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Development and Evaluation of a Truncated Recombinant NS3 Antigenbased Indirect ELISA for Detection of Pestivirus Antibodies in Sheep and Goats a
a
Semmannan Kalaiyarasu , Niranjan Mishra , Katherukamem a
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Rajukumar , Ram Kumar Nema & Sthita Pragnya Behera
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High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh, India Accepted author version posted online: 13 Aug 2014.Published online: 26 Sep 2014.
To cite this article: Semmannan Kalaiyarasu, Niranjan Mishra, Katherukamem Rajukumar, Ram Kumar Nema & Sthita Pragnya Behera (2015) Development and Evaluation of a Truncated Recombinant NS3 Antigen-based Indirect ELISA for Detection of Pestivirus Antibodies in Sheep and Goats, Journal of Immunoassay and Immunochemistry, 36:3, 312-323, DOI: 10.1080/15321819.2014.947433 To link to this article: http://dx.doi.org/10.1080/15321819.2014.947433
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Journal of Immunoassay and Immunochemistry, 36:312–323, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.947433
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DEVELOPMENT AND EVALUATION OF A TRUNCATED RECOMBINANT NS3 ANTIGEN-BASED INDIRECT ELISA FOR DETECTION OF PESTIVIRUS ANTIBODIES IN SHEEP AND GOATS
Semmannan Kalaiyarasu, Niranjan Mishra, Katherukamem Rajukumar, Ram Kumar Nema, and Sthita Pragnya Behera High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh, India
The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats. Keywords BVDV antibodies, goat, NS3 ELISA, sheep, VNT
INTRODUCTION Pestiviruses are important veterinary pathogens that cause economical losses to the livestock industry worldwide. Sheep and goat farming is a major source of earning of the farmers in many developing countries. Pestivirus infections in sheep and goats can cause a variety of clinical syndromes including reproductive failure, abortion, still birth, respiratory disease, poor growth rate, diarrhea, nervous signs, and muscular tremor.[1] The Pestivirus genus in the family Flaviviridae consists of four accepted species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus Address correspondence to Semmannan Kalaiyarasu, High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India. E-mail: [email protected]
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2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV).[2] Historically, all pestivirus isolates from sheep and goats were referred as BDV. But it is now a fact that border disease in sheep and goats can be caused by BVDV-1, BVDV-2, and BDV and in some countries it is caused exclusively by BVDV-1 or BVDV-2.[3–10] Transmission of pestivirus infection between sheep and goats and cattle in both ways has been demonstrated which is important for BVD control in cattle.[11–13] The pestivirus genome is a 12.3 kb long single stranded positive sense RNA containing a single ORF encoding about 4000 amino acids flanked by untranslated regions (UTR) at 5 and 3 ends.[14] The structural proteins are represented by capsid protein C and three envelope proteins namely, Erns , E1, and E2 and the remaining are non structural proteins. The structural proteins Erns , E2, and non structural protein NS3 are the immunodominant proteins eliciting antibody response following pestivirus infection. Serological diagnosis is still the most commonly used method for determining pestivirus infection in a herd. BVDV antigen ELISAs are commercially available, rapid and cost effective and are useful in the detection of persistently infected (PI) animals. However, maternally derived or vaccine antibodies can mask the antigen and lead to false negative ELISA results.[15,16] Hence, detection of pestivirus antibodies is still the most rapid and cost effective method for detection of exposure to pestiviruses in unvaccinated herds. Virus neutralization test (VNT) is considered as the gold standard test for serological diagnosis of pestivirus infection. However, it can be performed only in specialized laboratories besides being labor and time intensive. Since the NS3 protein is immunodominant and highly conserved among pestiviruses, it is a preferred target to develop immunoassays for detection of pestivirus antibodies. The major immunodominant regions of NS3 helicase domain is defined by amino acids 205–549 consisting of domain A (aa 205–369) and B (aa 368–549) and both the domains are of equal diagnostic significance.[17] Pestivirus infections are widely prevalent in sheep and goats in India with predominant occurrence of BVDV-1 and sporadic occurrence of BVDV2.[6,7,18] Although there are several NS3 ELISA kits available commercially for detection of BVDV antibodies in cattle, only a few have been validated for use in sheep and goats. Some manufacturers claim suitability of their kits to screen sheep and goats, but lack validation data to be useful in seroepidemiological studies in field conditions. Hence, the aim of this study was to develop an indirect ELISA using the 50KDa helicase domain of BVDV NS3 protein instead of full length NS3 protein (being used in commercial kits) for detection of pestivirus antibodies in sheep and goats and its evaluation by comparing the sensitivity and specificity with VNT as the reference test.
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MATERIALS AND METHODS
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Recombinant NS3 Antigen and Serum Samples Expression of a 50-kDa recombinant-NS3 antigen (Rc-NS3) in prokaryotic expression system containing the helicase domain of BVDV1 NS3, its characterization and purification have been described in our previous work.