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In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.

Myxomatosis is an infectious disease caused by the myxoma virus (MYXV), which has very high mortality rates in European wild rabbits (Oryctolagus cuniculus). While sporadic cases of myxomatosis have also been reported in some hare species, these lagomorphs are considered to have a low susceptibility to MYXV infection. In the present study, we describe the spatiotemporal evolution and main epidemiological findings of novel hare MYXV (ha-MYXV or MYXV-Tol) epidemics in Iberian hares (Lepus granatensis) in Spain. In the period 2018-2020, a total of 487 hares from 372 affected areas were confirmed to be MYXV-infected by PCR. ha-MYXV outbreaks were detected in most of the Spanish regions where the Iberian hare is present. The spatial distribution was not homogeneous, with most outbreaks concentrated in the southern and central parts of Spain. Consecutive outbreaks reported in the last two years suggest endemic circulation in Spain of this emerging virus. A retrospective study carried out just after the first epidemic period (2018-2019) revealed that the virus could have been circulating since June 2018. The number of outbreaks started to rise in July, peaked during the first half of August and October and then decreased sharply until January 2019. The apparent mean mortality rate was 55.4% (median: 70%). The results indicated high susceptibility of the Iberian hare to ha-MYXV infection, but apparent resistance in the sympatric hare species present in Spain and less infectivity in European rabbits. The novel ha-MYXV has had significant consequences on the health status of Iberian hare populations in Spain, which is of animal health and conservation concern. The present study contributes to a better understanding of ha-MYXV emergence and will provide valuable information for the development of control strategies. Further research is warranted to assess the impact of this emerging virus on wild lagomorph populations and to elucidate its ecological implications for Iberian Mediterranean ecosystems.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

West Nile Virus (WNV; family Flaviviridae, genus flavivirus) is a zoonotic arbovirus worldwide spread. Its genetic diversity has allowed the definition of at least seven lineages, being lineages 1 and 2 the most widely distributed. Western Mediterranean region has been affected by WNV since decades. In Spain, WNV is actively circulating, provoking annual outbreaks in birds, horses and lately in humans. Lineage 1 is responsible for outbreaks that occurred in central and southern regions, while lineage 2 has been recently described in wild birds in north-eastern part of the country. During 2017 season, a disease outbreak in captive raptors was reported in southern Spain and WNV was isolated from a dead northern goshawk. Full genome sequencing was followed by phylogenetic analyses and analyses of the amino acidic substitutions. This strain, named Spain/2017/NG-b, highly differs from those which have been circulating both in Spain and in the neighbouring Mediterranean countries, constituting a new distinct group, tentatively classified in a newly defined cluster 7 within the WNV clade 1a, supporting a new, independent introduction of the virus in the Western Mediterranean region from an unknown origin. Besides, circumstantial evidence indicates that this emerging WNV strain could be behind the subsequent outbreak occurred nearby in horses. Overall, the reinforcement of surveillance programs, especially in wild birds, is essential to early detect the circulation of WNV and other related flaviviruses that could cause outbreaks in wild or domestic birds, equine and human populations., This study was supported by the Spanish INIA-MAPA agreement EG17-141 and by the INIA grant E-RTA2015-00002-C02-00.

