Serological study of foot and mouth disease virus type A in India

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1 Rev. sci. tech. Off. int. Epiz., 1986, 5 (3), Serological study of foot and mouth disease virus type A in India L.M. BELWAL, R. PALANISAMY, K. NAGAIAH, A.P. KALANIDHI, H.M. JAGANNATHA, B.C. RAMANNA and V.A. SRINIVASAN* Summary: Serological comparisons among type A Indian FMD virus isolates from different parts of the country were made using the two-dimensional microneutralisation test. Current vaccine strains and candidate vaccine strains were employed for reference. More than one distinct antigenic group in type A FMD virus could be identified. A broad antigenic spectrum was exhibited by A India 7/82 followed by A 22 India 57/79 while A India 85/79 and A Holland showed narrow spectra. The antigenic diversity among type A viruses in India indicated that a proposed candidate vaccine strain needs to be thoroughly compared with representative isolates from various Indian territories to establish its wide antigenic spectrum as required for the vast sub-continent. KEYWORDS: Antigens - Aphthovirus - Cattle diseases - Disease control - India - Serotypes - Vaccines - Viral diseases - Virus neutralization. INTRODUCTION The control of foot and mouth disease (FMD) has to take into account the antigenic variation of the FMD virus. The study of antigenic variation among field viruses helps in understanding the epidemiology of FMD as also in the selection of suitable vaccine virus strains. Variation among type A strains has been reported by several authors (1, 3, 4, 12). In India, prevalence of types О, С and A FMD virus and their variants was first recorded by Seetharaman and Datt (11). Type Asia 1 was reported in 1957 (6). A quadrivalent vaccine was subsequently produced in the year While strains of Indian origin were incorporated for valences О, С and Asia 1, a Dutch type A strain was employed for the fourth valence (5). This strain has been designated as A 5 or A iq at various stages (5). However, for the sake of clarity, it is referred to as A Holland in the present study. Type A strains isolated from outbreaks in cattle vaccinated with the aforementioned vaccine were shown to be closer to the Middle Eastern subtype (5). Rweyemamu et al. (10) studied several Asian type A isolates from the Middle East and Indian sub-continent and observed that although the Indian type A FMD viruses could be related to Middle Eastern A 22 virus ( Iraq 24/64), they were quite divergent from each other. Limited strain differentiation studies showed that several Indian type A isolates were closer to subtype than to the vaccine strain A Holland (2). * Indian Immunologicals, Hyderabad , India.

2 724 The present study was undertaken to: i) elucidate the antigenic relationship among Indian type A strains; ii) evaluate the current vaccine strains and Hi) identify as candidate vaccine virus the strain(s) exhibiting a broad antigenic spectrum. Viruses MATERIALS AND METHODS Two type A virus isolates, A 22 India 57/79 (vaccine strain) and A Ind 25/81 were received from the Wellcome FMD Laboratory (Pirbright, UK) and twelve isolates were obtained from the World Reference Laboratory for FMD, Animal Virus Research Institute, Pirbright, UK. The virus isolates were received from the Wellcome FMD Laboratory as BHK 21 monolayer or suspension cell-adapted virus strains. The WRL supplied the isolates as calf thyroid (BTY), bovine kidney (BK), pig kidney (RS) or BHK cell culture adapted viruses. The vaccine strain A Holland was obtained from the Indian Veterinary Research Institute, Bangalore, as BHK adapted virus. All the virus strains were adapted to BHK 21 monolayer cells. The details of the viruses included in the study are given in Table I. TABLE I Type A Indian FMD virus isolates used in the study s. No. Strain Ident. WRL Ref. No. Place of origin District State Passage history at time of receipt Passage history at time of use 1. Ind 57/79 Dharmapuri Tamil Nadu RS1 BTY BHK1 2. A Ind 73/79 Mehboobnagar Andhra Pradesh RSI RSI BTY1 BHK1 E/T BHK8 Susp. 4 BHK1 RSI E/T BHK5 3. A Ind 54/79 Tirunelveli Tamil Nadu RS2 RS2 E/T BHK6 4. A Ind 7/82 Kaira Gujarat BK2 BTY2 BTY2 E/T BHK3 5. A Ind 25/81 Ahmedabad Gujarat BK1 BK1 E/T BHK5 6. A Ind 85/79 Bombay Maharashtra BHK1 BHK1 E/T BHK6 7. A Ind 36/82 Ahmednagar Maharashtra BTY2 BTY2 E/T BHK3 8. A Ind 37/82 Kolhapur Maharashtra BTY2 BTY2 E/T BHK3 9. A Ind 16/82 Jaipur Rajasthan BK2 BK2 E/T BHK3 10. A Ind 25/82 Jind Haryana BTY1 BTY1 E/T BHK3 11. A Ind 14/82 Hoshiarpur Punjab BTY1 BTY1 E/T BHK3 12. A Ind 29/82 Kangra Himachal Pradesh 13. A Ind 20/82 Jammu Jammu & Kashmir BK1 BTY1 BK2 BTY1 BK1 BTY1 BHK3 BK2 BTY1 BHK3 14. A Ind 3/77 Haringhata West Bengal BTY1 BTY1 E/T BHK6 15. A Holland Holland BHK5 BHK6 Susp. 7 BHK1 E/T : ether treated.

