Diagnosis Of Classical Swine Fever Research Articles

<b>Classical swine fever: Unveiling the complexity through a multifaceted approach</b>

Classical swine fever (CSF), sometimes referred to as hog cholera, is a highly contagious, virally-based, systemic illness that affects both domestic and wild pigs. The virus known as classical swine fever (CSFV) is a member of the Flaviviridae family, specifically the genus Pestivirus. This disease is thought to be endemic in many Asian countries that produce pork as well as in several countries in Central and South America, the Caribbean, and elsewhere. As previously indicated, depending on the virulence of the virus strain involved and several host circumstances, clinical indications of CSFV infection can vary greatly, ranging from abrupt fatality to an occult course. CSF diagnosis can be made by serological detection, antigen, RNA, and isolation. CSF's highly varied symptoms and post-mortem pathology resemble those of African swine fever. African swine fever (ASF). The kind of CSFV, the pig's age, and its susceptibility all affect the clinical symptoms. Pigs that contract CSFV, a highly infectious and economically significant virus. The great economic significance of the swine business makes the classical swine fever virus a potential bioterrorism threat. Live attenuated CSF vaccinations have been around for many years and are quite safe and effective. Controlling epidemics in CSFV-free zones requires quick action. Pigs that are impacted must be slaughtered, and the carcasses must be buried or burned.

Creation of a hybridization probe for the diagnosis of classical swine fever by the FISH method

The article presents data creation of the DNA probe labeled with fluorescein-12-dUTP for its subsequent use in in-situ hybridization in cryotomic histological sections from samples of internal organs of pigs. This diagnostic method is especially relevant for the inapparate course of infection, which contributes to maintaining the viral load inside the herd without showing pronounced clinical signs. In addition, the method helps to identify the viral genetic material in the pathological samples during the post-viremia period. In this article the stages of creating a probe consisting of 173 pairs of nucleotide bases corresponding to the site of the glycoprotein E2 gene of the classical swine fever virus are shown. The final DNA probe was tested on tonsils from pigs infected with the CSF virus as a positive control for the DNA probe and as a negative control for the DNA probe is tonsils from healthy pigs not vaccinated against CSF were used. Finally, we obtained positive results of using the DNA probe, reflecting its high specificity and selectivity in the studied samples of positive control. The data obtained by us formed the basis for our further research in the field of infectious pathology of farm animals.

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Comparison of three commercial enzyme-linked immunosorbent assays for the diagnosis of Classical Swine Fever

Classical swine fever (CSF) is a highly contagious viral disease that affects pigs and wild boars. The disease can cause significant economic losses in the pig industry and poses a threat to food security. Therefore, it is crucial to have effective surveillance programs to detect and control the disease. The study mentioned above aimed to compare three commercial ELISA kits for the detection of CSF antibodies. The results showed that all three kits had 100% congruence in undiluted samples, indicating that they are highly reliable for use as a screening test on routine samples. Additionally, the study found good reproducibility between technicians with no significant influence on the variation of results. However, the study also identified a low consistency of positive results (37.7%) for the kit IDvet Screen E2 in diluted samples, with significant variations in results from all three kits. This finding suggests that the other two kits, Herdchek E2 and Priocheck E2, may be better suited for detecting animals with low levels of antibodies, allowing for earlier detection of infected animals and the implementation of relevant control measures. In conclusion, the study demonstrates the importance of using high-quality commercial ELISA kits for the surveillance of CSF. The findings suggest that any of the three kits tested could be used as a reliable screening test on routine samples. However, for the detection of animals with low levels of antibodies, the Herdchek E2 and Priocheck E2 kits may be more effective

Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples.

African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping.

Development and Application of RAA Nucleic Acid Test Strip Assay and Double RAA Gel Electrophoresis Detection Methods for ASFV and CSFV.

