Downloaded from http://veterinaryrecord.bmj.com/ on February 18, 2016 - Published by group.bmj.com
Research
Research EDITORIAL
Controlling scrapie and bovine spongiform encephalopathy in goats Cristina Acín and José Luis Pitarch Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the accumulation in the central nervous system (CNS) of an anomalous isoform called prion protein (Prusiner 1982). Bovine spongiform encephalopathy (BSE) represents one of the most important food crises of past decades in Europe. BSE started in the UK, due to cattle consumption of feedstuff contaminated with prions (Wilesmith and others 1992). In Europe, efforts have focused on eradicating the disease in the bovine population. However, since sheep and goats are susceptible to prion diseases, eradication efforts in these species have to be considered; in fact, in 2005 the first case of natural BSE in a goat from France was diagnosed (Eloit and others 2005). These findings imply that BSE in small ruminants (particularly goats) could represent a danger for human health and therefore various strategies must be considered to avoid infection. It has been recognised that polymorphisms of the PRNP gene are responsible for some element of susceptibility or resistance to prion diseases. Studies in sheep have shown that the haplotype R171 (ARR sheep) confers resistance to the disease while the V136 variant (VRQ-sheep) is the most susceptible haplotype to acquire the disease. The EU
Cristina Acín, BVet, PhD, José Luis Pitarch, BVet, PhD, Research Centre for TSE and Emerging Transmissible Diseases, University of Zaragoza, Zaragoza 50013, Spain e-mail: [email protected]
166 | Veterinary Record | February 13, 2016
legislation currently in force has required the start of breeding programmes based on the selection of sheep that are resistant to classical scrapie disease. In goats the association between scrapie and PRNP genetics is different from that in sheep but it is only since 2004 that more research has been undertaken on goat TSEs. Research surrounding the caprine PRNP gene has revealed 17 silent mutations and 43 amino acid substitutions. Some allelic variations in the PRNP gene could potentially protect species against the disease, or at least extend the incubation period. Those identified are: S/G127, I/M142, G/D145, H/R143, N/S146, N/D146, R/H154, R/Q211 and Q/K222 (Goldmann and others 1996, 2011, Billinis and others 2002, Acutis and others 2006, Vaccari and others 2006, 2009, PapasavvaStylianou and others 2007, 2011, Barillet and others 2009, Gonzalez and others 2009, Serrano and others 2009, Bouzalas and others 2010, Hussain and others 2011, Acin and others 2013, Corbiere and others 2013, Maestrale and others 2015). With regards to BSE, few studies have addressed genetic resistance in goats, but the polymorphism I/M142 is associated with a slight increase in incubation periods after experimental BSE challenges (Goldmann and others 1996). An experimental oral transmission study with goat BSE was performed in goats carrying the mutations R/Q211 and Q/K222; this showed that while the polymorphism R/Q211 extends the incubation period, only the K222 allele seems to have a protective effect after oral infection (Aguilar-Calvo and others 2015). This suggests that the Q/K222
Downloaded from http://veterinaryrecord.bmj.com/ on February 18, 2016 - Published by group.bmj.com
Research (a)
(b)
FIG 1: Polymorphism at codon 222: (a) homozygote lysine (KK) genotype and (b) heterozygote lysine/glutamine (K/Q) genotype