[18] The concentration of purified Rc-NS3 protein was estimated using Qubit® fluorometer (Invitrogen, Eugene, OR, USA). A total of 569 serum samples, 463 from sheep and 106 from goats collected from seven different states of India (Andhra Pradesh, Gujarat, Madhya Pradesh, Punjab, Rajasthan, and Tamil Nadu) during a pestivirus surveillance program in the years 2005–2010 were used for testing. Known BVDV positive and negative serum samples were collected from BVDV-1 and BVDV-2 experimentally infected and control animals respectively and tested by VNT to confirm their status. Fifty sheep serum samples of known BDV antibody status (20 BDV antibody positive and 30 BDV antibody negative samples as determined by VNT) were kindly provided by Ana L. Garcıa-Perez, NEIKER Instituto Vasco de Investigacio´n y Desarrollo Agrario, Department of Animal Health, Derio (Bizkaia), Spain.and P. Becher, Institut fur Virologie, Giessen, Germany. Optimization of Serum Dilution and Rc-NS3 Antigen Concentration A checkerboard titration of Rc-NS3 antigen with BVDV or BDV antibody positive and negative sera was carried out for determining the optimum antigen concentration and serum dilution to be used in the test proper. Briefly, different dilutions of serum (1/10 to 1/80) and Rc-NS3 antigen concentrations (10–1280 ng per well) were added to the wells of ELISA plate in row wise and column wise, respectively. The combination that showed maximum positive/negative (P/N ratio) was selected for further development of NS3 ELISA. Bovine serum albumin (BSA), non fat dry milk powder (NFDM) and horse serum with LAH at various dilutions (1–5%) were evaluated as blocking agents. Various dilutions of anti-sheep and anti-goat HRPO conjugate (1/4000 to 1/10,000, Sigma-Aldrich, St. Louis, MO, USA) and substrate (TMB, Sigma-Aldrich) reaction times (5–15 min) were also tested to ascertain the optimal test conditions. Optimized Rc-NS3 ELISA Procedure The wells of ELISA plate (Maxisorp, Nunc, Roskilde, Denmark) were coated with 50 µL (320 ng) of purified NS3 recombinant protein in carbonate bicarbonate buffer and kept at 4◦ C overnight. After washing once with 300 µL of PBS, wells were blocked with 200 µL of blocking buffer containing
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3% NFDM (Propure, Amresco, Solon, OH, USA) in PBS and incubated at 37◦ C for 1 hr. The wells were washed thrice with 300 µL of PBST (1X PBS with 0.05% Tween) and 50 µL of 1:20 diluted (1% NFDM in PBST) serum samples were added to each well in duplicate including positive and negative controls. The plate was incubated for 90 min at 37◦ C and after washing thrice with 300 µL of PBST, 50 µL of anti-sheep and anti-goat HRPO conjugate (1:8000 diluted in1% NFDM in PBST) was added to each well for sheep and goat serum samples, respectively. Then the plate was incubated for 1 hr at 37◦ C and washed thrice with PBST. The substrate reaction was developed by adding 50 µL of TMB (Sigma) to each well and the reaction was stopped after 10 min with 0.4M H2 SO4. The optical density (O.D.) of each well was read at 450 nm in a multiwell ELISA reader (Tecan, Tokyo, Japan). Virus Neutralization Test (VNT) A total of 569 field sheep and goat sera were tested for presence of virus neutralizing antibodies against BVDV-1 or BVDV-2 by VNT. Briefly, the sera were heat inactivated at 56◦ C for 30 min, diluted 1:5 and were tested in triplicate by microplate indirect immuno peroxidase monolayer assay (IPMA) as described in our previous report[18] using 200 tissue culture infectious dose 50 (TCID50 ) of BVDV-1 cattle isolate Ind S-1449, BVDV-2 sheep isolate Ind 51966,[18] and SFT-R cells. The immunostaining was carried out using anti-BVDV goat polyclonal antibody (VMRD, Pullman, WA, USA), anti goat IgG-HRPO conjugate (Sigma), substrate H2 O2 with chromogen 3-amino-9-ethyl carbazole (Sigma) and the results were noted after examination under microscope. Appropriate positive, negative, cell and virus controls were included in each plate. The serum sample was considered positive for BVDV-1 and/or BVDV-2 neutralizing antibodies when cells mixed with serum at an initial dilution of 1:5 and virus did not show any pestivirus specific cytoplasmic staining, otherwise it was considered as negative. Determination of Cut-Off Value A total number of 619 serum samples from sheep and goats were subjected to Rc-NS3 ELISA and the cut-off value was calculated based on the results of BVDV/BDV VNT positive (n = 201) and negative (n = 418) serum samples. The ELISA cut-off value for determining the status (pestivirus antibody positive or negative) of the serum was calculated by the following formula: Cut-off value = Negative mean OD at 450 nm (n = 418) × 2 standard deviation
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Determination of Sensitivity and Specificity The sensitivity, specificity, and agreement of the Rc-NS3 ELISA were calculated by considering VNT as the reference standard using the following formulae:
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Sensitivity % = True positive/ True positive + False negative × 100 Specificity % = True Negative/ True negative + False positive × 100 Agreement % =
Kappa value =
True positive + True Negative /Total number of samples × 100 Observed agreement − Expected agreement / 1 − Expected agreement
Evaluation of NS3 ELISA for Detection of BDV Antibodies A total of 50 reference sheep serum samples (20 BDV antibody positive and 30 BDV negative by VNT) were also tested by the NS3 ELISA developed in this study and its sensitivity and specificity was determined. RESULTS Optimization of Rc-NS3 ELISA The Rc-NS3 antigen was tested for its utility in ELISA for detection of pestivirus antibodies in sheep and goat serum samples. The checkerboard titrations showed that maximum difference in O.D. values between reference positive and negative serum (P/N values) was obtained at 320 ng per well of antigen and 1:20 dilution of serum (Figure 1). The correlation between the absorbance and the antigen concentration (10–1280 ng/well) at different dilutions of serum (1/10, 1/20, 1/40, and 1/80) was calculated by regression method (r2 = 0.83, 0.9449, 0.9186, and 0.9327, respectively) using Microsoft Excel 2007 and maximum correlation coefficient found for 1/20 dilution followed by 1/80 dilution. When 418 BVDV/BDV antibody negative serum samples were tested by the Rc-NS3 ELISA, a mean O.D. value of 0.220 with a standard deviation of 0.089 was obtained. Hence, the cut-off value was set at O.D. value of 0.40. This was further supported by distribution plot of O.D. values obtained by ELISA and results of VNT positive (n= 201) and negative (n= 418) serum samples (Figure 2).
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1280
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320
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10 4.56912 6.5117 6.44487 4.80804 3.49744 2.17989 2.23121 1.29221 20 5.19524 6.07407 6.93038 5.73973 3.97101 2.81208 2.34328 1.8087 40 3.08929 3.21514 3.01449 1.74074 1.17677 1.14141 1.15104 1.90741 80 3.45306 2.51948 1.97664 2.05854 1.61735 1.16842 0.89305 2.80392 FIGURE 1 Checker board titration of Rc-NS3 antigen (10–1280 ng per well) with reference BVDV antibody positive and negative sera (1/10 to 1/80) for determining the optimum antigen concentration and serum dilution. 250 Number of Samples
200 150 100
Positive Negative
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. 20 20 –1 .3 0 1.
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P/N ratio
P/N ratio between serum dilution and antigen concentration 8 7 6 5 4 3 2 1 0
OD Value at 450 nm
FIGURE 2 Distribution plot of 418 VNT negative and 201 VNT positive serum samples in ELISA.