Background Resistance to colistin was an uncommon phenomenon traditionally linked to chromosome point mutations, but since the first description of a plasmid-mediated colistin-resistance in late 2015, transmissible resistance to colistin has become a Public Health concern. Despite colistin is considered as a human last resort antibiotic, it has been commonly used in swine industry to treat post-weaning diarrhoea in piglets. However, the progressively increase of colistin resistance during the last decade led to the Spanish Medicines and Healthcare Products Agency (AEMPS) to launch a strategic and voluntary plan aimed to reduce colistin consumption in pig production. Our longitudinal study (1998–2021) aimed to evaluate the trend of colistin resistance mediated through the mcr-1 mobile gene in Spanish food-producing pig population and compare it with published polymyxin sales data in veterinary medicine to assess their possible relationships. Results The first mcr-1 positive sample was observed in 2004, as all samples from 1998 and 2002 were mcr-1 PCR-negative. We observed a progressive increase of positive samples from 2004 to 2015, when mcr-1 detection reached its maximum peak (33/50; 66%). From 2017 (27/50; 54%) to 2021 (14/81; 17%) the trend became downward, reaching percentages significantly lower than the 2015 peak (p mcr-1 gene in PCR-positive samples showed a similar trend reaching the highest levels in 2015 (median: 6.6 × 104mcr-1 copies/mg of faeces), but decreased significantly from 2017 to 2019 (median 2.7 × 104, 1.2 × 103, 4.6 × 102mcr-1 copies/mg of faeces for 2017, 2018 and 2019, respectively), and stabilizing in 2021 (1.6 × 102mcr-1 copies/mg of faeces) with similar values than 2019. Conclusions Our study showed the decreasing trend of colistin resistance associated to mcr-1 gene, after a previous increase from among 2004–2015, since the European Medicines Agency and AEMPS strategies were applied in 2016 to reduce colistin use in animals, suggesting a connection between polymyxin use and colistin resistance. Thus, these plans could have been effective in mcr-1 reduction, reaching lower levels than those detected in samples collected 17 years ago, when resistance to colistin was not yet a major concern.

Myxomatosis is an infectious disease caused by myxoma virus (MYXV; genus Leporipoxvirus), which affects the European wild rabbit (Oryctolagus cuniculus) and sporadically brown hares (Lepus europaeus). Here, we describe the first outbreak of myxomatosis in Iberian hares (Lepus granatensis). Between mid-July and the end of September 2018, around 530 dead animals were detected in Iberian hare populations in southern Spain. The apparent mean mortality rate was 56.7%, and the estimated mean case fatality rate was 69.2%. Histopathological and molecular results confirmed MYXV infections in all hares analysed. To the authors' knowledge, this is the first myxomatosis outbreak causing a high mortality in hares and the first detailed characterization of a myxomatosis outbreak in the Iberian hare. The absence of cases in sympatric wild rabbits suggests differences in the susceptibility between both lagomorph species to the virus strain implicated in the outbreak. After the first case, the number of affected areas increased sharply affecting most of the Iberian Peninsula where the Iberian hare is present. Further studies are required to elucidate the origin of the implicated MYXV strain as well as to assess the impact of this outbreak on the Iberian hare populations.

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host–pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis‐like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5‐month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next‐generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population. info:eu-repo/semantics/acceptedVersion

International audience; The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Summary African horse sickness (AHS) is a disease of equids caused by African Horse Sickness Virus (AHSV) and is transmitted by Culicoides midges. AHS is endemic in sub-Saharan Africa, but during the past century, outbreaks of significant economic importance and elevated mortality have been recorded in Northern African countries, the Iberian and Arabian Peninsula, the Middle East and the Indian subcontinent. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Conventional reverse-transcriptase (RT) PCR (RT-PCR) and real-time RT-PCR (rRT-PCR) assays have improved the sensitivity and rapidity of diagnosing AHS, resulting in the adoption of these methods as recommended tests by the World Organisation for Animal Health (OIE). However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for AHS would improve the fast implementation of control policies. Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed in the field. LAMP assays are attractive molecular assays because they are simple to use, rapid, portable and have sensitivity and specificity within the range of rRT-PCR. This study describes the development of a novel RT-LAMP assay for the detection of AHSV. The AHSV RT-LAMP assay has an analytical sensitivity of 96.1% when considering an rRT-PCR cut-off value of CT > 36, or 91.3% when no rRT-PCR cut-off is applied. Diagnostic sensitivity and specificity were 100%. This assay provides for a rapid and low cost AHS diagnostic for use in the field.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977 to 2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