polymorphism is a good candidate for establishing a genetic breeding programme to eradicate TSE in goats. The GOAT-TSE-FREE project from the EMIDA ERA-NET (Anon 2014) has supplied supplementary results in favour of the K222 polymorphism. The scientists in the project group searched for more data on the effect of the K222 allele on the resistance for scrapie and BSE, both in goats as well as in especially suitable in vivo rodent and in vitro models. In these experiments the K222 allele resulted in a prolonging of the incubation period when scrapie and BSE were analysed, but did not have a great effect against caprine BSE in goats after intracerebral challenge (neither heterozygosis nor homozygosis) (Fig 1 a, b). This mirrors and reaffirms the results reported by Aguilar-Calvo and others (2015) in transgenic mice, and suggests that a breeding programme for resistance against TSE based on the selection of allele K222 carriers could efficiently prevent scrapie and even BSE infection. However, it may not be effective against caprine BSE. Unfortunately, the dimorphism found in ARR sheep that confers resistance to scrapie does not occur in goats, but we should not underestimate the relevance of the K222 polymorphism. Analysing the results of the goat-BSE and GOAT-TSE-FREE projects shows that this polymorphism is the most protective in natural host studies, prolongs incubation periods when cow BSE, goat BSE and scrapie is inoculated intracerebrally and orally, shows resistance in rodent bioassays when cow BSE or scrapie is inoculated, and poorly converts in vitro when conversion assays are performed. A limitation that should be considered is that the frequency of this K222 allele can be low in certain regions. This observation has been highlighted by Goldmann and
others (2016) in a paper summarised on p 168 in this week’s Veterinary Record, which also explores the difficulty in establishing a complete breeding programme in goats. Nevertheless, a gradual and careful selection of resistant animals with sufficient crossbreeding to prevent in-breeding has been possible in rare sheep breeds with very low R171 frequencies, and, in goats, the selection of resistant animals could provide an alternative to control scrapie and BSE. Currently, when a single animal is affected with scrapie the entire herd is culled. This has implications for social reaction and severely damages the farmer’s image and finances. In the future, analysis of all these results will contribute to proposing improved guidelines for controlling goat TSEs by selective breeding-culling strategies.
References
ACIN, C., MARTIN-BURRIEL, I., MONLEON, E., LYAHYAI, J., PITARCH, J. L., SERRANO, C., MONZON, M., ZARAGOZA, P. & BADIOLA, J. J. (2013) Prion protein gene variability in Spanish goats. Inference through susceptibility to classical scrapie strains and pathogenic distribution of peripheral PrP(sc). PLoS One 8, e61118 ACUTIS, P. L., BOSSER, A., PRIEM, J., RIINA, M. V., PELETTO, S., MAZZA, M., Casalone, C., Forloni, G., Ru, G. & Caramelli, M. (2006) Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks. Journal of General Virology 87, 1029-1033 AGUILAR-CALVO, P., FAST, C., TAUSCHER, K., ESPINOSA, J-C., GROSCHUP M. H., NADEEM, M. & OTHERS (2015) Effect of Q211 and K222 PRNP polymorphic variants in the susceptibility of goats to oral infection with goat bovine spongiform encephalopathy. Journal of Infectious Diseases 212, 664-672 ANON (2015) Goat TSE-FREE. www.goatbse.eu/site/ Accessed February 2, 2016 BARILLET, F., MARIAT, D., AMIGUES, Y., FAUGERAS R., CAILLAT, H., MOAZAMI-GOUDARZI, K. & OTHERS (2009) Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie. Journal of General Virology 90, 769-776 BILLINIS, C., PANAGIOTIDIS, C. H., PSYCHAS, V., ARGYROUDIS, S., NICOLAOU, A., LEONTIDES,