3 725 Antisera Guinea-pig antisera Anti 140S guinea-pig antisera against India 57/79 and A Holland were prepared in our laboratory while those against A India 7/82 and A India 85/79 were obtained from Dr G.N. Mowat of AVRI, Pirbright, UK. The antisera were raised as per the method described by Rweyemamu et al. (7). Briefly, BEI (binaryethyleneimine) inactivated and sucrose density gradient purified 140S antigen was emulsified with Freund's incomplete adjuvant and inoculated intramuscularly in the hind leg of guinea-pigs. A booster dose of similar 140S antigen was administered 21 days later. Ten days after the second dose, the guinea-pigs were bled by exsanguination and the sera pooled before use. Bovine vaccinate sera A group of 6 seronegative steers were vaccinated with appropriate monovalent vaccine and boosted with the same on the 21st day. The animals were bled on the 35th day and the sera were pooled before use. Serum neutralisation test The two-dimensional microneutralisation test system, as described by Rweyemamu et al. (8), was used. The 'r' values - the ratio of serum neutralisation titre for heterologous and homologous virus - were calculated from the mean titres of three replicate tests. For testing the significance of V values an estimated, pooled variance of (9) was used. RESULTS The 'r' values derived from replicate comparisons are furnished in Table II. The anti 140S guinea-pig antiserum raised against India 57/79 showed a narrow serological spectrum and all the viruses exhibited significant differences (r/1 at P = 0.01) when compared with India 57/79. The V values ranged from 0.01 to Two isolates, namely A India 54/79 and A India 73/79, were found to be related to India 57/79 when bovine vaccinate serum raised against India 57/79 was used. All other viruses were found to be significantly divergent from India 57/79 ('r' values ranging from 0.02 to 0.12). Strain A India 85/79 also showed a narrow serological spectrum and 'r' values significantly less than 1 (P = 0.01) were obtained against all the strains. Seven field strains were shown to be related to A India 7/82 when anti 140S guinea-pig serum was employed while the other seven were significantly divergent ('г' values ranging from 0.03 to 0.15). All the Indian field strains were shown to be divergent from A Holland when anti 140S guinea-pig serum was used. Significantly low (P/0.01) 'r' values were also obtained with bovine vaccinate serum except in the case of India 57/79 with which A Holland appeared to have an asymmetric relationship. Among the four strains used for reference, the strain A India 7/82 showed the broadest serological spectrum.

