Classical Swine Fever Virus Antigen Research Articles

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.

Pathology of the outbreak of subgenotype 2.5 classical swine fever virus in northern Vietnam

Classical swine fever (CSF) is an endemic disease in southeastern Asia and is one of the most important swine diseases in Vietnam. This study was conducted to characterize the pathology of natural cases of CSF in northern Vietnam in 2018 and their genetic prevalence. A total of 10 representative pigs were collected from four provinces (Hung Yen, Ha Noi, Quang Ninh and Thai Binh) during five outbreaks and examined pathologically. The gross and histopathological findings showed the disease was expressed as the acute or the subacute to chronic form of CSF, depending on the age of the animals. The most consistently observed lesions associated with infection by the classical swine fever virus (CSFV) included lymphoid depletions in tonsils, lymph node and spleen; histiocytic hyperplasia in spleen; cerebral haemorrhage; perivascular cuffing in the brain; renal erythrodiapedesis; urothelial vacuolation and degeneration and interstitial pneumonia. The immunohistochemical findings showed a ubiquitous CSFV antigen mainly in the monocytes/macrophages and in the epithelial and endothelial cells in various organs. CSFV neurotropism was also found in the small neurons of the cerebrum and the ganglia of the myenteric plexus. Analysis of the full‐length envelope protein (E2) genome sequence showed that all strains were genetically clustered into subgenotype 2.5, sharing a nucleotide identity of 94.0%–100.00%. Based on the results of this study, the strain was categorized as a moderately virulent CSFV.

Development of Plant-produced E2 Protein for Use as a Green Vaccine Against Classical Swine Fever Virus

Plants are promising host systems for recombinant protein production. However, progress in the commercialization of plant-made proteins (PMPs) has been slow. Only one PMP drug is commercially available. In this study, we explored the possibility of using plants to produce E2 of classical swine fever virus (CSFV) and the use of this plant-produced E2 as a vaccine. We designed high-level expression vectors for transgenic plants by considering the transcription, translation, and storage of E2 in the cell. We incorporated a cellulosebinding domain sequence into the expression vector as an affinity tag for cost-effective, one-step purification. Using this vector, we generated multiple lines of transgenic Arabidopsis thaliana plants expressing a fusion protein of E2 from CSFV at high levels (0.7% of total soluble proteins). ER-targeted E2 fusion protein was successfully purified via a one-step purification process using amorphous cellulose resin. Arabidopsis-produced E2 was recognized by an antibody that detects CSFV antigen. Finally, antisera from mice immunized with E2 fusion protein reacted strongly to the antigens in a CSFV antibody detection kit. Therefore, we propose that plant-produced E2 fusion proteins could be further developed for use as a green vaccine against CSFV in animals.

Standardization and application of rapid tests viz dot ELISA and vertical flow through tests for serodiagnosis of classical swine fever

The present study was carried out to develop rapid field-based immunoassays for onsite detection of Classical swine fever virus (CSFV) antibody in clinical serum sample. Purified CSFV antigen was coated onto nitrocellulose membranes housed in a plastic module with layers of absorbent filter pads underneath. Following addition of serum to be tested appearance of a red dot indicated the presence of antibodies to CSF virus in the sample tested. A total of 240 serum samples collected from different areas of North Eastern States of India were tested for the presence of CSFV antibody by Indirect ELISA, Dot ELISA and Vertical flow through test. The result of the Indirect ELISA could be obtained within 2.5 hours whereas the result in case of Dot ELISA and Vertical Flow through test was obtained within 1 hour and 15 minutes respectively. Dot-ELISA and Vertical flow through test could equally detect the CSFV antibody in samples possessing 1:4 and above ELISA titre. The specificity and sensitivity of Dot-ELISA and vertical flow through test after comparison with Indirect ELISA indicated 100% specific and 84.28% sensitive. These tests have the potential to be used as a field-based kit to assess the serodiagnosis of CSFV.

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.

Prevalence of classical swine fever in Karnataka, India.