S., PAPADOPOULOS, O. & SKLAVIADIS, T. (2002) Prion protein gene polymorphisms in natural goat scrapie. Journal of General Virology 83, 713-721 BOUZALAS, I. G., DOVAS, C. I., BANOS, G., PAPANASTASOPOULOU, M., KRITAS, S., OEVERMANN, A. & OTHERS (2010) Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie. Journal of General Virology 91, 1629-1634 CORBIÈRE, F., PERRIN-CHAUVINEAU, C., LACROUX, C., COSTES, P., THOMAS, M., BRÉMAUD, I. & OTHERS (2013) PrP-associated resistance to scrapie in five highly infected goat herds. Journal of General Virology 94, 241-245 ELOIT, M., ADJOU, K., COULPIER, M., FONTAINE, J. J., HAMEL, R., LILIN, T. & OTHERS (2005) BSE agent signatures in a goat. Veterinary Record doi:10.1136/ vr.156.16.523-b GOLDMANN, W., MARIER, E., STEWART, P., KONOLD, T., STREET, S., LANGEVELD, J., WINDL, O. & ORTIZ-PELAEZ, O. (2016) Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds. Veterinary Record doi:10.1136/vr.103521 GOLDMANN, W., MARTIN, T., FOSTER, J., HUGHES, S., SMITH, G., HUGHES, K., DAWSON, M. & HUNTER, N. (1996) Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. Journal of General Virology 77, 2885-2891 GOLDMANN, W., RYAN, K., STEWART, P., PARNHAM, D., XICOHTENCATL, R., FERNANDEZ, N., SAUNDERS, G., WINDL, O., GONZALEZ, L., BOSSERS, A. & FOSTER, J. (2011) Caprine prion gene polymorphisms are associated with decreased scrapie susceptibility in goat herds in the United Kingdom. Veterinary Research 42, 110 GONZALEZ, L., MARTIN, S., SISO, S., KONOLD, T., ORTIZ-PELAEZ, A., PHELAN, L. & OTHERS (2009) High prevalence of scrapie in a dairy goat herd: tissue distribution of disease-associated PrP and effect of PRNP genotype and age. Veterinary Research 40, 65 HUSSAIN, A., BABAR, M. E., IMRAN, M., HAQ, I. U. & JAVED, M. M. (2011) Detection of four novel polymorphisms in PrP gene of Pakistani sheep (Damani and Hashtnagri) and goats (Kamori and local hairy) breeds. Virology Journal 8, 246 MAESTRALE, C., CANCEDDA, M. G., PINTUS, D., MASIA, M., NONNO, R., RU, G. & OTHERS (2015) Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate. Journal of virology 89,10044-52 PAPASAVVA-STYLIANOU, P., KLEANTHOUS, M., TOUMAZOS, P., MAVRIKIOU, P. & LOUCAIDES, P. (2007) Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie. Veterinary Journal 173, 459-462 PAPASAVVA-STYLIANOU, P., WINDL, O., SAUNDERS, G., MAVRIKIOU, P., TOUMAZOS, P. & KAKOYIANNIS, C. (2011) PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie. Veterinary Journal 187, 245-250 PRUSINER, S. B. (1982) Novel proteinaceous infectious particles cause scrapie. Science, 216, 136-144 SERRANO, C., HAMMOUCHI, M., BENOMAR, A., LYAHYAI, J., RANERA, B., ACIN, C. & OTHERS (2009) PRNP haplotype distribution in Moroccan goats. Animal Genetics 40, 565-568 VACCARI, G., DI BARI, M. A., MORELLI, L., NONNO, R., CHIAPPINI, B., ANTONUCCI G. & OTHERS (2006) Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. Journal of General Virology 87, 1395-1402 VACCARI, G., PANAGIOTIDIS, C. H., ACIN, C., PELETTO, S., BARILLET, F., ACUTIS P. L. & OTHERS (2009) State-of-the-art review of goat TSEs in the European Union, with special emphasis on PrP genetics. Veterinary Research 40, 48 WILESMITH, J. W., RYAN, J. B., HUESTON, W. D. & HOINVILLE, L. J. (1992) Bovine spongiform encephalopathy: epidemiological features 1985 to 1990. Veterinary Record doi:10.1136/vr.130.5.90
doi: 10.1136/vr.i702 February 13, 2016 | Veterinary Record | 167
Downloaded from http://veterinaryrecord.bmj.com/ on February 18, 2016 - Published by group.bmj.com
Controlling scrapie and bovine spongiform encephalopathy in goats Cristina Acín and José Luis Pitarch Veterinary Record 2016 178: 166-167
doi: 10.1136/vr.i702 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/178/7/166
These include:
References Email alerting service
This article cites 18 articles, 3 of which you can access for free at: http://veterinaryrecord.bmj.com/content/178/7/166#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