Rabbit hemorrhagic disease (RHD) is a highly infectious disease in European rabbits (Oryctolagus cuniculus), caused by a virus belonging to the genus Lagovirus (RHDV; family Caliciviridae). In 2010, a new genotype of RHDV (RHDV2 or RHDVb, currently designated GI.2) emerged in France, affecting both domestic rabbits, even those vaccinated for the classical RHDV genotypes (currently designated GI.1) and wild rabbits. GI.2 was subsequently identified in other European countries. The aim of the present study was to monitor the GI.2 epidemic in wild rabbits in Andalusia (southern Spain) during the period 2013-2017. At the beginning of summer 2013, high mortalities were detected in wild rabbit populations in southern Spain. A total of 96 affected hunting or protected areas were surveyed. The first outbreak was observed on June 2013. The number of outbreaks sharply increased in 2013 and 2014, with a decreasing trend being observed during the following years. The spatial distribution of GI.2 was not homogeneous, since most of the detected outbreaks were concentrated in the western part of Andalusia. The outbreaks peaked in winter and spring and have been detected in the last five consecutive years, which suggests endemic circulation of GI.2 in wild rabbit populations in Spain. A total of 190 dead rabbits from 87 of the 96 areas surveyed were collected during the study period. Mortality affected rabbits of different age classes, including kittens. RT-PCR confirmed the presence of GI.2 RNA in the livers of 185 of the 190 (97.4%) rabbits. Phylogenetic analysis performed on eleven samples collected in different provinces of Andalusia between 2013 and 2017, showed high nucleotide identity with GI.2 strains Spain, France and Portugal. The results constitute an important step in understanding of the emergence and spread of GI.2 in this country and will provide valuable information for the development of surveillance programs in Europe.

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies. © 2012 The Author(s).

West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 59-Taq nuclease-39 minor groove binder DNA probe (TaqMan MGBE) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 1022-1023 pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.

Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification. © 2013 Elsevier B.V.

An increase in activity of two mosquito-borne flaviviruses, West Nile virus (WNV) and Usutu virus (USUV), has been reported in Europe in recent years. The current epidemiological situation calls for RT-PCR methods that are able to detect not only the widespread lineage 1 (L1) WNV, but also lineage 2 (L2) WNV. In addition, the presence in Europe of the closely related USUV requires methods that can identify these three flaviviruses and permit an efficient and accurate differential diagnosis. Here we describe a new one-step real-time multiplex RT-PCR that detects and differentiates efficiently WNV-L1, WNV-L2 and USUV in a single reaction. The assay is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen-quencher pairs. This enables the fluorescence emitted by each probe, characterized by distinct wavelengths, to be differentiated. This multiplex assay was very sensitive to all of the target viruses; in addition, there were no cross-reactions between the viruses and the assay did not react to any other phylogenetically or symptomatically related viruses. Quantitation was enabled through the use of in vitro-transcribed RNAs developed specifically for each virus as copy number standards. This new assay was validated using different types of experimental and field samples. © 2013 Elsevier B.V.

In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identifi ed Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent.

Porcine teschoviruses (PTVs) have been previously shown to be the most abundant cytopathic viruses found in swine feces. In the present study, the diversity of PTVs was studied, using PTV isolates collected between 2004 and 2009 in a wide territory in Spain. In order to characterize genetically the isolates, phylogeny reconstructions were made using maximum likelihood and Bayesian inference methods, based on the 1D (VP1) gene, and including sequences available in public databases. The phylogenetic trees obtained indicated that PTVs present 12 main lineages, 11 corresponding to the PTV serotypes described to date, and one lineage distinct from the rest.The geographic distribution of the different lineages does not seem to be strongly associated to particular territories, and co-circulation of multiple lineages was found in the same geographic areas. Nevertheless, some spatial structuring of the viral populations studied is indicated by the differences found between Spanish samples with respect to other European countries. A coalescent-based approach indicated that mutation may have been the main factor in originating the genetic diversity observed in the VP1 gene region. This study revealed a high diversity of teschoviruses circulating in the pig populations studied, and showed that molecular analysis of the complete VP1 protein is a suitable method for the identification of members of the porcine teschovirus group. However, further analyses are needed to clarify the geographical structuring of the different PTV populations. © 2011 Elsevier B.V.

