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AGRICULTURAL RESEARCH COMMUNICATION CENTRE
www.arccjournals.com/www.ijaronline.in
Indian J. Anim. Res., 49 (4) 2015 : 512-514
Print ISSN:0367-6722 / Online ISSN:0976 -0555
Diagnostic potential of polymerase chain reaction in detection of classical swine
fever virus infection in slaughtered pigs
M. Rout*, G. Saikumar and K. Nagarajan1
Division of Pathology,
Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, India.
Received: 06-04-2014 Accepted: 02-12-2014 DOI:10.5958/0976-0555.2015.00083.7
ABSTRACT
The present work was carried out to study the diagnostic potential of reverse transcription polymerase chain reaction (RT-
PCR) in detection of classical swine fever virus (CSFV) infection in slaughtered pigs of Bareilly, Uttar Pradesh during 2005.
A total of 1120 pigs were examined for CSF suggestive pathological lesions and tissue samples were tested for CSFV
genome by RT-PCR. Out of 1120 cases examined, 110 (9.82%) showed lesions suggestive for CSF. Based on pathological
findings, 58.18% (64/110) were categorized as acute, 16.36% (18/110) as chronic and 25.45% (28/110) as inapparent form
of CSF. Based on RT-PCR targeting 5’NTR and E2/NS2 region, the prevalence of CSF was found to be 4.82% (54/1120)
and 3.83% (43/1120), respectively. RT-PCR thus ensures a sensitive and specific detection of CSFV and hence can be
considered as a screening method of choice.
Key words: Classical swine fever, Diagnosis, Polymerase chain reaction, Slaughtered pigs.
*Corresponding author’s e-mail: drmrout@gmail.com. Address: Project Directorate on Foot and Mouth Disease, IVRI
Campus, Mukteswar, Nainital, 263138, Uttarakhand, India.
1Department of Veterinary Pathology, Madras Veterinary College, Vepery, Chennai–600 007, Tamil Nadu, India.
INTRODUCTION
Classical swine fever (CSF), a listed disease of the
Office International des Epizooties (OIE), is an important,
highly contagious and devastating animal disease of great
concern worldwide (Zhang et al., 2011a). CSF is caused by
Classical swine fever virus (CSFV), a member of the genus
Pestivirus within the family Flaviviridae, and engenders
important economic ramifications (Meyers and Thiel, 1996;
Zhang et al., 2011b). It is a leading cause of death in pigs and
endemic in India, but the real intensity of the problem is not
known as only outbreaks of acute disease are reported and
many cases of chronic and clinically inapparent forms remain
undiagnosed. In the present study, the diagnostic potential of
reverse transcription polymerase chain reaction (RT-PCR)
in detection of CSFV infection was evaluated by examination
of tissue samples of infected pigs from a slaughterhouse in
Bareilly, Uttar Pradesh during 2005.
MATERIALS AND METHODS
Tissue specimens were collected at random from
1120 pigs over a period of six months during 2005 from a
local abattoir in Bareilly, Uttar Pradesh. The carcasses were
examined for CSF suggestive pathological lesions and
lymphoid tissues were collected from mesenteric lymph
nodes, spleen and tonsil. Acute, chronic and inapparent forms
of CSF were observed based on standard gross lesions as
described by Van Oirschot (1999) and Moennig et al. (2003).
In brief, acute CSF is characterized by exudative
conjunctivitis, petechial or ecchymotic haemorrhages in the
skin, epiglottis, larynx, kidneys (turkey-egg kidney), in all
visceral organs along with pathognomonic splenic infarcts.
In chronic CSF, button-ulcers are present in the large intestine,
while thymic atrophy and exostoses at the costo-chondral
junctions of the ribs are the commonest lesions in inapparent
or late onset infections.
For evaluation of RT-PCR assay, total RNA was
extracted from spleen of all cases using TRIZOL reagent
(Invitrogen, USA) and stored at -700C. For RT-PCR, the
genomic region 5’NTR (pan-pestivirus specific) and E2/
NS2 (CSFV specific) were targeted for amplification of a
232 bp and 308 bp pr oduct, respectively. The
oligonucleotide primers targeting 5’NTR (HCVgt1sense
5’-CTGGGTGGTCTAAGTCCTGA-3’; HCVgt4 antisense
5’- TCAACTCCATGTG CCATGTA-3’) and E2/NS2
(HCVgp5-sense 5’- ATATATGC TCAAGGGCGAGT-3’;
HCVgp8-antisense 5’- ACAGCAG TAGTATCCATTTCT
TTA-3’) were as per Katz et al. (1993). Reverse
transcription was carried out in a 20 µl reaction volume
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Volume 49 Issue 4 (August 2015) 513
FIG 1: RT-PCR amplicons of 232 bp of CSFV targeting 5’ NTR
region
M 1 2 3 4 5 6 7
FIG 2: RT-PCR amplicons of 308 bp of CSFV targeting E2/NS2
region
M 1 2 3
using 2 µg of total RNA, 10 pmol specific reverse primer
HCVgt4 / HCVgp8 and 200 un its of M-MLVRT
(Invitrogen, USA) as instructed. Two microlitre of cDNA
was amplified in a RT-PCR mixture of 25 µl volume
containing 5X PCR buffer, 25 mM MgCl2, 10 mM dNTP
mix, 10 pmol forward primer HCVgt1 / HCVgp5, 10 pmol
reverse primer HCVgt4 / HCVgp8 and 2.5 u/µl Taq DNA
polymerase (Fermentas) in a thermal cycler (Techne
Genius, USA). After initial denaturation at 940C for 5
minutes, 35 amplification cycles were conducted with each
cycle consisting of 940C for 45 seconds (denaturation),
590C (for 5’NTR) / 580C (for E2/NS2) for 45 seconds
(annealing), and 720C for 45 seconds (extension), followed
by a 10 minutes final extension at 720C. The confirmation
of specific RT-PCR amplicons was done by 1.5% agarose
gel electrophoresis containing ethidium bromide (0.5µg/
µl).