Research
Research EDITORIAL
Controlling scrapie and bovine spongiform encephalopathy in goats Cristina Acín and José Luis Pitarch Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the accumulation in the central nervous system (CNS) of an anomalous isoform called prion protein (Prusiner 1982). Bovine spongiform encephalopathy (BSE) represents one of the most important food crises of past decades in Europe. BSE started in the UK, due to cattle consumption of feedstuff contaminated with prions (Wilesmith and others 1992). In Europe, efforts have focused on eradicating the disease in the bovine population. However, since sheep and goats are susceptible to prion diseases, eradication efforts in these species have to be considered; in fact, in 2005 the first case of natural BSE in a goat from France was diagnosed (Eloit and others 2005). These findings imply that BSE in small ruminants (particularly goats) could represent a danger for human health and therefore various strategies must be considered to avoid infection. It has been recognised that polymorphisms of the PRNP gene are responsible for some element of susceptibility or resistance to prion diseases. Studies in sheep have shown that the haplotype R171 (ARR sheep) confers resistance to the disease while the V136 variant (VRQ-sheep) is the most susceptible haplotype to acquire the disease. The EU
Cristina Acín, BVet, PhD, José Luis Pitarch, BVet, PhD, Research Centre for TSE and Emerging Transmissible Diseases, University of Zaragoza, Zaragoza 50013, Spain e-mail: [email protected]
166 | Veterinary Record | February 13, 2016
legislation currently in force has required the start of breeding programmes based on the selection of sheep that are resistant to classical scrapie disease. In goats the association between scrapie and PRNP genetics is different from that in sheep but it is only since 2004 that more research has been undertaken on goat TSEs. Research surrounding the caprine PRNP gene has revealed 17 silent mutations and 43 amino acid substitutions. Some allelic variations in the PRNP gene could potentially protect species against the disease, or at least extend the incubation period. Those identified are: S/G127, I/M142, G/D145, H/R143, N/S146, N/D146, R/H154, R/Q211 and Q/K222 (Goldmann and others 1996, 2011, Billinis and others 2002, Acutis and others 2006, Vaccari and others 2006, 2009, PapasavvaStylianou and others 2007, 2011, Barillet and others 2009, Gonzalez and others 2009, Serrano and others 2009, Bouzalas and others 2010, Hussain and others 2011, Acin and others 2013, Corbiere and others 2013, Maestrale and others 2015). With regards to BSE, few studies have addressed genetic resistance in goats, but the polymorphism I/M142 is associated with a slight increase in incubation periods after experimental BSE challenges (Goldmann and others 1996). An experimental oral transmission study with goat BSE was performed in goats carrying the mutations R/Q211 and Q/K222; this showed that while the polymorphism R/Q211 extends the incubation period, only the K222 allele seems to have a protective effect after oral infection (Aguilar-Calvo and others 2015). This suggests that the Q/K222
Downloaded from http://veterinaryrecord.bmj.com/ on February 18, 2016 - Published by group.bmj.com
Research (a)
(b)
FIG 1: Polymorphism at codon 222: (a) homozygote lysine (KK) genotype and (b) heterozygote lysine/glutamine (K/Q) genotype