In recent years, West Nile virus (WNV) has re-emerged in the Western Mediterranean region. As a result, the number of complete WNV genome sequences available from this region has increased, allowing more detailed phylogenetic analyses, which may help to understand the evolutionary history of WNV circulating in the Western Mediterranean. To this aim, the present work describes six new complete WNV sequences from recent outbreaks and surveillance in Italy in 2008–2009 and in Spain in 2008 and 2010. Comparison with other sequences from different WNV clusters within lineage 1 (clade 1a) confirmed that all Western Mediterranean WNV isolates obtained since 1996 (except one from Tunisia, collected in 1997) cluster in a single monophyletic group (here called ‘WMed’ subtype). The analysis differentiated two subgroups within this subtype, which appear to have evolved from earlier WMed strains, suggesting a single introduction in the area, and further dissemination and evolution. Close similarities between WNV variants circulating in consecutive years, one in Spain, between 2007 and 2008, and another in Italy between 2008 and 2009, suggest that the virus possibly overwinters in Western Mediterranean sites. The NS3249-proline genotype, recently proposed as a virulence determinant for WNV, has arisen independently at least twice in the area. Overall, these results indicate that the frequent recurrence of outbreaks caused by phylogenetically homogeneous WNV in the Western Mediterranean since 1996 is consistent with a single introduction followed by viral persistence in endemic foci in the area, rather than resulting from independent introductions from exogenous endemic foci.

Performance of a real-time reverse-transcription polymerase chain reaction method for the rapid, simple and reliable detection of porcine teschovirus (PTV) was assessed. The method was based on the use of a set of oligonucleotides consisting of two specific primers and a fluorogenic TaqMan-MGB probe. Reverse transcription and PCR reactions were performed sequentially in one step. As a result the whole procedure was simple and rapid, taking less than 3. h for completion. The method reacted in a dose-dependent manner with prototype strains for the eleven known PTV serotypes (PTV1-11), with higher analytical sensitivity than other gel-based RT-PCR methods described, which were performed in parallel to allow for a comparison. The assay did not cross-react with other related viruses or porcine viruses tested. The diagnostic performance of the method was analyzed using a panel of field samples consisting of pig fecal and pig slurry samples. As a conclusion, this technique is adequate and convenient for porcine teschovirus detection, both for diagnosis as well as in environmental investigations. © 2011 Elsevier B.V.

In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.

