Five groups of ten animals were vaccinated with a combined vaccine containing foot and mouth disease (FMD), rabies, Pasteurella multocida and Clostridium chauvoei antigens and individual component vaccines containing FMD, rabies, Pasteurella multocida and Clostridium chauvoei antigens, respectively. Serological response of the calves was assayed on days 21 and 90 post vaccination. There was no significant variation in the serological response elicited by individual component vaccines and combined vaccine containing all four antigens.Pet skupina po deset životinja cijepljeno je različitim kombinacijama vakcina. Prva je skupina cijepljena polivalentnim cjepivom koje je sadržavalo antigene virusa slinavke i šapa, virusa bjesnoće te bakterija Pasteurella multocida i Clostridium chauvoei. Ostale su skupine cijepljene pojedinačnim antigenima, pa je tako druga skupina cijepljena antigenima virusa slinavke i šapa, treća antigenima virusa bjesnoće, četvrta antigenima bakterije Pasteurella multocida, a peta antigenima bakterije Clostridium chauvoei. Serološki odgovor svih životinja provjeren je 21. i 90. dana nakon cijepljenja. Na osnovi serološkoga odgovora nisu utvrđene značajne razlike među različitim načinima cijepljenja

Guddeti Srinivas Reddy

Koppunuru Ananda Rao

Ueli Kihm

Villuppanoor Alwar Srinivasan

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37Serological response of bovines to combined vaccinecontaining foot and mouth disease virus, rabies virus,Pasteurella multocida and Clostridium chauvoei antigensVilluppanoor Alwar Srinivasan1*, Guddeti Srinivas Reddy1,Koppunuru Ananda Rao1, and Ueli Kihm21 Indian Immunologicals, Rakshapuram, Gachibowli, India2 Federal Veterinary Office, Liebefeld-Bern, SwitzerlandSRINIVASAN, V. A., G. S. REDDY, K. A. RAO, U. KIHM: Serological responseof bovines to combined vaccine containing foot and mouth disease virus, rabiesvirus, Pasteurella multocida and Clostridium chauvoei antigens. Vet arhiv 71,37-45, 2001.ABSTRACTFive groups of ten animals were vaccinated with a combined vaccine containing foot and mouthdisease (FMD), rabies, Pasteurella multocida and Clostridium chauvoei antigens and individualcomponent vaccines containing FMD, rabies, Pasteurella multocida and Clostridium chauvoeiantigens, respectively. Serological response of the calves was assayed on days 21 and 90 postvaccination. There was no significant variation in the serological response elicited by individualcomponent vaccines and combined vaccine containing all four antigens.Key words: food and mouth disease, rabies, Pasteurella multocida, Clostridium chauvoei,prophylaxis, combined antigens, serology, vaccinationIntroductionRegular prophylactic vaccination of dairy animals against infectiousdiseases in developing countries has become an important input to maintainmilk production and to reduce economic losses. In an exercise to reducethe cost of vaccination, many workers attempted simultaneous vaccinationwith foot and mouth disease (FMD) and haemorrhagic septicaemia (HS)(JOSEPH and HEDGER, 1984), FMD and rinderpest (RP) (HEDGER et al., 1986;VETERINARSKI ARHIV  71 (1), 37-45, 2001*  Contact address:Dr.Villuppanoor Alwar Srinivasan, Indian Immunologicals, Rakshapuram, Gachibowli, Hyderabad - 500 019, India,Tel: +91 40 3000 894, Fax: +91 40 3000 213, E-mail: raksha2@hd1.vsnl.netinISSN 0372-5480Printed in Croatia38SRINIVAS et al., 1996) and vaccination with combined vaccines containingFMD and rabies antigens (DANNACHER et al., 1987; PALANISAMY et al., 1992).The use of combined vaccines depends on the epidemiological situationand vaccination regimes practiced in a particular region. In many Southand South-East Asian Countries FMD, HS and Black Quarter (BQ) vaccinesare commonly used