S.S. Patel*, N.M. Shah, H.C. Chauhan, B.S. Chandel,
M.D. Shrimali, 
A.C. Patel, K.B. Patel, M.A. Patel, B.K Patel, M.G. Patel, J.K. Kala and Manish Rajgor

Department of Veterinary Microbiology, Veterinary College, S.D.A.U., Sardarkrushinagar, India.

Abstract

Bluetongue (BT) is an infectious, non-contagious disease of domestic and wild ruminants. Bluetongue virus (BTV) causes severe disease in sheep, which is transmitted by insect vector belonging to Culicoides spp. It is particularly a viral disease of sheep, occasionally affecting cattle, buffaloes, goats, camels and other wild ruminants. Out of 377 (364-blood, 5-spleen and 8-pooled Culicoides) samples 110 (29.18%) and 28 (7.42%) were found positive for BTV antigen by s-ELISA and BT-AGID respectively. Species wise incidence by s-ELISA recorded was 48.20 per cent in sheep, 57.14 per cent in goats and 2.60 per cent in cattle however, none of the blood sample found positive from buffalo and camel. Specieswise incidence by BT-AGID recorded was 12.23 per cent in sheep and 15.71 per cent in goats however, none of the blood sample found positive for BTV antigen from cattle, buffalo and camel. Higher incidence seen in goats by both the test. s-ELISA proved to be the most sensitive in detecting BTV antigen than BT-AGID. Considering s-ELISA as the reference test, the relative sensitivity, specificity and overall agreement between both the tests were 25.45 per cent, 100 per cent and 78.24 per cent respectively.

Keywords: Bluetongue virus, antigen, s-ELISA, BT-AGID.

INTRODUCTION

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease, principally of sheep but many domestic and wild animals are also affected by this disease3. The severity of BT in sheep varies depending on the virus, the breed of sheep and environmental stress. Infection in cattle and goats is usually sub-clinical. Etiological agent of the disease belongs to the genus Orbivirus in the family Reoviridae. Twenty seven serotypes of BTV have so far been recognized worldwide and many more may be prevalent in regions where no survey has been made so far10. BTV infection occurs in most of the tropical, semitropical and temperate region of the world in parallel with the distribution of its vectors. The regions which are considered endemic areas of bluetongue virus are inhabited by 2/3 of sheep and cattle population around the globe between 400 North and 350 South9. The disease is characterized by high fever, excessive salivation, swollen lips and tongue, petechial hemorrhage, congestion and small ulcer in the mucous membrane of mouth and conjunctiva, coronitis and reproductive disorders leading to abortion or congenital deformities. The disease is a cause for serious concern to the livestock industry.

Studies on Bluetongue in Gujarat is concerned various workers have reported the existence of Bluetongue in Gujarat based on detection of group specific BTV antibodies, Serotypes specific antibodies, antigen detection by AGID, RT-PCR based detection and isolation of BTV2,4,6. However, no systemic studies based on surveillance on detection of  BTV in wide host ranges including sheep, goats, cattle, buffaloes and camels, using sandwich ELISA has been carried out.

MATERIAL AND METHODS    

During the present investigation, s-ELISA and BT-AGID were used for the detection of BTV group specific antigen from the blood samples, Spleen samples and pooled Culicoides. A total of 364 blood, five spleen and eight pooled Culicoides samples were collected from different places of Gujarat for detection of BTV antigen.

 Preparation of samples:

The blood samples mixed in lysis buffer in 2:1 proportion were kept at 40C for overnight and used in the test. The tissue materials were ground in mortar and pestle in sterile 1X PBS (pH 7.4) to make 10 per cent (w/v) tissue suspension followed by sonication and centrifuged at 10,000X g for 30 minutes and the resulting supernatant was used. The Culicoides were ground in mortar and pestle in sterile 1X PBS (pH 7.4) to make suspension and sonication and used for the test.

 s-ELISA:

The test was performed as per the standard protocol developed at BTV Lab, IVRI, Mukteswar8 for the detection of BTV antigen from blood, Culicoides and spleen samples.  

  1. Capture antibody (50 ml rabbit HIS to whole BTV-23 purified) was coated on to wells of ELISA plate at 1:2000 dilutions in carbonate bicarbonate buffer, pH 9.6 for 40C overnight.
  2. The unbound capture antibodies were removed by three washing with washing buffer.
  3. Un-occupied places in the wells were blocked by adding 100 ml of blocking buffer. The plated was incubated at 370C for 1h with continuous shaking.
  4. Following incubation and washing, 50 ml positive antigen (1:10 dilution), negative antigen (1:10 dilution) and test samples (1:2 dilution) were added in respective wells and incubated at 370C for 1h with continuous shaking. No antigen was added in the blank control wells.
  5. After washing (as described above), 50 ml of detection antibody diluted 1:400 in blocking buffer, was added in wells. Plate was incubated at 370C for 1h after which washing was done as mentioned above. No detection antibody was added in the conjugate control wells.
  6. Fifty ml conjugate (rabbit anti-guinea pig immunoglobin conjugated to HRPO, Dakopad Corp., USA), diluted to 1:2000 in blocking buffer was added and the plate was incubated at 370C for 1h with continuous shaking.
  7. After washing, 50 ml of freshly prepared substrate/chromogen mixture was added to wells and plate was kept at 370C for 10-15 min for color development.
  8. The color reaction was stopped by adding 50 ml of 1M H2SO4 in all wells and optical density (OD) was measured at a wavelength of 492 nm on an ELISA reader.
  9. Test samples showing double or more than double OD492 of the mean negative control value are considered as positive.

