an island can occur either by incoming shipments of infected animals or animal products such as semen, or by aerial dispersion of competent vectors from infected areas. Since import records did not show any evidence of ruminants or ruminant semen being shipped from infected areas to the Balearic Islands in the three months before the onset of the outbreak, this did not seem to be a likely source. Previous epidemiological studies have shown that the wind is capable of transporting the 1 to 3 mm long Culicoides midges great distances as a kind of ‘aerial plankton’. According to Wittmann and Bayliss (2000), Culicoides species can be transported as aerial plankton when the wind speed ranges from 3 to 11 m/second. Air currents were considered to be one of the most probable sources for the introduction of bluetongue into Israel (Braverman and Chechik 1993), Queensland, Australia (Ward 2000) and Cyprus (Polydorou 1980). The aim of the present study was to analyse the possibility that bluetongue was introduced into the Balearic Islands via the aerial transport of Culicoides species from the infected areas of Sardinia, Corsica, Tunisia or Algeria. The climatic factors that could have affected the transport of Culicoides species during the period from July to September 2000, were studied, taking into account the chronology of the appearance of the different foci of infection in the Mediterranean, the incubation period and the length of the viraemia in ruminants, and the eclipse phases of the bluetongue virus in the vectors. Six parameters were considered from weather stations at Mao, Portocolom and Pollenca on an hourly basis: relative humidity, mean temperature, mean wind speed and direction, and maximum wind speed and direction. These local meteorological parameters were obtained from automatic weather stations of the Servicio Nacional de Meteorologia. The data were linked with daily surface maps showing weather fronts and aviation model maps (AVN) showing the direction of air streams in the western Mediterranean. The AVN maps were designed by the US National Center for Environmental Prediction. All the maps used were obtained from the Servei d’Informacio Meteorologica ‘InfoMet’ of Barcelona University. Starting with these data, the days which fulfilled the following conditions were selected: the daily surface maps and the AVN maps showing air streams which came from the areas where serotype 2 had previously been detected (Sardinia, Corsica, Tunisia and Algeria), and air streams which were measured in the three weather stations and maintained a minimum speed of 3 m/second for a period of more than two hours. During the period studied, only four days, August 10, 17, 18 and 30, fulfilled these conditions. Backward isentropic trajectories were then carried out for those days for 24 and 36 hours through the Instituto Nacional de Metereologia. This method is based on the simulation and analysis of meteorological data, and has previously shown good agreement with observational data (Reiff and others 1986). The trajectories were designed following the methodology of the integrated Veterinary Record (2004) 155, 460-461

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.

Analysis of the spatial variation of Bovine tuberculosis disease risk in Spain (2006-2009).

In this study we explored the spatial variation of Bovine tuberculosis (BTB) risk of being positive, new positive or persistently positive, as well as the risk of eliminating BTB in positive herds throughout Spain from 2006 to 2009 by means of hierarchical Bayesian models. The results of the models showed that the risk of infection (positive or new positive herds), persistence and elimination was lower in counties located in north and north-eastern of Spain, and in the Balearic and Canary islands than in the rest of the country. In some counties the risk of positivity was high during the four years of study, whereas there were others where the risk of positivity was high only in some of the years. With regard to the risk of persistence of BTB positive herds, counties located in the central, western and south-western part of the country had a higher risk in the three studied periods. This study has identified some specific areas of increased BTB risk in Spain, information that is useful for disease management.

Seroprevalence and risk factors associated with Toxoplasma gondii in domestic pigs from Spain.

Serum samples from 2970 (1400 sows, 1570 fattening) pigs, from 100 farms in the 10 main swine production regions in Spain were tested for antibodies against T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT 1:25 or higher) were detected in 492 pigs (16.6%, 9.7% in fattening pigs and 24.2% in sows). The herd prevalence was 85.0% (95% CI: 78-92) and within-farm prevalence ranged from 2.9% to 92.8% (median=17.6%). Statistically significant differences were observed among sampling regions with seroprevalence significantly higher in pigs from Valencia Community (27.3%), Extremadura (23.3%) and Catalonia (21.2%). A generalized estimating equations model indicated that the risk factors associated with T. gondii seroprevalence were: age, sows compared to fattening pigs (OR=2.9; 95% CI=1.83-4.53), lack of rodent control (OR=1.9; 95% CI=1.04-3.60) and presence of cats (OR=1.6; 95% CI=1.12-2.34). The seroprevalence observed in the present study indicates a widespread, although variable, exposure to T. gondii among domestic pigs in Spain, which might have important implications for public health. Management measures including control of rodents and cats on the farms could help to reduce the observed prevalence levels in Spain.