African swine fever (ASF) is an acute, severe and hemorrhagic infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Since the outbreak of the disease in China in 2018, it has brought a great impact on China’s pig industry. Classical swine fever (CSF) is an acute contact infectious disease of pigs caused by classical swine fever virus (CSFV) infection. Clinically, acute CSF usually shows persistent high fever, anorexia, extensive congestion and bleeding of the skin and mucosa, which are similar to ASF. It is of great significance to prevent, control and accurately detect ASF and CSF in pig farms. In this study, Recombinase aided amplification (RAA) technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of ASFV/CSFV. The sensitivity and preliminary clinical application results showed that the RAA test strip established in this study could detect recombinant plasmids containing ASFV/CSFV gene fragments as low as 103 copies/µL. The minimum detection limits of virus DNA/cDNA were 10 and 12 pg respectively, and there was no cross-reaction with other porcine viruses. The specificity of the method was good. We used 37–42 clinical samples to evaluate the performance of our established method, and the positive concordance rates with conventional PCR were 94.1 and 57.1%, respectively. In addition, ASFV and CSFV double RAA agarose gel electrophoresis detection methods were established. The results showed that the method had good specificity. The detection limit of this method is 106 copies for ASFV p72 gene recombinant plasmid and 105 copies for CSFV NS5B Gene recombinant plasmid. The use of this method for clinical material detection was consistent with the PCR method. In summary, the developed method of RAA-strip assay for ASFV and CSFV realized the visual detection of pathogens, and the developed method of dual RAA agarose gel electrophoresis assay for ASFV and CSFV realized the simultaneous detection of two pathogens in one reaction, with good specificity, high sensitivity and rapid reaction rate, which was expected to be clinically feasible for the differential diagnosis of ASF and CSF provided technical support.

Immunohistochemical analysis of the distribution of classical swine fever (CSF) viral antigen in boar-pig hybrids and pigs four weeks after infection

We detected the classical swine fever virus (CSFV) antigen in three boar-pig hybrids (hybrids) and three pigs. All animals were experimentally infected with CSFV strain JPN/27/2019 to optimize diagnostic sampling and risk assessment of virus dissemination. Two hybrids died 17- and 19-days post-inoculation (dpi). The other animals were euthanized at 28 dpi. The detection of CSFV antigen at 28 dpi in epithelial cells of the apocrine sweat and sebaceous glands in the skin, salivary glands, mucosal epithelial cells in the rectum, and epithelial cells in the kidney and urinary bladder, suggests that CSFV persists in these tissues and spreads via sweat, saliva, feces, and urine for at least 4 weeks. These findings reveal that hybrids and pigs represent a high risk of virus dissemination four weeks after infection with CSFV strain JPN/27/2019. Prominent CSFV antigens were also detected in hair follicles of the skin. These results suggest that postmortem sampling of animal skin may be effective for CSF diagnosis and can be used to develop a rapid and easy diagnostic method using hair follicles.

Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives.

Classical swine fever (CSF) is a highly contagious viral disease of pigs, including wild boar. It is regarded as one of the major problems in the pig industry as it is still endemic in many regions of the world and has the potential to cause devastating epidemics, particularly in countries free of the disease. Rapid and reliable diagnosis is of utmost importance in the control of CSF. Since clinical presentations of CSF are highly variable and may be confused with other viral diseases in pigs, laboratory diagnosis is indispensable for an unambiguous diagnosis. On an international level, well-established diagnostic tests of CSF such as virus isolation, fluorescent antibody test (FAT), antigen capture antibody enzyme-linked immunosorbent assay (ELISA), reverse-transcription polymerase chain reaction (RT-PCR), virus neutralization test (VNT), and antibody ELISA have been described in detail in the OIE Terrestrial Manual. However, improved CSF diagnostic methods or alternatives based on modern technologies have been developed in recent years. This review thus presents recent advances in the diagnosis of CSF and future perspectives.

A chemiluminescence immunoassay for rapid detection of classical swine fever virus E2 antibodies in pig serum samples.