BT-AGID:

The BTV antibody test kits used for the present study were made available by courtesy of Dr. M. M. Jochim, President, Veterinary Diagnostic Technology Incorporation, USA. All the sonicated blood cells, spleen and Culicoides suspensions were tested for the presence of BTV antigen by AGID Test. 0.9% Agarose was prepared and add six ml of molten gel was poured in each petridish (60×15mm) and allowed to solidify on a horizontal plane. A pattern consisting of a center well surrounded by six well was made using an immunodiffusion template. Each well had a diameter of four mm and the center to center distance between wells was 6.4 mm. Each well was sealed with one drop of molten gel. The central well was charged with sonicated sample as an antigen and all the peripheral wells were charged with different dilution of BTV antiserum (1:1, 1:2, 1:4, 1:8, 1:16 and 1:32). The charged petridishes were incubated at room temperature under humid condition for 72 hours before pronouncing the sample as negative or positive. The Positive sample petridishes were washed in normal saline for 24 hours followed by distilled water for 24 hours. The gel was pressed and dried under five layer thick whatman No.1 filter paper cover at room temperature for 3 days. The petridishes were stained with coomassie brilliant blue for 10 minutes, destaining with destaining solution for 10 minutes, air dried and examined for precipitation lines.

Results

Overall incidence:

A total of 377 samples (364-blood, 5-spleen and 8-pooled Culicoides) screened for detection of BTV antigen by s-ELISA and BT-AGID. Of these, 110 (29.18%) and 28 (7.42%) samples were found positive respectively (Fig 1, 2). Among the 364 blood samples screened for BTV antigen by s-ELISA and BT-AGID. Of these, 109 (29.94%) and 28 (7.69%) samples were found positive respectively  and out of five spleen samples, one calf spleen sample (20%) was found positive by s-ELISA and none of the spleen sample found positive by BT-AGID. where as none of the samples found positive from eight pooled Culicoides samples by both the test. Present finding was in agreement with earlier studies which detected 5 to 9 % BTV antigen by BT-AGID1, 5. However, In contrast to the present findings, reported 92.86 per cent and 100.00 per cent BTV antigen by BTID12,13.

Specieswise incidence of BTV:  

Of 364 blood samples were screened and Specieswise incidence by s-ELISA recorded was 67 (48.20%) in sheep, 40 (57.14%) in goats and 2 (2.60%) in cattle however, none of the blood sample found positive from buffalo and camel. Specieswise incidence by BT-AGID recorded was 17 (12.23%) in sheep and 11 (15.71%) in goats however, none of the blood sample found positive from cattle, buffalo and camel (Table-1). Similarly, none of the blood sample positive from camel7 and cattle5 and 11.11 per cent incidence reported in sheep2. While in contrary to the present findings 9.09 per cent and Zero per cent incidence reported in sheep and goats respectively5 and higher incidence rate 9.21% reported in buffaloes2.

Comparison of s-ELISA and BT-AGID for detection of BTV antigen:

Performance of the s-ELISA and BT-AGID for the detection of BTV antigen was compared. Cross tabulation of s-ELISA and BT-AGID considering s-ELISA as reference test was recorded as per method described by7 to determine relative sensitivity and specificity of BT-AGID. Considering s-ELISA as the reference test, the relative sensitivity, specificity and overall agreement between both the tests were 25.45 per cent, 100 per cent and 78.24 per cent respectively (Table-2).

CONCLUSIONS

  1. Specieswise incidence of BTV antigen indicated that the goats were more susceptible to BTV infection than sheep, cattle, buffaloes and camels.
  2. None of the buffaloes and camels blood and pooled Culicoides yielded positive results in s-ELISA and BT-AGID.
  3. s-ELISA is more sensitive then BT-AGID for detection of BTV antigen.

