Transboundary and Emerging Diseases
ORIGINAL ARTICLE
Detection and Seroprevalence of Foot and Mouth Disease in Sheep and Goats in Punjab, Pakistan Saif Ur-Rehman1, M. Arshad1, I. Hussain1 and Z. Iqbal2 1 2
Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan Department of Parasitology, University of Agriculture, Faisalabad, Pakistan
Keywords: foot and mouth disease; sheep; goats; Punjab; Pakistan; seroprevalence; NSP-ELISA; risk factors Correspondence: Saif Ur-Rehman. Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan. Tel./Fax: 00923217628262; E-mail: [email protected] Received for publication September 6, 2013 doi:10.1111/tbed.12194
Summary Foot and mouth disease (FMD) is a highly contagious disease of ruminants that causes huge economic losses around the globe. However, the prevalence of FMDV in small ruminants has been overlooked in Pakistan. A seroepidemiological study was conducted in Chakwal, Faisalabad and Khanewal districts of Punjab, Pakistan to determine the prevalence of FMD in sheep and goats. A total 1200 serum samples were collected from sheep (n = 180) and goats (n = 920) and were subjected to 3ABC non-structural protein enzyme-linked immunosorbent assay for detection of antibodies against non-structural proteins of FMD virus. Samples collected from clinical cases were also confirmed for FMDV using RT-PCR. The overall seroprevalence of FMD in sheep and goats was 21% (n = 252) while 19.44% (n = 35) in sheep and 21.27% (n = 217) prevalence was recorded in goats. Highest seroprevalence (32.5%) was observed in southern Punjab (Khanewal), followed by (25.75%) central Punjab (Faisalabad) and the lowest seroprevalence (4.75%) was detected in northern Punjab (Chakwal). There was no statistically significant difference in seroprevalence between sheep and goats. Among different risk factors tested, age and sex were found to be significantly associated with the prevalence of disease while pregnancy and herd type had no association with the prevalence of the disease. This study illustrate that FMD is highly prevalent in sheep and goats in Punjab. Therefore, a broader study is needed to ascertain the countrywide prevalence of FMD in small ruminants.
Introduction Foot and mouth disease (FMD) is a severe, highly contagious viral disease of animals. All cloven footed animal species are susceptible to the disease. Foot and mouth disease is caused by Aphthovirus genus, which is the member of the Picornaviridae family (Reuckert, 1996). FMDV consists of an RNA genome, which is positive-sense and singlestranded. The size of the genome is about 8.5 kilobases, and it is bordered by four structural proteins to make an icosahedral shape of the capsid (Marvin and Barry, 2004). On the basis of serology, FMDV is grouped into seven different types (O, A, C, Asia 1, SAT1, SAT2 and SAT3), which are further divided into numerous subtypes, and there is no cross-protection among these serotypes (Kitching et al., 2005). The most prevalent serotypes in
Pakistan are O (70%), Asia-I (25%) and A (4.67%), which cause approximately US $ 60.00 million per annum losses to the farmers (Zulfiqar, 2003). Foot and mouth disease is commonly mild or unobvious in adult sheep and goats but occasionally, in case of young animals, the mortality rate is relatively significant (Kitching and Hughes, 2002). Generally, sheep and goats exhibit quiet signs of disease, which are usually overlooked by the farmer (Mishra and Ghei, 1983). Foot and mouth disease can be diagnosed by isolating the virus or by the detection of FMD viral antigen or nucleic acid in samples of vesicular fluid or epithelial tissue. Diagnosis can also be made by detecting specific antibodies against FMDV in the serum while detection of antibodies against non-structural proteins (NSPs) of the virus can be used to differentiate infected and vaccinated animals (DIVA).
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
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It is established that the disease in sheep and goats has been overlooked in Pakistan as disease is often observed in a milder form in small ruminants than in cattle and buffaloes. Therefore, this study was designed to determine the seroprevalence of FMD in sheep and goats in Punjab, Pakistan and to identify various risk factors associated with prevalence of the disease. Materials and Methods Study area and sample size To ascertain the seroprevalence among sheep and goats in Punjab, the serum samples were collected from three districts representing the whole province. Blood samples were collected from sheep and goats in three districts of the Punjab, that is, Chakwal (representing North Punjab), Faisalabad (representing Central Punjab) and Khanewal (representing South Punjab). To validate the study, sample size was calculated using formula for simple random sampling (Thrusfield, 2008), n¼
1:962 Pexp ð1 Pexp Þ d2
1:962 0:5 ð1 0:1Þ 0:052 n ¼ 384
Collection of samples Serum samples from 400 sheep and goats were collected from each of the three districts. Sheep and goats were sampled proportional to their total population in each district. Equal numbers of samples were collected from each Tehsil/ Town of the selected districts. The samples were labelled properly, and information regarding species, age, sex, pregnancy status and herd type was recorded. Measurement of antibodies against non-structural protein of FMD The sera samples were tested using FMD 3ABC-ELISA SVANOVIRâ kit (Svanova Diagnostic, Uppsala, Sweden) following the procedure described by kit manufacturer. The test sera, negative and positive controls sera were diluted and added to 96-well ELISA plates coated with 3ABC antigen. After 30 min incubation at 37°C, washing of ELISA plates was performed three times with washing 26
Data analysis The data were statistically analysed using software portable Minitab, version 16.1 (Minitab Inc, State College, PA, USA). Chi-square test was employed to detect any significant association of risk factors with that of FMD infection. Identification of FMDV
where, n is number of samples; Pexp is expected prevalence; d is desired absolute precision. Expected prevalence was kept at 50%, while desired absolute precision at 5% and level of confidence at 95%. n¼
buffer. In the next step, peroxidase conjugated anti-ruminant antibodies were added to the plate and incubated for another 30 min. Washing was repeated, and substrate (Tetramethyl Benzidine) was added, and plates were incubated at room temperature for 30 min under dark conditions. The reaction was terminated by the addition of 1 M sulphuric acid as stopping solution. The optical density (OD) of the samples was measured at 405 nm, and the results were calculated by dividing the corrected OD value of the test serum by that of corrected OD value of positive control. A sample with OD ≥48% was considered positive and OD 0.05) different, v2 (1 df) = 0.204, P = 0.652.
