Enhanced passive surveillance for early detection of African and classical swine fevers.

African swine fever (ASF) and classical swine fever (CSF) are transboundary animal diseases (TADs) of pigs. Much effort and resources are regularly put into preventing these diseases' introduction in free areas. Passive surveillance activities bring the highest chances for the early detection of TAD incursions because they are routinely and widely conducted at farms, and because these activities focus on the time between introduction and when the first sample is sent for diagnostic testing. The authors proposed the implementation of an enhanced passive surveillance (EPS) protocol based on collecting data through participatory surveillance actions using an objective and adaptable scoring system to aid the early detection of ASF or CSF at the farm level. The protocol was applied in two commercial pig farms for ten weeks in the Dominican Republic, which is a CSF- and ASF-infected country. This study was a proof of concept, based on the EPS protocol to aid detection of substantial variations in the risk score triggering testing. One of the followed farms had score variation, which triggered testing of the animals, although the test results were negative. The study enables assessment of some of the weaknesses associated with passive surveillance and provides lessons applicable to the problem. Results demonstrate the potential for overcoming some issues preventing the broad application of EPS protocols and suggest that standardised approaches may contribute to the early detection of CSF and ASF introductions.

La peste porcine africaine (PPA) et la peste porcine classique (PPC) sont des maladies animales transfrontalières touchant les porcs. De nombreux efforts et ressources sont régulièrement alloués pour prévenir l'introduction de ces maladies dans des zones indemnes. Les activités de surveillance passive offrent les meilleures perspectives de détection précoce des incursions de maladies animales transfrontalières parce qu'elles sont menées de manière systématique et exhaustive dans les élevages, et parce qu'elles se concentrent sur la période entre l'introduction de la maladie et le moment où le premier échantillon est envoyé au laboratoire pour analyse. Les auteurs proposent la mise en oeuvre d'un protocole de surveillance passive renforcée fondé sur la collecte de données via des actions de surveillance participative utilisant un système de notation objectif et adaptable, en vue d'une détection précoce de la PPA et de la PPC dans les élevages. Ce protocole a été appliqué en République dominicaine, pays infecté par la PPA et la PPC, dans deux élevages porcins commerciaux pendant dix semaines. Cette étude était destinée à valider le principe de la méthode et se fondait sur le protocole de surveillance passive renforcée pour mieux détecter les variations substantielles de la note de risque qui conduisent à tester les animaux. L'un des élevages suivis a présenté une variation de cette note, ce qui a conduit à tester les animaux mais les tests se sont révélés négatifs. L'étude permet d'évaluer certaines des faiblesses associées à la surveillance passive et apporte des enseignements applicables à ce problème. Les résultats illustrent le potentiel de l'approche à surmonter certaines des problématiques empêchant l'application extensive des protocoles de surveillance passive renforcée. Ils suggèrent également que des approches normalisées pourraient contribuer à la détection précoce des cas d'introduction de la PPC et de la PPA.

La peste porcina africana (PPA) y la peste porcina clásica (PPC) son enfermedades animales transfronterizas que afectan al cerdo. Periódicamente se dedican grandes esfuerzos y cuantiosos recursos a evitar que estas enfermedades penetren en zonas que están exentas de ellas. Las actividades de vigilancia pasiva son las más eficaces para detectar con prontitud toda incursión de enfermedades animales transfronterizas, no solo por la regularidad y amplitud con que se llevan a cabo en las explotaciones, sino también porque inciden específicamente en el intervalo entre la penetración de una enfermedad y el momento en que se envía la primera muestra para que sea sometida a pruebas de diagnóstico. Los autores propusieron que se aplicara un protocolo de vigilancia pasiva reforzada que reposaba en la obtención de datos mediante actividades de vigilancia participativa, empleando para ello un sistema objetivo y adaptable de puntuación que ayudaba a detectar con prontitud la presencia en las explotaciones de PPA o PPC. Dicho protocolo fue aplicado a lo largo de diez semanas en dos explotaciones porcinas industriales de la República Dominicana, país en el que ambas infecciones están presentes. El estudio, que sirvió para poner a prueba la idea, pasaba por la aplicación del protocolo de vigilancia pasiva reforzada para ayudar a detectar variaciones sustanciales de la puntuación del nivel de riesgo que activa la realización de pruebas. En una de las explotaciones estudiadas se produjo una variación de la puntuación, cosa que activó la realización de pruebas en los animales, aunque estas arrojaron resultado negativo. El estudio aquí descrito permite evaluar algunos de los puntos débiles de la vigilancia pasiva y extraer enseñanzas aplicables al problema. Los resultados demuestran que es posible salvar algunas de las dificultades que impiden la aplicación generalizada de protocolos de vigilancia pasiva reforzada y dejan pensar que quizá el uso de planteamientos normalizados pueda ayudar a detectar con prontitud los casos de penetración de PPC o PPA.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus.