Aim:The present study was conducted to know the current scenario of classical swine fever (CSF) in Bengaluru Urban, Bengaluru Rural, Chikkaballapur, Madikeri, Mandya, Bagalkot, Gadag, Yadgir, Koppal, and Bidar districts of Karnataka with the using of both antigen and antibody ELISA.Materials and Methods:We collected 218 sera and 121 blood samples from pigs from 10 different districts of Karnataka. Screening of sera for CSF IgG antibody and whole blood for CSF virus antigen were carried out using the CSF virus (CSFV) antibody and antigen ELISA kits, respectively.Results:The mean seroprevalence was 41% (89/218) and prevalence of CSFV antigen in blood samples was 32% (39/121) for the 10 districts of Karnataka. Seroprevalence of 61%, 29%, 20%, and 21%; and antigen prevalence of 40%, 50%, 13%, and 12% were recorded for Bangalore, Mysore, Belgaum, and Gulbarga divisions of Karnataka, respectively.Conclusions:The study revealed an alarmingly high prevalence of CSF, both for the antigen (32%) and antibody (41%) in Karnataka. Southern Karnataka has the highest seroprevalence (61% in Bangalore and 29% in Mysore divisions), which confirms the endemicity of the disease in that region. This could be attributed to the intensive pig farming practices in the region as compared to Northern Karnataka (Seroprevalence of 20% in Belgaum and 21% in Gulbarga divisions), where the commercial pig farming is still in infantile stages.

Detection of Classical Swine Fever Virus Antigen and Nucleic Acid on Blood of Experimentally Infected Piglets

Detection of Classical Swine Fever Virus Antigen and Nucleic Acid on Blood of Experimentally Infected Piglets

Virus Load in Pigs Affected with Different Clinical Forms of Classical Swine Fever

Classical swine fever (CSF) is an endemic disease in India, but the real magnitude of the problem is not known as only outbreaks of acute CSF are reported and many cases of chronic and clinically inapparent forms of the disease, which manifest a confusing clinical picture, remain undiagnosed. The real status of classical swine fever virus (CSFV) infection can only be known by testing pigs with highly specific and sensitive diagnostic assays. To obtain the baseline prevalence of CSFV infection among pigs in an endemic region where no vaccination was being performed, a real-time PCR assay was used to detect viral genetic material in tissue samples collected from a slaughterhouse in the northern state of Uttar Pradesh in India. In total, 1120 slaughtered pigs were examined for the presence of CSF suggestive pathological lesions and tissues from suspected cases were tested for the presence of CSFV antigen and nucleic acids by indirect immuno-peroxidase test and real-time PCR, respectively. Based on the detection of viral genetic material in the tonsils, the prevalence of CSFV infection among slaughtered pigs was found to be 7.67%. Pigs detected positive for viral genome by quantitative real-time PCR assay when categorized into different forms of CSF, depending upon the pathological lesions observed, the viral load in the tonsils of some of the pigs with chronic or clinically inapparent form of the disease was similar to that detected in pigs with acute CSF. The results of the study suggested that the risk posed by pigs with chronic disease or those infected but showing no clinical disease may be relatively higher as they can transmit the virus to new susceptible hosts over a longer period of time.

Prevalence of Classical Swine Fever Virus in India: A 6-Year Study (2004-2010)

A study was undertaken regarding the prevalence of classical swine fever virus (CSFV) antibodies and antigens in sera and suspected tissue samples of domestic pigs. The samples were received between January 2004 and September 2010. A total of 594 serum samples from 12 states and 287 tissue samples from 13 states of India were tested using commercial enzyme-linked immunosorbent assay (ELISA) kits. The mean prevalence of CSFV antibodies in suspected sera was 63.3% (376/594), whereas 76.7% (220/287) of the suspected samples were found to contain CSFV antigen. The high prevalence of CSFV antibodies suggests that the disease is endemic in the country. This baseline data will be of use in the formulation of control and eradication programmes.

Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen

Classical swine fever (CSF) is a highly contagious and severe viral disease of swine resulting in substantial production losses in different farming systems in many regions of the world. The accurate and rapid detection of CSF outbreaks is reliant on sensitive and specific laboratory testing and is a key component of disease control. Specific detection of CSF virus can be achieved by virus isolation in tissue culture, antigen capture or the detection of viral RNA using molecular techniques. In order to reduce the time taken to achieve a diagnostic result and simplify testing methods, an antigen capture ELISA using immunomagnetic beads (IMB) as the solid phase was developed and compared to a microplate-based antigen capture (AC)-ELISA. The IMB-ELISA has up to 64-fold greater analytical sensitivity than the AC-ELISA and initial estimates of diagnostic sensitivity and specificity are 100%. The IMB-ELISA has a highly robust, rapid and stable test format and is simpler to perform than the AC-ELISA. The IMB-ELISA has the added advantage that a result can be sensitively and specifically determined by eye, lending it to the possibility of adaptation to a near-to-field test with minimal equipment or expertise needed.

Value of Skin Punch Biopsies for the Diagnosis of Acute Classical Swine Fever

The objective of this study was to determine if skin punch biopsies are appropriate for the diagnosis of classical swine fever. For this purpose, 6 wild boars and 2 domestic pigs were experimentally infected with the highly virulent classical swine fever virus (CSFV) Koslov and 5 domestic pigs with a CSFV field isolate (genotype 2.3 Uelzen) derived from wild boar. Skin biopsy specimens were tested using virus isolation, real-time reverse transcription polymerase chain reaction (rtRT-PCR), and fluorescent antibody test (FAT) on cryosections. Whereas CSFV Koslov was first detected at 4 days postinfection (DPI) by rtRT-PCR and virus isolation, FAT failed to detect CSFV antigen until 9 DPI. In domestic pigs infected with CSFV 2.3 Uelzen, viral RNA and CSFV were detected at 7 DPI. FAT was negative until 11 DPI. CSFV antigen was detected in endothelial cells of the vascular plexus in the upper dermis as shown by confocal laser-scanning microscopy and double labeling with von Willebrand factor. At 18 DPI, CSFV antigen was present diffusely in capillaries and spindle shaped cells of the dermis, multifocally within keratinocytes of the epidermis and in numerous cells of the inner and outer root sheath epithelium, hair bulb, and intravascular leukocytes. The rtRT-PCR proved to be the test with the highest sensitivity followed by virus isolation and FAT. Taken together, this study demonstrates that skin is easy to sample antemortem and is also suitable as postmortem tissue, and suggests that rtRT-PCR of skin should be included for CSF diagnosis in the acute period of disease.

Porcine interleukin-3 enhances DNA vaccination against classical swine fever

DNA vectors can be used to deliver vaccine antigens that stimulate effective protective immunity in mice, but in larger, outbred animal species, the protective efficacy is lower or large doses of DNA are required. These data demonstrate that porcine interleukin-3 (IL-3) when delivered to pigs by DNA vector or in low doses as recombinant protein, can enhance antibody responses to classical swine fever virus antigen expressed from co-delivered DNA, and improve the protective efficacy of the DNA vaccine. The effect was further enhanced when IL-3 was expressed as a fusion protein with the potyvirus coat protein. The adjuvant effect of IL-3 was compared to that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 was shown to be at least as efficacious as GM-CSF. The response to IL-3 is novel and suggests, that at least in pigs, IL-3 could be used as an adjuvant for DNA vaccines.

Nictitating Membrane as a Potentially Useful Postmortem Diagnostic Specimen for Classical Swine Fever

The gold standard for diagnosis of classical swine fever (CSF) is cell culture virus isolation combined with reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent antibody test (FAT) in cryosections of tonsils, spleen, various lymph nodes, ileum, and kidney. Autolytic and heterolytic samples render correct FAT evaluation difficult and can even yield false-negative or ambiguously positive results. To extend the spectrum of CSF diagnostic specimens, the authors tested whether the nictitating membrane (NM) might be a useful adjunct diagnostic specimen in wild boars and domestic pigs. To accomplish this, results of virus isolation, FAT, and RT-PCR were compared on NM samples and lymphoid tissues, which are the routine specimens of choice for CSF diagnosis. Wild boars (n = 30) and domestic pigs (n = 8) were experimentally challenged with various CSF virus (CSFV) strains or isolates of different virulence. The FAT revealed CSFV antigen in surface and tubular adenoid epithelium as well as in lymphatic follicles of the NM. In wild boars and domestic pigs with CSF, a strong agreement was found between results of FAT, virus isolation, and RT-PCR on NM and lymphoid tissues. These results suggest that NM is a useful additional specimen that can provide valuable data for postmortem diagnosis of CSF. The NM is relatively easy to sample at necropsy, and postmortem autolysis and heterolysis of this tissue is minimal compared with internal organs.

