Trop. Anita. Hlth Prod. (1988) 20, 109-113

D E T E C T I O N O F G O A T P O X A N T I G E N A N D A N T I B O D Y B Y T H E C O U N T E R I M M U N O E L E C T R O P H O R E S I S T E S T

BHASKAR SHARMA, B. S. NEGI, A. B. PANDEY, S. K. BANDYOPADHYAY, H. SHANKAR AND M. P. YADAV

Indian Veterinary Research Institute, Mukteswar-Kumaon-263138, UP, India

SUMMARY

The counterimmunoelectrophoresis (CIE) test was standardised for the detection o f goat pox antigen and antibody using inactivated antigens. The chloroform inactivated and live antigens were equally sensitive for detection of goat pox precipitins. The precipitinogens of goat pox virus (GPV) were found to be soluble in nature. The CIE test was quick as well as more sensitive than the agar gel precipitation test for detection of GPV antibody/antigen. The CIE employing inactivated antigen has been used for the first time in the detection of GPV antibodies~antigens.

INTRODUCTION

The agar gel precipitation test (AGPT) has been used for the detection of goat pox (GP) antigen and antibodies (Sharma and Dhanda, 1971; Pandey and Singh, 1972 and Kitching, Hammond and Black, 1986). This test takes two to three days to complete and its sensitivity is also poor. Recently counterimmunoelectro- phoresis (CIE) has been used for the detection of sheep pox antigen and antibodies (Puranchand, Rao, Garg and Singh, 1985). We here report the application of the CIE test for detecting goat pox antigen and antibody using inactivated antigen.

MATERIALS AND METHODS

Apparently healthy goats of either sex aged from six to twelve months and having no history of goat pox, either purchased from goat pox free areas or raised in the Divisional sheds, were bled to collect serum and tested for the presence of antibody to goat pox antigen by the AGPT. Animals negative for antibodies were used for preparation of hyperimmune serum (HIS) and propagation of the virus.

The Sambalpur strain of goat pox virus (GPV) was used for infection of goats for antigen preparation, for preparation of vaccine and postvaccinal challenge (Bandyopadhyay, Gajendragad, Dhal, Gupta and Yadav, 1984). The virus was maintained by skin to skin transfer in goats. Prevaccination, post-vaccination (killed GP vaccine), post-challenge and convalescent goat pox sera were collected for GPV antibody screening.

HIS against GPV was raised in goats by infecting them initially by the intradermal route using 10 -I dilution of virus suspension. On recovery from the disease these goats were inoculated with 5 ml of the above virus suspension by the subcutaneous route after adding an equal volume of Freund's complete adjuvant and thereafter nine times with 5 ml of virus suspension with equal volume of Freund's incomplete adjuvant at weekly intervals. Serum was collected 10 days after the last inoculation. It was inactivated at 56°C for 30 rain in a water bath and kept at -20"C till use.

The antigen was prepared from goat pox lesions collected between seven and eight days post-infection. For crude antigen the skin lesions were washed three

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times with PBS (pH 7-2) and then triturated in a pestle and mortar with sterile sand to make a 30% suspension in PBS. The suspension was frozen and thawed three times before centrifuging at 2,000 rpm for 20 rain at 4°C. The supernatant, after concentration with PEG 6,000 in a dialysis bag for 20 rain at 4°C, was used as antigen as such as well as after heat inactivation at 60°C for 30rain. Chloroform treated antigen was prepared by mixing equal volumes of chloroform (Analar grade) and crude antigen followed by vigorous shaking. It was kept for 60 rain at 4°C with intermittent shaking and centrifuged at 2,000 rpm for 20 rain. The upper aqueous phase was collected and used as antigen. It was stored at -20°C before use.

The heat and chloroform treated antigens were inoculated into healthy goats intradermally to test the infectivity. The crude and chloroform treated antigens prepared as above were centrifuged at 35,000 g for i h in a refrigerated centrifuge to pellet the virus. These two antigens were also passed through an 0.45/~ filter. The filtrates supernatant as well as resuspended pellet (in i ml NSS) were also used in the CIE for the detection of GPV antigen activity in them. The CIE test was carried out in 1% agarose (SRL low gelling and high EEO agarose) in 0-04 M pH 8.6 barbitone buffer at 20 v/cm for 45 rain on microscope glass slides. The AGPT was carried out according to Uppal and Nilakantan (1970) using the above antigens.

