seroprevalence of foot-and-mouth disease in bovine ...cases were found in 3 to

Livestock Research International | July-September, 2015 | Vol 3 | Issue 3 | Pages 58-63 © 2015 Jakraya Publications (P) Ltd

LIVESTOCK RESEARCH INTERNATIONAL

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ORIGINAL ARTICLE

Seroprevalence of Foot-and-Mouth Disease in Bovine Population of a District of Uttarakhand, India Anand Mohana∗, Ajay K. Upadhyayb, Mahesh Kumarc and Neelam Kushwahad. Department of Veterinary Epidemiology and Preventive Medicine, G.B. Pant University of Agriculture and Technology, Pantnagar- 263 145, Uttarakhand, India. Present Address: aAssistant Professor, Veterinary Epidemiology and Preventive Medicine, KNP College of Veterinary Science, Shirval- 412 801, Satara, Maharashtra, India. bAssociate Professor, Veterinary Public Health, cProfessor, Veterinary Medicine, G.B. Pant University of Agriculture and Technology, Pantnagar- 263 145, Uttarakhand, India. dHospital Registrar, Teaching Veterinary Clinical Complex, College of Veterinary and Animal Science, Udgir, Latur, Maharashtara, India. *Corresponding Author: Anand Mohan Email: [email protected] Received: 17/09/2015 Revised: 26/09/2015 Accepted: 27/09/2015

Abstract Biannual vaccination and sero-monitoring of FMD under the scheme

FMDCP is being undertaken by government of India. Serological test viz. LPBE and NSP-ELISA are being used to monitor the immune status host and virus multiplication in bovine population. Present study was undertaken to differentiate vaccinated and infected animals by NSP-ELISA in bovine population. Bovine sera from Udham Singh Nagar were screened for identification of active multiplication of FMD virus. A total of 25.28% bovine population included in this study, infected with FMD while 14.15% in transition stage of disease. The prevalence of FMD in cattle population (31.03%) was found higher than that of buffalo population (7.58%). Age of animals had influence on disease distribution, as maximum number of FMD cases were found in 3 to <5 yrs age groups (51.52%) followed by ≥5 yrs (33.04%), 1 to <3 yrs (23.08%) and minimum in animals of <1 yr age group (4.35%). Study revealed that 60.22% of bovine population became immune to FMD infection by repeated vaccination. Still there is huge gap in herd immunity and susceptibility to FMD in concern population. There is need to stick with FMDCP to reduce this gap to such an extent that virus multiplication in animal population is discontinue. Keywords: Bovine, Foot-and-Mouth disease, NSP-ELISA, Seroprevalence.

1. Introduction Foot-and-Mouth disease (FMD) is an acute,

vesicular disease of cloven-hoofed ruminants, pigs and wild animals (Alexandersen and Mowat, 2005). It is caused by foot-and-mouth disease virus (FMDV), classified in the genus Aphthovirus, a member of the Picornaviridae. Diagnosis of FMD includes; isolation of virus, demonstration of viral antigen or nucleic acid, etc. in clinical samples likes tissue or fluid. Detection of host immune response in term of antibodies against surface (structural) or non-structural proteins (NSPs) are commonly used tests for screening the herd. Virus isolation is the most reliable diagnostic method, but it is labor intensive, time-consuming, and requires properly equipped facilities. Moreover detection of virus-specific antibody is convenient for diagnosis for epidemiological purpose. OIE (2012) prescribed liquid phase blocking ELISA (LPBE), solid phase competitive ELISA and virus neutralization tests for international trade. In India an indigenously developed LPBE is used extensively to monitor post vaccination antibody response (Biswal et al., 2012), but this test doesn’t

differentiate between vaccinated and infected animals. For discrimination, non-structural protein (NSP) was incorporated in ELISA. It is based on principle that, viral replication during infection results in the production of a number of NSPs, of which some are immunogenic. Antibodies to viral NSPs are indicators of virus multiplication or infection, irrespective of vaccination status of animal (Bergmann et al., 2005). NSPs, unlike structural proteins, are highly conserved and therefore, are not serotype specific. On the other hand detection of antibodies in host indicative of virus multiplication irrespective of serotype involved (Kweon et al., 2003).