4 726 TABLE II Serological comparison among Indian type A FMD viruses Virus State T.N. T.N. Guj. Guj. Mah. Mah. Mah. Dist. Dharma- Tirunel- Kaira Ahmeda- Bombay Ahmed- Kolhapuri veli bad nagar pur Strain Ind A Ind A Ind A Ind A Ind A Ind A Ind Antiserum Ref : 57/79 54/79 7/82 25/81 85/79 36/82 37/82 India 57/ * 0.01* 0.01* 0.01* 0.01* 0.01* India 57/ * 0.02* 0.06* 0.03* 0.12* (bovine vaccinate serum) A India 7/ * 0.06* * 1.00 A India 85/ * 0.03* 0.05* 0.02* * 0.15* (Anti I40S A Holland 0.01* 0.01* 0.01* 0.01* 0.01* 0.04* 0.02* A Holland * 0.09* 0.09* 0.09* 0.09* 0.09* (bovine vaccinate serum) * Significantly different from 1 at P = 0.01.

5 727 I л HI Y II (cont.) Virus A.P. J & К H.P. Punjab Haryana Rajasthan W. Bengal Mehboob- Jammu Kangra Hoshiar- Jind Jaipur Harin- Holland nagar pur ghata A Ind A Ind A Ind A Ind A Ind A Ind A Ind A Holland Antiserum 73/79 20/82 29/82 14/82 25/82 16/82 3/77 India 57/ * 0.01* 0.01* 0.01* 0.01* 0.01* 0.01* 0.01* India 57/ * 0.02* 0.04* 0.06* 0.04* 0.10* 0.02* (bovine vaccinate serum) A India 7/ * * * 0.03* A India 85/ * 0.1* 0.07* 0.16* 0.10* 0.10* 0.03* 0.03* A Holland 0.01* 0.02* 0.01* 0.01* 0.02* 0.02* 0.01* 1.00 A Holland 0.09* 0.09* 0.09* 0.09* 0.09* 0.09* 0.09* 1.00 (bovine vaccinate serum)

6 728 DISCUSSION The results of the study indicated that more than one distinct antigenic group within the serotype A may be prevalent in India. It can be observed that the strains India 57/79 and A India 85/79 have narrow serological spectra. The strain India 57/79, isolated from Southern India, appears to be related only to other strains of Southern Indian origin, namely A India 54/79 and A India 73/79. The strains from Maharashtra, namely A India 36/82 and A India 37/82, are divergent from A India 85/79 (r<l at P = 0.01), an isolate from Maharashtra which presents a narrow immunogenic spectrum against a majority of strains and cannot be considered as a candidate vaccine strain. The strain A India 7/82, an isolate from Western India, exhibited a broader serological spectrum than the other two Indian strains ( India 57/79 and A India 85/79) and A Holland. While the strains from Southern parts of India were neutralised poorly (r < 1 at P = 0.01) by antiserum A India 7/82, all the strains from Western and North Western parts of India, except A India 14/82 and A India 36/82, showed closer antigenic relationship to this strain. Although the strain A Holland exhibits an asymmetric relationship with India 57/79, its extremely narrow serological spectrum in relation to Indian isolates suggests that a more appropriate type A strain of Indian origin should be incorporated in the quadrivalent vaccine. However, the wide diversity and complex interrelationship exhibited by Indian type A viruses in the present study supports the earlier observation by Rweyemamu et al. and makes the choice of an appropriate vaccine strain difficult. Keeping in mind that the uncontrolled livestock movements from one region to another play a major role in the spread of disease in India, it may be possible that the immunity provided by a single representative vaccine strain may be endangered if a variant strain is introduced. Therefore it is suggested that, in order to provide the broad immunogenic spectrum required, the type A strain to be incorporated in the quadrivalent vaccine should be evaluated against representative isolates from various Indian territories and the immunocompetence of the vaccine strain should be monitored continuously against the field strains. * * * ÉTUDE SÉROLOGIQUE DU VIRUS APHTEUX DE TYPE A EN INDE. L.M. Belwal, R. Palanisamy, K. Nagaiah, A.P. Kalanidhi, H.M. Jagannatha, B.C. Ramanna et V.A. Srinivasan. Résumé : Une étude sérologique comparative de souches de virus aphteux de type A isolées dans différentes régions de l'inde a été réalisée par la microméthode de neutralisation bidimensionnelle. L'étude comparative a porté sur les souches actuellement utilisées ou proposées pour la préparation de vaccins, et a permis d'identifier plusieurs groupes antigéniques différents parmi les virus aphteux de type A. La souche A Inde 7/82 présente un large spectre antigénique, suivie par la souche A 22 Inde 57/79, tandis que les souches A Inde 85/79 et A Hollande se caractérisent par des spectres étroits. La diversité antigénique des virus de type A en Inde montre qu'une souche proposée comme souche vaccinale doit être comparée en détail avec des souches représentatives isolées dans les différentes régions du pays pour s'assurer que son spectre antigénique est assez large pour couvrir l'ensemble du sous-continent.