Relative Sensitivity and Specificity of Rc-NS3 ELISA The sensitivity and specificity of the Rc-NS3 ELISA in detecting BVDV antibodies was determined by comparison with the gold standard VNT. The Rc-NS3 ELISA showed a sensitivity and specificity of 91.02%, 94.59% in case of sheep and 100%, and 94.57% in goats, respectively. The agreement rate between the two tests was 93.3% (sheep) and 95.3% (goat) (Table 1). When 569 (463 sheep and 106 goat) serum samples were tested by Rc-NS3 ELISA, 168 (36.28%) sheep and 19 (17.92%) goats were found positive for BVDV antibodies (Table 2). As a whole, the RC-NS3 ELISA showed a sensitivity of 91.71% and specificity of 94.59% among sheep and goats. Testing of same
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TABLE 1 Relative sensitivity and specificity of Rc-NS3 ELISA with BVDV virus neutralization test based on 463 sheep and 106 goat serum samples
Positive
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Rc- NS3 ELISA
Prevalence a TP, b FP, c FN,
Positive Negative Total Sensitivity Specificity Agreement Kappa value VNT Rc-NS3 ELISA
Sheep
Goat
VNT
VNT
Negative
Total
152a 16b 168 15c 280d 295 167 296 463 91.02% (95% CI-85.71, 94.48) 94.59% (95% CI-91.4, 96.65) 93.3% (95% CI-90.65, 95.24) 0.856 (95% CI-0.81, 0.91) 36.07 % 36.29 %
Positive
Negative
Total
14a 5b 19 0c 87d 87 14 92 106 100 (95% CI-78.47,100) 94.57 (95% CI-87.9, 97.66) 95.28% (95% CI- 89.43, 97.97) 0.82 (95% CI-0.67, 0.97) 13.21 % 17.92 %
and d TN
serum samples by VNT revealed that 167 (36.07%) sheep and 14 (13.21%) goats were positive for BVDV neutralizing antibodies (Table 2). Hence, a very good correlation between Rc-NS3 ELISA and VNT was observed while determining the prevalence of BVDV antibodies in sheep and goats. Additionally, when we compared the test results of 50 serum samples of known BDV antibody status, it showed a sensitivity of 85% and specificity of 86.6% (Table 3). DISCUSSION Prevalence of pestivirus infections in sheep and goats have been reported worldwide with seroprevalence rates ranging from 5–50% in various countries.[1] This poses a serious challenge to the efforts of BVD control in cattle being operational currently in several countries, since transmission of pestiviruses from sheep and goats to cattle has been demonstrated. In view of the recent isolation and identification of BVDV-1 and BVDV-2 in Indian sheep and goats,[6,7,18] and future preparedness for BDV detection, there is an urgent need of active surveillance for pestivirus infections in small ruminants. ELISA is employed commonly for sero-epidemiological studies, as it is rapid, inexpensive, sensitive, and useful for mass screening. NS3 antibody ELISA is commonly used for detection of pestivirus antibodies in cattle, sheep, and goats during surveillance and control. Despite of availability of several commercial NS3 antibody ELISA kits, only scant information is available with regard to their validation in sheep and goat serum samples. Hence, the aim of the present study was development of a recombinant NS3 antigen based antibody ELISA and its evaluation for serological diagnosis of pestivirus infection in sheep and goats.
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Andhra Pradesh Gujarat Madhya Pradesh Punjab Rajasthan Tamil Nadu Total
States
126 43 0 20 211 63 463
Individuals tested 100 0 0 5 43 19 167
Positive
VNT
Sheep
79.37 0.00 0.00 25.00 20.38 30.16 36.07
% 90 3 0 5 52 18 168
Positive 71.43 6.98 0.00 25.00 24.64 28.57 36.28
%
Rc-NS3 ELISA
2 0 75 0 27 2 106
Individuals tested
1 0 11 0 0 2 14
Positive
VNT
50 0 14.67 0 0 100 13.21
%
1 0 14 0 2 2 19
50 0 18.67 0 7.41 100 17.92
%
Rc-NS3 ELISA Positive
Goats
TABLE 2 Comparison between Rc-NS3 ELISA and VNT in determining the prevalence of BVDV antibodies among sheep and goats (n = 569) in different states
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TABLE 3 Performance of Rc-NS3 ELISA with BDV reference serum VNT Positive
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Rc- NS3 ELISA
a TP, b FP, c FN,
Positive Negative Total Sensitivity Specificity Agreement Kappa value
Negative
17a 4b 3c 26d 20 30 85% (95% CI-63.96, 94.76) 86.67 (95% CI-70.32, 94.69) 86% (95% CI-73.81, 93.05) 0.711 (95% CI-0.512, 0.909)
Total 21 29 50
and d TN
Different forms and sources of antigen have earlier been used to develop ELISA such as whole virus particles,[19] BVDV infected cell culture extracts,[20] viral antigens immobilized with monoclonal antibodies,[21,22] or recombinant proteins produced in bacteria.[23,24] Prokaryotic expression of NS3 antigen and its application for detection of BVDV antibodies in cattle has been reported by several workers.[17,24–26] In this study, we report the development of an indirect ELISA using BVDV Rc-NS3 antigen expressed in E. coli. Rc-NS3 antigen (50 kDa) used in our study consisted of 384 amino acids from C- terminus of full-length NS3 protein carrying the whole of domain B (aa 368–549) and a part of domain A (aa 205–369) as defined earlier.[17] Use of smaller helicase domain not only avoids the diagnostic non-significant part of NS3 region but also has been found useful in detection of BVDV antibodies in cattle and buffalos.[27] An indirect ELISA was optimized to evaluate the Rc-NS3 protein for its potential use as antigen in diagnosis. The ELISA showed clear differences in the absorbance values between known BVDV antibody positive and negative sera from sheep and goats at appropriate dilutions of antigen and antibody. After optimization of the ELISA, BVDV antibody negative serum samples from sheep and goats (n=418) as determined by the reference virus neutralization test were employed for deciding the cut-off value (0.40) which was further verified by distribution analysis of antibody positive and negative serum samples. Since VNT is considered as the gold standard test for serological diagnosis of pestivirus infection (BVDV/BDV), the performance of the developed Rc-NS3 indirect ELISA was compared with VNT using 619 field serum samples (463 from sheep and 106 from goats and 50 reference serum). When compared with BVDV VNT, the ELISA exhibited a high sensitivity (91.02% and 100%) and specificity (94.59% and 94.57%) with a good correlation rate (93.3% and 95.3%) with kappa value of 0.855 and 0.821 for sheep and goat, respectively, indicating efficiency of Rc-NS3 ELISA in detecting BVDV antibodies. However, the sensitivity of Rc-NS3 ELISA was
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found a little bit lower than VNT. It is well known that appearance of BVDV neutralizing antibodies occur earlier than the non neutralizing antibodies in natural infection.[28] NS3 ELISA detects antibodies against BVDV nonstructural NS3 protein, whereas VNT detects neutralizing antibodies against E2 structural protein. Moreover, 100% correlation with VNT may not be possible with ELISA due to the preferential detection of only IgG isotype in ELISA and ability of detection of both IgG and IgM by VNT thereby increased sensitivity found for VNT. These reasons may explain why a slight lower sensitivity of Rc-NS3 ELISA was observed in our study when compared to VNT. A slightly lower sensitivity and specificity of Rc-NS3 ELISA in detecting BDV antibodies was observed. This can be explained by the fact that the NS3 recombinant antigen was generated from a BVDV isolate. However, these results proved that Rc-NS3 ELISA using 50 kDa of NS3 protein instead of 80 KDa of NS3 full-length non structural protein (being used by commercial kits) can be used for detection of BVDV/BDV antibodies in sheep and goats during sero-epidemiological studies. In the present study, the prevalence rate of BVDV antibodies was 36.5% in sheep and 17.9% in goats by the Rc-NS3 ELISA, while the prevalence rate was 36.06% in sheep and 13.2% in goats by VNT indicating that efficacy of Rc-NS3 ELISA compared very well with VNT. A similar range of seroprevalence of BVDV infection in sheep and goats in India has been reported earlier.[29] Varying rates of sensitivity and specificity between commercial NS3 antibody ELISA and VNT have been reported earlier while studying pestivirus seroprevalence in sheep and goats. A study using Ceditest NS3 ELISA (Ceditest® , Cedi Diagnostics, Lelystad, Netherlands) found a pestivirus seroprevalence rate of 45% in sheep, compared to a prevalence rate of 28% found by VNT.[30] Although the producer of ELISA kit claimed a sensitivity and specificity rate of 99% with serum, milk, and plasma samples of cattle, the kit had not been validated with serum samples from sheep and goats and hence, a high percentage of false positive results were found. In another study, a variation in pestivirus antibody prevalence rates between ELISA (48.12%) and VNT (42.89%) was observed, when 401 sheep serum samples were tested by Institut Pourquier NS3 ELISA kit (Institut Pourquier, Montpellier, France).[31] These results indicated that commercial NS3 antibody ELISAs developed and validated for detection of BVDV antibodies in cattle using full length NS3 protein should be validated before use in sheep and goats. Being an inexpensive and rapid method, the developed and validated Rc-NS3 ELISA reported in this study should be found useful during sero-surveillance and sero-monitoring of pestivirus infections in sheep and goats.