The RNA genome of FMDV comprises four structural protein genes 1A, 1B, 1C and 1D which make up the viral capsid and eight non-structural protein genes L, 2A, 2B, 2C, 3A, 3B, 3C and 3D responsible for proteolytic cleavage and viral replication (Clavijo et al., 2004). Different NSPs are evaluated alone or combination with other (Inoue et al., 2006; Mohapatra et al., 2011; Yadin et al., 2007) in ELISA for diagnosis of FMD. Indirect-trapping ELISA

Anand Mohan et al…Seroprevalence of Foot-and-Mouth Disease in Bovine Population


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using monoclonal antibodies to trap NSP 3ABC FMDV polypeptide were developed with 100% sensitivity (De Diego et al., 1997). The high specificity and sensitivity made this test an ideal tool for FMD eradication and control programs. Among the several ELISA assays developed by different investigators to distinguish infected animals from uninfected naive or vaccinated animals the detection of antibodies to the non-structural-polypeptide 3ABC was found to be the most reliable marker of infection (Clavijo et al., 2004; De Diego et al., 1997). In addition of that, antibodies to 3ABC appeared early after infection and were detected for longer periods than antibodies to any other of the NSPs examined (Mackay et al., 1998a). In India, an indigenously produced 3AB/3ABC-ELISA kit is extensively used to monitor virus circulation and clearance in vaccinated areas.

2. Material and Methods 2.1 Area Under Study and Sample Collection

The study population selected for sample collection comes under the government of India Foot-and-mouth disease control program (FMDCP) and hence animals in this area were regularly vaccinated. On other hand, selected animals supposed to be having protective antibody against FMD. A total of 269 blood samples of cattle and buffaloes of Udham Singh Nagar district of Uttarakhand, India were collected aseptically by jugular venepuncture in vacutainers. Details of samples in respect to species, sex and age was mentioned in Table 1. Sera were separated from blood samples by placing tubes in slanting position for 1 h at room temperature followed by 4 h at 4°C. Thereafter tubes were centrifuged at 3000x g for 5 min. A clear supernatant on top of tubes were collected and stored in aliquots at -20°C in the deep freeze till used.

2.2 NSP-ELISA

Prokaryotic expressed recombinant 3AB3 NSP, positive serum, negative serum and anti-bovine-HRP conjugate were procured from Project Directorate on FMD (PDFMD), Mukteshwer, India. The procedure recommended by PDFMD was followed. Briefly, polystyrene immuno plates (Nunc, Maxisorp) were coated with the recombinant protein, 50 µl/well (50 ng/well, diluted in 0.2 M carbonate-bicarbonate buffer, pH 9.6) and incubated at 4°C for overnight. After thawing at 37°C for 15 min, three continuous washing were given without holding time with wash buffer (2.5 mM, dihydrate sodium dihydrogen phosphate; 2.93% w/v, NaCl; 0.05% w/v, Tween-20; pH 7.2-7.4). Meanwhile, in low binding perpex plates, negative and positive control serum were diluted respectively, 1:20 and 1:10 in diluents buffer (3% w/v, skimmed milk powder; 10% v/v, chicken serum; 0.01% v/v, E.Coli lysate in washing buffer). In duplicate, 100 µl diluted test serum, positive serum and negative serum were transferred to coated plates. For background control

only 100 µl of diluents buffer was dispensed without any serum. Again three washing were given with 3 min soaking period for each washing after incubation at 37°C for 1 h. Antigen-antibody complexes were traced by anti-bovine-HRP conjugate, diluted in diluents buffer (1:2000) and used as 50 µl/well incubated for 1 hr at 37°C in a plate shaker. Again three washing were given with 3 min soaking period for each washing after incubation at 37°C for 1 h. Fresh prepared substrate solutions, 50 µl/well were used for development of color at 37°C without shaking for 15 min. Reactions were stopped by 1 M H2SO4, 50 µl/well. Optical density of plates was taken at 492 nm.