The antibody-based prevalence of FMDV in sheep and goats was 21.27% and 19.44%, respectively. However, this difference in prevalence of antibodies to FMDV in two species was statistically non-significant v2 (1 df) = 0.204, P = 0.652. Among sheep of three districts, prevalence was highest in sheep of Khanewal (31.94%) followed by Faisalabad (25%) and Chakwal (7.14%). Similar pattern of prevalence was observed in goats, highest prevalence in goats of Khanewal (32.62%) followed by Faisalabad (25.8%) and Chakwal (4.11%) (Table 1). The prevalence of antibodies to FMDV among various age groups of sheep and goats was studied. Total sampled animals were divided into five age groups (i) 6–12 months of age (n = 401), (ii) 13–18 months of age (n = 141), (iii) 19–24 months of age (n = 195), (iv) 25–36 months of age (n = 214) and (v) more than 36 months of age (n = 249) to determine the possible association of disease with age of animals. Highest seroprevalence was found in animals belonging to group v (39.36%) followed by animals in group iv (33.64%), animals in group iii (17.44%), animals in group i (8.91%) and animals in group ii (8.51%). Seroprevalence of groups iii, iv and v was found significantly different from that of group i and group ii, v2 (4 df) = 76.848, P = 0.000 (Table 2). The antibody-based prevalence against FMDV in male sheep and goats (n = 445) was 11.46%, compared with 26.26% for females (n = 755), (Table 3). This difference in the seroprevalence in male and female animals was statistically highly significant v2 (1 df) = 26.276, P = 0.000.
Prevalence of antibodies to FMDV in pregnant and nonpregnant female sheep and goats was also studied. Prevalence in pregnant animals (n = 94) was 27.65%, while in non-pregnant animals (n = 661), it was 26.48% (Table 4). However, this difference in the prevalence was statistically non-significant v2 (1 df) = 0.034, P = 0.854. In sheep and goats kept with large ruminants, that is, mixed herds (n = 294), 22.11% prevalence was detected while it was 20.64% among sheep and goats kept alone (n = 906). Seroprevalence in sheep and goats kept with large ruminants was numerically high but statistically nonsignificant v2 (1 df) = 0.187, P = 0.665 (Table 5). RT-PCR results indicated that four of 13 field samples were positive for FMDV. All these four positive samples were taken from goats of district Khanewal. For further confirmation, PCR products were purified and were sequenced in both forward and reverse directions. The BLAST results confirmed the presence of FMDV in clinical samples. The BLAST search showed 97% homology with FMD-type-O-Tur/27/2011 Accession Number JX40500. Discussion The current project was designed to determine the seroprevalence of FMD in sheep and goats in province of Punjab, Pakistan. Determination of existing disease status through prevalence studies is important to estimate the disease load in a population and to design control policies. Sheep and goat population of investigated areas is generally kept in
Table 2. Comparative age-related seroprevalence of foot and mouth disease in three districts of the Punjab Age group (Prev.%) Animal species
Samples taken (Age group distribution)
6–12 months
13–18 months
19–24 months
25–36 months
>36 months
Sheep Goat Total
180 (61, 22, 37, 34, 26) 1020 (340, 119, 158, 180, 223) 1200 (401, 141, 195, 214, 249)
6 (9.61)a 30 (8.82)a 36 (8.98)a
2 (9.09)a 10 (8.4)a 12 (8.51)a
5 (13.5)b 29 (18.35)b 34 (17.44)b
11 (32.35)c 61 (33.89)c 72 (33.64)c
11 (42.31)d 87 (39.01)d 98 (39.36)d
Values with different superscript alphabets in a row are significantly (P < 0.05) different, v2 (4 df) = 76.848, P = 0.000.