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CONCLUSION
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The sensitivity and specificity of Rc-NS3 indirect ELISA developed and evaluated in this study correlated well with the reference virus neutralization test for detection of BVDV antibodies in sheep and goats and correlated moderately in detecting BDV antibodies. Being a simple and rapid test, the developed NS3 ELISA should be found useful during sero-surveillance and sero-monitoring of pestivirus infections in sheep and goats. FUNDING N. Mishra was supported by a grant (BT/PR4415/AAQ/01/165/2003) from Department of Biotechnology, Govt. of India, New Delhi. REFERENCES 1. Nettleton, P.F.; Gilray, J.A.; Russo, P.; Dlissi, E. Border disease of sheep and goats. Vet. Res. 1998, 29, 327–340. 2. King, A.M.Q.; Lefkowitz, E.; Adams, M.J.; Carstens, E.B. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, 1st Ed. Elsevier, San Diego, CA, 2011, pp 1003–1020. 3. Czopowicz, M.; Kaba, J.; Schirrmeter, H.; Bagnick, E.; Szalus-Jordanow, O.; Nowicki, M.; Witkowski, L.; Frymus T. Serological evidence for BVDV-1 infection in goats in Poland- Short communication. Acta. Vet. Hung . 2011, 59, 399–404. 4. Kim, I.J.; Hyun, B.H.; Shin, J.H.; Lee, K.K.; Lee, K.W.; Cho, K.O.; Kang, M.I. Identification of bovine viral diarrhoea virus type 2 in Korean native goat (Capra hircus). Virus. Res. 2006, 121,103–106. 5. Krametter-Froetscher, R.; Loitsch, A.; Kohler, H.; Schleiner, A.; Schiefer, P.; Moestl, K.; Golja, F.; Baumgartner, W. Prevalence of antibodies to pestiviruses in goats in Austria. J Vet. Med. B. Infect. Dis. Vet. Public. Health. 2006, 53, 48–50. 6. Mishra, N.; Dubey, R.; Rajukumar, K.; Tosh, C.; Tiwari, A.; Pitale, S.S.; Pradhan, H.K. Genetic and antigenic characterization of bovine viral diarrhea virus type 2 isolated from Indian goats (Capra hircus). Vet. Microbiol. 2007, 124, 340–347. 7. Mishra, N.; Pitale, S.S.; Rajukumar, K.; Prakash, A.; Behera, S.P.; Nema, R.K.; Dubey, S.C. Genetic variety of bovine viral diarrhea virus 1 strains isolated from sheep and goats in India. Acta. Virol. 2012, 56, 209–215. 8. Pratelli, A.; Martella, V.; Cirone, F.; Buonavoglia, D.; Elia, G.; Tempesta, M.; Buonavoglia, C. Genomic characterization of pestiviruses from lambs and kids in Southern Italy. J. Virol. Methods. 2001, 94, 81–85. 9. Sullivan, D.G.; Chang, G.J.; Akkina, R.K. Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep. Virus. Res. 1997, 47 , 19–29. 10. Valdazo-Gonzalez, B.; Alvarez-Martinez, M.; Greiser-Wilke, I. Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain. Vet. Microbiol. 2006, 117 , 141–153. 11. Carlsson, U. Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine viral diarrhea virus. Vet. Rec. 1991, 128, 145–147. 12. Carlsson, U.; Belak, K. Border disease virus transmitted to sheep and cattle by a persistently infected ewe: epidemiology and control. Acta. Vet. Scand. 1994, 35, 79–88. 13. Loken, T. Ruminant pestivirus infections in animals other than cattle and sheep. Vet. Clin. North. Am. Food. Anim. Pract. 1995, 3, 597–614. 14. Meyers, G.; Thiel, H.J. Molecular characterization of pestiviruses. Adv. Virus. Res. 1996, 47 , 53–118. 15. Palfi, V.; Houe, H.; Philipsen, J. Studies on the decline of bovine virus diarrhoea virus (BVDV) maternal antibodies and detectability of BVDV in persistently infected calves. Acta. Vet.Scand. 1993, 34, 105–107.
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16. Zimmer, G.M.; Van Maanen, C.; De Goey, I.; Brinkhof, J;, Wentink, G.H. The effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples. Vet. Microbiol. 2004, 100, 145–149. 17. Deregt, D.; Dubovi, E.J.; Jolley, M.E.; Nguyen, P.; Burton, K.M.; Gilbert, S.A. Mapping of two antigenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus. Vet. Microbiol. 2005, 108, 13–22. 18. Mishra, N.; Pitale, S.S.; Pradhan, H.K. Genetic analysis and expression of NS3 gene of bovine viral diarrhea virus type 1 from Indian for detection of antibodies in cattle. J. Appl. Anim. Res. 2008, 33, 99–103. 19. Chu, H.J.; Zee, Y.C.; Adrans A.A.; Dai K. Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhoea virus in bovine sera. Vet. Microbiol. 1985, 10, 325–333. 20. Howard, C.J.; Clarke, M.C.; Brownlie, J. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhea virus (BVDV) in cattle sera. Vet. Microbiol. 1985, 10, 359–369. 21. Cho, H.J.; Masri, S.A.; Deregt, D.; Yeo, S.G.; Golsteyn Thomas E.J. Sensitivity and specificity of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea virus antibody in cattle. Can. J. Vet. Res. 1991, 55, 56–59. 22. Moennig, V.; Leder, L.; Greiser-Wilke, I.; Frey, H.R.; Liess, B. A new enzyme immunoassay for the detection of antibodies against the bovine viral diarrhea virus. Tierarztl. Prax. Ausg. G. Grosstiere. Nutztiere. 1991, 19, 35–38. 23. Kwang, J.; Bolin, S.R.; Littledike E.T. Bovine viral diarrhea serologic diagnostic reagents prepared from bacterially expressed recombinant proteins. J. Vet. Diagn. Invest. 1995, 7 , 143–145. 24. Lecomte, C.; Pin, J.J.; De Moerlooze, L.; Vandenbergh, D.; Lambert, A.F.; Pastoret, P.P.; Chappuis, G. ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies. Vet. Microbiol. 1990, 23, 193–201. 25. Reddy, J.R.; Kwang, J.; Okwumabua, O.; Kapil, S.; Loughin, T.M.; Lechtenberg, K.F.; Chengappa, M.M.; Minocha, H.C. Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle. Vet. Microbiol. 1997, 57 , 119–133. 26. Vanderheijden, N.; De Moerlooze, L.; Vandenbergh, D.; Chappuis, G.; Renard, A.; Lecomte, C. Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection. J. Gen. Vir . 1993, 74, 1427–1431. 27. Bhatia, S.; Sood, R.; Mishra, N.; Pattnaik, B.; Pradhan, H.K. Development and evaluation of a MAb based competitive-ELISA using helicase domain of NS3 protein for sero- diagnosis of bovine viral diarrhea in cattle and buffaloes. Res. Vet. Sci. 2008, 85, 39–45. 28. Bolin, S.R. Viral and viral protein specificity of antibodies induced in cows persistently infected with noncytopathic bovine viral diarrhea virus after vaccination with cytopathic bovine viral diarrhea virus. Am. J. Vet. Res. 1988, 49, 1040–1044. 29. Mishra, N.; Rajukumar, K.; Tiwari, A.; Nema, R.K.; Behera, S.P.; Satav, J.S.; Dubey, S.C. Prevalence of Bovine viral diarrhoea virus (BVDV) antibodies among sheep and goats in India. Trop. Anim. Health. Prod. 2009, 41, 1231–1239. 30. Orsel, K.; Antonis, A.F.; Oasterloo, J.C.; Vellema, P.; Van der Meer, F.J. Seroprevalence of antibodies against pestiviruses in small ruminants in The Netherlands. Tijdschr. Diergeneeskd, 2009, 134, 380–384. 31. Yazici, Z.; Serdar, M.S.; Gumusova, S.O.; Albayrak, H. Molecular diagnosis and seroepidemiology of pestiviruses in sheep. Veterinarski. Arhiv. 2012, 82, 35–45.
