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Frontiers in pharmacology2010; 1; 122; doi: 10.3389/fphar.2010.00122

Edaphic and Phytochemical Factors as Predictors of Equine Grass Sickness Cases in the UK.

Abstract: Equine dysautonomia or equine grass sickness (EGS), as it is more commonly known, is a usually fatal disease of equids of uncertain etiology, although associated with grazing, that affects the autonomic and enteric nervous system. Lowered gastrointestinal motility, leading to paralysis of the gut, is one of the main symptoms of EGS. Previous studies have implicated anaerobic bacteria, notably Clostridium botulinum, but what triggers the severe bacterial infestations remains enigmatic. We hypothesized that a detailed comparison of soil mineral and botanical composition of EGS and control sites would yield new insights into the causation of the disease. Results: Between March 2007 and September 2008, soil, plant, and water samples from a total of 23 EGS sites and 11 control sites were studied. Metal and non-metal element levels of the soil and herbage samples were assessed. Significantly, EGS sites had higher levels of soil nitrogen, and significantly higher levels of iron, lead, arsenic, and chromium in the herbage. Toxic Ranunculus spp. (buttercups) were found in abundance at every EGS site, making ingestion plausible. Conversely, neurotoxin-producing cyanobacteria were not found in any of the water samples analyzed. Conclusions: The significantly higher levels of iron and heavy metals found in herbage growing in EGS sites, in addition to toxic Ranunculus species, suggest that previously unknown triggers are involved in a multi-factorial EGS etiology. Our results also show that cyanobacteria on the other hand, are unlikely to be a factor in EGS. Consequently, the concomitant presence of two (or more) factors could be the trigger for an outbreak of EGS.
Publication Date: 2010-10-25 PubMed ID: 21833167PubMed Central: PMC3153002DOI: 10.3389/fphar.2010.00122Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research study investigates the relationship between soil minerals, the chemical compounds of plants, and Equine Grass Sickness (EGS), a grave disease affecting horses. Through comparing EGS impacted areas with uncontaminated locations, the study discovers that higher levels of specific heavy metals in plants and certain poisonous plant types could have a role in causing EGS.

Research Methodology

  • The study was conducted between March 2007 and September 2008, with soil, plant, and water samples collected from 23 EGS sites and 11 control sites.
  • The collected samples were studied for the presence and levels of multiple elements, both metal and non-metal.

Key Findings

  • Significantly, EGS sites had elevated levels of soil nitrogen and higher levels of iron, lead, arsenic, and chromium in plants on the site.
  • Ranunculus species, or buttercups, that are known to be toxic were found abundantly at each EGS site, implying that animals grazing at these sites could easily ingest them.
  • Conversely, no neurotoxin-producing cyanobacteria were found in any of the water samples taken from either EGS or control sites, thus dismissing the possibility of their role in causing EGS.

Conclusions

  • The results of the study point to the fact that higher levels of certain heavy metals in plants coupled with the presence of toxic Ranunculus species could serve as triggers for EGS, thus suggesting a complex multi-factorial cause for the disease.
  • The absence of cyanobacteria in all water samples suggests that they are unlikely to be a factor contributing to the onset of EGS.
  • It is proposed that the presence of two or more factors might be needed to trigger an episode of EGS, indicating that individual factors in isolation may not be sufficiently causative.

Cite This Article

APA
Edwards SE, Martz KE, Rogge A, Heinrich M. (2010). Edaphic and Phytochemical Factors as Predictors of Equine Grass Sickness Cases in the UK. Front Pharmacol, 1, 122. https://doi.org/10.3389/fphar.2010.00122

Publication

ISSN: 1663-9812
NlmUniqueID: 101548923
Country: Switzerland
Language: English
Volume: 1
Pages: 122

Researcher Affiliations

Edwards, Sarah E
  • Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London London, UK.
Martz, Kathrin E
    Rogge, Anja
      Heinrich, Michael