During the analysis of the bluetongue virus serotype 1 (BTV-1) strain Algeria 2006 grown in Vero cells at our laboratory (Laboratorio Central de Veterinaria [LCV]), false-positive results for epizootic hemorrhagic disease virus (EHDV) were detected when the reverse transcription-PCR (RT-PCR) technique previously described by Aradaib et al. was used (1). Bluetongue outbreaks caused by BTV-1 in Algeria and Morocco at the end of 2006 represented a high risk of introduction of this serotype in Spain. To assure the capabilities of LCV routine diagnostic procedures for detection of this BTV-1 strain, the BTV-1 Algeria 2006 isolate was obtained from the European Reference Laboratory. The virus was grown in Vero cells and analyzed with the BTV-specific RT-PCR assay previously described by Toussaint et al. (2), confirming the suitability of the method for sensitive detection of this viral strain. It was also tested to discard the concomitant presence of EHDV and other pathogens. EHDV detection was carried out with Aradaib's multiplex BTV-EHDV RT-PCR but using only EHDV-specific primers. Surprisingly, an amplification fragment of the specific size was obtained (Fig. ​(Fig.1)1) when assaying BTV-1 Algeria 2006 in EHDV-specific RT-PCR with the external primers E1 and E4 (targeted at nucleotides [nt] 175 to 194 and 543 to 562, respectively, of the EHDV NS1 gene). However, no amplification was produced using this PCR product in nested PCR with the internal primers E2 and E3 (targeted at nt 216 to 235 and 421 to 440, respectively). The DNA fragment size, established by sequence, was 384 bp and, consequently, could not be distinguished from the EHDV NS1 gene expected specific band (388 bp) on agarose gels. The sequence of this DNA fragment was compared with that in the GenBank database using BLASTN (NCBI), and high homology (94.8%) was found within a region of the VP2 gene (nt 2080 to 2464) belonging to the BTV-1 South Africa reference isolate (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ585122","term_id":"118420304","term_text":"AJ585122"}}AJ585122). In order to explain the BTV-1 Algeria 2006 unspecific amplification with primers specific for E1 and E4 EHDV, the nucleotide sequence around this VP2 region was determined in the BTV-1 Algeria 2006 isolate. A comparative study showed that the 3′ end of E1 and E4 oligonucleotides shared 9 and 5 nt, respectively, with the sequence of the BTV-1 Algeria 2006 VP2 gene (Fig. ​(Fig.2).2). This finding could justify the unexpected amplification, as it is known that correct annealing of the 3′ terminus of the primer with a target template is very important for efficient polymerase extension. Even though the same homology is observed in the 3′ end of the E1 and VP2 sequences of other BTV-1 isolates (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AJ585122","term_id":"118420304","term_text":"AJ585122"}}AJ585122 and {"type":"entrez-nucleotide","attrs":{"text":"X55800","term_id":"297132","term_text":"X55800"}}X55800), only 2 nt of the E4 3′ end are shared, making false-positive results with these BTV-1 strains unlikely. No significant homology between the primers and reference strains of other BTV serotypes has been found. FIG. 1. Agarose gel showing DNA products obtained in the EHDV-specific RT-PCR with external primers E1 and E4 (1st RT-PCR) and in the nested PCR with internal primers E2 and E3 (Nested PCR). C+, EHDV used as a positive control in the reaction; BT-1, BTV-1 ... FIG. 2. Comparison of the BTV-1 Algeria 2006 VP2 gene, the partial sequence, and oligonucleotides E1 and E4. Coincident nucleotides are indicated with an asterisk. E4c, E4 oligonucleotide complementary sequence. The BTV-1 sequence is numbered according to {"type":"entrez-nucleotide","attrs":{"text":"AJ585122","term_id":"118420304","term_text":"AJ585122"}} ... EHDV and BTV infect wild and domestic ruminants and share transmission vectors (Culicoides spp.), making it essential to have a differential diagnosis of these viruses in areas where both viruses are present. Furthermore, the absence of EHDV and other pathogens must be proven in virus seeds employed in the development of BTV vaccines. Therefore, it is necessary to perform an EHDV-specific detection test on several occasions for samples containing BTV-1. Only a few RT-PCR methods for EHDV detection have been reported, and Aradaib's method of detection is probably the most widely used. This has encouraged us to communicate these results showing the requirement of confirmatory tests, as nested PCR or sequencing, when an EHDV-positive result is obtained using Aradaib's RT-PCR method.

African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for its detection; however, these methods are cumbersome and difficult to apply when large numbers of samples are to be tested, as in the case of epizootics. To overcome this problem, a real-time RT-PCR method has been developed, based on a 5'-Taq nuclease-3′-minor groove binder-DNA probe (TaqMan MGB) for detection of a wide range of AHSV serotypes and strains designed to the highly conserved region of the VP7 gene (segment 7). The method was able to detect all prototype strains from the 9 known serotypes of the virus, with a high analytical sensitivity; no cross-reactions were observed with other orbiviruses or with other viruses affecting horses. The diagnostic sensitivity was assessed using a panel of AHSV-positive tissue samples from an epizootic that occurred in Spain between 1987 and 1990. This method, which can be performed in 96-well format, is suitable for large-scale surveillance of AHSV in areas where it can potentially spread.

SINCE first being detected in Spain in July 2007, bluetongue virus (btv) serotype 1 (btv-1) has caused severe losses in livestock. However, little is known about the impact of this newly introduced virus on susceptible wildlife that is being exposed to the virus. This short communication describes

A highly sensitive and specific one-step multiplex RT-PCR assay has been developed and standardised for the simultaneous and differential detection of the most important vesicular viruses affecting livestock foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV). The method uses three primer sets, each one specific for the corresponding virus, selected to detect of all serotypes of FMD and VS. The detection range was confirmed by examination of a collection of 31 isolates of the three target viruses. The specificity of the assay was also demonstrated by testing other related viruses, uninfected cell line cultures and healthy pig tissues. The testing of blood and serum samples from animals infected experimentally proved that the method can be useful for early diagnosis of the diseases, even before the first vesicular lesions are visualized in the infected pigs. An assessment of the performance of the multiplex RT-PCR was carried out using a panel of more than 100 samples from animals infected experimentally, showing the suitability of the method for a rapid (less than 6 h), sensitive and specific differential diagnosis in clinical samples. Additionally, a uniplex RT-PCR for VSV, that amplifies the two viral serotypes, was also developed and tested as a rapid tool for the diagnosis of this vesicular disease. © 2007 Elsevier B.V. All rights reserved.