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Development and Evaluation of a Truncated Recombinant NS3 Antigenbased Indirect ELISA for Detection of Pestivirus Antibodies in Sheep and Goats a
a
Semmannan Kalaiyarasu , Niranjan Mishra , Katherukamem a
a
Rajukumar , Ram Kumar Nema & Sthita Pragnya Behera
a
a
High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh, India Accepted author version posted online: 13 Aug 2014.Published online: 26 Sep 2014.
To cite this article: Semmannan Kalaiyarasu, Niranjan Mishra, Katherukamem Rajukumar, Ram Kumar Nema & Sthita Pragnya Behera (2015) Development and Evaluation of a Truncated Recombinant NS3 Antigen-based Indirect ELISA for Detection of Pestivirus Antibodies in Sheep and Goats, Journal of Immunoassay and Immunochemistry, 36:3, 312-323, DOI: 10.1080/15321819.2014.947433 To link to this article: http://dx.doi.org/10.1080/15321819.2014.947433
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Journal of Immunoassay and Immunochemistry, 36:312–323, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.947433
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DEVELOPMENT AND EVALUATION OF A TRUNCATED RECOMBINANT NS3 ANTIGEN-BASED INDIRECT ELISA FOR DETECTION OF PESTIVIRUS ANTIBODIES IN SHEEP AND GOATS
Semmannan Kalaiyarasu, Niranjan Mishra, Katherukamem Rajukumar, Ram Kumar Nema, and Sthita Pragnya Behera High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh, India
The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats. Keywords BVDV antibodies, goat, NS3 ELISA, sheep, VNT
INTRODUCTION Pestiviruses are important veterinary pathogens that cause economical losses to the livestock industry worldwide. Sheep and goat farming is a major source of earning of the farmers in many developing countries. Pestivirus infections in sheep and goats can cause a variety of clinical syndromes including reproductive failure, abortion, still birth, respiratory disease, poor growth rate, diarrhea, nervous signs, and muscular tremor.[1] The Pestivirus genus in the family Flaviviridae consists of four accepted species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus Address correspondence to Semmannan Kalaiyarasu, High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal, Madhya Pradesh 462021, India. E-mail: [email protected]
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2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV).[2] Historically, all pestivirus isolates from sheep and goats were referred as BDV. But it is now a fact that border disease in sheep and goats can be caused by BVDV-1, BVDV-2, and BDV and in some countries it is caused exclusively by BVDV-1 or BVDV-2.[3–10] Transmission of pestivirus infection between sheep and goats and cattle in both ways has been demonstrated which is important for BVD control in cattle.[11–13] The pestivirus genome is a 12.3 kb long single stranded positive sense RNA containing a single ORF encoding about 4000 amino acids flanked by untranslated regions (UTR) at 5 and 3 ends.[14] The structural proteins are represented by capsid protein C and three envelope proteins namely, Erns , E1, and E2 and the remaining are non structural proteins. The structural proteins Erns , E2, and non structural protein NS3 are the immunodominant proteins eliciting antibody response following pestivirus infection. Serological diagnosis is still the most commonly used method for determining pestivirus infection in a herd. BVDV antigen ELISAs are commercially available, rapid and cost effective and are useful in the detection of persistently infected (PI) animals. However, maternally derived or vaccine antibodies can mask the antigen and lead to false negative ELISA results.[15,16] Hence, detection of pestivirus antibodies is still the most rapid and cost effective method for detection of exposure to pestiviruses in unvaccinated herds. Virus neutralization test (VNT) is considered as the gold standard test for serological diagnosis of pestivirus infection. However, it can be performed only in specialized laboratories besides being labor and time intensive. Since the NS3 protein is immunodominant and highly conserved among pestiviruses, it is a preferred target to develop immunoassays for detection of pestivirus antibodies. The major immunodominant regions of NS3 helicase domain is defined by amino acids 205–549 consisting of domain A (aa 205–369) and B (aa 368–549) and both the domains are of equal diagnostic significance.[17] Pestivirus infections are widely prevalent in sheep and goats in India with predominant occurrence of BVDV-1 and sporadic occurrence of BVDV2.[6,7,18] Although there are several NS3 ELISA kits available commercially for detection of BVDV antibodies in cattle, only a few have been validated for use in sheep and goats. Some manufacturers claim suitability of their kits to screen sheep and goats, but lack validation data to be useful in seroepidemiological studies in field conditions. Hence, the aim of this study was to develop an indirect ELISA using the 50KDa helicase domain of BVDV NS3 protein instead of full length NS3 protein (being used in commercial kits) for detection of pestivirus antibodies in sheep and goats and its evaluation by comparing the sensitivity and specificity with VNT as the reference test.