The tests were considered valid; as mean absorbance of the positive control wells were 1.0 with ±20% coefficient of variation, mean absorbance of negative control serum were <0.4 and OD of background control were lied in between 0.00 to 0.05. Test serum was considered positive if T/P ratio more than 0.5, under suspicion if T/P ratio fall in between 0.4-0.5 and negative if it was less than 0.4.

3. Results 3.1 Overall and Species-Wise Seroprevalence

Total 269 bovine samples were screen for the identification of virus multiplication or infection. The result showed that, 25.28% bovine population infected with FMD and 60.22% free from infection while 14.15% animal in transition stage of disease (Table 1). The FMDV infection was found higher in cattle (31.03%) as compared to buffaloes (7.58%). This difference in infection level in two species were statistically significant (p<0.05). Doubtful cases were more in cattle (16.26%) than buffalo (9.10%) but this was not statistically significant (p>0.05). In contrast to this, comparatively higher number of buffaloes (83.33%) were free from infection than cattle (52.71%) and this was also statistically significant (p<0.05).

3.2 Sex-Wise Seroprevalence

T/P ratio for NSP antibody in 27.15% and 13.51% respectively in females and males animals were higher than 0.5. In other words these animals were positive for FMD, although this difference level of virus infection were not statistically significant at 5% level of significance. For transition stage of FMD infection, T/P ratio of NSP antibody was kept between 0.4-0.5. it was found that, 15.52% and 8.11% animals from female and male in transition stage of FMD, moreover this was also not significant (p>0.05) statistically. On the other hand, 78.38% male and 57.33% female bovine animals were free of FMD infection as its T/P ratio were less than 0.4, and it was statistically significant (p<0.05).

3.3 Age-Wise Seroprevalence

Among different age groups of animals, maximum numbers of FMD positive cases were found



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Table 1: Seroprevalence of FMD in Udham Singh Nagar (Uttarkhand) with respect to species, sex and age of bovine by using NSP ELISA.

Category No. of samples NSP-ELISA Infected Doubtful Not infected

Species Cattle 203 63 (31.03) 33 (16.26) 107 (52.71) Buffalo 66 5 (07.58) 6 (09.10) 55 (83.33) Sex Female 232 63 (27.15) 36 (15.52) 133 (57.33) Male 37 5 (13.51) 3 (08.11) 29 (78.38) Age <1 yrs 23 1 (04.35) 2 (08.70) 20 (86.96) 1 - <3 yrs 52 12 (23.08) 6 (11.54) 34 (65.38) 3 - <5 yrs 79 17 (51.52) 13 (16.46) 49 (62.03) ≥5 yrs 115 38 (33.04) 18 (15.65) 59 (51.30) Overall Total 269 68 (25.28) 39 (14.50) 162 (60.22)

Figures in parenthesis are respective percentage. Test sera are considered positive if T/P ratio more than 0.5, under suspicion if T/P ratio fall in between 0.4-0.5 and negative if it was less than 0.4.

in 3 to <5 yrs (51.52%) followed by ≥5 yrs (33.04%), 1 to <3 years (23.08%) and minimum in <1 yr age group (4.35%). These differences were statistically significant at 5% level of significance. Similarly, 16.46%, 15.65%, 11.54% and 8.70% animals respectively in age groups 3 to <5 yrs, ≥5 yrs, 1 to <3 yrs and <1 yr were in transition stage of FMD, but it was not statistically significant (p>0.05). Effect of age of animals on immunity against FMD in bovine population were followed inverse relation of age as maximum animals in age group <1 yr (86.96%) immune to infection followed by 1 to <3 yrs (65.38%), 3 to <5 yrs (62.03%) and ≥5 yrs (51.30%). Statistically these differences in immunity were also significant (p<0.05).

4. Discussion

Biannual vaccination along with segregation of infected animals from health groups is commonly followed in India for control of FMD. Vaccines used for FMD consists of purified preparations of inactivated virions and therefore host induces antibodies almost exclusively to the structural proteins of virions provided that vaccine batch is significantly free of NSPs. For screening of animals it is mandatory to differentiate vaccinated animals from infected one. The demonstration of specific antibodies to structural proteins in non-vaccinated animals is indicative of prior infection with FMDV. However tests for antibodies to some NSPs of FMDV are useful in providing evidence of previous or current viral replication in the hosts, irrespective of their vaccination status.

