Evaluation of single step TaqMan real-time PCR assay lateral to conventional RT-PCR and antigen-capture ELISA for pre-clinical detection of classical swine fever virus

- Evaluation of Single Step TaqMan Real-time PCR Assay Lateral to Conventional RT-PCR and Antigen-Capture ELISA for Pre-Clinical.
- Detection of Classical swine fever virus.
- Classical swine fever (CSF) is a highly contagious and devastating viral disease, causing serious losses in the pig industry worldwide.
- In the present study, a fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase real time PCR assay (RT-qPCR)was evaluated parallel to conventional RT-PCR and antigen capture ELISA to detect Classical swine fever virus (CSFV) in the pre-clinical phase of the disease.
- Single step RT-qPCR confirmed the presence of CSFV nucleic acid in blood, nasal swabs, ocular swabs as well as in tonsillar scrapings in the pre-clinical phase.
- Thus, TaqMan based RT-qPCR assay can be used as an efficient assay for rapid CSFV detection at pre-clinical phase of the disease to contain the disease from in-contact infected pigs to susceptible population..
- Classical swine fever virus, pre- clinical detection, TaqMan RT-qPCR, RT-PCR, antigen capture-ELISA.
- Classical swine fever (CSF)is a highly contagious, economically devastating disease of domestic pigs, wild boars and pygmy hogs notifiable to the World Organization for Animal Health (OIE)(Depner et al., 1994;.
- Dewulf et al., 2004).
- The disease is caused by Classical Swine Fever virus (CSFV), a positive-sense, enveloped virus belonging to the genus Pestivirus of the family Flaviviridae (https://talk.ictvonline.org/ictv.
- Ribbens et al., 2004.
- Blome et al., 2017).
- The principal mode of entry of CSFV in pigs under natural infections is the oro-nasal route although other possible routes such as the conjunctival, genital mucous membranes and skin abrasions have been described(Floegel et al., 2000.
- The incubation period of CSFV is typically 3-10 days following an infection (Postel et al., 2018)..
- Depending upon the virulence of CSFV, host age, status of individual or herd immunity, CSFV exhibits anacute, chronic and persistent disease mode in host animals (Isoda et al., 2020).Typical clinical signs of CSFV in natural infections include high fever (>40.
- et al., 2018).
- However, these symptoms are seldom visible in animals infected with strains of varied virulence and infected animals might develop a mild, chronic or unapparent form of the disease (Tarradas et al., 2014).
- Rapid, sensitive and specific pre-clinical diagnostic methods are necessary for early identification of infected herds to contain further spread of the disease and to control CSF epidemics.
- Detection of CSFV in live animals has been performed traditionally by a combination of antigen detection and virus isolation using blood samples (Kaden et al., 1999).
- Antibody against CSFV can be detected by virus neutralization test or by antibody-ELISA but infected antibody appears 2-3 weeks of post infection (Ganges et al., 2020).Therefore, to provide a precise diagnosis in the face of an outbreak, conventional methods of antigen detection (antigen capture ELISA)is practically not always feasible because of its low sensitivity..
- As a routine diagnostic tool, RT-PCR targeting a highly conserved viral gene is more sensitive in early detection of CSFV during the incubation period (OIE 2019)..
- In such situations, real-time PCR protocols (RT-qPCR) helps to increase the throughput, reduces the chance of carryover contamination and disables post-PCR processing as a potential source of error (Hoffmann et al., 2005.
- Ciglenečki et al., 2008)..
- In the present study, we evaluated a single step TaqMan based real-time RT-PCR (RT-qPCR) in parallel to conventional RT- PCR and antigen capture ELISA(Ag- ELISA)for pre-clinical detection of CSFV in infected pigs from natural outbreaks reported from Assam..
- In each CSFV affected farm/unit, animals were categorized as per pre-clinical (Group I), early clinical (Group II) and late clinical phase (Group III) of the disease based on clinical parameters(Table 1)previously described by Mittelholzer et al., (2000)with some modifications..
- Biological samples such as nasal and ocular swabs, tonsillar scrapings and whole blood collected from each animal at pre-clinical and late clinical phase of the disease were processed and tested by single step RT-qPCR, nested RT-PCR and Ag-ELISA..
- Haematological analysis was carried out in a total of 70 blood samples collected at pre- clinical (n=30) and late clinical phase (n=30) of the disease.
- Detection of CSFV antigen and nucleic acid.
- Detection of CSFV antigen in clinical samples was done using CSFV antigen test kit (IDEXXCSFVAg Serum Plus Test, IDEXX Laboratories, USA) following manufacturer’s instruction.
- For detection of CSFV nucleic acid, viral RNA was extracted using the QIAamp RNA Kit (QIAgen, Hilden, Germany) according to the manufacturer’s instructions.
- Single-step TaqManRT-qPCR was performed to detect CSFV genome as per the method described by Hoffmann et al., (Hoffmann et al., 2005).
- Animals in the pre-clinical phase was categorized as Group I and consists of in- contact pigs that have not exhibited any CSFV specific clinical symptoms post outbreak.
- Pigs in the early-clinical phase that presented an acute infection within day 1-4 post CSFV infection was categorized as Group II..
- Infected pigs presenting a late clinical phase.
- Hematological examination revealed marked leucopaenia and thrombocytopaenia in the blood samples collected from in-contact animals at pre-clinical phase of the disease (Table 2).
- In the pre-clinical phase, all the in- contact pigs apparently seemed to be healthy showed drop in TLC, total platelet count and lymphocyte percentage in comparison to healthy pigs.