        References

        This article includes 86 references
        1. Aiello S. E.. The Merck Veterinary Manual. .
        2. Bai Y., Benn M. H., Majak W., McDiarmid R.. Extraction and HPLC determination of ranunculin in species of the buttercup family. J. Agric. Food Chem. 44, 2235–2238.
          doi: 10.1021/jf950626mgoogle scholar: lookup
        3. Benoni G., Cuzzolin L., Zambreri D., Donini M., Del Soldato P., Caramazza I.. Gastrointestinal effects of single and repeated doses of ferrous sulphate in rats. Pharmacol. Res. 27, 73–80.
          doi: 10.1006/phrs.1993.1007pubmed: 8456060google scholar: lookup
        4. Bidar G., Garçon G., Pruvot C., Dewaele D., Cazier F., Douay F., Shirali P.. Behaviour of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity. Environ. Pollut. 147, 546–553.
          doi: 10.1016/j.envpol.2006.10.013pubmed: 17141383google scholar: lookup
        5. Böhnel H., Wernery U., Gessler F.. Two cases of equine grass sickness with evidence for soil-borne origin involving botulinum neurotoxin. J. Vet. Med. B Infect. Dis. Vet. Public Health 50, 178–182.
        6. Bonora A., Tosi B., Donini A., Botta B., Bruni A.. Elicitor-induced accumulation of protoanemonin in Caltha palustris L.. J. Plant Physiol. 131, 489–494.
        7. Brej T.. Heavy metal tolerance in Agropyron repens (L.) P. Bauv. populations from the Legnica copper smelter area, Lower Silesia. Acta Soc. Bot. Pol. 67, 325–333.
        8. Bullen J. J., Rogers H. J., Spalding P. B., Ward C. G.. Iron and Infection: the heart of the matter. FEMS Immunol. Med. Microbiol. 43, 325–330.
          doi: 10.1016/j.femsim.2004.11.010pubmed: 15708305google scholar: lookup
        9. Carpenter D. O.. The public health significance of metal neurotoxicity. Cell. Mol. Biol. 14, 591–597.
          pmc: PMC11566966pubmed: 7641222
        10. Chorus I., Batram J.. Toxic Cyanobacteria in Water: a Guide to Their Public Health Consequences, Monitoring and Management. London, England: E&FN Spon (on behalf of WHO).
        11. Codd G. A., Morriso F., Metcalf J. S.. Cyanobacterial toxins: risk management for health protection. Toxicol. Appl. Pharmacol. 203, 264–272.
          doi: 10.1016/j.taap.2004.02.016pubmed: 15737680google scholar: lookup
        12. Collier D. S., Collier S. O., Rossdale P. D.. Grass sickness – the same old suspects but still no convictions!. Equine Vet. J. 35, 540–542.
          pubmed: 11720023
        13. Cooper M R, Johnson A. W.. Poisonous Plants in Britain and their Effects on Animals and Man. London, England: Her Majesty's Stationery Office.
        14. Cottrell D. F., McGorum B. C., Pearson G. T.. The neurology and enterology of equine grass sickness: a review of basic mechanisms. Neurogastroenterol. Motil. 11, 79–92.
        15. Craig A. M., Pearson E. G., Meyer C., Schmitz J. A.. Clinicopathologic studies of tansy ragwort toxicosis in ponies: sequential serum and histopathological changes. J. Equine Vet. Sci. 11, 261–262, 264–271.
        16. De Ponti F., D'Angelo L., Forster R., Einaudi A., Crema A.. Effect of iron succinyl-protein complexes on gastrointestinal motility in the fasting dog. Digestion 50, 72–81.
          doi: 10.1159/000200742pubmed: 1804735google scholar: lookup
        17. Dowler L. E., Siciliano P. D.. Prediction of hourly pasture dry matter intake in horses. J. Equine Vet. Sci. 29, 354–355.
        18. Duke J. A.. Handbook of Energy Crops. .
        19. EC Commission. Regulation No. 1881/2006. .
        20. Edouard N., Fleurance G., Martin-Rossel W., Duncan P., Dulphy J. P., Grange S., Baumont R., Dubroeucq H., Pérez-Barbería F. J., Gordon I. J.. Voluntary intake and digestibility in horses: effect of forage quality with emphasis on individual variability. Animal 2, 1526–1533.
          doi: 10.1017/S1751731108002760pubmed: 22443911google scholar: lookup