BACKGROUND: Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. METHODS: The CSFV Erns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). RESULTS: Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. CONCLUSION: The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. RESULTS: The one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/µL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. CONCLUSION: A one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

Classical swine fever (CSF): towards new challenges / La peste porcine classique (PPC) : vers de nouveaux défis

Classical swine fever (CSF) is a highly contagious swine-specific disease which may have a huge economic impact for porcine production. CSF is caused by a virus belonging to the Pestivirus genus, which has expanded for the past 5 years with the discovery of new species whose genetic proximity to the CSF virus could further complicate laboratory diagnosis. The various forms of the disease, and in particular the increased frequency of attenuated forms, linked to an evolution of CSF virus strains towards a reduction in their virulence, delay clinical diagnosis. Thus, a long period may elapse before an outbreak is detected, allowing the virus to circulate longer, with the risk of spreading to distant geographical areas. Efforts must be maintained in terms of surveillance and diagnostic tools development in order to detect CSF virus infection early and thus limit the spread of the disease and facilitate control measures.

La peste porcine classique (PPC) est une maladie très contagieuse, spécifique des suidés, qui continue à constituer une menace pour la production porcine malgré un statut indemne de la plupart des pays de l'Union européenne. La PPC est causée par un virus de la famille des Flaviviridae appartenant au genre Pestivirus, en extension depuis les cinq dernières années avec la découverte de nouvelles espèces, notamment chez le porc ou autres animaux de rente dont la proximité génétique avec le virus de la PPC pourrait davantage compliquer le diagnostic de laboratoire. La diversité des formes de la maladie, et notamment la fréquence accrue des formes atténuées et inapparentes liée à une évolution des souches du virus de la PPC vers une réduction de leur virulence, retarde le diagnostic clinique. Ainsi, une longue période peut s'écouler avant la détection d'un foyer, permettant au virus de la PPC de circuler plus longuement, avec le risque de diffuser vers des zones géographiques éloignées des premiers cas d'infection. Les efforts doivent être maintenus en termes de surveillance et de développement d'outils de diagnostic afin de détecter précocement une infection par le virus de la PPC et ainsi limiter la propagation de la maladie et faciliter les mesures de contrôle.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR.

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/µL for CSFV and 36 copies/µL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Evaluation of surface glycoproteins of classical swine fever virus as immunogens and reagents for serological diagnosis of infections in pigs: a recombinant Newcastle disease virus approach.

Classical swine fever (CSF) is an important viral disease of domestic pigs and wild boar. The structural proteins E2 and Erns of classical swine fever virus (CSFV), which participate in the attachment of the virion to the host cell surface and its subsequent entry, are immunogenic. The E2 and Erns proteins are used for diagnosis and the development of vaccines against CSFV infection in swine. Newcastle disease virus (NDV) has been successfully used as a viral vector to express heterologous proteins. In the present study, the E2 and Erns proteins of CSFV were expressed in cell culture as well as embryonated chicken eggs, using recombinant NDV (rNDV). Rescued rNDV expressing the E2 and Erns proteins induced the production of CSFV-neutralizing antibodies upon intranasal vaccination of pigs. Serum samples from vaccinated animals were found to neutralize both homologous and heterologous CSFV strains. Furthermore, rNDV expressing the E2 and Erns proteins of CSFV was used to develop an indirect ELISA, which was used to measure the the antibody titers of randomly collected serum samples. The results suggested that the ELISA based on rNDV-expressed E2 and Erns proteins could be used to screen for CSFV infections. This study shows that rNDV-based expression of CSFV antigens is potentially applicable for development of vaccines and diagnostic tests for CSFV infection. This approach could be an economically favorable alternative to the existing vaccine and diagnostics for CSFV in pigs.