ACKNOWLEDGEMENTS

The authors are thankful to Dr. Mondal and Dr. Sanchay Bishwas Scientist, Tissue culture lab, division of Virology, Mukteswar and Director of CADRAD, IVRI, Izatnagar for their kind help and cooperation

REFERANCES:

  1. Abu Elzein, E.M.E. Rapid detection of bluetongue virus antigen in the sera and plasma of camels, sheep and cattle in the Sudan, using the Gel Immunodiffusion Test. Arch. Virol., 1984; 79: 131-134.
  2. Agrawal, S. M. Seroepidemiology and Isolation of Bluetongue virus in relation to prevalence of Culicoides vectors in Gujarat state. M.V.Sc. Thesis submitted to Sardarkrushinagar Dantiwada Agricultural University., 2009; pp 53-55
  3. Akita, G.Y., Ianconescu, M., MacLachlan, N.J., Osburn, B.I. Bluetongue disease in dogs associated with contaminated vaccine. Vet. Rec., 1994; 134 (11): 283-284.
  4. Chandel, B.S. Seroepidemiology, isolation and pathogenicity of Bluetongue virus. Ph.D. Thesis submitted to Gujarat Agricultural University., 1996; pp 60-61.
  5. Chauhan, H.C. Seroepidemiology of Bluetongue in different species of Livestock. M.V.Sc. Thesis, Submitted to Sardarkrushinagar Dantiwada Agricultural University. 2003; pp 67-68.
  6. Chauhan, H.C., Chandel, B.S., Gerdes, T., Vasava, K.A., Patel, A.R., Jadhav,K.M., Kher, H.N. Detection of group and serotype specific antibodies to Bluetongue virus in Buffaloes in Gujarat, India. Buffalo Bull., 2005; 24(2): 29-36.
  7. Khimaniya, K.N. Seroepidemiology and Attempt to isolate Bluetongue virus from Camels. M.V.Sc. Thesis Submitted to Sardarkrushinagar Dantiwada Agricultural University. 2009; pp 53-54.
  8. Knegi, k., Biswas, S.K., Ankan, D., Sing, B., Mondal, B. A polyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally J. Virol. Methods., 2009; 160(1-2):189-192.
  9. Ozawa, Y. Bluetongue and related orbiviruses: Overview of the world situation. Prog. Clin. Biol. Res., 1985; 178: 13-20.
  10. Prasad, G., Minakshi, M.Y., Mann, S. Bluetongue virus infection in India: a review. Indian J. Anim.Sci., 2000; 70: 103-109.
  11. Martin, S.W. The evaluation of tests. Canadian J. Comp. Med., 1977; 41: 19-25.
  12. Wilson, A.A., Babu, M., Palaniswamy, K.S., Ebenezer, D. A report on Bluetongue in Tamil Nadu. Indian Vet. J., 1999; 76: 953-955.
  13. Wilson, A.A., Jayapal, G. Kathirchelvan, M. Report on combined outbreak of Bluetongue and sheep pox in Sivagangai District. Indian Vet. J., 2001; 78: 1-3.
  14.  Table 1:- Specieswise detection of BTV antigen from blood samples by s-ELISA and BT-AGID
Species of    animal
No. of  blood sample Tested
Sample found positive by

s-ELISA

Percentage

 (%)

Sample found positive by

BT-AGID

Percentage (%)
Sheep
139
67
48.20
17
12.23
Goats
70
40
57.14
11
15.71
Cattle
77
02
2.60
00
00.00
Buffaloes
40
00
00.00
00
00.00
Camels
38
00
00.00
00
00.00
Total
364
109
29.95
28
7.69

 

Table 2: Comparative evaluation of s-ELISA and BT-AGID for detection of BTV antigen

Test                  BT-AGID
Positive Negative
      s-ELISA Positive 28 82
Negative 00 267
Sensitivity (%) 25.45
Specificity (%) 100.00
Overall agreement (%) 78.24

 

 

28                                                                     267

Sensitivity (%)   =   ——- x 100    =   25.45,     Specificity (%)   =   —— x 100    =   100.00

110                                                                      267

295

Overall agreement (%) =   ——- x 100    =   78.24

377

Fig. 1: Microtitre ELISA Plate showing results of s-ELISA

Wells A1, A2, B1, B2: Positive control,               Wells C1, C2, D1, D2: Antigen blank

Wells E1, E2, F1, F2:   Negative control,              Wells G1, G2, H1, H2: Conjugate control

Rest of the Wells from A3-H12: test samples in vertical duplicate.

Wells A3, B3; A4, B4 etc.:- BTV antigen positive samples

Wells E8, F8; E12, F12 etc.:- BTV antigen negative samples

Fig. 1: Microtitre ELISA Plate showing results of s-ELISA

Wells A1, A2, B1, B2: Positive control,               Wells C1, C2, D1, D2: Antigen blank

Wells E1, E2, F1, F2:   Negative control,              Wells G1, G2, H1, H2: Conjugate control

Rest of the Wells from A3-H12: test samples in vertical duplicate.

Wells A3, B3; A4, B4 etc.:- BTV antigen positive samples

Wells E8, F8; E12, F12 etc.:- BTV antigen negative samples

           Placement of samples

Central well (S): Blood sample

Peripheral wells (1 to 6): Known antiserum                  

                        Well No.    Dilution of antiserum

1:                   1:1

2:                   1:2

3:                   1:4

4:                    1:8

5:                    1:16

6:                    1:32