        21. Evans W. C.. Bracken thiaminase-mediated neurotoxic syndromes. Bot. J. Linn. Soc. 1, 113–131.
        22. Fischer W. J., Garthwaite I., Miles C. O., Ross K. M., Aggen J. B., Chamberlin A. R., Towers N. R., Dietrich D. R.. Congener-independent immunoassay for microcystins and nodularins. Environ. Sci. Technol. 35, 4849–4856.
          doi: 10.1021/es011182fpubmed: 11775161google scholar: lookup
        23. Forbes J. C., Watson R. D.. Plants in Agriculture. Cambridge: Cambridge University Press, 68–80.
        24. French N. P., McCarthy H. E., Diggle P. J., Proudman C. J.. Clustering of equine grass sickness cases in the United Kingdom: a study considering the effect of position-dependent reporting on the space-time K-function. Epidemiol. Infect. 133, 343–348.
          doi: 10.1017/S0950268804003322pmc: PMC2870255pubmed: 15816161google scholar: lookup
        25. Friebe A., Schulz M., Kück P., Schnabl H.. Phytotoxins from shoot extracts and root exudates of Agropyron repens seedlings. Phytochemistry 38, 1157–1159.
        26. Garrett L. A., Brown R., Poxton I. R.. A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness. Vet. Microbiol. 87, 81–88.
          doi: 10.1016/S0378-1135(02)00018-4pubmed: 12079749google scholar: lookup
        27. Giasson P., Jaouich A., Cayer P., Gagné S., Moutoglis P., Massicotte L.. Enhanced phytoremediation: a study of mycorrhizoremediation of heavy metal-contaminated soil. Remediation 17, 97–110.
          doi: 10.1002/rem.20115google scholar: lookup
        28. Gilmour J. S., Jolly G. M.. Some aspects of the epidemiology of equine grass sickness. Vet. Rec. 95, 77–80.
          doi: 10.1136/vr.95.4.77pubmed: 4439627google scholar: lookup
        29. Götz M. E., Künig G., Riederer P., Youdim M. B. H.. Oxidative stress: free radical production in neuronal degeneration. Pharmacol. Ther. 63, 37–12.
          doi: 10.1016/0163-7258(94)90055-8pubmed: 7972344google scholar: lookup
        30. Hallberg L., Brune M., Rossander L.. The role of vitamin C in iron absorption. Int. J. Vitam. Nutr. Res. Suppl. 30, 103–108.
          pubmed: 2507689
        31. Hilbe M., Guscetti S., Wunderlin S., Ehrensperger F.. Synaptophysin: an immunohistochemical marker for animal dysautonomias. J. Comp. Pathol. 132, 223–227.
          doi: 10.1016/j.jcpa.2004.07.002pubmed: 15737349google scholar: lookup
        32. Hsu P. C., Guo Y. L.. Antioxidant nutrients and lead toxicity. Toxicology 180, 33–44.
          doi: 10.1016/S0300-483X(02)00380-3pubmed: 12324198google scholar: lookup
        33. Hubbard C. E.. Grasses: A Guide to Their Structure, Identification, Uses and Distribution. London: Penguin Books Ltd.
        34. Hudson N., Mayhew I., Pearson G.. Presence of in vitro electrical activity in the ileum of horses with enteric nervous system pathology: equine dysautonomia (grass sickness). Auton. Neurosci. 99, 119–126.
          doi: 10.1016/S1566-0702(02)00065-6pubmed: 12241086google scholar: lookup
        35. Hudson N. P. H., Pirie R. S.. Four cases of equine grass sickness: acute, subacute, chronic and surviving chronic grass sickness. Equine Vet. Educ. 17, 19–26.
        36. Hunter L. C., Miller J. K., Poxton I. R.. The association of Clostridium botulinum type C with equine grass sickness: a toxicoinfection?. Equine Vet. J. 31, 492–499.
        37. Järup L.. Hazards of heavy metal contamination. Br. Med. Bull. 68, 167–182.
          doi: 10.1093/bmb/ldg032pubmed: 14757716google scholar: lookup
        38. John H. A., Marrs J., Laffling A. J.. Investigation of the susceptibility of equine autonomic neuronal cell lines, clonally derived from the same paravertebral ganglion, to toxic plasma from equine dysautonomia (grass sickness) cases. Toxicol. In Vitro 14, 459–465.
          doi: 10.1016/S0887-2333(00)00037-0pubmed: 10963962google scholar: lookup
        39. Liang J., Karamanos R. E.. “DTPA extraction for iron,” in Soil Sampling and Methods of Analysis. ed Carter M. R. (Boca Raton, FL: CRC Press; ), 87–90.