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MATERIALS AND METHODS
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Recombinant NS3 Antigen and Serum Samples Expression of a 50-kDa recombinant-NS3 antigen (Rc-NS3) in prokaryotic expression system containing the helicase domain of BVDV1 NS3, its characterization and purification have been described in our previous work.[18] The concentration of purified Rc-NS3 protein was estimated using Qubit® fluorometer (Invitrogen, Eugene, OR, USA). A total of 569 serum samples, 463 from sheep and 106 from goats collected from seven different states of India (Andhra Pradesh, Gujarat, Madhya Pradesh, Punjab, Rajasthan, and Tamil Nadu) during a pestivirus surveillance program in the years 2005–2010 were used for testing. Known BVDV positive and negative serum samples were collected from BVDV-1 and BVDV-2 experimentally infected and control animals respectively and tested by VNT to confirm their status. Fifty sheep serum samples of known BDV antibody status (20 BDV antibody positive and 30 BDV antibody negative samples as determined by VNT) were kindly provided by Ana L. Garcıa-Perez, NEIKER Instituto Vasco de Investigacio´n y Desarrollo Agrario, Department of Animal Health, Derio (Bizkaia), Spain.and P. Becher, Institut fur Virologie, Giessen, Germany. Optimization of Serum Dilution and Rc-NS3 Antigen Concentration A checkerboard titration of Rc-NS3 antigen with BVDV or BDV antibody positive and negative sera was carried out for determining the optimum antigen concentration and serum dilution to be used in the test proper. Briefly, different dilutions of serum (1/10 to 1/80) and Rc-NS3 antigen concentrations (10–1280 ng per well) were added to the wells of ELISA plate in row wise and column wise, respectively. The combination that showed maximum positive/negative (P/N ratio) was selected for further development of NS3 ELISA. Bovine serum albumin (BSA), non fat dry milk powder (NFDM) and horse serum with LAH at various dilutions (1–5%) were evaluated as blocking agents. Various dilutions of anti-sheep and anti-goat HRPO conjugate (1/4000 to 1/10,000, Sigma-Aldrich, St. Louis, MO, USA) and substrate (TMB, Sigma-Aldrich) reaction times (5–15 min) were also tested to ascertain the optimal test conditions. Optimized Rc-NS3 ELISA Procedure The wells of ELISA plate (Maxisorp, Nunc, Roskilde, Denmark) were coated with 50 µL (320 ng) of purified NS3 recombinant protein in carbonate bicarbonate buffer and kept at 4◦ C overnight. After washing once with 300 µL of PBS, wells were blocked with 200 µL of blocking buffer containing
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3% NFDM (Propure, Amresco, Solon, OH, USA) in PBS and incubated at 37◦ C for 1 hr. The wells were washed thrice with 300 µL of PBST (1X PBS with 0.05% Tween) and 50 µL of 1:20 diluted (1% NFDM in PBST) serum samples were added to each well in duplicate including positive and negative controls. The plate was incubated for 90 min at 37◦ C and after washing thrice with 300 µL of PBST, 50 µL of anti-sheep and anti-goat HRPO conjugate (1:8000 diluted in1% NFDM in PBST) was added to each well for sheep and goat serum samples, respectively. Then the plate was incubated for 1 hr at 37◦ C and washed thrice with PBST. The substrate reaction was developed by adding 50 µL of TMB (Sigma) to each well and the reaction was stopped after 10 min with 0.4M H2 SO4. The optical density (O.D.) of each well was read at 450 nm in a multiwell ELISA reader (Tecan, Tokyo, Japan). Virus Neutralization Test (VNT) A total of 569 field sheep and goat sera were tested for presence of virus neutralizing antibodies against BVDV-1 or BVDV-2 by VNT. Briefly, the sera were heat inactivated at 56◦ C for 30 min, diluted 1:5 and were tested in triplicate by microplate indirect immuno peroxidase monolayer assay (IPMA) as described in our previous report[18] using 200 tissue culture infectious dose 50 (TCID50 ) of BVDV-1 cattle isolate Ind S-1449, BVDV-2 sheep isolate Ind 51966,[18] and SFT-R cells. The immunostaining was carried out using anti-BVDV goat polyclonal antibody (VMRD, Pullman, WA, USA), anti goat IgG-HRPO conjugate (Sigma), substrate H2 O2 with chromogen 3-amino-9-ethyl carbazole (Sigma) and the results were noted after examination under microscope. Appropriate positive, negative, cell and virus controls were included in each plate. The serum sample was considered positive for BVDV-1 and/or BVDV-2 neutralizing antibodies when cells mixed with serum at an initial dilution of 1:5 and virus did not show any pestivirus specific cytoplasmic staining, otherwise it was considered as negative. Determination of Cut-Off Value A total number of 619 serum samples from sheep and goats were subjected to Rc-NS3 ELISA and the cut-off value was calculated based on the results of BVDV/BDV VNT positive (n = 201) and negative (n = 418) serum samples. The ELISA cut-off value for determining the status (pestivirus antibody positive or negative) of the serum was calculated by the following formula: Cut-off value = Negative mean OD at 450 nm (n = 418) × 2 standard deviation
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Determination of Sensitivity and Specificity The sensitivity, specificity, and agreement of the Rc-NS3 ELISA were calculated by considering VNT as the reference standard using the following formulae:
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Sensitivity % = True positive/ True positive + False negative × 100 Specificity % = True Negative/ True negative + False positive × 100 Agreement % =
Kappa value =
True positive + True Negative /Total number of samples × 100 Observed agreement − Expected agreement / 1 − Expected agreement
Evaluation of NS3 ELISA for Detection of BDV Antibodies A total of 50 reference sheep serum samples (20 BDV antibody positive and 30 BDV negative by VNT) were also tested by the NS3 ELISA developed in this study and its sensitivity and specificity was determined. RESULTS Optimization of Rc-NS3 ELISA The Rc-NS3 antigen was tested for its utility in ELISA for detection of pestivirus antibodies in sheep and goat serum samples. The checkerboard titrations showed that maximum difference in O.D. values between reference positive and negative serum (P/N values) was obtained at 320 ng per well of antigen and 1:20 dilution of serum (Figure 1). The correlation between the absorbance and the antigen concentration (10–1280 ng/well) at different dilutions of serum (1/10, 1/20, 1/40, and 1/80) was calculated by regression method (r2 = 0.83, 0.9449, 0.9186, and 0.9327, respectively) using Microsoft Excel 2007 and maximum correlation coefficient found for 1/20 dilution followed by 1/80 dilution. When 418 BVDV/BDV antibody negative serum samples were tested by the Rc-NS3 ELISA, a mean O.D. value of 0.220 with a standard deviation of 0.089 was obtained. Hence, the cut-off value was set at O.D. value of 0.40. This was further supported by distribution plot of O.D. values obtained by ELISA and results of VNT positive (n= 201) and negative (n= 418) serum samples (Figure 2).
