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Global emergence and re-emergence of Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus which causes a highly contagious enteric disease, have led to several studies addressing its variability. The aim of this study was to characterize the infection of weaned pigs with Swine enteric coronavirus (SeCoV) -a chimeric virus most likely originated from a recombination event between PEDV and Transmissible gastroenteritis virus, or its mutant Porcine respiratory coronavirus-, and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals; piglets in groups B, C and D were orally inoculated with a PEDV variant (B and D) or SeCoV (C), while piglets in group A were mock inoculated and maintained as controls. At day 20 post-infection all groups were exposed to rPEDV-SeCoV; thus, group D was subjected to a homologous re-challenge, groups B and C to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively) and group A was primary challenged (-/rPEDV-SeCoV). Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposition to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in feces.

Rotaviruses (RVs) are classified into nine species or groups (RVA-RVD and RVF-RVJ). RVA, RVB and RVC are well-recognized as etiological agents of enteric disease on swine farms and have been identified in all countries with a relevant pork production. Contrarily, RVH has only been identified on swine farms from Japan and more recently from Brazil, USA, South Africa and Vietnam but not yet in Europe. The occurrence of RVH was investigated in 103 Spanish pig herds. Nine farms were positive and we achieved a complete nucleotide sequences in three isolates while another RVH isolate was partially sequenced. Mean nucleotide identities with the RVH sequences available in GenBank ranged between 69.4 and 93.7 %. Phylogenetically, all genomic segments of Spanish RVH isolates clustered closely with other porcine RVH strains but were distantly related to human RVH as well as bat RVH strain. To the best of our knowledge this is the first report of RVH on swine farms in Europe including its characterization by means of complete genome sequencing. info:eu-repo/semantics/acceptedVersion

9 páginas., A large-scale study was carried out to determine the prevalence of antibodies againstPestivirusspecies in wild ruminants and describe their spatial variation in mainland Spain. Serum samples of 1,874 wild ruminants from different regions of this country were collected between the years 2000 and 2017. A total of 6.6% (123/1,874) animals showed antibodies againstPestivirusby both blocking ELISA (bELISA) and virus neutralization tests (VNT). The prevalence of antibodies against pestiviruses was different both among species and regions. Seroprevalence by species was 30.0% (75/250) in Southern chamois (Rupicapra pyrenaica), 7.0% (25/357) in fallow deer (Dama dama), 2.5% (10/401) in red deer (Cervus elaphus), 2.4% (8/330) in Iberian wild goat (Capra pyrenaica), 1.1% (4/369) in roe deer (Capreolus capreolus) and 0.8% (1/130) in mouflon (Ovis aries musimon), not detecting seropositivity (0/37) in Barbary sheep (Ammotragus lervia). The results confirm that exposure to pestiviruses was detected throughout mainland Spain, with significantly higher seroprevalence in Northern regions associated with the presence of Southern chamois. This indicates an endemic circulation of pestiviruses in Southern chamois and a limited circulation of these viruses in the remaining wild ruminant species during the last two decades, thus suggesting that non-chamois species are not truePestivirusreservoirs in Spain. Nonetheless, the high spatial spread of these viruses points out that new epidemic outbreaks in naive wild ruminant populations or transmission to livestock may occur, evidencing the usefulness of monitoring pestiviruses in wild ruminants, especially at the wildlife-livestock interface., The present work has benefited from the financial aid of research grants funded by MINECO (AGL2016-76358-R and CGL2017-89866-R). S. Jiménez-Ruiz holds a PhD contract from the UCLM co-supported by the European Social Fund (2018/12504).

Este artículo se encuentra disponible en la siguiente URL: https://onlinelibrary.wiley.com/doi/epdf/10.1111/tbed.13737 En este artículo de investigación también participan: Marta Cerdà Cuéllar, Teresa Ayats, Jaume Jordá, Clara Marín y Santiago Vega. This is the pre-peer reviewed version of the following article: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, which has been published in final form at https://doi.org/10.1111/tbed.13737. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Este es el pre-print del siguiente artículo: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, que se ha publicado de forma definitiva en https://doi.org/10.1111/tbed.13737. Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented. Preprint

A retrospective evaluation of PEDV positive samples recovered in Spain before and after the re‐emergence of this coronavirus in several European countries was carried out. We described for the first time recombinant SeCoV circulating in Spain between 1993 and 2014 and its misidentification as PEDV when diagnostic assays based on the S‐protein or S‐gene of the PEDV were used. The complete S‐gene sequence of 7 Spanish SeCoV and 30 PEDV Spanish isolates was phylogenetically analysed including the S‐gene sequences of the three SeCoV and a representative selection of the PEDV strains with complete genome sequences available in the GenBank. The tree showed a common ancestor for the S‐gene of the PEDV and SeCoV, but no evolution from any known PEDV clade was shown for the SeCoV strains. Moreover, complete genome sequences were obtained from 23 PEDV strains recovered in Spanish swine farms since 2014. The phylogenetic tree showed the INDEL type genogroup of these Spanish strains, supporting the lower pathogenicity of this genogroup since no significant economic losses were reported in the affected Spanish swine farms. Four subgroups were detected among PEDV strains in Spain, closely related to the recent European strains. Moreover, eight of the most recent Spanish PEDV isolates formed a subclade together with three European strains from 2015, showing a new evolution branch with a recombinant virus. info:eu-repo/semantics/acceptedVersion