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
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Table 3. Comparative sex-related seroprevalence of foot and mouth disease in three districts of the Punjab Sheep (Prev.%) Area sampled
Samples taken
Chakwal
S (37♂, 47♀) G (119♂, 197♀) S (16♂, 8♀) G (141♂, 235♀) S (15♂, 57♀) G (117♂, 211♀) S (68♂, 112♀) G (377♂, 643♀)
Faisalabad Khanewal Total
Goat (Prev.%)
Male
Female
Male
Female
2 (5.41)
4 (8.51)
6 (5.04)
7 (3.55)
3 (18.75)
3 (37.5)
23 (16.31)
74 (31.49)
5 (33.33)
18 (31.57)
12 (10.26)
95 (45.02)
10 (14.71)a
25 (22.32)b
41 (10.88)a
176 (27.37)b
Values with different superscript alphabets in a row are significantly (P < 0.05) different, v2 (1 df) = 26.276, P = 0.000.
Table 4. Comparative seroprevalence of foot and mouth disease in three districts of the Punjab on the basis of pregnancy status Sheep
Goat
Pregnant
Non-pregnant
Pregnant
Non-pregnant
Area sampled
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Chakwal Faisalabad Khanewal Total
8 3 10 21
1 (12.5) 1 (33.33) 3 (30) 5 (23.8)a
39 5 47 91
3 (7.69) 2 (40) 15 (31.9) 20 (21.97)a
19 26 28 73
2 (10.53) 10 (38.46) 9 (32.14) 21 (28.77)a
178 209 183 570
5 (2.81) 64 (30.62) 86 (46.99) 155 (27.19)a
Values with same superscript alphabets in a row are not significantly (P > 0.05) different, v2 (1 df) = 0.034, P = 0.854.
Table 5. Comparative seroprevalence of foot and mouth disease in three districts of the Punjab on the basis of herd type Type of herd
Sheep and Goats
Kept with ruminants
large
Location
Samples taken
Positive (Prev.%)
Samples taken
Positive (Prev.%)
Chakwal Faisalabad Khanewal Total
290 294 322 906
12 (4.14) 72 (24.49) 103 (31.99) 187 (20.64)a
110 106 78 294
7 (6.36) 31 (29.24) 27 (34.61) 65 (22.11)a
Values with same superscript alphabets in a row are significantly or not significantly (P > 0.05) different, v2 (1 df) = 0.187, P = 0.665.
poor management and housing conditions that favours the spread of contagious diseases. Moreover, deprived nutrition compromises the immune system of animals and favours the disease spread. On the basis of NSP-ELISA, high prevalence of antibodies to FMDV indicates towards the possible hyperendemic nature of disease in selected districts. Results also indicate that there are no significant differences between the prevalence of disease in two species. This non-significant 28
difference in the prevalence of antibodies to FMDV in two species may be attributed to the fact that both sheep and goats are mostly reared together and have equal chances of contracting disease. Similar disease pattern was also recorded in different reports. In Southern Jordan, the seroprevalence of FMD in sheep and goat at individual species level was recorded to be 10.4% and 6.3%, whereas seroprevalence at herd level was 44.7% and 33.3%, respectively (Al-Majali et al., 2008). In another epidemiological study in Uganda following an outbreak of FMD indicated prevalence of antibodies to FMDV NSP up to 14% in goats and 22% in sheep (Balinda et al., 2009). This dissimilarity in seroprevalence of FMD in different countries may be due to disparity in climatic conditions and ecology and an overall exposure to the virus. Results indicate that prevalence of antibodies to FMDV was significantly different in three selected districts of Punjab. The differences in percentage positivity of animals can be associated with animal population density and climatic conditions of these regions/districts. The greater time-span that FMDV has been endemic in the southern and central districts may account for the higher prevalence of disease in these areas. Relatively, low prevalence in northern district may be due to the fact that the northern parts of the province have got relatively small sheep and goat population.