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1280
640
320
160
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10 4.56912 6.5117 6.44487 4.80804 3.49744 2.17989 2.23121 1.29221 20 5.19524 6.07407 6.93038 5.73973 3.97101 2.81208 2.34328 1.8087 40 3.08929 3.21514 3.01449 1.74074 1.17677 1.14141 1.15104 1.90741 80 3.45306 2.51948 1.97664 2.05854 1.61735 1.16842 0.89305 2.80392 FIGURE 1 Checker board titration of Rc-NS3 antigen (10–1280 ng per well) with reference BVDV antibody positive and negative sera (1/10 to 1/80) for determining the optimum antigen concentration and serum dilution. 250 Number of Samples
200 150 100
Positive Negative
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P/N ratio
P/N ratio between serum dilution and antigen concentration 8 7 6 5 4 3 2 1 0
OD Value at 450 nm
FIGURE 2 Distribution plot of 418 VNT negative and 201 VNT positive serum samples in ELISA.
Relative Sensitivity and Specificity of Rc-NS3 ELISA The sensitivity and specificity of the Rc-NS3 ELISA in detecting BVDV antibodies was determined by comparison with the gold standard VNT. The Rc-NS3 ELISA showed a sensitivity and specificity of 91.02%, 94.59% in case of sheep and 100%, and 94.57% in goats, respectively. The agreement rate between the two tests was 93.3% (sheep) and 95.3% (goat) (Table 1). When 569 (463 sheep and 106 goat) serum samples were tested by Rc-NS3 ELISA, 168 (36.28%) sheep and 19 (17.92%) goats were found positive for BVDV antibodies (Table 2). As a whole, the RC-NS3 ELISA showed a sensitivity of 91.71% and specificity of 94.59% among sheep and goats. Testing of same
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TABLE 1 Relative sensitivity and specificity of Rc-NS3 ELISA with BVDV virus neutralization test based on 463 sheep and 106 goat serum samples
Positive
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Rc- NS3 ELISA
Prevalence a TP, b FP, c FN,
Positive Negative Total Sensitivity Specificity Agreement Kappa value VNT Rc-NS3 ELISA
Sheep
Goat
VNT
VNT
Negative
Total
152a 16b 168 15c 280d 295 167 296 463 91.02% (95% CI-85.71, 94.48) 94.59% (95% CI-91.4, 96.65) 93.3% (95% CI-90.65, 95.24) 0.856 (95% CI-0.81, 0.91) 36.07 % 36.29 %
Positive
Negative
Total
14a 5b 19 0c 87d 87 14 92 106 100 (95% CI-78.47,100) 94.57 (95% CI-87.9, 97.66) 95.28% (95% CI- 89.43, 97.97) 0.82 (95% CI-0.67, 0.97) 13.21 % 17.92 %
and d TN
serum samples by VNT revealed that 167 (36.07%) sheep and 14 (13.21%) goats were positive for BVDV neutralizing antibodies (Table 2). Hence, a very good correlation between Rc-NS3 ELISA and VNT was observed while determining the prevalence of BVDV antibodies in sheep and goats. Additionally, when we compared the test results of 50 serum samples of known BDV antibody status, it showed a sensitivity of 85% and specificity of 86.6% (Table 3). DISCUSSION Prevalence of pestivirus infections in sheep and goats have been reported worldwide with seroprevalence rates ranging from 5–50% in various countries.[1] This poses a serious challenge to the efforts of BVD control in cattle being operational currently in several countries, since transmission of pestiviruses from sheep and goats to cattle has been demonstrated. In view of the recent isolation and identification of BVDV-1 and BVDV-2 in Indian sheep and goats,[6,7,18] and future preparedness for BDV detection, there is an urgent need of active surveillance for pestivirus infections in small ruminants. ELISA is employed commonly for sero-epidemiological studies, as it is rapid, inexpensive, sensitive, and useful for mass screening. NS3 antibody ELISA is commonly used for detection of pestivirus antibodies in cattle, sheep, and goats during surveillance and control. Despite of availability of several commercial NS3 antibody ELISA kits, only scant information is available with regard to their validation in sheep and goat serum samples. Hence, the aim of the present study was development of a recombinant NS3 antigen based antibody ELISA and its evaluation for serological diagnosis of pestivirus infection in sheep and goats.
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Andhra Pradesh Gujarat Madhya Pradesh Punjab Rajasthan Tamil Nadu Total
States
126 43 0 20 211 63 463
Individuals tested 100 0 0 5 43 19 167
Positive
VNT
Sheep
79.37 0.00 0.00 25.00 20.38 30.16 36.07
% 90 3 0 5 52 18 168
Positive 71.43 6.98 0.00 25.00 24.64 28.57 36.28
%
Rc-NS3 ELISA
2 0 75 0 27 2 106
Individuals tested
1 0 11 0 0 2 14
Positive
VNT
50 0 14.67 0 0 100 13.21
%
1 0 14 0 2 2 19
50 0 18.67 0 7.41 100 17.92
%
Rc-NS3 ELISA Positive
Goats
TABLE 2 Comparison between Rc-NS3 ELISA and VNT in determining the prevalence of BVDV antibodies among sheep and goats (n = 569) in different states
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TABLE 3 Performance of Rc-NS3 ELISA with BDV reference serum VNT Positive
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Rc- NS3 ELISA
a TP, b FP, c FN,
Positive Negative Total Sensitivity Specificity Agreement Kappa value
Negative
17a 4b 3c 26d 20 30 85% (95% CI-63.96, 94.76) 86.67 (95% CI-70.32, 94.69) 86% (95% CI-73.81, 93.05) 0.711 (95% CI-0.512, 0.909)
Total 21 29 50
and d TN
Different forms and sources of antigen have earlier been used to develop ELISA such as whole virus particles,[19] BVDV infected cell culture extracts,[20] viral antigens immobilized with monoclonal antibodies,[21,22] or recombinant proteins produced in bacteria.[23,24] Prokaryotic expression of NS3 antigen and its application for detection of BVDV antibodies in cattle has been reported by several workers.[17,24–26] In this study, we report the development of an indirect ELISA using BVDV Rc-NS3 antigen expressed in E. coli. Rc-NS3 antigen (50 kDa) used in our study consisted of 384 amino acids from C- terminus of full-length NS3 protein carrying the whole of domain B (aa 368–549) and a part of domain A (aa 205–369) as defined earlier.[17] Use of smaller helicase domain not only avoids the diagnostic non-significant part of NS3 region but also has been found useful in detection of BVDV antibodies in cattle and buffalos.[27] An indirect ELISA was optimized to evaluate the Rc-NS3 protein for its potential use as antigen in diagnosis. The ELISA showed clear differences in the absorbance values between known BVDV antibody positive and negative sera from sheep and goats at appropriate dilutions of antigen and antibody. After optimization of the ELISA, BVDV antibody negative serum samples from sheep and goats (n=418) as determined by the reference virus neutralization test were employed for deciding the cut-off value (0.40) which was further verified by distribution analysis of antibody positive and negative serum samples. Since VNT is considered as the gold standard test for serological diagnosis of pestivirus infection (BVDV/BDV), the performance of the developed Rc-NS3 indirect ELISA was compared with VNT using 619 field serum samples (463 from sheep and 106 from goats and 50 reference serum). When compared with BVDV VNT, the ELISA exhibited a high sensitivity (91.02% and 100%) and specificity (94.59% and 94.57%) with a good correlation rate (93.3% and 95.3%) with kappa value of 0.855 and 0.821 for sheep and goat, respectively, indicating efficiency of Rc-NS3 ELISA in detecting BVDV antibodies. However, the sensitivity of Rc-NS3 ELISA was