An epidemiological surveillance programme was carried out to assess exposure and spatiotemporal patterns of selected pathogens (Brucella spp., Mycobacterium avium subsp. paratuberculosis (MAP), Mycoplasma agalactiae, Pestivirus and bluetongue virus (BTV)) in Iberian ibex (Capra pyrenaica) from Andalusia (southern Spain), the region with the largest population of this species. A total of 602 animals in five distribution areas were sampled during 2010–2012 (P1) and 2013–2015 (P2). The Rose Bengal test (RBT) and complement fixation test (CFT) were used in parallel to detect anti‐Brucella spp. antibodies. Commercial ELISAs were used to test for antibodies against the other selected pathogens. Sera positive for BTV and Pestivirus by ELISA were tested by serum neutralization test (SNT) to identify circulating serotypes/genotypes. The overall seroprevalences were as follows: 0.4% for Brucella spp. (2/549; CI 95%: 0.1–1.3) (14/555 positive by RBT; 2/564 by CFT), 0.5% for MAP (3/564; CI 95%: 0.1–1.5), 5.7% for M. agalactiae (30/529; CI 95%: 3.9–8.0), 11.1% for Pestivirus (58/525; CI 95%: 8.5–14.1) and 3.3% for BTV (18/538; CI 95%: 2.0–5.2). Significantly higher seropositivity to both M. agalactiae and BTV was observed in P1 compared with P2. Spatiotemporal clusters of high seroprevalence were also found for M. agalactiae in four of the five sampling areas in 2010, and for BTV in one of five areas in 2012. Specific antibodies against BTV‐4, BDV‐4 and BVDV‐1 were confirmed by SNT. Our results indicate that the Iberian ibex may be considered spillover hosts of Brucella spp. and MAP rather than true reservoirs. The prevalence of antibodies against M. agalactiae and BTV suggests spatiotemporal variation in the circulation of these pathogens, while Pestivirus has a moderately endemic circulation in Iberian ibex populations. Our study highlights the importance of long‐term surveillance for a better understanding of the spatiotemporal distribution of shared infectious diseases and providing valuable information to improve control measures at the wildlife–livestock interface. info:eu-repo/semantics/acceptedVersion

Porcine circovirus 3 (PCV‐3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV‐3 infection dynamics. Finally, to elucidate PCV‐3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV‐3 DNA was extracted and amplified with a conventional PCR. For the PCV‐3 PCR‐positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV‐3 PCR‐positive samples from different years, different time‐points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV‐3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long‐term infections, since 5 out of 10 PCV‐3 PCR‐positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV‐3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV‐3 PCR‐positive samples was moderate to low. All partial and complete PCV‐3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV‐3 infection, showing high frequency of detection in this animal species. Furthermore, PCV‐3 can be found in different tissues of wild boar and is apparently able to cause persistent infection. Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria. Grant Number: E‐RTA2017‐00007‐00‐00

The implementation of biosecurity measures in the animal health and production context is quite broad and aims at limiting the risk of introduction and spread of diseases. Veterinarians play a major role in biosecurity as key informants on the subject for cattle holders, key players in terms of disease prevention/control and eradication programs, as well as key risk factor in terms of disease dissemination. Many biosecurity studies have highlighted professional visitors such as veterinary practitioners as representing a high-risk factor in terms of disease introduction in animal facilities but, to date, very few studies have focused on the implementation level of biosecurity measures by veterinarians. An online survey was implemented in three European countries (Belgium, France and Spain) to assess the behaviour of rural veterinarians towards biosecurity, as well as their implementation level of the biosecurity measures. A descriptive analysis of data and a scoring system were applied to assess the implementation level of measures. The influence of different factors on the implementation level of biosecurity measures was investigated through a negative binomial regression model. The study identified different strengths, weaknesses, possible constraints and solutions in terms of veterinary perspectives. Veterinarians are considered as key informants by the farmers and could therefore play a more active role in terms of guidance and improvement of biosecurity at farm level. Based on the survey outcomes, two factors seemed to influence significantly the implementation level of measures: the country where he/she practices and the veterinarian's perception level of biosecurity. The biosecurity stages with the lowest application level, therefore representing the biggest threats, were bio-exclusion (increasing the risk of disease introduction) and biocontainment (increasing the risk of inter-herd transmission). info:eu-repo/semantics/acceptedVersion