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
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Significantly higher seroprevalence was observed in older animals than young ones. This difference in prevalence may just be an outcome of the opportunities to get in contact with the disease, the older the animal, the higher the chance of being in contact with the disease. The low seroprevalence of FMD recorded in young animals could also be associated with the low frequency of exposure, and the causes of increased susceptibility to old animals might be due to malnutrition, poor immunity and poor management system. These findings concord with the findings of Al-Majali et al. (2008) who found that the prevalence of FMD in sheep older than 5 years of age was significantly higher than that in younger sheep. Similar to that in sheep, the prevalence of FMD in goats older than 5 years of age was significantly higher than that in younger goats. Significantly higher seroprevalence of FMD was found in female sheep and goats as compared with male animals. Similarly, in another epidemiological investigation in Ethiopia, the prevalence rate of FMD among female animals was found higher (15.7%) than in males (8.27%) (Gelaye et al., 2009). The greater percentage in females might be due to the physiological stresses that include oestrus, pregnancy and lactation which are known to affect their resistance to infection (Susan and Asamays, 1998). Lower seroprevalence in males could also be attributed to the fact that male sheep and goats are usually provided with better nutrition than female in studied areas. Another possible reason may be that the females are kept for an extended duration of time as compared to males to promote production exercises, which may ultimately result in high prevalence of antibodies to FMDV in female animals. Study shows that there was no relation of seroprevalence of FMD with the pregnancy status of female sheep and goats. Present study also indicates that there was no significance difference in the seroprevalence of FMD between sheep and goats that are kept alone and that are kept with larger animals. In conflict with our results, in an epidemiological investigation in Jordan, mixed farming was found as risk factor to seropositivity of FMD in sheep and goats (AlMajali et al., 2008). RT-PCR detected FMD virus in four of 13 field samples (vesicular epithelium and vesicular fluid). It was for the first time that the detection of FMD virus was carried out from the samples of sheep and goat origin. This low percentage positivity by RT-PCR may be attributed to the fact that there are no clear signs and symptoms of the disease and animals showing any oral lesions were sampled for detection of virus. The 5′UTR region of FMD virus is a conserved region among all serotypes. These sequences cannot explain evolutionary relationship among FMD viruses. However, the sequences showed maximum homology to FMD-type O isolates, which is in accordance with results of
FMDV in Small Ruminants
Waheed et al. (2011) who detected 31 samples as FMDtype O of 32 samples. In conclusion, the results demonstrate that the prevalence of foot and mouth disease is awfully high in the sampled sheep and goat population, and there was no significant difference in the prevalence of FMD in sheep and goats. Seroprevalence of FMD was significantly associated with sampling locales. Highest seroprevalence was observed in Southern Punjab followed by Central and Northern Punjab. Regular vaccination of FMD is recommended in sheep and goats in the studied areas as sheep and goats are usually ignored for vaccination. The present study demonstrates that FMD is highly prevalent in sheep and goats in Punjab. Therefore, a broader study is required to find out the countrywide prevalence of FMD in small sheep and goats. Acknowledgements We are grateful to Endowment Fund Secretariat, University of Agriculture, Faisalabad for financial support to conduct this research. We are also grateful to District Livestock Officers of studied districts for their support in collection of samples. Conflicts of Interest The authors declare there is no conflict of interest. References Al-Majali, A. M., K. Jawasreh, and A. A. Nsour, 2008: Epidemiological studies on foot and mouth disease and paratuberculosis in small ruminants in Tafelah and Ma’an, Jordan. Small Rumin. Res. 78, 197–201. Balinda, S. N., K. Tjornehoj, V. B. Muwanika, A. K. Sangula, F. N. Mwiine, C. Ayebazibwe, C. Masembe, H. R. Siegismund, and S. Alexandersen, 2009: Prevalence estimates of antibodies towards foot-and-mouth disease virus in small ruminants in Uganda. Transbound. Emerg. Dis. 56, 362–371. Gelaye, E., G. Ayelet, T. Abera, and K. Asmare, 2009: Seroprevalence of foot and mouth disease in Bench Maji zone, Southwestern Ethiopia. J. Vet. Med. Anim. Health 1, 05–10. Kitching, R. P., and C. J. Hughes, 2002: Clinical variation in foot and mouth disease: sheep and goats. Rev. Sci. Tech. 21, 505– 512. Kitching, R. P., A. M. Hutber, and M. V. Thrusfield, 2005: A review of foot and mouth disease with special consideration for the clinical and epidemiological factors relevant to predictive modelling of the disease. Vet. J. 169, 197–209. Marvin, J. G., and B. Barry, 2004: Foot-and-Mouth disease. Clin. Microbiol. Rev. 17, 465–493.
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Mishra, K. C., and J. C. Ghei, 1983: Target sites of apthovirus infection in Sikkim local goats. Ind. Vet. Med. J. 7, 227–228. Reid, S. M., N. P. Ferris, G. H. Hutchings, A. R. Samuel, and N. J. Knowles, 2000: Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods 89, 167–176. Reuckert, R. R., 1996: Picornaviridae: the viruses and their replication. In: Fields, B. N., D. M. Knipe and P. M. Howley (eds), Fields Virology. 3rd edn, pp. 609–654. Lippincott/Raven, Philadelphia. Susan, E., and A. Asamays, 1998: The Merck Veterinary Manual, 8th edn. Whitehouse Stat N J Merck and Co. inc. 1879, National Publishing, Inc. Philadelphia, PA.