Strategy for efficient generation of numerous full-length cDNA clones of classical swine fever virus for haplotyping.

BACKGROUND: Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection. RESULTS: In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order. CONCLUSIONS: This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.

Rope-based oral fluid sampling for early detection of classical swine fever in domestic pigs at group level.

BACKGROUND: Non-invasive sampling techniques based on the analysis of oral fluid specimen have gained substantial importance in the field of swine herd management. Methodological advances have a focus on endemic viral diseases in commercial pig production. More recently, these approaches have been adapted to non-invasive sampling of wild boar for transboundary animal disease detection for which these effective population level sampling methods have not been available. In this study, a rope-in-a-bait based oral fluid sampling technique was tested to detect classical swine fever virus nucleic acid shedding from experimentally infected domestic pigs. RESULTS: Separated in two groups treated identically, the course of the infection was slightly differing in terms of onset of the clinical signs and levels of viral ribonucleic acid detection in the blood and oral fluid. The technique was capable of detecting classical swine fever virus nucleic acid as of day 7 post infection coinciding with the first detection in conventional oropharyngeal swab samples from some individual animals. Except for day 7 post infection in the "slower onset group", the chances of classical swine fever virus nucleic acid detection in ropes were identical or higher as compared to the individual sampling. CONCLUSIONS: With the provided evidence, non-invasive oral fluid sampling at group level can be considered as additional cost-effective detection tool in classical swine fever prevention and control strategies. The proposed methodology is of particular use in production systems with reduced access to veterinary services such as backyard or scavenging pig production where it can be integrated in feeding or baiting practices.

Hunters' acceptability of the surveillance system and alternative surveillance strategies for classical swine fever in wild boar - a participatory approach.

BACKGROUND: Surveillance measures can only be effective if key players in the system accept them. Acceptability, which describes the willingness of persons to contribute, is often analyzed using participatory methods. Participatory epidemiology enables the active involvement of key players in the assessment of epidemiological issues. In the present study, we used a participatory method recently developed by CIRAD (Centre de Coopération Internationale en Recherche Agronomique pour le Développement) to evaluate the functionality and acceptability of Classical Swine Fever (CSF) surveillance in wild boar in Germany, which is highly dependent on the participation of hunters. The acceptability of alternative surveillance strategies was also analyzed. By conducting focus group discussions, potential vulnerabilities in the system were detected and feasible alternative surveillance strategies identified. RESULTS: Trust in the current surveillance system is high, whereas the acceptability of the operation of the system is medium. Analysis of the acceptability of alternative surveillance strategies showed how risk-based surveillance approaches can be combined to develop strategies that have sufficient support and functionality. Furthermore, some surveillance strategies were clearly rejected by the hunters. Thus, the implementation of such strategies may be difficult. CONCLUSIONS: Participatory methods can be used to evaluate the functionality and acceptability of existing surveillance plans for CSF among hunters and to optimize plans regarding their chances of successful implementation.

Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study.

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 "Margarita" and the recent field isolate CSF1058 "Pinar del Rio". Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

Bead-based suspension array for simultaneous differential detection of five major swine viruses.

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/µL (corresponding to 0.5-6.8 fg/µL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

Differential detection of classical swine fever virus challenge strains in C-strain vaccinated pigs.