Detection of classical swine fever virus in boar semen by reverse transcription-polymerase chain reaction.

A seminested reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of classical swine fever virus (CSFV) in semen. Five boars were inoculated intranasally with CSFV isolate propagated in PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Semen and serum samples were collected twice weekly for 63 days postinoculation (dpi). Samples were tested for the presence of antibodies to CSFV by an enzyme-linked immunosorbent assay and for the presence of CSFV nucleic acid by seminested RT-PCR. Antibodies to CSFV could be detected as early as 7 dpi in 1 boar, and all 5 infected boars were found positive by 14 dpi. CSFV from boar semen was infrequently identified by virus isolation compared with seminested RT-PCR. CSFV nucleic acid was detected in semen by seminested RT-PCR as early as 7 dpi in 3 infected boars and persistently thereafter in all 5 infected boars until 63 dpi. When separated fractions of CSFV-contaminated semen were analyzed by the seminested RT-PCR, the CSFV nucleic acid was detected mainly in seminal fluid and occasionally in nonsperm cells. CSFV antigen was also detected in nonsperm cells from semen smear by immunohistochemistry. Thus, infection via semen, specially through CSFV-infected seminal fluid, seems to be a major route of transmission of CSFV.

Detection of Classical Swine Fever Virus in the Ovaries of Experimentally Infected Sows

Six sows were infected intranasally with a Korean isolate of classical swine fever virus (CSFV). The distribution of virus in ovarian tissues was then assessed for 21 days by in-situ hybridization and immunohistochemistry. CSFV was detected in the ovaries between 7 and 21 days post-inoculation (dpi) by both methods, but the labelling was particularly intense and widespread at 7dpi. CSFV nucleic acid and antigen were located almost exclusively within the cytoplasm of cells shown by haematoxylin and eosin staining to be macrophages, which were numerous in atretic follicles. Small numbers of CSFV nucleic acid-positive cells with distinctly round morphology and oval nuclei, resembling monocytes, were also observed in the blood vessels of sows at 7 and 14dpi. CSFV nucleic acid and antigen were not observed in primordial, primary or secondary follicles from infected sows at 7, 14 or 21dpi. The results suggest that CSFV replicates in circulating peripheral monocytes and gains access to ovarian tissues from the bloodstream, and that this contributes to the distribution of CSFV in macrophages throughout the atretic follicles.

Detection of the classical swine fever virus antigen following experimental infection

In this experiment, twelve pigs were infected by intramuscular inoculation of virulent virus strains of classical swine fever (Baker, Kansas, BAI autochthonic isolate). Three animals survived infection without any clinical symptoms and were sacrificed 60 days post infection (p.i). Nine animals developed an acute form of classical swine fever and died between 8 and 15 days p.i. Rectal temperature was monitored daily along with the clinical symptoms and usual hematological parameters. Viremia was determined by two immunoenzyme-ELISA tests (CHEKIT CSF VIRUS DR BOMMELIAG, Switzerland, and CSFV-IDEXX, USA). Pathomorphological changes were determined post mortem and tissues were examined for the presence of virus antigen by a fluorescent antibody - based test and' immunoenzyme test. In infected animals viremia occured between 3 and 5 days post infection. Virus antigens were detected in 73.03% of the examined blood samples using the Dr Bommeli test and in 56.18% using the IDEXX test. Viremia was not detected in animals that survived infection. Between day 3 and 5 p. i. leukopenia, lymphopenia and thrombocytopenia were observed in eight infected animals. Viral •antigens were confirmed by the fluorescent antibody based test in 72.2 % of the examined tissues, by the IDEXX test in 84.4% and by the Dr Bommeli test in 100% samples.

Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge.

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise ( S/ N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation ( n=47) averaged a S/ N ratio of 5.34. In contrast, samples negative by virus isolation ( n=96) demonstrated a mean S/ N ratio of 0.16. At S/ N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.