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Thrusfield, M., 2008: Surveys: sampling, Types of sampling. Veterinary Epidemiology, 3rd edn, pp. 228–242. Blackwell publishing company, State Avenue, Ames, IA. Waheed, U., S. Parida, Q. M. Khan, M. Hussain, K. Ebert, J. Wadsworth, S. M. Reid, G. H. Hutchings, M. Mahapatra, D. P. King, D. J. Paton, and N. J. Knowles, 2011: Molecular characterisation of foot-and-mouth disease viruses from Pakistan, 2005–2008. Transbound. Emerg. Dis. 58, 166–172. Zulfiqar, M., 2003: Draft Report for Development of National Disease Control Policy for Foot and Mouth Disease in Pakistan under the FAO Project “Support for Emergency Prevention and Control of Main Trans-boundary Animal Diseases in Pakistan Rinderpest, FMD, PPR”
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
ORIGINAL ARTICLE
Detection and Seroprevalence of Foot and Mouth Disease in Sheep and Goats in Punjab, Pakistan Saif Ur-Rehman1, M. Arshad1, I. Hussain1 and Z. Iqbal2 1 2
Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan Department of Parasitology, University of Agriculture, Faisalabad, Pakistan
Keywords: foot and mouth disease; sheep; goats; Punjab; Pakistan; seroprevalence; NSP-ELISA; risk factors Correspondence: Saif Ur-Rehman. Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan. Tel./Fax: 00923217628262; E-mail: [email protected] Received for publication September 6, 2013 doi:10.1111/tbed.12194
Summary Foot and mouth disease (FMD) is a highly contagious disease of ruminants that causes huge economic losses around the globe. However, the prevalence of FMDV in small ruminants has been overlooked in Pakistan. A seroepidemiological study was conducted in Chakwal, Faisalabad and Khanewal districts of Punjab, Pakistan to determine the prevalence of FMD in sheep and goats. A total 1200 serum samples were collected from sheep (n = 180) and goats (n = 920) and were subjected to 3ABC non-structural protein enzyme-linked immunosorbent assay for detection of antibodies against non-structural proteins of FMD virus. Samples collected from clinical cases were also confirmed for FMDV using RT-PCR. The overall seroprevalence of FMD in sheep and goats was 21% (n = 252) while 19.44% (n = 35) in sheep and 21.27% (n = 217) prevalence was recorded in goats. Highest seroprevalence (32.5%) was observed in southern Punjab (Khanewal), followed by (25.75%) central Punjab (Faisalabad) and the lowest seroprevalence (4.75%) was detected in northern Punjab (Chakwal). There was no statistically significant difference in seroprevalence between sheep and goats. Among different risk factors tested, age and sex were found to be significantly associated with the prevalence of disease while pregnancy and herd type had no association with the prevalence of the disease. This study illustrate that FMD is highly prevalent in sheep and goats in Punjab. Therefore, a broader study is needed to ascertain the countrywide prevalence of FMD in small ruminants.
Introduction Foot and mouth disease (FMD) is a severe, highly contagious viral disease of animals. All cloven footed animal species are susceptible to the disease. Foot and mouth disease is caused by Aphthovirus genus, which is the member of the Picornaviridae family (Reuckert, 1996). FMDV consists of an RNA genome, which is positive-sense and singlestranded. The size of the genome is about 8.5 kilobases, and it is bordered by four structural proteins to make an icosahedral shape of the capsid (Marvin and Barry, 2004). On the basis of serology, FMDV is grouped into seven different types (O, A, C, Asia 1, SAT1, SAT2 and SAT3), which are further divided into numerous subtypes, and there is no cross-protection among these serotypes (Kitching et al., 2005). The most prevalent serotypes in
Pakistan are O (70%), Asia-I (25%) and A (4.67%), which cause approximately US $ 60.00 million per annum losses to the farmers (Zulfiqar, 2003). Foot and mouth disease is commonly mild or unobvious in adult sheep and goats but occasionally, in case of young animals, the mortality rate is relatively significant (Kitching and Hughes, 2002). Generally, sheep and goats exhibit quiet signs of disease, which are usually overlooked by the farmer (Mishra and Ghei, 1983). Foot and mouth disease can be diagnosed by isolating the virus or by the detection of FMD viral antigen or nucleic acid in samples of vesicular fluid or epithelial tissue. Diagnosis can also be made by detecting specific antibodies against FMDV in the serum while detection of antibodies against non-structural proteins (NSPs) of the virus can be used to differentiate infected and vaccinated animals (DIVA).
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
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Saif-ur-Rehman et al.
It is established that the disease in sheep and goats has been overlooked in Pakistan as disease is often observed in a milder form in small ruminants than in cattle and buffaloes. Therefore, this study was designed to determine the seroprevalence of FMD in sheep and goats in Punjab, Pakistan and to identify various risk factors associated with prevalence of the disease. Materials and Methods Study area and sample size To ascertain the seroprevalence among sheep and goats in Punjab, the serum samples were collected from three districts representing the whole province. Blood samples were collected from sheep and goats in three districts of the Punjab, that is, Chakwal (representing North Punjab), Faisalabad (representing Central Punjab) and Khanewal (representing South Punjab). To validate the study, sample size was calculated using formula for simple random sampling (Thrusfield, 2008), n¼
1:962 Pexp ð1 Pexp Þ d2
1:962 0:5 ð1 0:1Þ 0:052 n ¼ 384
Collection of samples Serum samples from 400 sheep and goats were collected from each of the three districts. Sheep and goats were sampled proportional to their total population in each district. Equal numbers of samples were collected from each Tehsil/ Town of the selected districts. The samples were labelled properly, and information regarding species, age, sex, pregnancy status and herd type was recorded. Measurement of antibodies against non-structural protein of FMD The sera samples were tested using FMD 3ABC-ELISA SVANOVIRâ kit (Svanova Diagnostic, Uppsala, Sweden) following the procedure described by kit manufacturer. The test sera, negative and positive controls sera were diluted and added to 96-well ELISA plates coated with 3ABC antigen. After 30 min incubation at 37°C, washing of ELISA plates was performed three times with washing 26
Data analysis The data were statistically analysed using software portable Minitab, version 16.1 (Minitab Inc, State College, PA, USA). Chi-square test was employed to detect any significant association of risk factors with that of FMD infection. Identification of FMDV
where, n is number of samples; Pexp is expected prevalence; d is desired absolute precision. Expected prevalence was kept at 50%, while desired absolute precision at 5% and level of confidence at 95%. n¼
buffer. In the next step, peroxidase conjugated anti-ruminant antibodies were added to the plate and incubated for another 30 min. Washing was repeated, and substrate (Tetramethyl Benzidine) was added, and plates were incubated at room temperature for 30 min under dark conditions. The reaction was terminated by the addition of 1 M sulphuric acid as stopping solution. The optical density (OD) of the samples was measured at 405 nm, and the results were calculated by dividing the corrected OD value of the test serum by that of corrected OD value of positive control. A sample with OD ≥48% was considered positive and OD 0.05) different, v2 (1 df) = 0.204, P = 0.652.