polymorphism is a good candidate for establishing a genetic breeding programme to eradicate TSE in goats. The GOAT-TSE-FREE project from the EMIDA ERA-NET (Anon 2014) has supplied supplementary results in favour of the K222 polymorphism. The scientists in the project group searched for more data on the effect of the K222 allele on the resistance for scrapie and BSE, both in goats as well as in especially suitable in vivo rodent and in vitro models. In these experiments the K222 allele resulted in a prolonging of the incubation period when scrapie and BSE were analysed, but did not have a great effect against caprine BSE in goats after intracerebral challenge (neither heterozygosis nor homozygosis) (Fig 1 a, b). This mirrors and reaffirms the results reported by Aguilar-Calvo and others (2015) in transgenic mice, and suggests that a breeding programme for resistance against TSE based on the selection of allele K222 carriers could efficiently prevent scrapie and even BSE infection. However, it may not be effective against caprine BSE. Unfortunately, the dimorphism found in ARR sheep that confers resistance to scrapie does not occur in goats, but we should not underestimate the relevance of the K222 polymorphism. Analysing the results of the goat-BSE and GOAT-TSE-FREE projects shows that this polymorphism is the most protective in natural host studies, prolongs incubation periods when cow BSE, goat BSE and scrapie is inoculated intracerebrally and orally, shows resistance in rodent bioassays when cow BSE or scrapie is inoculated, and poorly converts in vitro when conversion assays are performed. A limitation that should be considered is that the frequency of this K222 allele can be low in certain regions. This observation has been highlighted by Goldmann and
others (2016) in a paper summarised on p 168 in this week’s Veterinary Record, which also explores the difficulty in establishing a complete breeding programme in goats. Nevertheless, a gradual and careful selection of resistant animals with sufficient crossbreeding to prevent in-breeding has been possible in rare sheep breeds with very low R171 frequencies, and, in goats, the selection of resistant animals could provide an alternative to control scrapie and BSE. Currently, when a single animal is affected with scrapie the entire herd is culled. This has implications for social reaction and severely damages the farmer’s image and finances. In the future, analysis of all these results will contribute to proposing improved guidelines for controlling goat TSEs by selective breeding-culling strategies.
References
ACIN, C., MARTIN-BURRIEL, I., MONLEON, E., LYAHYAI, J., PITARCH, J. L., SERRANO, C., MONZON, M., ZARAGOZA, P. & BADIOLA, J. J. (2013) Prion protein gene variability in Spanish goats. Inference through susceptibility to classical scrapie strains and pathogenic distribution of peripheral PrP(sc). PLoS One 8, e61118 ACUTIS, P. L., BOSSER, A., PRIEM, J., RIINA, M. V., PELETTO, S., MAZZA, M., Casalone, C., Forloni, G., Ru, G. & Caramelli, M. (2006) Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks. Journal of General Virology 87, 1029-1033 AGUILAR-CALVO, P., FAST, C., TAUSCHER, K., ESPINOSA, J-C., GROSCHUP M. H., NADEEM, M. & OTHERS (2015) Effect of Q211 and K222 PRNP polymorphic variants in the susceptibility of goats to oral infection with goat bovine spongiform encephalopathy. Journal of Infectious Diseases 212, 664-672 ANON (2015) Goat TSE-FREE. www.goatbse.eu/site/ Accessed February 2, 2016 BARILLET, F., MARIAT, D., AMIGUES, Y., FAUGERAS R., CAILLAT, H., MOAZAMI-GOUDARZI, K. & OTHERS (2009) Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie. Journal of General Virology 90, 769-776 BILLINIS, C., PANAGIOTIDIS, C. H., PSYCHAS, V., ARGYROUDIS, S., NICOLAOU, A., LEONTIDES,