Out of the seven, only three serotypes, viz; ‘O’, ‘A’ and ‘Asia-1 were reported since 1995 in India (Shah et al., 2011). Infected animals, whether symptomatic or not, usually elicit antibodies to both the FMDV structural capsid proteins and NSPs. Although vaccinated animals, which are exposed to infection,

might become persistently infected with FMD without ever showing clinical signs (Mackay et al., 1998b). Since NSPs are exclusively synthesized during the FMDV replication, the presence of anti NSP antibodies in animal sera is a clear indication of either past or present viral activity. Assays to detect responses against the NSP of FMD virus are able to detect antibodies to all serotypes of the virus and are therefore useful serological test and not restricted to serotype of FMDV (Kweon et al., 2003). NSP-ELISA Tests are used that measure antibodies to the non-structural proteins of FMDV, since these are specific for infection and are not induced by vaccination, providing the vaccines used have been formulated using FMD antigens that have been purified to remove most of the non-structural viral proteins (Kitching, 2002).

4.1 Overall Seroprevalence

In the study population, 25.28% bovine population infected with FMD as his sera have antibody titre against NSP above the cut off value. According to the annual report submitted by OIE/FAO Reference Laboratories for FMD in the 5th annual meeting at New Delhi in 2009, a survey of 32,000 cattle in different regions of India had revealed an overall NSP seroprevalence of 31%, with ranges from 6-46% at a state level. In adjoining states Utter Predesh, 27.75% bovine tested were found positive for antibody to NSP (Singh, 2010). In Haryana, percentage of animals with anti-NSP 3A-specific antibodies was found to be significantly higher in cattle than buffalo (Kumar et al., 2007). Small ruminants which did not show clinical signs were also reported to circulated disease and endemic area. In Odisha 38% goat population was found to positive for NSP antibody (Ranabijuli et al., 2010). Likewise in adjoining country (Bhutan), 17.6%

Anand Mohan et al…Seroprevalence of Foot

Livestock Research International | July© 2015 Jakraya Publications (P) Ltd

Fig 1: Ninety well plate showing the result of NSP ELISA, Each Serum samples areWells marked with + = Positive samples, Wells marked with Background controls, Rest wells were used for samples in duplicate. cattle populations were infected with 2011).

Animal which had NSP T/P ration in transition stage of disease were 14.15% of total screened animals. However 60.22% bovine population free from FMD infection. Since India is endemic for FMD, therefore possibility of virus multiplication in vaccination animals even in vaccinated animals which may provide NSP antibody. Moreover vaccine used is thought to be free from NSP proteins but vaccine manufactured in India varies from NSP free to traces of NSP. In addition to that, there is no separate claim on absence of NSP in vaccine by manufacturer. In addition to that repeated vaccination with contaminated residual nonproteins, contained in less pure FMD vaccine purified FMD vaccine may produce antibody against NSP antibody (Lee et al., 2006). Occasional animals which had received more than ten vaccinations had NSP antibody profiles which were similar to those seen following infection (Mackay et al., 1998a).

4.2 Species-Wise Seroprevalence

Comparison of NSP antibodies among revealed cattle (31.03%) were more commonly infected with FMD virus than buffalo (7.58%). This may be indicative of presence of high resistance of buffaloes

Seroprevalence of Foot-and-Mouth Disease in Bovine Population


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Fig 1: Ninety well plate showing the result of NSP ELISA, Each Serum samples are taken in duplicate wells; = Positive samples, Wells marked with - = Negative samples, Wells marked with

Background controls, Rest wells were used for samples in duplicate.

cattle populations were infected with FMD (Duka et al.,

Animal which had NSP T/P ration in transition stage of disease were 14.15% of total screened animals. However 60.22% bovine population free from FMD infection. Since India is endemic for FMD, therefore

ication in vaccination animals even in vaccinated animals which may provide NSP antibody. Moreover vaccine used is thought to be free from NSP proteins but vaccine manufactured in India varies from NSP free to traces of NSP. In addition

no separate claim on absence of NSP in vaccine by manufacturer. In addition to that repeated vaccination with contaminated residual non-structural proteins, contained in less pure FMD vaccine purified FMD vaccine may produce antibody against NSP

Occasional animals which had received more than ten vaccinations had NSP antibody profiles which were similar to those seen

1998a).