The antibody-based prevalence of FMDV in sheep and goats was 21.27% and 19.44%, respectively. However, this difference in prevalence of antibodies to FMDV in two species was statistically non-significant v2 (1 df) = 0.204, P = 0.652. Among sheep of three districts, prevalence was highest in sheep of Khanewal (31.94%) followed by Faisalabad (25%) and Chakwal (7.14%). Similar pattern of prevalence was observed in goats, highest prevalence in goats of Khanewal (32.62%) followed by Faisalabad (25.8%) and Chakwal (4.11%) (Table 1). The prevalence of antibodies to FMDV among various age groups of sheep and goats was studied. Total sampled animals were divided into five age groups (i) 6–12 months of age (n = 401), (ii) 13–18 months of age (n = 141), (iii) 19–24 months of age (n = 195), (iv) 25–36 months of age (n = 214) and (v) more than 36 months of age (n = 249) to determine the possible association of disease with age of animals. Highest seroprevalence was found in animals belonging to group v (39.36%) followed by animals in group iv (33.64%), animals in group iii (17.44%), animals in group i (8.91%) and animals in group ii (8.51%). Seroprevalence of groups iii, iv and v was found significantly different from that of group i and group ii, v2 (4 df) = 76.848, P = 0.000 (Table 2). The antibody-based prevalence against FMDV in male sheep and goats (n = 445) was 11.46%, compared with 26.26% for females (n = 755), (Table 3). This difference in the seroprevalence in male and female animals was statistically highly significant v2 (1 df) = 26.276, P = 0.000.
Prevalence of antibodies to FMDV in pregnant and nonpregnant female sheep and goats was also studied. Prevalence in pregnant animals (n = 94) was 27.65%, while in non-pregnant animals (n = 661), it was 26.48% (Table 4). However, this difference in the prevalence was statistically non-significant v2 (1 df) = 0.034, P = 0.854. In sheep and goats kept with large ruminants, that is, mixed herds (n = 294), 22.11% prevalence was detected while it was 20.64% among sheep and goats kept alone (n = 906). Seroprevalence in sheep and goats kept with large ruminants was numerically high but statistically nonsignificant v2 (1 df) = 0.187, P = 0.665 (Table 5). RT-PCR results indicated that four of 13 field samples were positive for FMDV. All these four positive samples were taken from goats of district Khanewal. For further confirmation, PCR products were purified and were sequenced in both forward and reverse directions. The BLAST results confirmed the presence of FMDV in clinical samples. The BLAST search showed 97% homology with FMD-type-O-Tur/27/2011 Accession Number JX40500. Discussion The current project was designed to determine the seroprevalence of FMD in sheep and goats in province of Punjab, Pakistan. Determination of existing disease status through prevalence studies is important to estimate the disease load in a population and to design control policies. Sheep and goat population of investigated areas is generally kept in
Table 2. Comparative age-related seroprevalence of foot and mouth disease in three districts of the Punjab Age group (Prev.%) Animal species
Samples taken (Age group distribution)
6–12 months
13–18 months
19–24 months
25–36 months
>36 months
Sheep Goat Total
180 (61, 22, 37, 34, 26) 1020 (340, 119, 158, 180, 223) 1200 (401, 141, 195, 214, 249)
6 (9.61)a 30 (8.82)a 36 (8.98)a
2 (9.09)a 10 (8.4)a 12 (8.51)a
5 (13.5)b 29 (18.35)b 34 (17.44)b
11 (32.35)c 61 (33.89)c 72 (33.64)c
11 (42.31)d 87 (39.01)d 98 (39.36)d
Values with different superscript alphabets in a row are significantly (P < 0.05) different, v2 (4 df) = 76.848, P = 0.000.