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found a little bit lower than VNT. It is well known that appearance of BVDV neutralizing antibodies occur earlier than the non neutralizing antibodies in natural infection.[28] NS3 ELISA detects antibodies against BVDV nonstructural NS3 protein, whereas VNT detects neutralizing antibodies against E2 structural protein. Moreover, 100% correlation with VNT may not be possible with ELISA due to the preferential detection of only IgG isotype in ELISA and ability of detection of both IgG and IgM by VNT thereby increased sensitivity found for VNT. These reasons may explain why a slight lower sensitivity of Rc-NS3 ELISA was observed in our study when compared to VNT. A slightly lower sensitivity and specificity of Rc-NS3 ELISA in detecting BDV antibodies was observed. This can be explained by the fact that the NS3 recombinant antigen was generated from a BVDV isolate. However, these results proved that Rc-NS3 ELISA using 50 kDa of NS3 protein instead of 80 KDa of NS3 full-length non structural protein (being used by commercial kits) can be used for detection of BVDV/BDV antibodies in sheep and goats during sero-epidemiological studies. In the present study, the prevalence rate of BVDV antibodies was 36.5% in sheep and 17.9% in goats by the Rc-NS3 ELISA, while the prevalence rate was 36.06% in sheep and 13.2% in goats by VNT indicating that efficacy of Rc-NS3 ELISA compared very well with VNT. A similar range of seroprevalence of BVDV infection in sheep and goats in India has been reported earlier.[29] Varying rates of sensitivity and specificity between commercial NS3 antibody ELISA and VNT have been reported earlier while studying pestivirus seroprevalence in sheep and goats. A study using Ceditest NS3 ELISA (Ceditest® , Cedi Diagnostics, Lelystad, Netherlands) found a pestivirus seroprevalence rate of 45% in sheep, compared to a prevalence rate of 28% found by VNT.[30] Although the producer of ELISA kit claimed a sensitivity and specificity rate of 99% with serum, milk, and plasma samples of cattle, the kit had not been validated with serum samples from sheep and goats and hence, a high percentage of false positive results were found. In another study, a variation in pestivirus antibody prevalence rates between ELISA (48.12%) and VNT (42.89%) was observed, when 401 sheep serum samples were tested by Institut Pourquier NS3 ELISA kit (Institut Pourquier, Montpellier, France).[31] These results indicated that commercial NS3 antibody ELISAs developed and validated for detection of BVDV antibodies in cattle using full length NS3 protein should be validated before use in sheep and goats. Being an inexpensive and rapid method, the developed and validated Rc-NS3 ELISA reported in this study should be found useful during sero-surveillance and sero-monitoring of pestivirus infections in sheep and goats.
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CONCLUSION
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The sensitivity and specificity of Rc-NS3 indirect ELISA developed and evaluated in this study correlated well with the reference virus neutralization test for detection of BVDV antibodies in sheep and goats and correlated moderately in detecting BDV antibodies. Being a simple and rapid test, the developed NS3 ELISA should be found useful during sero-surveillance and sero-monitoring of pestivirus infections in sheep and goats. FUNDING N. Mishra was supported by a grant (BT/PR4415/AAQ/01/165/2003) from Department of Biotechnology, Govt. of India, New Delhi. REFERENCES 1. Nettleton, P.F.; Gilray, J.A.; Russo, P.; Dlissi, E. Border disease of sheep and goats. Vet. Res. 1998, 29, 327–340. 2. King, A.M.Q.; Lefkowitz, E.; Adams, M.J.; Carstens, E.B. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, 1st Ed. Elsevier, San Diego, CA, 2011, pp 1003–1020. 3. Czopowicz, M.; Kaba, J.; Schirrmeter, H.; Bagnick, E.; Szalus-Jordanow, O.; Nowicki, M.; Witkowski, L.; Frymus T. Serological evidence for BVDV-1 infection in goats in Poland- Short communication. Acta. Vet. Hung . 2011, 59, 399–404. 4. Kim, I.J.; Hyun, B.H.; Shin, J.H.; Lee, K.K.; Lee, K.W.; Cho, K.O.; Kang, M.I. Identification of bovine viral diarrhoea virus type 2 in Korean native goat (Capra hircus). Virus. Res. 2006, 121,103–106. 5. Krametter-Froetscher, R.; Loitsch, A.; Kohler, H.; Schleiner, A.; Schiefer, P.; Moestl, K.; Golja, F.; Baumgartner, W. Prevalence of antibodies to pestiviruses in goats in Austria. J Vet. Med. B. Infect. Dis. Vet. Public. Health. 2006, 53, 48–50. 6. Mishra, N.; Dubey, R.; Rajukumar, K.; Tosh, C.; Tiwari, A.; Pitale, S.S.; Pradhan, H.K. Genetic and antigenic characterization of bovine viral diarrhea virus type 2 isolated from Indian goats (Capra hircus). Vet. Microbiol. 2007, 124, 340–347. 7. Mishra, N.; Pitale, S.S.; Rajukumar, K.; Prakash, A.; Behera, S.P.; Nema, R.K.; Dubey, S.C. Genetic variety of bovine viral diarrhea virus 1 strains isolated from sheep and goats in India. Acta. Virol. 2012, 56, 209–215. 8. Pratelli, A.; Martella, V.; Cirone, F.; Buonavoglia, D.; Elia, G.; Tempesta, M.; Buonavoglia, C. Genomic characterization of pestiviruses from lambs and kids in Southern Italy. J. Virol. Methods. 2001, 94, 81–85. 9. Sullivan, D.G.; Chang, G.J.; Akkina, R.K. Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep. Virus. Res. 1997, 47 , 19–29. 10. Valdazo-Gonzalez, B.; Alvarez-Martinez, M.; Greiser-Wilke, I. Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain. Vet. Microbiol. 2006, 117 , 141–153. 11. Carlsson, U. Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine viral diarrhea virus. Vet. Rec. 1991, 128, 145–147. 12. Carlsson, U.; Belak, K. Border disease virus transmitted to sheep and cattle by a persistently infected ewe: epidemiology and control. Acta. Vet. Scand. 1994, 35, 79–88. 13. Loken, T. Ruminant pestivirus infections in animals other than cattle and sheep. Vet. Clin. North. Am. Food. Anim. Pract. 1995, 3, 597–614. 14. Meyers, G.; Thiel, H.J. Molecular characterization of pestiviruses. Adv. Virus. Res. 1996, 47 , 53–118. 15. Palfi, V.; Houe, H.; Philipsen, J. Studies on the decline of bovine virus diarrhoea virus (BVDV) maternal antibodies and detectability of BVDV in persistently infected calves. Acta. Vet.Scand. 1993, 34, 105–107.
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