The recent spread of highly pathogenic H5N1 avian influenza (AI) has made it important to develop highly sensitive diagnostic systems for the rapid detection of AI genome and the differentiation of H5N1 variants in a high number of samples. In the present paper, we describe a high-throughput procedure that combines automated extraction, amplification, and detection of AI RNA, by an already described TaqMan real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay targeted at the matrix (M) protein gene of AI virus (AIV). The method was tested in cloacal and tracheal swabs, the most common type of samples used in AI surveillance, as well as in tissue and fecal samples. A robotic system (QIAGEN Biosprint 96) extracted RNA and set up reactions for RRT-PCR in a 96-well format. The recovery of the extracted RNA was as efficient as that of a manual RNA extraction kit, and the sensitivity of the detection system was as high as with previously described nonautomated methods. A system with a basic configuration (one extraction robot plus two real-time 96-well thermocyclers) operated by two persons could account for about 360 samples in 5 hr. Further characterization of AI RNA-positive samples with a TaqMan RRT-PCR specific for H5 (also described here) and/or N1 was possible within 2 hr more. As this work shows, the system can analyze up to 1400 samples per working day by using two nucleic acid extraction robots and a 384-well-format thermocycler.

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5′-Taq nuclease-3′ minor groove binder (TaqMan© MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 × 10−3 tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease. © INRA, EDP Sciences, 2004.

This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.

At the end of September 2000, clinical symptoms of Bluetongue appeared in sheep flocks of the Balearic Islands (Spain). The presence of the BTV serotype 2 in tissue and blood samples of affected animals was confirmed by laboratory techniques. A systematic vaccination were carried out in affected areas using a live monovalent serotype 2 vaccine available from Onderstepoort laboratory (South Africa). In order to perform epidemiological studies, a new method to differentiate between the NS1 genes of BTV-2 affecting the Balearic islands and that of the Onderstepoort commercial live virus vaccine (monovalent, serotype 2) has been developed. This procedure is based on the use of an RT-PCR, followed by restriction endonuclease analysis. Epidemiological data of a study carried out in the period January-October 2001 using this procedure are included. © 2002 Elsevier Science B.V. All rights reserved.

Rapid detection of AIV is essential in disease outbreak situations, when the rapid identification of viremic animals is crucial to preventing the spread of disease. Viremia can be demonstrated in tracheal or cloacal swabs, tissues (mainly from the respiratory and gastrointestinal tracts) and faecal samples of infected animals both by virus isolation or nucleic acid detection methods. Although virus isolation in embryonated chicken eggs is reliable and sensitive, it is cumbersome and takes longer to perform than nucleic acid-based detection assays. Several reverse transcription (RT)-PCR methods have previously been described for this purpose but conventional RT-PCR methods are difficult to apply when very large numbers of samples are to be analyzed. Recently, realtime PCR protocols for the detection of AIV have become available. This system allows rapid diagnosis and is amenable for automation, which allows high throughput sample monitoring.

This work describes the development of a real-time RT-PCR (RRT-PCR) procedure for detection of the N1 gene from avian influenza virus (AIV), based on the use of specific primers and a TaqMan-MGB (minor groove binder) probe. Nucleotide sequences of the neuraminidase type 1 gene from a collection of H5N1 Eurasian strains of AIV were aligned using ClustalW software. Conserved regions were located and used to design specific primers and a TaqMan-MGB probe using Primer Express software. A one-step RRT-PCR method was optimized using RNA from the Turkey 2005 H5N1 strain of AIV and can be completed in about 2 hr once the RNA is extracted from the sample. The specificity of the assay was assessed with non-N1 AIV strains, another related avian virus, and different avian tissue samples from healthy animals. Sensitivity was determined using 10-fold serial dilutions of the H5N1 Turkey 2005 strain and was compared with the generic RRT-PCR detection method, targeted at the matrix protein gene of AIV, commonly used at the Spanish AIV National Reference Laboratory. The N1 detection method proved to be even more sensitive than the generic (matrix-based) method, allowing a very quick confirmation (or discarding) of any Eurasian N1 strain when a positive result was obtained with the matrix RRT-PCR assay. Combined with RRT-PCR tests for general detection of AIV and H5 typing in use at the NRL, the procedure here described allows characterizing of any H5N1 Eurasian AIV strain in a field sample within a working day.

