Studies on the duration of immunity induced in cattle after natural FMD infection and post vaccination with bivalent oil vaccine

Aim: To detect the proper time for vaccination of infected calves after the onset of clinical signs and revaccination for previously vaccinated animals with bivalent FMD montanide ISA206 inactivated vaccine. Materials and Methods: Twenty calves naturally infected with foot and mouth disease (FMD) showing clinical signs represented by lesions on the tongue, buccal mucosa and feet. Samples were collected from these calves including tongue epithelial and oropharyngeal fluid (OP). The causative virus was isolated from such samples through inoculation of baby mice and typed by indirect Sandwich ELISA and was confirmed in 2 tongue epithelia samples using PCR. Results: The serum antibody titers were determined using SNT and ELISA in the sera of the 20 infected calves revealing that the infected farms should be vaccinated with inactivated bivalent FMD vaccine for (type O and A) Adjuvanted with montanide ISA206 after 32 weeks from the appearance of clinical signs. In addition the FMD immune status was monitored in 2 farms in EL-Fayoum and El-Sharkia Governorates where 50 calves in each farm were vaccinated with the bivalent FMD inactivated vaccine adjuvaned with montanide ISA206 and serum samples were collected monthly from these animals to determine their immune status using SNT and ELISA. The obtained results showed that vaccinated calves with the bivalent FMD montanide ISA206 inactivated vaccine should be revaccinated after 36 weeks post vaccination. Conclusions: naturally infected calves should be vaccinated on the 32 weeks post infection while vaccinated calves should be revaccinated on the 36 weeks post vaccination


Introduction
days and remain at protective titers [4].The duration of antibody response in cattle experimentally infected Foot and Mouth disease (FMD) is a highly with FMDV lasted for a period of 40 weeks.The infectious disease of ungulates primarily cattle, sheep, maximum antibody titer was reached at 10 weeks post goats and pigs.It also affects wild animals such as th infection followed by steady reduction to the 4 month buffaloes and deer [1].Foot-and-mouth disease virus post infection [5].Neutralizing antibodies persisted (FMDV) is the etiologic agent of one of the most for 18 months in convalescent cattle experimentally devastating diseases that can affect cloven-hoofed infected with FMDV.The serum neutralizing livestock.Infection with FMDV causes an acute antibodies rose to high titers within 7 to 10 days after disease that spreads very rapidly and is characterized infection of cattle with type 'O' FMD virus.Such level by fever, lameness and vesicular lesions on the feet, remained high for 4 months; while the virus could be tongue, snout and teats, with high morbidity but low mortality [ [9] was used for application of by serum neutralization test (SNT) and Enzyme linked serum neutralization test.Immunosorbant Assay (ELISA).These animals were vaccinated with the local inactivated bivalent oil FMD Serum neutralization test (SNT): It was performed vaccine with a dose of 2 ml injected subcutaneously in using the micro titer technique as described by [10].each calf.