with different vaccination regimes. Rabies in bovinesis also a serious problem in many developing countries. Vaccination withrabies is advocated in bovines in endemic areas as many workers haverecorded outbreaks in bovines (RAMANNA et al., 1991; MURIUKI et al., 1994;KIM et al., 1994; SINGH et al., 1995). The efficacy of combined FMD, HS andBQ antigens has been studied by REDDY et al. (1997). The presentinvestigation was undertaken to study the serological response of bovinesto combined vaccine containing FMD, rabies, Pasteurella multocida andClostridium chauvoei antigens.Materials and methodsVaccinesFMD virus strains O, A, C and Asial grown on BHK 21 suspensioncell cultures and inactivated with Binaryethylenimine (BEI) were used toformulate the vaccine. The vaccine contained aluminium hydroxide geland saponin. Potency of the vaccine was determined as per ANONYMOUS(1993) and each valency had a potency of ≥ 3 PD50.Rabies antigen containing CVS strain of rabies grown on BHK21suspension cell cultures and inactivated with BEI. The vaccine containedaluminium hydroxide and tested for potency as per ANONYMOUS (1993).The antigen used for vaccine production had a potency ≥ 2.5 IU per dose.Pasteurella multocida (HS). Formalin inactivated, purified andconcentrated bacterin with 3 mg dry mass was used to formulate the vaccine.The vaccine containing aluminium hydroxide gel was tested as perANONYMOUS (1989).Clostridium chauvoei (BQ). Fully grown cultures of Clostridiumchauvoei are inactivated with formalin, purified and concentrated and usedto formulate the vaccine. Antigens containing 200 haemolytic units perdose are incorporated. The vaccine containing aluminium hydroxide gelV. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 200139was tested as per ANONYMOUS (1993).Combined vaccine (FMD, HS, BQ, Rabies). The combined aqueousvaccine was formulated incorporating antigen payloads similar to that ofindividual component vaccines.Five groups of 10 animals each were vaccinated withFMD+HS+BQ+Rabies, FMD, HS, BQ and Rabies vaccines in dosevolumes of 4 ml, 3 ml, 2 ml, 2ml, and 1 ml respectively by intramuscularroute. One group of five animals was kept as controls.Five groups of 10 animals each were vaccinated withFMD+HS+BQ+Rabies, FMD, HS, BQ and Rabies vaccine in dose volumesof 4 ml, 3 ml, 2 ml, 2 ml and 1 ml respectively by intramuscular route.Animals vaccinated with FMD component were given a booster dose onthe 21st day. One group of five animals was kept as controls.Serum from blood samples collected on day 0, day 21 and day 90 postvaccination (dpv) were separated, inactivated at 56 °C for 30 min. andstored at -20 °C until further use.Serological assayFood and mouth disease. The serological response was measured bymicro-serum neutralization test described by GOLDING et al. (1976) usingIB-RS2 cells. Titres were calculated as per the method described by KARBER(1931) and expressed as log10 SN50 values.Rabies. Antibody titres were determined by rapid fluorescent focusinhibition test (RFFIT) as per the method described by SMITH et al. (1973).SAD strain of Rabies virus and BHK21 monolayer cells were used in thetest. Titres are expressed as International Units (IU) per ml after comparingthe RFFIT titres of serum samples with International standard preparation.Haemorrhagic septicaemia. ELISA test was performed on serumsamples to determine the serological response elicited by all the vaccinesas per the method described by NATALIA et al. (1992) with minor modifications– Flat-bottom PVC plates and orthophenylene diamine (OPD) as substratewere used in the test, and readings were taken at 492 nm wavelength.Titres were obtained by multiplying the corrected optical density (OD)values (sample OD - background OD (Ag+Conjugate) with serum dilutionfactor.V. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 200140Black quarter. Antibody titres were measured by ELISA test describedby CRICHTON et al. (1990) with minor modifications – HRPO-labelled rabbitanti-bovine conjugate (Sigma) was used at 1/3500 dilution OPD was usedas substrate. OD was measured at 492 nm wavelength. Titres werecalculated similarly to HS ELISA test.Serological response to different vaccines was compared by usingstandard statistical methods described by SNEDECOR and COCHRAN (1980).Results and discussionSerological response of calves to food and mouth disease virus antigensO, A, C, Asia in FMD vaccine and FMD, rabies, HS, BQ combined vaccineis presented in Table 1.Immunity to FMD is conferred by serum neutralising antibody. Indirectestimation of the log10 SN50 values which equates with 50% protection(PD) has been assessed, which is referred to as 50% protective antibody(PA). The PA50 values for type O, A, C and Asia were calculated as 1.62,0.99, 1.05 and 1.45, respectively (unpublished data). These values correlatewell with observations made by PAY and HINGLEY (1992), using British cattle.The PA50 values of type O in our studies (unpublished data) indicate lowerV. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 20011 = FMD+HS+BQ+Rabies; 2 = FMD; 3 =  HS; 4 =BQ; 5 = Rabies;  6 = ControlTable 1. Serological response of calves to FMD antigen (log10 SN50 values) Mean titres ± SE    Virus types     O A C Asia l Vacc.    dpv  N 0 21 90 0 21 90 0 21 90 0 21 90 1 10 <0.61 2.4  ±0.21 2.34 ±0.4 <0.61 1.98 ±0.21 2.20 ±0.3 <0.61 1.86 ±0.21 1.86 ±0.3 <0.61 2.31 ±0.30 2.40 ±0.21 2 10 <0.61 2.36 ±0.34 2.41 ±0.34 <0.61 1.86 ±0.3 2.11 ±0.2 <0.61 1.75 ±0.32 1.86 ±0.11 <0.61 2.2 ±0.3 2.46 ±0.36 3 10 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 4 10 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 5 10 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 6 5 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61 <0.61  41V. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 2001values when compared to the figures mentioned by PAY and HINGLEY (1992).This could be attributed to animal variation. PAY and HINGLEY (1992)suggested global PA50 values of 1.26 for all virus strains. Although PA50values provide some indication regarding the indirect measure of protectivelevels, an attempt was made to derive a logical conclusion by comparingchallenge test data and SN titres in our laboratory. Comparison of SNtitres and challenge test results indicate that more than 80%, of animalswith SN titres above l.41 and 1.61 were protected when challenged withtype Asial and type O, respectively, whereas challenge using type C andtype A viruses showed that more than 95% of animals with SN titres above1.01 were protected. These tests were carried out on 85, 53, 101 and 129animals using type O, C, A and Asia challenge viruses, respectively. Theabove data also indicate that animals with SN titres for type O above 1.61,Table 2. Serological response of calves to rabies antigen (IU/ml)1 =  FMD+HS+BQ+Rabies; 2 = FMD; 3 = HS; 4 = BQ; 5 = Rabies;  6 = Control Mean antibody titres ± SE Vacc. dpv     N 0 21 90 1 10 < 0.12 8.5±2.2 9.06±3.1 2 10 < 0.12 8.35±1.5 8.61±2.3 3 10 < 0.12 < 0.12 < 0.12 4 10 < 0.12 < 0.12 < 0.12 5 10 < 0.12 < 0.12 < 0.12 6 5 < 0.12 < 0.12 < 0.12  and Asial above 1.41, can be considered as protective titres, and that SNtitres above 1.01 for type C and A can be considered as protective titres.In the present study, the SN titres for all four valences are higher thanthe protective level on 90 dpv, and both groups of animals administeredwith FMD vaccine and combined vaccine showed no significant differences(P>0.05) (Table 1).42There