S., PAPADOPOULOS, O. & SKLAVIADIS, T. (2002) Prion protein gene polymorphisms in natural goat scrapie. Journal of General Virology 83, 713-721 BOUZALAS, I. G., DOVAS, C. I., BANOS, G., PAPANASTASOPOULOU, M., KRITAS, S., OEVERMANN, A. & OTHERS (2010) Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie. Journal of General Virology 91, 1629-1634 CORBIÈRE, F., PERRIN-CHAUVINEAU, C., LACROUX, C., COSTES, P., THOMAS, M., BRÉMAUD, I. & OTHERS (2013) PrP-associated resistance to scrapie in five highly infected goat herds. Journal of General Virology 94, 241-245 ELOIT, M., ADJOU, K., COULPIER, M., FONTAINE, J. J., HAMEL, R., LILIN, T. & OTHERS (2005) BSE agent signatures in a goat. Veterinary Record doi:10.1136/ vr.156.16.523-b GOLDMANN, W., MARIER, E., STEWART, P., KONOLD, T., STREET, S., LANGEVELD, J., WINDL, O. & ORTIZ-PELAEZ, O. (2016) Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds. Veterinary Record doi:10.1136/vr.103521 GOLDMANN, W., MARTIN, T., FOSTER, J., HUGHES, S., SMITH, G., HUGHES, K., DAWSON, M. & HUNTER, N. (1996) Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. Journal of General Virology 77, 2885-2891 GOLDMANN, W., RYAN, K., STEWART, P., PARNHAM, D., XICOHTENCATL, R., FERNANDEZ, N., SAUNDERS, G., WINDL, O., GONZALEZ, L., BOSSERS, A. & FOSTER, J. (2011) Caprine prion gene polymorphisms are associated with decreased scrapie susceptibility in goat herds in the United Kingdom. Veterinary Research 42, 110 GONZALEZ, L., MARTIN, S., SISO, S., KONOLD, T., ORTIZ-PELAEZ, A., PHELAN, L. & OTHERS (2009) High prevalence of scrapie in a dairy goat herd: tissue distribution of disease-associated PrP and effect of PRNP genotype and age. Veterinary Research 40, 65 HUSSAIN, A., BABAR, M. E., IMRAN, M., HAQ, I. U. & JAVED, M. M. (2011) Detection of four novel polymorphisms in PrP gene of Pakistani sheep (Damani and Hashtnagri) and goats (Kamori and local hairy) breeds. Virology Journal 8, 246 MAESTRALE, C., CANCEDDA, M. G., PINTUS, D., MASIA, M., NONNO, R., RU, G. & OTHERS (2015) Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate. Journal of virology 89,10044-52 PAPASAVVA-STYLIANOU, P., KLEANTHOUS, M., TOUMAZOS, P., MAVRIKIOU, P. & LOUCAIDES, P. (2007) Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie. Veterinary Journal 173, 459-462 PAPASAVVA-STYLIANOU, P., WINDL, O., SAUNDERS, G., MAVRIKIOU, P., TOUMAZOS, P. & KAKOYIANNIS, C. (2011) PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie. Veterinary Journal 187, 245-250 PRUSINER, S. B. (1982) Novel proteinaceous infectious particles cause scrapie. Science, 216, 136-144 SERRANO, C., HAMMOUCHI, M., BENOMAR, A., LYAHYAI, J., RANERA, B., ACIN, C. & OTHERS (2009) PRNP haplotype distribution in Moroccan goats. Animal Genetics 40, 565-568 VACCARI, G., DI BARI, M. A., MORELLI, L., NONNO, R., CHIAPPINI, B., ANTONUCCI G. & OTHERS (2006) Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. Journal of General Virology 87, 1395-1402 VACCARI, G., PANAGIOTIDIS, C. H., ACIN, C., PELETTO, S., BARILLET, F., ACUTIS P. L. & OTHERS (2009) State-of-the-art review of goat TSEs in the European Union, with special emphasis on PrP genetics. Veterinary Research 40, 48 WILESMITH, J. W., RYAN, J. B., HUESTON, W. D. & HOINVILLE, L. J. (1992) Bovine spongiform encephalopathy: epidemiological features 1985 to 1990. Veterinary Record doi:10.1136/vr.130.5.90
doi: 10.1136/vr.i702 February 13, 2016 | Veterinary Record | 167
Downloaded from http://veterinaryrecord.bmj.com/ on February 18, 2016 - Published by group.bmj.com
Controlling scrapie and bovine spongiform encephalopathy in goats Cristina Acín and José Luis Pitarch Veterinary Record 2016 178: 166-167
doi: 10.1136/vr.i702 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/178/7/166
These include:
References Email alerting service
This article cites 18 articles, 3 of which you can access for free at: http://veterinaryrecord.bmj.com/content/178/7/166#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