Skin lesions of GP collected from experimentally or naturally infected goats between two and 16 days after their appearance were processed to prepare crude suspensions and after chloroform extraction as given above before testing in AGPT and CIE for GPV antigen detection.

RESULTS

The chloroform and heat treated antigens were found to be inactivated as none of these produced pox lesions in susceptible goats. The chloroform treated antigen showed up to three lines of precipitation in the CIE whereas crude antigen gave only one line. Chloroform treated and crude healthy goat skin suspension did not show any precipitin line in the CIE.

In the AGPT two precipitins developed against all the three antigens (crude, heat treated and chloroform treated) within 48 h at 37"C when 1% Noble agar in 0.85% NaC1 was used with 5 mm distance between wells; a third line developed after recharging. Chloroform treated and crude healthy goat skin antigens did not produce any line against HIS. The supernatant but not the pellet obtained after centrifuging crude chloroform treated antigens gave precil~itin reactions in CIE and AGPT. The filtrate obtained after fitering chloroform treated and crude GPV antigens through a 0.45 # filter also gave a line of precipitation when tested in CIE.

The sensitivity of CIE as compared to AGPT was determined by screening 604 sera of goats (convalescent, prevaeeination, post-vaccination and post- challenge) for the presence of precipitins. While only 29 out of the 604 (4.8%) samples were positive by the AGPT, 121/604 (20-03%) were positive in the CIE test (Table I). The CIE was also better than the AGPT for detection of goat pox antigen in skin lesions of goats (Table II). The results of the comparative efficacy of live and chloroform inactivated GPV antigens in the detection of antibodies (Table III) indicate that both the antigens were equally sensitive. However, when skin lesions processed for detection of antigen against HIS, chloroform extraction

DETECTION OF GOAT POX ANTIGEN AND ANTIBODY

TABLE I

Comparative efficacy of CIE and AGPT in detection of GPV antibody

Serum samples CIE AGPT

Convalescent 35/216 (16.20%) 6/216 (2.77%) Prevaccination 1/58 (1-72%) 0/58 (0%) Post-vaccination 64/276 (23.18%) 8/276 (2.89%) Post-challenge 21/54 (38-88%) 15/54 (27.77%)

Total 121/604(20-03)% 29/604(4.8%)

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TABLE II

Comparative efficacy of CIE and AGPT in detection of GPV antigen in skin lesions I

Number of samples

Test No. tested No. +ve % +ve

AGPT 74 30 40.5 CIE 74 38 51.3 AGPT 522 14 27.0 CIE 521 22 42.3

~The tests were performed on identical samples of 2-16 day old goat pox lesions after natural/experimental infection. 2These samples were tested without PEG concentration.

TABLE III

Comparative efficacy of live and killed GPV antigens in detecting goat pox antibodies by CIE test on identical

samples

Number of sera samples

Type of antigen Tested Found +ve % +ve

Live 20 9 45 Chloroform

inactivated 20 9 45

of the antigen resulted in more positive reactions compared with live crude antigen.

DISCUSSION

The commonly used method for diagnosing goat pox infection in the laboratory has been the AGPT. The test is, however, leSS sensitive and takes a comparatively long time for completion. The CIE test could detect the presence of precipitins in 20-03% sera as compared to 4-8% detected by AGPT, thus

112 SHARMA er AL.

clearly showing CIE to be more sensitive than AGPT. We have recently used ELISA and found it to be more sensitive in detecting GPV antibody (Sharma, Negi, Yadav, Shankar and Pandey, 1988). However, the CIE has the merit of ease, simplicity as also rapidity and was found to be slightly better for detection of GPV antigen than the ELISA. The sensitivity of CIE over AGPT was more marked in detection of antibodies than antigen. This is probably low antibody levels developed in GP after natural infection or vaccination as the use of potent HIS and concentrated antigen increased the sensitivity of AGFT considerably (Table II). The presence of precipitation reaction with the supernate after ultracentrifugation and in the filtrate after filtration through 0-45/z membrane filter and absence of the reaction when virus pellet was used indicate that the GPV precipitins are soluble antigens. The goat pox and sheep pox virus antigens have been shown to be soluble in nature (Pandey and Singh, 1972).

The antigens used in this study were killed as the treatments viz. heat and chloroform abolished the infectivity of the test antigens. The chloroform extracted antigen was comparable to live crude antigen in detection of GPV antibodies but gave higher positive results when antigen from skin lesions of goats were prepared by this method for detection of GPV antigen.