was no significant differences (P>0.05) when the results of 0.21and 90 dpv antibody titres of animals vaccinated with rabies vaccine aloneand FMD, HS, BQ, rabies combined vaccines were compared (Table 2).DANNACHER et al. (1987) and PALANISAMY et al. (1992) reported that therewas no difference in the serological response to rabies antigen when cattlewere injected with rabies + FMD antigens.The immune response of calves to Pasteurella multocida andClostridium chauvoei antigens measured by ELISA test is shown in Table3. Comparison of antibody titres of calves vaccinated with HS vaccinealone, BQ vaccine alone, and FMD, HS, BQ, rabies combined vaccineshowed no significant differences (p>0.05). Similar observations weremade by REDDY et al. (1997) when HS and BQ antigens were combined withFMD virus antigens. An identical serological response was recorded byJOSEPH and HEDGER (1984) when HS vaccine was injected simultaneouslywith FMD vaccine. Serological response to FMD antigens when injectedsimultaneously with HS (JOSEPH and HEDGER, 1984), RP (SRINIVAS et al.,1996) or HS and BQ antigens (REDDY et al., 1997) remained altered and therewas no evidence of antigenic competition.V. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 20011 = FMD+HS+BQ+Rabies; 2 = FMD; 3 = HS; 4 = BQ; 5 = Rabies;  6 = ControlTable 3. Selorogical response of calves to Pasteurella multocida andClostridium chauvoei antigens (ELISA titres)± SE43Serum antibody titres (ELISA) for animals vaccinated with Pasteurellamultocida vaccine and combined vaccine containing FMD+HS+BQ++Rabies antigens indicated that both tile vaccines induced similar response.CHANDRASEKARAN et al. (1994) compared serum antibody titres as measuredby ELISA, and protection of buffaloes challenged with virulent organisms.Serum antibody titres above 54 ELISA units appeared to be indicative ofprotective titres. In the present report the serum antibody titres in animalsvaccinated with HS combined vaccine and individual component vaccineshowed protective titres.Results of the present study indicated that the combined vaccinecontaining antigens of, FMD virus, rabies virus, Pasteurella multocidaand Clostridium chauvoei produced and formulated according to ourprocedure can be used safely in countries where all these diseases areendemic, and that this procedure is likely to reduce the cost of vaccination.ReferencesANONYMOUS (1989): Recommended Diagnostic Techniques and requirement forBiological products. Vol. I. Office International Des Epizooties. Paris.ANONYMOUS (1993): British Pharmacopoeia (Veterinary), HMSO, London.CHANDRASEKARAN, S., L. KENNET, P. C. YEAP, N. MUNIANDY, B. RANI, T. K. S.MUKKUR (1994): Relationship between active protection in vaccinated buffaloesagainst haemorrhagic septicaemia and passive mouse protection test or serum antibodytitres. Vet. Microbiol. 41, 303-309.CRICHTON, R., J. SOLOMON, A. M. BARTON (1990): The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containingClostridium chauvoei antigens. Biologicals 18, 49-54.DANNACHER, G., J. M. BLANCOV, M. COUDERT, M. FEDIDA (1987): Report sessionof Research Group Standing Technical Committee on European Commission. Controlof FMD (FAO), Lyon, France.GOLDING, S. M., R. S. HEDGER, P. TALBOT, J. WATSON (1976): Radialimmunodiffusion and serum neutralization techniques for the assay of antibodies toswine vesicular disease. Res. Vet Sci. 20, 142-147.HEDGER, R. S., W. P. TAYLOR, I. T. R. BARNETT, R. REIK, D. HARPHAN (1986):Simultaneous vaccination of cattle against foot and mouth disease and rinderpest.Trop. Anim. Hlth. Prod. 18, 21-25.V. A. Srinivasan et al.: Serological response of bovines to combined vaccineVet arhiv 71  (1), 37-45, 200144 Vet arhiv 71  (1), 37-45, 2001JOSEPH, P. G., R. S. HEDGER (1984): Serological response of cattle to simultaneousvaccination against foot and mouth disease and haemorrhagic septicaemia. Vet Rec.114, 494-496.KARBER, G. (1931): Beitrag Zur Kollektiven Behandlung pharmakologischerReihenversuche. Arch. Exp. Pathol. and Pharmakol. 