The high performance of chemiluminescence immunoassays (CLIAs) in diagnosis has been gradually recognized in recent years, but their application in the diagnosis of classical swine fever (CSF) has not been reported. Here, a recombinant E2 (rE2) protein and a peroxidase-conjugated monoclonal antibody (MAb G5) were used to develop a competition-based chemiluminescence immunoassay (cCLIA) for rapid and accurate detection of E2-specific antibodies in pig serum. To evaluate the feasibility of cCLIA in the diagnosis of CSF, we developed a competition-based enzyme-linked immunosorbent assay (cELISA) as a control. Under the optimum test conditions, cCLIA showed a higher signal-to-noise ratio than that of the control cELISA. The best signal-to-noise ratios of cCLIA and cELISA were 70 and 17, respectively. Then, the diagnostic performance of the two assays was compared by examining a panel of pig serum samples (n=285) with a confirmed status, and cCLIA showed higher diagnostic sensitivity (Dn) and diagnostic specificity (Dp) values than those of cELISA. The Dn and Dp of cCLIA were 97.49% and 96.08%, respectively, and those of cELISA were 93.97% and 94.12%, respectively. Furthermore, cCLIA can provide results within 20min, whereas the control cELISA requires at least 1hr. According to these findings, the newly developed cCLIA has potential application in the diagnosis of CSF and offers an alternative approach for efficient and rapid detection of E2-specific antibodies.

Inactivation of Classical Swine Fever Virus in Porcine Serum Samples Intended for Antibody Detection.

Shipping of serum samples that were taken from pigs infected with classical swine fever (CSF) virus is frequently requested with the objective of serological analyses, not only for diagnostic purposes but also for exchange of reference materials that are used as control material of diagnostic assays. On the basis of the fact that an outbreak with CSF is associated with enormous economic losses, biological safety during the exchange of reference material is of great importance. The present study aimed to establish a pragmatic approach for reliable CSF virus (CSFV) inactivation in serum without impairing antibody detection. Considering the fact that complement inactivation through heating is routinely applied, the basic idea was to combine heat treatment with the dilution of serum in a detergent containing buffer in order to facilitate the inactivation process. The results show that treatment of serum samples with phosphate buffered saline-Tween20 (final concentration = 0.15%) along with incubation at 56 °C for 30 min inactivated CSFV and such treatment with ≤ 0.25% PBS-Tween20 does not impair subsequent antibody detection by ELISA or virus neutralization test. This minimizes the risk of virus contamination and represents a valuable contribution to a safer CSF diagnosis on a national and international level.

Approbation of RT-qPCR test kit for differential diagnosis of African and Classical swine fever

The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.

A TaqMan real time probe assay for specific detection and quantification of lapinized vaccine strain of classical swine fever virus

Classical swine fever (CSF) is a highly contagious viral disease having a tremendous socio-economic impact. In CSF endemic countries such as India, control strategies depend heavily on the use of a live attenuated vaccine. The persistence of vaccine virus in tissue/blood of vaccinated animals may interfere with the diagnosis when RT-PCR assay is used for CSF diagnosis. A TaqMan based real-time RT-PCR assay was developed with primers and probe targeting the NS5B region of the lapinized CSFV (vaccine strain) used in India for detection and quantification of lapinized CSFV. The real-time TaqMan RT-PCR assay presented in the current study provided a rapid, specific and sensitive diagnostic tool for the detection of LCSFV and for quantification of virus load in various organs. This real time RT-PCR assay can differentiate the infected from vaccinated animals and quantification assay can be used for post-vaccination studies.

Recent emergence of a novel porcine pestivirus: interference with classical swine fever diagnosis?

Emerging Microbes & Infections (2017) 6, e19; doi:10.1038/emi.2017.5; published online 12 April 2017

Localization of classical swine fever virus antigen in young piglets by immunohistochemistry

The present study investigated the diagnosis of classical swine fever in piglets of four to six months using gross lesions, histopathology and immunohistochemistry. Gross examination revealed hemorrhagic kidneys, lymph nodes and spleen along with button shaped ulcer in intestine. Histopathological examination revealed focal to diffuse haemorrhages in the red pulp of spleen with depletion of the lymphocytes and multiple ulcerative lesions with necrosis of the epithelial mucosa in large intestine. Immunohistochemically, the viral antigen was demonstrated in the bronchial epithelial cells, mononuclear cells in spleen, intestine and Purkinje cells of the brain.