RESULTS AND DISCUSSION
Of 1120 cases examined, 110 (9.82%) cases
showed lesions suggestive for CSF. Based on pathological
findings, 58.18% (64/110) cases were categorized as acute
CSF, 16.36% (18/110) as chronic and 25.45% (28/110) as
inapparent form of CSF. Based on the detection of viral
genome using RT-PCR targeting 5’NTR and E2/NS2
genomic region, the prevalence of CSF was found to be
4.82% (54/1120) and 3.83% (43/1120), respectively. The
RT-PCR amplicons were observed at expected 232 bp for
5’NTR (Fig. 1) and 308 bp for E2/NS2 region (Fig. 2).
CSF in its acute form generally results in high morbidity
and mortality. But the chronic and inapparent forms are
very difficult to diagnose as the pathological lesions are
often diverse and misleading. The histopathologic changes
in the lymphoid tissues of pigs with chronic CSFV infection
are non-specific (Benson, 1952; Urman et al., 1962).
Secondary bacterial and parasitic infections are frequently
observed in pigs with chronic CSF. In the clinically
inapparent form, characteristic clinical signs are not
manifested. Unlike in acute CSF, it is virtually impossible
to make a clinical diagnosis in case of chronic or persistent
CSF because of the wide variability of clinical signs and
pathological lesions (Mengeling, 1970). Conclusive
diagnosis of CSF is therefore not possible without suitable
laboratory confirmation (Young, 1970), because of the
variable and atypical clinical signs that may sometimes
resemble bovine viral diarrhoea (BVD) (Terpstra and
Wensvoort, 1988). This might be one of the probable
causes of relatively more number of samples found positive
in 5’NTR-PCR as compared to E2/NS2-PCR. As 5’NTR
is conserved among all pestiviruses, whereas E2/NS2 is
CSFV specific; hence those samples found positive for
5’NTR, but negative for E2/NS2, might be suspected for
BVDV or border disease virus (BDV) infection that needs
further investigation. Both BVDV and BDV have
previously been reported to cause clinically inapparent
infections in pigs (Gaede, 2002). The CSF confirmed cases
classified as per the gross lesions are presented in Table
1. In this study, RT-PCR technique was found to be a
viable, rapid and sensitive method for detection of CSFV
in infected pigs, therefore, can be used as an essential
element of the laboratory armoury towards confirmatory
diagnosis of CSF, when misleading and non-specific
clinical picture create confusion. Much progress in CSF
molecular diagnosis has been made till date, which will
undoubtedly accelerate the way of approach in disease
diagnosis in future.
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514 INDIAN JOURNAL OF ANIMAL RESEARCH
TABLE 1: Gross pathological findings in classical swine fever (CSF) confirmed cases
Attributes Acute CSF cases (total 59) Percentage
Haemorrhagic lymph nodes 59 100
Pulmonary haemorrhage 22 37.28
Splenic infarcts 19 32.20
Kidney haemorrhage 10 16.94
Intestinal haemorrhage 7 11.86
Cutaneous haemorrhage 6 10.16
Cardiac haemorrhage 4 6.77
Gastric haemorrhage 4 6.77
Epiglottal haemorrhage 3 5.08
Urinary bladder haemorrhage 2 3.38
Necrotic tonsillitis 2 3.38
Exudative conjunctivitis 1 1.69
Brain haemorrhage 1 1.69
Attributes Chronic CSF cases (total 10) Percentage
Ulcers in caecum and colon 10 100
Regressing splenic infarcts 8 80
Blotching effect on the ear 2 20
Pale or gelatinized lymph nodes 1 10
Attributes Atypical/inapparent CSF cases (total 17) Percentage
Thymic atrophy 17 100
Swollen pale lymph nodes 16 94.11
Exostoses at the costo-chondral junctions of ribs No case detected 0
ACKNOWLEDGEMENTS
The authors gratefully acknowledge and thank the
Director, IVRI, Izatnagar for supporting the study in terms
of funds and facilities. Due acknowledgement is also extended
to the slaughterhouse owners and workers who helped in
providing samples.
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