162, 480-483.KIM, J. E, E. K. HWANG, Y. J. LEE, J. W. PARK, S. M. BAE, H. J. SOHN, Y. D. YOON(1994): An outbreak of rabies in cattle in Korea. Korean Journal of Veterinary PublicHealth 18, 365-371.MURIUKI, S. K., J. G. WANDERA, M. J. NJENGA, E. R. MUTIGA, A. THAIGAH, O. N.NJAGI, A. M. UTURI (1994): An outbreak of rabies in a diary farm in Kiambu district,Kenya. Bulletin, Animal Health and Production in Africa 42, 265-266.NATALIA, L., B. PATTEN, A. SYAMSUDIN (1992): Evaluation of’ bovine antibodyresponses to Haemorrhagic Septicaemia vaccine using ELISA and PMPT. Pasteurellosisin Production animals. Australian Centre for International Agricultural Research,Canberra.PALANISAMY, R., B. C. RAMANNA, K. ANANDA RAO, V. A. SRINIVASAN (1992):Combined vaccination of cattle against FMD and rabies. Microbiologica 15, 45-50.PAY, T. W. F., P. HINGLEY (1992): A potency test method for foot and mouth diseasevaccine based on the serum neutralising antibody response produced in cattle. Vaccine10, 707-713.RAMANNA, B. C., G. S. REDDY, V. A. SRINIVASAN (1991): All outbreak of rabies incattle and use of tissue culture rabies vaccine during the outbreak. J. of Comm. Dis.23, 283-285.REDDY, G. S, P. X. ANTHONY, V. A. SRINIVASAN (1997): Serological response tocombined vaccination of cattle against foot and mouth disease, haemorrhagicsepticaemia and black quarter diseases. Indian J. Anim. Sci. 67, 585-586.SINGH, R., D. C. SHUKLA, P. N. KHANNA, K .P. SINGH, M. L. MEHROTRA (1995):An outbreak of rabies in cattle and buffaloes in Uttar Pradesh. Indian J. Anim. Sci. 65,166-168.SMITH, J. S., P. A. YAGAR, G. M. BAER (1973): Laboratory techniques in Rabies. 3rdedition. WHO, Geneva.SNEDECOR, G. W., W. G. COCHRAN (1980): Statistical Methods, 7th edition. The IowaState University Press, Iowa USA.SRINIVAS, K., N. G. RAMESHBABU, RAMAKANT, M. RAJASEKHAR, M. S.VIJAYAKUMAR, T. M. LAXMINARAYANA, V. A. SRINIVASAN (1996):Simultaneous vaccination against foot and mouth and rinderpest diseases. Indian J.Virol. 12, 33-37.Received: 19 August 1998Accepted: 20 February 2001V. A. Srinivasan et al.: Serological response of bovines to combined vaccine45Vet arhiv 71  (1), 37-45, 2001V. A. Srinivasan et al.: Serological response of bovines to combined vaccineSRINIVASAN, V. A., G. S. REDDY, K. A. RAO, U. KIHM:  Serološki odgovorgoveda  na cijepljenje polivalentnim cjepivom protiv slinavke i šapa, bjesnoće,hemoragijske septikemije i šuštavca. Vet. arhiv 71, 37-45, 2001.SAŽETAKPet skupina po deset životinja cijepljeno je različitim kombinacijama vakcina. Prva je skupinacijepljena polivalentnim cjepivom koje je sadržavalo antigene virusa slinavke i šapa, virusa bjesnoćete bakterija Pasteurella multocida i Clostridium chauvoei. Ostale su skupine cijepljene pojedinačnimantigenima, pa je tako druga skupina cijepljena antigenima virusa slinavke i šapa, treća antigenimavirusa bjesnoće, četvrta antigenima bakterije Pasteurella multocida, a peta antigenima bakterijeClostridium chauvoei. Serološki odgovor svih životinja provjeren je 21. i 90. dana nakon cijepljenja.Na osnovi serološkoga odgovora nisu utvrđene značajne razlike među različitim načinima cijepljenja.Ključne riječi: slinavka i šap, bjesnoća, Pasteurella multocida,Clostridium chauvoei, profilaksa,polivalentna vakcina, serologija

Serološki odgovor goveda na cijepljenje polivalentnim cjepivom protiv slinavke i šapa, bjesnoće, hemoragijske septikemije i šuštavca.

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