Since the chloroform and heat inactivated antigens retained their antigenicity for the precipitation test, it is suggested that these treatments may be used to prepare GPV antigen for CIE and AGP tests so as to avoid the risk of spread of virus from the laboratory. Use of inactivated antigen in CIE and AGP tests for diagnosis of goat pox has been demonstrated for the first time. There also does not appear to be any other report of using CIE for demonstration of goat pox antigen/antibodies.

Recently Kitching et al. (1986) have shown that sheep pox, goat pox, sheep and goat pox and lumpy skin disease virus share a common precipitating antigen. We had also observed cross reaction among goat pox, sheep pox and contagious ecthyma virus in CIE and ELISA using adsorbed and unadsorbed HIS (Anon, 1986). This suggests that CIE can be used for diagnosis of goat pox, sheep pox and contagious ecthyma viruses using antigen/antisera of either of these viruses.

ACKNOWLEDGEMENTS

The authors wish to thank Dr P. N. Bhat, Director of the Institute for providing facilities for the work and G. C. Chaudhary and L. C. Shah for technical assistance.

Accepted for publication April 1987

REFERENCES

A~ON (1986). Annual report of the Division of Virology, IVRI, Mukteswar, UP, India. BANDYOPADHYAY, S. K., GAJENDRAGAD, M. R., DHAL, N. K., GUFrA, A. R., & YADAV, M. P.

(1984). Indian Journal @Animal Science, 55, 961-964o KrrCHIN6, R. P., HAl~4ObrD, J. M. & BLACK, D. N. (1986). Journal of General Virology, 67,

139-148. PANDEY, R. & Sn~GH, I. P. (1972). Acta Virologica, 16, 41-46. PURANCHAt~, RAO, V. D. P., GARG, S. K., & SINGH, I. P. (1985). British Veterinary Journal, 141,

124-128. SI-IARMA, S. N. & DHANDA, M. R. (1971). Indian Journal of Animal, Science, 41, 267-272. SHARMA, B., NEGI, B. S., YADAV, M. P., SHANKAR, H. & PAbrDEY, A. B. (1988). Acta Virologica,

(In press). UI~AL, P. K. & NILAKAbrrAN, P. R. (1970). Journal of Hygiene (Cambridge), 68, 349-359.

DETECTION OF GOAT POX ANTIGEN AND ANTIBODY 113

DETECTION DE L'ANTIGENE ET DE L'ANTICORPS VARIOLE CAPRINE PAR UN TEST DE CONTRIMMUNO-ELECTROPHORESE

R~um6--Le test de contrimmuno-~lectrophor~se (CIE) a ~t~ standardis~ pour la d~tection de l'antig~ne et de l'anticorps variole caprine avec des antig~nes inaetiv~s. Les antig~nes vivants et inactiv~s par le chloroforme sont de sensibilit~ ~quivalente pour la d~tection des pr~cipitines variole caprine. On a montr~ que ces pr~cipitog~nes du virus variole eaprine ( W C ) 6taient de nature soluble. Le CIE est rapide et plus sensible que le test de precipitation en g~lose pour la d~tection des antig~nes et anticorps W C . C'est la premiere lois que le CIE mettant en oeuvre tm antig~,ne inactiv~ a ~t~ utili~.

DETECCION DEL ANTIGENO Y ANTICUERPOS DE VIRUELA CAPRINA MEDIATE LA PRUEBA DE CONTRAINMUNOELECTROFORESIS

R e l m e n - - S e estandariz6 la prueba de contrainmunoeleetroforesis, para la detecci6n del antigeno y anticuerpos del virus de la viruela caprina, usando antigenos inaetivados. El antigeno inactivado con eloroformo y el antigeno vivo, fueron igualmente sensitivos para la deteeci6n de precipitinas de viruela caprina. Los preeipit6genos del virus de la viruela caprina se encontraron que eran solubles. La prueba de contrainmunoeleetroforesis rue m~s rapida y mas sensitiva que la precipitaci6n en agar gelatina para la deteeci6n de anticuerpos/antigenos del virus de la viruela eaprina. La prueba de contrainmunoeleetroforesis con antigeno inaetivado ha sido utilizada por vez primera en la detecci6n de anticuerpos/antfgenos del virus de la viruela caprina.

BIOTECHNOLOGY 1989

First Notice

An international conference on the application of biotechnology to livestock in developing countries is to be held in the University of Edinburgh, 4th to 9th September, 1989.

Further details from the conference organiser:--

Mr A. G. Hunter, CTVM, Easter Bush, Roslin, Midlothian, EH25 9RG, Scotland, UK.