Indirect enzyme linked immune sorbent assay
Suckling baby mice: Sixty suckling Swiss Albino (ELISA): ELISA and its reagents were prepared mice of 2-3 days old were used for isolation and according to [11] used to follow up the immune titration of FMD virus through the inoculation response in animals.intraperitoneal (I/P).They were supplied by Indirect Sandwich ELISA: Indirect Sandwich ELISA Veterinary Serum and Vaccine Research Institute and its reagents were prepared according to [12] used (VSVRI) -Abassia-Cairo.
for typing the isolated virus.FMD virus: Locally isolated foot and mouth disease PCR: It was applied on the tongue epithelial and OP virus (FMDV/O /93/Aga), (FMDV/A/Egypt/2006) of 1 using 2 specific primers against A, O to confirm typing cattle origin typed and sub-typed at the FMD of the 2 infected calves according to [13]  collected tongue epithelium; and oesopharyngeal fluid recording peaks of FMD antibodies in infected cattle through the intrapretonial inoculation of baby mice between 15-16 weeks post infection.Also [24] stated revealed positive results represented by paralysis of that the clinical signs decline with the appearance of the hind limbs of all inoculated mice in agreement with circulating FMD specific antibody at around 4 to 5 [16] who isolated the FMDV from infected animals in days post infection.The titer of these antibodies baby mice concluded that mice are suitable models to remained within the protective level (1.5 log /ml for 10 study the pathogenicity of FMD.SNT and 1.9 log for ELISA) as reported by [17] up to 10 Identification and serotyping of isolated FMDV 36 weeks for type O and 32 weeks for type A. from collected samples of naturally infected calves by Vaccination of calves with the locally produced Indirect Sandwich ELISA revealed that 5 out of 20 bivalent FMD vaccine adjuvanted with Montanid ISA infected calves were typed as FMDV serotype A while 206 induced higher antibody titers than the 15 isolates were typed serotype O (Table-3).These recommended protective level (1.5 log for SNT and 10 results were confirmed using PCR on 2 samples as 1.9 log for ELISA) for both of type A and O as 10 examples (1 sample type O and 1 sample type A) as estimated by SNT and ELISA (Table-5 and Fig. 3&4) shown in photo (1&2).In this respect the use of th on the 4 week post vaccination to record peak titers by Indirect Sandwich ELISA and PCR for identification the 12 week post vaccination for both types.These and typing of FMDV was agreed with [12,[17][18][19][20][21][22].
antibody titers remained within the protective level up Studying the immune status in naturally infected to 36 weeks post vaccination in the 2 farms under calves as evaluated by SNT and ELISA (Table-4& Fig. studies.1&2) showed that the peak of circulating FMD type A These results agree with those of [7,25,26] who and O antibodies were recorded on the 16 weeks post indicated that Montanide ISA 206 achieving early infection.Similar findings were obtained by [23] T B F T B F T B F T B F T B F T B F T B F T B F T B F + + -+ + + + ---+ + + -T B F T B F T B F T B F T B F T B F T B F T B F T B F + Photo-1.PCR for TE and OP positive band in 800bp (serotype A) L: Ladder, TE: Tongue epithelial of sample NO. 1, +ve: positive control, -ve : negative sample

Materials and Methods Samples of virus isolation: Oesophageal Pharyngeal
The mean by Veterinary Serum and Vaccine Research Institute to protective serum antibody titers against FMD in be used for vaccination of calves in the 2 mentioned farms.calves vaccinated with double oil emulsion

Table - 1
using the Department Veterinary Serum and Vaccine Research demonstrated primers in Table-1.Institute, Abbasia, Cairo and confirmed by world reference Laboratories, Pirbright, United Kingdom. .Primers used in the study.

Table - 2
. FMD lesions in naturally infected cattle

Table - 3
. Isolation and identification of FMDV from different samples by baby mice inoculation and ELISA Studies on the duration of immunity induced in cattle after natural FMD infection and post vaccination with bivalent oil vaccine *WPI= week post infection

Table - 5
. Mean FMD serum neutralizing antibody titers in vaccinated calves with the bivalent oil FMD *WPV= week post vaccination N.B: SNT titer was expressed by log10 Studies on the duration of immunity induced in cattle after natural FMD infection and post vaccination with bivalent oil vaccine protective titers and longer lasting immunity.Also Acknowledgements [27] found that the immune response of vaccinated Authors are thankful to Prof. Dr. Seham Abd ELgoats with DOE Montanide ISA 206 vaccines Rashid Director of VSVRI, Prof. Dr. Sayed Zedan persisted for 36 weeks post vaccination.In addition, Deputy of VSVRI and all members of FMD [28] suggested that high potency vaccines adjuvanted department specially Prof. Dr. Magdy Abd El-Aty with Montanide ISA 206 can promote long lasting head of FMD department, VSVRI.immunity.The obtained results showed that the protective Competing interests titers of FMD antibodies were persisted up to 32 weeks Authors declares that they have no competing interests.post infection in naturally infected calves and up to 36 Cairo, 13-18 February.searched naturally infected animals and follow up the 6.Matsumato, M., McKercher, P.D. and Nusbaum, K.E.antibody titers by SNT and ELISA on the serum of (1978).Secretory antibody response in cattle infected infected animals.Samir Mohamed analysed the Data with FMDV.Am.J. Vet.Res., 39 (7): 1081-1087.
th viruses.5 Report of Int.Comm. on taxonomy of protective immune levels to the non-protective values.on the serum of vaccinated animals.Wael Mossad th Of the 13 Arab Vet.Conger.,