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Table 3. Comparative sex-related seroprevalence of foot and mouth disease in three districts of the Punjab Sheep (Prev.%) Area sampled
Samples taken
Chakwal
S (37♂, 47♀) G (119♂, 197♀) S (16♂, 8♀) G (141♂, 235♀) S (15♂, 57♀) G (117♂, 211♀) S (68♂, 112♀) G (377♂, 643♀)
Faisalabad Khanewal Total
Goat (Prev.%)
Male
Female
Male
Female
2 (5.41)
4 (8.51)
6 (5.04)
7 (3.55)
3 (18.75)
3 (37.5)
23 (16.31)
74 (31.49)
5 (33.33)
18 (31.57)
12 (10.26)
95 (45.02)
10 (14.71)a
25 (22.32)b
41 (10.88)a
176 (27.37)b
Values with different superscript alphabets in a row are significantly (P < 0.05) different, v2 (1 df) = 26.276, P = 0.000.
Table 4. Comparative seroprevalence of foot and mouth disease in three districts of the Punjab on the basis of pregnancy status Sheep
Goat
Pregnant
Non-pregnant
Pregnant
Non-pregnant
Area sampled
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Samples done
Positve (Prev.%)
Chakwal Faisalabad Khanewal Total
8 3 10 21
1 (12.5) 1 (33.33) 3 (30) 5 (23.8)a
39 5 47 91
3 (7.69) 2 (40) 15 (31.9) 20 (21.97)a
19 26 28 73
2 (10.53) 10 (38.46) 9 (32.14) 21 (28.77)a
178 209 183 570
5 (2.81) 64 (30.62) 86 (46.99) 155 (27.19)a
Values with same superscript alphabets in a row are not significantly (P > 0.05) different, v2 (1 df) = 0.034, P = 0.854.
Table 5. Comparative seroprevalence of foot and mouth disease in three districts of the Punjab on the basis of herd type Type of herd
Sheep and Goats
Kept with ruminants
large
Location
Samples taken
Positive (Prev.%)
Samples taken
Positive (Prev.%)
Chakwal Faisalabad Khanewal Total
290 294 322 906
12 (4.14) 72 (24.49) 103 (31.99) 187 (20.64)a
110 106 78 294
7 (6.36) 31 (29.24) 27 (34.61) 65 (22.11)a
Values with same superscript alphabets in a row are significantly or not significantly (P > 0.05) different, v2 (1 df) = 0.187, P = 0.665.
poor management and housing conditions that favours the spread of contagious diseases. Moreover, deprived nutrition compromises the immune system of animals and favours the disease spread. On the basis of NSP-ELISA, high prevalence of antibodies to FMDV indicates towards the possible hyperendemic nature of disease in selected districts. Results also indicate that there are no significant differences between the prevalence of disease in two species. This non-significant 28
difference in the prevalence of antibodies to FMDV in two species may be attributed to the fact that both sheep and goats are mostly reared together and have equal chances of contracting disease. Similar disease pattern was also recorded in different reports. In Southern Jordan, the seroprevalence of FMD in sheep and goat at individual species level was recorded to be 10.4% and 6.3%, whereas seroprevalence at herd level was 44.7% and 33.3%, respectively (Al-Majali et al., 2008). In another epidemiological study in Uganda following an outbreak of FMD indicated prevalence of antibodies to FMDV NSP up to 14% in goats and 22% in sheep (Balinda et al., 2009). This dissimilarity in seroprevalence of FMD in different countries may be due to disparity in climatic conditions and ecology and an overall exposure to the virus. Results indicate that prevalence of antibodies to FMDV was significantly different in three selected districts of Punjab. The differences in percentage positivity of animals can be associated with animal population density and climatic conditions of these regions/districts. The greater time-span that FMDV has been endemic in the southern and central districts may account for the higher prevalence of disease in these areas. Relatively, low prevalence in northern district may be due to the fact that the northern parts of the province have got relatively small sheep and goat population.