Seroprevalence and risk factors of swine influenza in Spain.

Swine influenza is caused by type A influenza virus. Pigs can be infected by both avian and human influenza viruses; therefore, the influenza virus infection in pigs is considered an important public health concern. The aims of present study were to asses the seroprevalence of swine influenza subtypes in Spain and explore the risk factors associated with the spread of those infections. Serum samples from 2151 pigs of 98 randomly selected farms were analyzed by an indirect ELISA for detection of antibodies against nucleoprotein A of influenza viruses and by the hemagglutination inhibition (HI) using H1N1, H1N2 and H3N2 swine influenza viruses (SIV) as antigens. Data gathered in questionnaires filled for each farm were used to explore risk factors associated with swine influenza. For that purpose, data were analyzed using the generalized estimating equations method and, in parallel by means of a logistic regression. By ELISA, 92 farms (93.9%; CI(95%): 89.1-98.7%) had at least one positive animal and, in total, 1340/2151 animals (62.3%; CI(95%): 60.2-64.3%) were seropositive. A total of 1622 animals (75.4%; CI(95%): 73.6-77.2%) were positive in at least one of the HI tests. Of the 98 farms, 91 (92.9%; CI(95%): 87.7-98.1%) had H1N1 seropositive animals; 63 (64.3%; CI(95%): 54.6-73.9%) had H1N2 seropositive pigs and 91 (92.9%; CI(95%): 87.7-98.1%) were positive to H3N2. Mixed infections were detected in 88 farms (89.8; CI(95%): 83.7-95.9%). Three risk factors were associated with seroprevalences of SIV: increased replacement rates in pregnancy units and, for fatteners, existence of open partitions between pens and uncontrolled entrance to the farm.

Causes of Morbidity in Wild Raptor Populations Admitted at a Wildlife Rehabilitation Centre in Spain from 1995-2007: A Long Term Retrospective Study

Background Morbidity studies complement the understanding of hazards to raptors by identifying natural or anthropogenic factors. Descriptive epidemiological studies of wildlife have become an important source of information about hazards to wildlife populations. On the other hand, data referenced to the overall wild population could provide a more accurate assessment of the potential impact of the morbidity/mortality causes in populations of wild birds. Methodology/Principal Findings The present study described the morbidity causes of hospitalized wild raptors and their incidence in the wild populations, through a long term retrospective study conducted at a wildlife rehabilitation centre of Catalonia (1995–2007). Importantly, Seasonal Cumulative Incidences (SCI) were calculated considering estimations of the wild population in the region and trend analyses were applied among the different years. A total of 7021 birds were analysed: 7 species of Strigiformes (n = 3521) and 23 of Falconiformes (n = 3500). The main causes of morbidity were trauma (49.5%), mostly in the Falconiformes, and orphaned/young birds (32.2%) mainly in the Strigiformes. During wintering periods, the largest morbidity incidence was observed in Accipiter gentillis due to gunshot wounds and in Tyto alba due to vehicle trauma. Within the breeding season, Falco tinnunculus (orphaned/young category) and Bubo bubo (electrocution and metabolic disorders) represented the most affected species. Cases due to orphaned/young, infectious/parasitic diseases, electrocution and unknown trauma tended to increase among years. By contrast, cases by undetermined cause, vehicle trauma and captivity decreased throughout the study period. Interestingly, gunshot injuries remained constant during the study period. Conclusions/Significance Frequencies of morbidity causes calculated as the proportion of each cause referred to the total number of admitted cases, allowed a qualitative assessment of hazards for the studied populations. However, cumulative incidences based on estimated wild raptor population provided a more accurate approach to the potential ecological impact of the morbidity causes in the wild populations.

Simulated airborne spread of Aujeszky's disease and foot-and-mouth disease

The atmospheric dispersion of virus was simulated using a computer model which had been developed for predicting the dispersion of toxic gases from chemical engineering plants. The results were compared with data from four outbreaks in which virus was believed to have been transported by air: two outbreaks of foot-and-mouth disease in the United Kingdom in 1967 and outbreaks of Aujeszkys disease in Yorkshire in 1981 to 1982 and Indiana in 1988. There was relatively good agreement with most of these data. The paper shows that the model could be useful in an emergency because the risk of virus spread could be predicted in real time.