Mycoplasma hyopneumoniae(M. hyopneumoniae) is the primary pathogen of enzooticpneumonia, a chronic respiratory disease in pigs. Infections occur worldwide andcause major economic losses to the pig industry. The present paper reviews the cur-rent knowledge onM. hyopneumoniaeinfections, with emphasis on identificationand analysis of knowledge gaps for optimizing control of the disease. Close contactbetween infected and susceptible pigs is the main route ofM. hyopneumoniaetrans-mission. Management and housing conditions predisposing for infection or diseaseare known, but further research is needed to better understandM. hyopneumoniaetransmission patterns in modern pig production systems, and to assess the impor-tance of the breeding population for downstream disease control. The organism isprimarily found on the mucosal surface of the trachea, bronchi and bronchioles. Dif-ferent adhesins and lipoproteins are involved in the adherence process. However, aclear picture of the virulence and pathogenicity ofM. hyopneumoniaeis still missing.The role of glycerol metabolism, myoinositol metabolism and theMycoplasmaIgbinding protein—MycoplasmaIg protease system should be further investigated fortheir contribution to virulence. The destruction of the mucociliary apparatus,together with modulating the immune response, enhances the susceptibility ofinfected pigs to secondary pathogens. Clinical signs and severity of lesions dependon different factors, such as management, environmental conditions and likely alsoM. hyopneumoniaestrain. The potential impact of strain variability on disease sever-ity is not well defined. Diagnostics could be improved by developing tests that maydetect virulent strains, by improving sampling in live animals and by designing ELI-SAs allowing discrimination between infected and vaccinated pigs. The currentlyavailable vaccines are often cost-efficient, but the ongoing research on developingnew vaccines that confer protective immunity and reduce transmission should becontinued, as well as optimization of protocols to eliminateM. hyopneumoniaefrompig herds. info:eu-repo/semantics/acceptedVersion

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica ) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free‐ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high‐virulence (Cadí‐6) and low‐virulence (Freser‐5) BDV strains was performed. Pregnant and non‐pregnant animals with and without antibodies against BDV were included in each group. Cadí‐6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser‐5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí‐6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí‐6 group. Chamois from both groups presented lesions in brain but the ones infected with the low‐virulence Freser‐5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low‐virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross‐protection in chamois against high‐virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations. info:eu-repo/semantics/acceptedVersion

Circulation of highly pathogenic avian influenza (HPAI) viruses poses a continuousthreat to animal and public health. After the 2005–2006 H5N1 and the 2014–2015H5N8 epidemics, another H5N8 is currently affecting Europe. Up to August 2017,1,112 outbreaks in domestic and 955 in wild birds in 30 European countries havebeen reported, the largest epidemic by a HPAI virus in the continent. Here, the mainepidemiological findings are described. While some similarities with previous HPAIvirus epidemics were observed, for example in the pattern of emergence, significantdifferences were also patent, in particular the size and extent of the epidemic. Eventhough no human infections have been reported to date, the fact that A/H5N8 hasaffected so far 1,112 domestic holdings, increases the risk of exposure of humansand therefore represents a concern. Understanding the epidemiology of HPAIviruses is essential for the planning future surveillance and control activities. info:eu-repo/semantics/publishedVersion

Porcine circovirus 3 (PCV3) is a new species of the Circovirus genus, which has recently been associated with different clinical syndromes. Its presence has been reported in different countries of North and South America, Asia and recently also Europe (Poland). However, different from the other continents, no European PCV3 sequence is currently available in public databases. There is a strong need of epidemiological data and full-genome sequences from Europe because of its relevance in the understanding of PCV3 molecular epidemiology and control. To fill this lack of information, samples collected in Denmark, Italy and Spain in 2016 and 2017 were screened for PCV3. Of the Danish samples, 36 of 38 the lymph nodes, six of 20 serum samples and two of 20 lung samples tested positive. Similarly, 10 of 29 lungs, 20 of 29 organ pools, six of 33 sera and one of eight nasal swabs tested PCV3 positive in Italy. Fourteen of 94 serum pools from seven of 14 Spanish farms were also positive. Despite the convenience nature of the sampling prevents any precise prevalence estimation, the preliminary screening of the data from three European countries confirmed a rather wide PCV3 distribution in Europe. Furthermore, the analysis of the six obtained complete European PCV3 genomes and their comparison with the public available sequences seems to support a remarkable worldwide PCV3 circulation. These results underline once more the urgency of more extensive epidemiological studies to refine the current knowledge on PCV3 evolution, transmission, spreading patterns and impact on pig health. info:eu-repo/semantics/acceptedVersion