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 25–30
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Significantly higher seroprevalence was observed in older animals than young ones. This difference in prevalence may just be an outcome of the opportunities to get in contact with the disease, the older the animal, the higher the chance of being in contact with the disease. The low seroprevalence of FMD recorded in young animals could also be associated with the low frequency of exposure, and the causes of increased susceptibility to old animals might be due to malnutrition, poor immunity and poor management system. These findings concord with the findings of Al-Majali et al. (2008) who found that the prevalence of FMD in sheep older than 5 years of age was significantly higher than that in younger sheep. Similar to that in sheep, the prevalence of FMD in goats older than 5 years of age was significantly higher than that in younger goats. Significantly higher seroprevalence of FMD was found in female sheep and goats as compared with male animals. Similarly, in another epidemiological investigation in Ethiopia, the prevalence rate of FMD among female animals was found higher (15.7%) than in males (8.27%) (Gelaye et al., 2009). The greater percentage in females might be due to the physiological stresses that include oestrus, pregnancy and lactation which are known to affect their resistance to infection (Susan and Asamays, 1998). Lower seroprevalence in males could also be attributed to the fact that male sheep and goats are usually provided with better nutrition than female in studied areas. Another possible reason may be that the females are kept for an extended duration of time as compared to males to promote production exercises, which may ultimately result in high prevalence of antibodies to FMDV in female animals. Study shows that there was no relation of seroprevalence of FMD with the pregnancy status of female sheep and goats. Present study also indicates that there was no significance difference in the seroprevalence of FMD between sheep and goats that are kept alone and that are kept with larger animals. In conflict with our results, in an epidemiological investigation in Jordan, mixed farming was found as risk factor to seropositivity of FMD in sheep and goats (AlMajali et al., 2008). RT-PCR detected FMD virus in four of 13 field samples (vesicular epithelium and vesicular fluid). It was for the first time that the detection of FMD virus was carried out from the samples of sheep and goat origin. This low percentage positivity by RT-PCR may be attributed to the fact that there are no clear signs and symptoms of the disease and animals showing any oral lesions were sampled for detection of virus. The 5′UTR region of FMD virus is a conserved region among all serotypes. These sequences cannot explain evolutionary relationship among FMD viruses. However, the sequences showed maximum homology to FMD-type O isolates, which is in accordance with results of
FMDV in Small Ruminants
Waheed et al. (2011) who detected 31 samples as FMDtype O of 32 samples. In conclusion, the results demonstrate that the prevalence of foot and mouth disease is awfully high in the sampled sheep and goat population, and there was no significant difference in the prevalence of FMD in sheep and goats. Seroprevalence of FMD was significantly associated with sampling locales. Highest seroprevalence was observed in Southern Punjab followed by Central and Northern Punjab. Regular vaccination of FMD is recommended in sheep and goats in the studied areas as sheep and goats are usually ignored for vaccination. The present study demonstrates that FMD is highly prevalent in sheep and goats in Punjab. Therefore, a broader study is required to find out the countrywide prevalence of FMD in small sheep and goats. Acknowledgements We are grateful to Endowment Fund Secretariat, University of Agriculture, Faisalabad for financial support to conduct this research. We are also grateful to District Livestock Officers of studied districts for their support in collection of samples. Conflicts of Interest The authors declare there is no conflict of interest. References Al-Majali, A. M., K. Jawasreh, and A. A. Nsour, 2008: Epidemiological studies on foot and mouth disease and paratuberculosis in small ruminants in Tafelah and Ma’an, Jordan. Small Rumin. Res. 78, 197–201. Balinda, S. N., K. Tjornehoj, V. B. Muwanika, A. K. Sangula, F. N. Mwiine, C. Ayebazibwe, C. Masembe, H. R. Siegismund, and S. Alexandersen, 2009: Prevalence estimates of antibodies towards foot-and-mouth disease virus in small ruminants in Uganda. Transbound. Emerg. Dis. 56, 362–371. Gelaye, E., G. Ayelet, T. Abera, and K. Asmare, 2009: Seroprevalence of foot and mouth disease in Bench Maji zone, Southwestern Ethiopia. J. Vet. Med. Anim. Health 1, 05–10. Kitching, R. P., and C. J. Hughes, 2002: Clinical variation in foot and mouth disease: sheep and goats. Rev. Sci. Tech. 21, 505– 512. Kitching, R. P., A. M. Hutber, and M. V. Thrusfield, 2005: A review of foot and mouth disease with special consideration for the clinical and epidemiological factors relevant to predictive modelling of the disease. Vet. J. 169, 197–209. Marvin, J. G., and B. Barry, 2004: Foot-and-Mouth disease. Clin. Microbiol. Rev. 17, 465–493.
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Mishra, K. C., and J. C. Ghei, 1983: Target sites of apthovirus infection in Sikkim local goats. Ind. Vet. Med. J. 7, 227–228. Reid, S. M., N. P. Ferris, G. H. Hutchings, A. R. Samuel, and N. J. Knowles, 2000: Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods 89, 167–176. Reuckert, R. R., 1996: Picornaviridae: the viruses and their replication. In: Fields, B. N., D. M. Knipe and P. M. Howley (eds), Fields Virology. 3rd edn, pp. 609–654. Lippincott/Raven, Philadelphia. Susan, E., and A. Asamays, 1998: The Merck Veterinary Manual, 8th edn. Whitehouse Stat N J Merck and Co. inc. 1879, National Publishing, Inc. Philadelphia, PA.
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Thrusfield, M., 2008: Surveys: sampling, Types of sampling. Veterinary Epidemiology, 3rd edn, pp. 228–242. Blackwell publishing company, State Avenue, Ames, IA. Waheed, U., S. Parida, Q. M. Khan, M. Hussain, K. Ebert, J. Wadsworth, S. M. Reid, G. H. Hutchings, M. Mahapatra, D. P. King, D. J. Paton, and N. J. Knowles, 2011: Molecular characterisation of foot-and-mouth disease viruses from Pakistan, 2005–2008. Transbound. Emerg. Dis. 58, 166–172. Zulfiqar, M., 2003: Draft Report for Development of National Disease Control Policy for Foot and Mouth Disease in Pakistan under the FAO Project “Support for Emergency Prevention and Control of Main Trans-boundary Animal Diseases in Pakistan Rinderpest, FMD, PPR”
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