Close relationship of ruminant pestiviruses and classical Swine Fever virus.

To determine why serum from small ruminants infected with ruminant pestiviruses reacted positively to classical swine fever virus (CSFV)–specific diagnostic tests, we analyzed 2 pestiviruses from Turkey. They differed genetically and antigenically from known Pestivirus species and were closely related to CSFV. Cross-reactions would interfere with classical swine fever diagnosis in pigs.

Pathology and molecular diagnosis of classical swine fever in Mizoram.

Aim:Clinical histopathological and molecular diagnosis of classical swine fever disease in pigs of Mizoram.Materials and Methods:Totally, 31 clinically suspected pigs from 6 districts of Mizoram were examined, and clinical symptoms were recorded. Detailed post mortem examination of all the 31 dead animals was conducted, and gross changes were recorded. Tissue samples were collected for histopathological examination and molecular diagnosis. The collected tissues (tonsil, lymph nodes, spleen) were also processed for RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the specific gene fragments of classical swine fever virus (CSFV).Results:Clinical examination of all the 31 suspected pigs revealed typical clinical signs of CSF. All the animals also showed typical gross and microscopic lesions of CSF. RT-PCR on tissue samples amplified the 421bp, 449bp and 735bp region of 5´NCR, non-structural protein 5B and Erns gene regions of CSFV, respectively. Nested PCR for internal region of E2 gene also amplified the expected product of 271bp using PCR product of whole E2 region as template DNA.Conclusion:CSF is highly endemic disease in Mizoram. The viral strains circulating in this region are highly virulent. The disease can be diagnosed specifically using RT-PCR.

Specific ligands for classical swine fever virus screened from landscape phage display library

Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 1010 transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF.

Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field

Classical swine fever (CSF) is a highly contagious viral disease and may cause heavy economic loss to farmers. The rapid, simple and accurate diagnosis of the disease at the frontline, for example on the farms of concern is crucial for disease control. This study describes the development and evaluation of a new loop-mediated isothermal amplification (LAMP) assay coupled with lateral flow dipstick (LFD) for the detection of classical swine fever virus (CSFV). This RT-LAMP-LFD assay combines the efficient one-step isothermal amplification of CSF viral RNA and the simplicity of the LFD to read the results within two to five minutes. Seven genotypes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.1), but not genotype 3.4, were successfully detected by the RT-LAMP-LFD assay, indicating that the method has a broad range of detection and can be applied in different geographical areas where CSFV strains belonging to these genotypes are present. The performance of this RT-LAMP-LFD assay was similar to that of the real-time RT-PCR. The analytical sensitivity was about 100copies per reaction when testing two genotypes (1.1 and 2.3). No cross-reactivity to non-CSFV pestiviruses was observed. This RT-LAMP-LFD assay can be a useful novel tool for the rapid, simple and economic diagnosis of classical swine fever in the field.

Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR

High sensitivity, minor risk of cross-contamination and in particular the rapid reaction time make quantitative real-time polymerase chain reaction (qPCR) assays well suited for outbreak investigations as well as for monitoring epidemics of pathogens.In this study qPCR assays for three highly contagious animal diseases, namely foot-and-mouth-disease (FMD), influenza A (IA) and classical swine fever (CSF) have been developed. Furthermore, an amplification control targeting 18S ribosomal RNA was included. Each assay was validated with samples from infected animals using three different standard qPCR-machines in two thermal profiles: one standard and one high-speed approach, respectively. The high-speed PCR assays allowed the reliable diagnosis of FMD, influenza A and CSF in less than 28min with an analytical sensitivity of at least 200 genome copies/μl in every case, with slight differences regarding reaction time and sensitivity for the individual PCR-cycler instruments. Therefore, the newly established rapid RT-PCR systems will be a valuable method for the monitoring and control of these three important viruses and will be a robust option for the development of novel molecular pen-side tests.