West Nile virus (WNV) is a mosquito-borne flavivirus which causes important morbidity and mortality in birds, horses and humans. In the Western Mediterranean region, WNV causes sporadic, self-limited outbreaks, with few or no human cases. Here we report the characterization of two recent Western Mediterranean WNV isolates, obtained in Spain in 2007 from two golden eagles. Complete genome sequence comparisons revealed high identity between these isolates and close relationship with other Western Mediterranean WNV strains isolated since 1996. Phylogenetic analysis within this group indicated that two distinct phylogenetic groups have emerged from earlier strains. Pathogenicity analysis in mice showed that the Spanish isolate is less pathogenic than other strains either from the Western Mediterranean (Morocco 2003) or from North America (NY'99). Changes in amino acid position NS3-249 (claimed as a virulence marker) did not influence the pathogenicity observed., This study was supported by grants from INIA (OT01-002), Fondo de Investigación Sanitaria (FIS PI07/1309) and Junta de Comunidades de Castilla–La Mancha JCCM (PAC08-0296-7771).

An analysis of the SalI restriction pattern of African swine fever virus DNA showed that the SalI recognition sites did not change after more than 100 virus passages in porcine macrophages. The virus strain BA71V, obtained from the virus isolate BA71 by adaptation to grow in VERO cells, differed from the nonadapted virus in two deletions with a length of 2.5 and 7 kb located close to the DNA ends. A restriction analysis of several virus clones obtained from a naturally infected pig revealed length heterogeneity in both variable regions. A comparison of SalI restriction maps from 23 African swine fever virus field isolates (8 African, 11 European, and 4 American) has shown that the virus genome consists of a central region with a constant length of about 125 kb and two variable regions located close to the DNA ends with a length of 38-47 kb for the left DNA end, and 13-16 kb for the right DNA end. The total length of ASF virus DNA varied between 178 (BA71) and 189 (MOZ64) kb. The 23 African swine fever virus isolates were classified into five groups, according to the arrangement of the SalI sites in the central region. Four groups contained only African isolates, whereas all the European and American isolates belonged to the same group. This distribution of isolates suggests that all non-African virus field isolates have a common origin.

Salmonella Kentucky is commonly found in poultry and rarely associated with human disease. However, a multidrug-resistant (MDR) S. Kentucky clone [sequence type (ST)198] has been increasingly reported globally in humans and animals. Our aim here was to assess if the recently reported increase of S. Kentucky in poultry in Spain was associated with the ST198 clone and to characterize this MDR clone and its distribution in Spain. Sixty-six isolates retrieved from turkey, laying hen and broiler in 2011–2017 were subjected to whole-genome sequencing to assess their sequence type, genetic relatedness, and presence of antimicrobial resistance genes (ARGs), plasmid replicons and virulence factors. Thirteen strains were further analysed using long-read sequencing technologies to characterize the genetic background associated with ARGs. All isolates belonged to the ST198 clone and were grouped in three clades associated with the presence of a specific point mutation in the gyrA gene, their geographical origin and isolation year. All strains carried between one and 16 ARGs whose presence correlated with the resistance phenotype to between two and eight antimicrobials. The ARGs were located in the Salmonella genomic island (SGI-1) and in some cases (bla SHV-12 , catA1, cmlA1, dfrA and multiple aminoglycoside-resistance genes) in IncHI2/IncI1 plasmids, some of which were consistently detected in different years/farms in certain regions, suggesting they could persist over time. Our results indicate that the MDR S. Kentucky ST198 is present in all investigated poultry hosts in Spain, and that certain strains also carry additional plasmid-mediated ARGs, thus increasing its potential public health significance.