        40. Mares D.. Antimicrobial activity of protoanemonin, a lactone from ranunculaceous plants. Mycopathologia 98, 133–140.
          doi: 10.1007/BF00437648pubmed: 3587338google scholar: lookup
        41. Marrs J., Small J., Milne E. M., John H. A.. Liver and Biliary System Pathology in Equine Dysautonomia (Grass Sickness). J. Vet. Med. A Physiol. Pathol. Clin. Med. 48, 243–255.
        42. McCarthy H. E., French N. P., Edwards G. B., Miller K., Proudman C. J.. Why are certain premises at increased risk of equine grass sickness? A matched case–control study. Equine Vet. J. 36, 130–134.
          doi: 10.2746/0425164044868594pubmed: 15038435google scholar: lookup
        43. McCarthy H. E., Proudman C. J., French N. P.. Epidemiology of equine grass sickness: a literature review (1909–1999). Vet. Rec. 149, 293–300.
          doi: 10.1136/vr.149.10.293pubmed: 11570789google scholar: lookup
        44. McGorum B. C., Fry S. C., Wallace G., Coenen K., Robb J., Williamson G., Aruoma O. I.. Properties of herbage in relation to equine dysautonomia: biochemical composition and antioxidant and prooxidant actions. J. Agric. Food Chem. 48, 2346–2352.
          doi: 10.1021/jf991101npubmed: 10888548google scholar: lookup
        45. McGorum B. C., Wilson R., Pirie R. S., Mayhew I. G., Kaur H., Aruoma O. I.. Systemic concentrations of antioxidants and biomarkers of macromolecular oxidative damage in horses with grass sickness. Equine Vet. J. 35, 121–126.
          doi: 10.2746/042516403776114225pubmed: 12638786google scholar: lookup
        46. Mez K., Beattie K. A., Codd G. A., Hanselmann K., Hauser B., Naegeli H., Preisig H. R.. Identification of a microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland. Eur. J. Phycol. 32, 111–117.
        47. Milne E., McGorum B.. Grass Sickness in Horses. .
        48. Milne E. M., Fintl C., Hudson N. P. H., Pearson G. T., Mayhew I. G., Hahn C. N.. Observations on the interstitial cells of Cajal and neurons in a recovered case of equine dysautonomia (grass sickness). J. Comp. Pathol. 133, 33–40.
          doi: 10.1016/j.jcpa.2005.01.004pubmed: 15904924google scholar: lookup
        49. Ministry of Agriculture, Fisheries and Food (MAFF). The Analysis of Agricultural Materials, 3rd Edn Technical Bulletin RB427. London, UK: HMSO, 98–194.
        50. Murphy T., Lawson A., Nalewajko C., Murkin H., Ross L., Oguma K., McIntyre T.. Algal toxins-initiators of avian botulism?. Environ. Toxicol. 15, 558–567.
        51. Nasu T., Toda H., Shibata H.. Differential inhibitory mechanism of Fe2+ and Fe3+ on contraction of ileal longitudinal smooth muscle. Pharmacol. Res. 43, 95–102.
          doi: 10.1006/phrs.2000.0751pubmed: 11207072google scholar: lookup
        52. Nehru B., Anand P.. Oxidative damage following chronic aluminum exposure in adults and pup rat brains. J. Trace Elem. Med. Biol. 19, 203–208.
          pubmed: 16325537
        53. Newell C. A., Anderson L. A., Phillipson J. D.. Herbal Medicines. London, England: Pharmaceutical Press.
        54. Newton J. R., Hedderson E. J., Adams V. J., McGorum B. C., Proudman C. J., Wood J. L. N.. An epidemiological study of risk factors associated with the recurrence of equine grass sickness (dysautonomia) on previously affected premises. Equine Vet. J. 6, 105–112.
          pubmed: 15038431
        55. Nomura M., Uehara K., Harada K., Uemura E., Iga A., Kawano T., Nishikado A., Saito K., Nakaya Y., Ito S.. Impairment of gastrointestinal motility by nitrate administration: evaluation based on electrogastrographic changes and autonomic nerve activity. Aliment. Pharmacol. Ther. 20, 118–124.
        56. Nunn F. G., Pirie R. S., McGorum B., Werney U., Poxton I. R.. Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanized and surviving cases of chronic grass sickness. Res. Vet. Sci. 83, 82–84.