- to pre-clinical phase while the granulocyte count dropped in the late clinical phase compared to pre-clinical phase (Fig.
- Maximum samples (73% in pre-clinical.
- in late clinical) were found positive in single step RT-qPCR, followed by E2 gene based nested RT-PCR (60% in pre-clinical.
- 26% in late clinical) and CSFV Ag-ELISA (40% in pre-clinical.
- Again, maximum CSFV positive cases (70-73%) were detected in samples collected in the pre- clinical phase as compared to 40-53% positive samples in the late clinical phase..
- In whole blood, CSFV RNA was detected by single step RT-qPCR upto late clinical phase (40%) but at pre-clinical phase maximum samples (73%) were found to be positive (Fig..
- Whereas, in tonsillar scrapings, nasal and ocular swabs, CSFV nucleic acid was detected at pre-clinical phase and percent positive was 70%, 53% and 60% respectively.
- Although nested RT-PCR could detect biological samples collected at the pre- clinical phase of the disease (Fig.
- Laboratory investigation results of the present study clearly showed that both viral antigen and nucleic acid could be detected in all clinical samples (blood, nasal swabs, ocular swabs and tonsillar scrapings) collected at pre- clinical phase.
- However, at late clinical phase CSFV nucleic acid could be detected only in blood and in tonsillar scrapings..
- Table.1 Categorization of clinical symptoms of CSF in pre-clinical, early clinical and late clinical phase as per days post infection (dpi).
- Pre-clinical.
- Table.3 Detection of CSFV in various clinical samples in pre-clinical and late clinical phases.
- Pre-clinical phase (No.
- Late clinical phase (No.
- ±6.475 Pre-clinical.
- clinical phase.
- Fig.1 Total Platelets and leukocyte count (TLC) in control healthy pigs and CSFV infected pigs in pre-clinical and late clinical phase.
- Fig.2 Differential leukocyte count (DLC) in healthy pigs and CSFV infected pigs in pre-clinical and late clinical phase.
- Fig.3 Amplification curves of CSFV using TaqMan RT-qPCR from blood samples and tonsillar scrapings collected at pre-clinical phase of the disease.
- Fig.4 Nested RT-PCR amplification of CSFV-E2 gene from different clinical samples collected at pre-clinical phase..
- 195 Dewulf et al., (2004) stated that the nested RT-PCR is suitable for early detection of CSFV in blood on average 2.8 days earlier than the isolation of CSF virus.
- In contrast, TaqMan probe-based RT-qPCR is an accurate, rapid and reliable assay for the detection of CSFV.
- Experimental study carried out by Everett et al., (2010) demonstrated detection of viral nucleic acid in blood at preclinical phase prior to excretion of virus at nasal secretions.
- It is interesting to note from the present study that viral RNA could be efficiently detected in blood, tonsillar scrapping, nasal and ocular swabs using Rt- qPCR with maximum positivity (70-73%) from the CSFV infected animal in the pre- clinical phase prior revealing any prominent CSFV specific clinical signs.
- In blood as well as in tonsil high percentage of virus positive samples was identified up to mid clinical phase of CSF infection.
- Present findings thus emphasize the importance of blood, tonsillar scrapings, nasal and ocular swabs as suitable clinical samples for pre-clinical diagnosis of CSFV infection by TaqMan RT-qPCR assays..
- Blood samples and tonsillar scrapings could be the clinical material of choice for detecting CSF wild type virus infection at pre-clinical or early stage of the disease (Donahue et al., 2012).
- On the other hand, low percentage of CSFV antigen detected by Ag-ELISA in blood further proved that Ag-ELISA is not suitable diagnostic tool for early clinical detection (Shivaraj et al., 2013).Negative result in Ag- ELISA does not eliminate true positive cases..
- Along with early clinical signs, hematological examination in-contact pigs can be an indirect way for presumptive diagnosis of CSFV infection.
- The present study showed two-to- three-fold decrease in leukocyte and thrombocyte counts in pre-clinical phase in all age groups of affected pigs.
- Remarkably, thrombocytopenia appeared quite ahead of clinical manifestation of the disease and even 4-5 days prior to detection of CSFV in blood.
- It was postulated that peripheral platelet depletion is due to disseminated intravascular coagulation and phagocytosis of activated/damaged platelets(Calderón et al., 1998.
- Bautista et al., 2002).
- Finding of the present study suggests that no marked thrombocytopenia and leukopenia persist at late clinical phase of CSFV infection but can be distinctive at the pre or early clinical phase of the disease.
- In summary, the single-step RT-qPCR assay is a simple, fast, highly sensitive and specific method for the pre-clinical detection of CSFV in clinical samples.
- The protocol presented is easy to perform and can be used for rapid detection of CSFV infected pigs or wild boars..
- Our findings could be valuable in order to diagnose the pre-clinical cases of CSF infection in an endemic locality..
- The authors would like to thank the Department of Biotechnology, Government of India, for providing financial help to carry out the research under the project entitled, ‘DBT Network project on Classical Swine fever with special reference to North Eastern Region’ and subproject entitled, ‘Classical swine fever:.
- Surveillance and monitoring in some North Eastern States and Establishment of National Classical Swine fever virus Repository’..
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- Evaluation of Single Step TaqMan Real-time PCR Assay Lateral to Conventional RT-PCR and Antigen-Capture ELISA for Pre-Clinical Detection of Classical swine fever virus

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