Seroprevalence Comparison of NSP antibodies among species

cattle (31.03%) were more commonly infected with FMD virus than buffalo (7.58%). This may be indicative of presence of high resistance of buffaloes

against FMD than cattle. Similarly, Hassanein (2011) detected 36.9% buffalo tested hagainst NSP in comparison to 40.21% cattle tested have antibody against NSP. Not only serology but isolation of FMDV from buffaloes was also reported to be less than cattle (Ghoneim et al., 2010)

4.3 Sex-Wise Seroprevalence

Animals belong to different sex had different level of FMD infection, although these difference were not statistical significant (p>0.05). Similar observation was made by Gelaye et al. (2009) in Bench Maji zone, Southwestern Ethiopia. Although, Sarker in Bangladesh assessed that sex influence the FMD infection in animal, but his observation based on questionnaire rather than any specific tests used.

4.4 Age-Wise Seroprevalence

Somehow age of animals influences the FMD infection in different age groups in the present study. Maximum animals in 3 to <5 yrs (51.52%) age group were positive for NSP antibody followed by (33.04%) and 1 to <3 yrs (23.08%) age groups while minimum animals in <1 yr age groups (4.35%). In early stage of life, maternal derived antibodies provide

Disease in Bovine Population

taken in duplicate wells; = Negative samples, Wells marked with B -

against FMD than cattle. Similarly, Hassanein et al. (2011) detected 36.9% buffalo tested have antibody against NSP in comparison to 40.21% cattle tested have antibody against NSP. Not only serology but isolation of FMDV from buffaloes was also reported to be less

2010).

Seroprevalence Animals belong to different sex had different

level of FMD infection, although these difference were not statistical significant (p>0.05). Similar observation

(2009) in Bench Maji zone, Although, Sarker et al. (2011)

in Bangladesh assessed that sex influence the FMD infection in animal, but his observation based on questionnaire rather than any specific tests used.

Seroprevalence Somehow age of animals influences the FMD

ge groups in the present study. Maximum animals in 3 to <5 yrs (51.52%) age group were positive for NSP antibody followed by ≥5 yrs (33.04%) and 1 to <3 yrs (23.08%) age groups while minimum animals in <1 yr age groups (4.35%). In early

ernal derived antibodies provide



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protection for 3-4 months (Nicholls et al., 1984) if dam is vaccinated. Thereafter primary vaccination at 4 month of age is protecting to further 6 to 12 months depending on vaccination preparation. These two factors may be responsible for lowest seroprevalence of NSP in age group less than one year. Along the advancement of age, chances of getting repeated exposure of FMDV is increased, although some of workers reported that repeated vaccination of inactivated virus vaccine may produce NSP antibody (Yadin et al., 2007), secondly FMD vaccine may contain NSP as no manufacturer warrant NSP free vaccine. All these may be responsible for increasing trend of NSP seroprevalence as age of animals increases and highest were found in 3 to <5 yrs age groups. In contrast to this, Potzsch et al. (2006) reported NSP serology was not association with age of animals. 5. Conclusions

Present study concluded that FMDV multiplications are going on in susceptible population

with significant proportion. Immunity generated with biannual vaccination was still below the level of herd immunity which is supposed to be achieved to restrict the virus multiplication in the population. Further maximum exposure and hence multiplications of virus were in the cattle than buffalo population when animal was in the peak production life. Therefore there is need of rigorous monitoring and vaccination of all susceptible population which also include sheep, goat swine and wild reservoir.

Acknowledgements

The authors are thankful to the FMD Regional Centre, Mathura-281 001 and Dean, College of Veterinary and Animal sciences, Director Experimental station, GBPUA&T, Pantnagar-263 145, U.S. Nagar (Uttarakhand), for supplying the necessary facility to conduct this study.

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seroprevalence of foot-and-mouth disease in bovine ...cases were found in 3 to

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