7 729 MOTS-CLÉS : Antigènes - Aphthovirus - Inde - Maladies des bovins - Maladies virales - Prophylaxie - Séroneutralisation - Sérotypes - Vaccins. * * * ESTUDIO SEROLÓGICO DEL VIRUS AFTOSO DE TIPO A EN INDIA. L.M. Belwal, R. Palanisamy, K. Nagaiah, A.P. Kalanidhi, H.M. Jagannatha, B.C. Ramanna y V.A. Srinivasan. Resumen : Se ha efectuado un estudio serológico comparativo de cepas de virus aftoso de tipo A aisladas en distintas regiones de la India empleando el micrométodo de neutralización bidimensional. El estudio comparativo se refirió a las cepas que se utilizan o proponen actualmente para preparar vacunas, con lo que se pudieron identificar varios y distintos grupos antigénicos entre los virus aftosos de tipo A. La cepa A India 7/82 presenta un amplio espectro antigénico, seguida por la cepa A 22 India 57/79, mientras que las cepas A India 85/79 y A Holanda se caracterizan por espectros estrechos. La diversidad antigénica de los virus de tipo A en India demuestra que debe compararse en detalle una cepa propuesta como cepa vacunal con cepas representativas aisladas en las diferentes regiones del país para cerciorarse que su espectro antigénico es lo bastante amplio para cubrir la totalidad del subcontinente. PALABRAS CLAVE : Aftovirus - Antígenos - Control - Enfermedades de bovinos - Enfermedades víricas - India - Seroneutralización - Serotipos - Vacunas. * REFERENCES 1. ANDERSON E.C., ANDERSON J. & DOUGHTY W.J. (1974). The foot and mouth disease virus subtype variation in Kenya. J. Hyg., Camb., 73, ANON. (1979). Annual report of the Project-Coordinator, All India Coordinated Research Project on Epidemiology of FMD, Indian Council of Agricultural Research, pp ARROWSMITH A.E.M. (1975). Variations among strains of type A foot and mouth disease virus in the Eastern Mediterranean region. J. Hyg., Camb., 71, BROOKSBY J.В., HENDERSON W.M. & GALLOWAY I.A. (1948). Strains of virus of foot and mouth disease recovered from outbreaks in Mexico. Proc. Soc. exp. Biol., N.Y., 69, DATT N.S. (1984). Epidemiology of virus types of foot and mouth disease in India versus use of vaccine. Proc. of IX Workshop of All India Coordinated Research Project for EpidemioIogicalStudies on Foot and Mouth Disease, Feb. 11th to 14th, 1984, DHANDA M.R., GOPALAKRISHNAN V.R. & DHILLON H.S. (1957). Note on the occurrence of atypical strains of foot and mouth disease virus in India. Ind. J. vet. Sci., 27, RWEYEMAMU M.M., BOOTH J.C. & PAY T.W.F. (1977). Neutralisation kinetics studies with type SAT 2 FMD virus strains and factors that influence the rate and pattern of neutralisation. J. Hyg., Camb., 78,

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