BACKGROUND: Control of classical swine fever (CSF) by vaccination ideally requires that field strain infection can be detected irrespective of the vaccination status of the herd. To inform on the usefulness of molecular tests compatible with genetic Differentiation of Infected from Vaccinated Animals (DIVA) principles when using live-attenuated vaccines, tonsil homogenates from a vaccination-challenge experiment were analyzed using a differential real-time qRT-PCR for the C-strain vaccine or real-time qRT-PCR assays developed to specifically detect the challenge strains used. RESULTS: In animals with high or moderate levels of blood viraemia, which were not, or not fully, protected by vaccination, challenge virus RNA was readily detected in tonsil homogenates. In three out of the seven vaccinated animals that had high or moderate viraemia, the vaccine strain RNA also could be detected but at lower levels. Lower but varying levels of challenge and/or vaccine virus RNA were detected in tonsil homogenate samples from animals with no or low-level viraemia, and in groups solely consisting of such animals, no transmission of infection to naïve in-contact animals occurred. In one group of animals that were vaccinated 3 days prior to challenge, viraemia levels varied from high to absent and transmission of challenge virus to naïve in-contact animals occurred. The DIVA assay revealed challenge virus in all tonsil homogenates from this group, even in those animals that did not have viraemia and were protected from clinical disease by vaccination. Such animals, particularly in a low biosecurity/informal farm setting, could constitute a risk for disease control in the field. CONCLUSIONS: Genetic DIVA testing is useful for detecting the presence of field virus infection especially in non-viraemic animals without overt clinical signs but which are incompletely protected by vaccination. Such tests could particularly be useful to inform decisions prior to and during cessation of a control strategy that employs vaccination.

Enhancing classical swine fever virus identification: the advantages of Field-LAMP testing.

Classical swine fever virus (CSFV) identification has witnessed significant advancements with the development of rapid reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays. However, conventional RT-LAMP assays for CSFV diagnosis are hindered by a laborious RNA extraction step. Moreover, the need for thermal incubators and expensive micropipettes has limited their application in field settings. Addressing these challenges, our study presents a groundbreaking solution-an electro-free and point-of-care (POC) tool known as the field-LAMP assay-for the rapid clinical detection of CSFV. By eliminating the RNA extraction requirement, advancing the colorimetric read-out and lyophilized reaction reagents, our field-LAMP assay streamlines the diagnostic process, saving valuable time and effort. This novel approach also overcomes the dependency on electric-dependent thermal incubators and expensive micropipettes, making it practical and accessible for use in the field. The successful development of the field-LAMP assay marks a significant milestone in CSFV detection. This electro-free and POC tool offers several advantages, including its ability to deliver rapid results without compromising accuracy, facilitating prompt response and containment measures.

Novel approach for detecting classical swine fever virus from swabs of wild boar cut tails using nested real-time PCR.

We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Classical swine fever.

Classical swine fever is a serious and economically important transboundary disease threatening pig production globally. The infection may occur in backyard pigs, feral pig populations and domestic pigs. Whereas there are proven control strategies for the latter pig population, control in backyard pigs with poor biosecurity settings or in wild boar populations of high density still poses a problem in some parts of the world. Laboratory diagnostic methods, efficacious vaccines and contingency plans are in place in most industrialised countries. So far modified live vaccines (MLV) are still the first choice for rapid and reliable immune protection. Since antibodies elicited by conventional MLV cannot be distinguished from antibodies after natural infection, considerable efforts are put into the development of a live marker vaccine accompanied by a serological test. Nevertheless, some remaining gaps with respect to the diagnosis of and vaccination against classical swine fever have been identified.

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies.

Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.

Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals.

The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit* CSF-Marker, an Erns ELISA. However, this test does not allow differentiation between antibodies directed against ruminant pestiviruses and those against CSFV. Therefore, it is not suitable for use with the chimeric marker vaccines tested. The PrioCHECK CSFV Erns was the only ELISA suitable for use in DIVA with marker vaccines containing Erns proteins from ruminant pestiviruses. However, this test was less sensitive and selective than the E2-ELISAs and cannot be recommended.