          doi: 10.1016/j.rvsc.2006.10.010pubmed: 17156804google scholar: lookup
        57. Ochoa R., de Valendia S.. Equine grass sickness: serologic evidence of association with Clostridium perfringens type A enterotoxin. Am. J. Vet. Res. 39, 1049–1051.
          pubmed: 208433
        58. Ochoa R., Gomez S., Bustos F., Trajos E.. Enterotoxaemia in the horse. Preliminary report on its reproduction by Clostridium perfringens toxin. Rev. Inst. Colomb. Agrop. 9, 15–49.
        59. Paesano R., Pietropaoli M., Gessani S., Valenti P.. The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia. Biochimie 91, 44–51.
          doi: 10.1016/j.biochi.2008.06.004pubmed: 18601971google scholar: lookup
        60. Pagotto C., Rémy N., Legret M., Le Cloirec P.. Heavy metal pollution of road dust and roadside soil near a major rural highway. Environ. Technol. 22, 307–319.
          doi: 10.1080/09593332208618280pubmed: 11346288google scholar: lookup
        61. Palacios H., Iribarren I., Olalla M. J., Cala V.. Lead poisoning of horses in the vicinity of a battery recycling plant. Sci. Total Environ. 290, 81–89.
          doi: 10.1016/S0048-9697(01)01066-Xpubmed: 12083718google scholar: lookup
        62. Panciera R. J., Martin T., Burrows G. E., Taylor D. S., Rice L. E.. Acute oxalate poisoning attributable to ingestion of curly dock (Rumex crispus) in sheep. J. Am. Vet. Med. Assoc. 196, 1981–1984.
          pubmed: 2365622
        63. Pérez Gutiérrez R. M., Vargas Solís R.. Smooth muscle relaxing properties of the hexanic extract of freshwater algae Oscillatoria limnetica and Hydrodictyon reticulatum. Bol. Latinoam. Caribe Planta Med. Aromat. 6, 30–35.
        64. Pirie R. S.. Grass sickness. Clin. Tech. Equine Pract. 5, 30–36.
        65. Pool W. A.. “Grass disease” in horses. Vet. Rec. 8, 23–30.
        66. Poxton I. R., Hunter L., Lough H., Miller K.. Is equine grass sickness (mal seco?) a form of botulism?. Anaerobe 5, 291–293.
          doi: 10.1006/anae.1999.0213google scholar: lookup
        67. Prieto J. M., Recio M. C., Giner R. M., Máñez S., Rios J. L.. Pharmacological approach to the pro-and anti-inflammatory effects of Ranunculus sceleratus L.. J. Ethnopharmacol. 89, 131–137.
          doi: 10.1016/S0378-8741(03)00271-Xpubmed: 14522444google scholar: lookup
        68. Ressler C.. Isolation and identification from common vetch of the neurotoxin β-cyano-l-anine, a possible factor in neurolathyrism. J. Biol. Chem. 237, 733–735.
          pubmed: 14491568
        69. Rocke T. E.. “The global importance of avian botulism,” in Waterbirds Around the World. eds Boere G. V., Galbraith C. A., Stroud D. A. (Edinburgh, UK: The Stationery Office; ), 422–426.
        70. Rose F.. The Wild Flower Key: How to Identify Wild Plants, Trees and Shrubs in Britain and Ireland (revised edition). London, UK: Penguin Books Ltd.
        71. Sobko T., Reinders C. I., Jansson E., Norin E., Midtvedt T., Lundberg J. O.. Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide 13, 272–278.
          doi: 10.1016/j.niox.2005.08.002pubmed: 16183308google scholar: lookup
        72. Spoerke D. G., Smolinske S. C.. Toxicity of Houseplants. Boston: CRC Press, 153.
        73. Stace C. A.. New Flora of the British Isles, 2nd Edn. Cambridge: Cambridge University Press, 1165.
        74. Stanek A., Gadowska-Cicha A., Gawron K., Wielkoszynski T., Adamek B., Cieslar G., Wiczkowski A., Sieron A.. Role of nitric oxide in physiology and pathology of the gastrointestinal tract. Mini Rev. Med. Chem. 8, 1549–1560.
          doi: 10.2174/138955708786786462pubmed: 19075811google scholar: lookup
        75. Thagard P.. How Scientists Explain Disease. Princeton, NJ: Princeton University Press, 101–117.
        76. Tocher J. F.. Grass sickness in horses. Trans. R. Highl. Agric. Soc. Scotl. 36, 65–83.
        77. Tocher J. F., Brown W., Tocher J. W., Buxton J. B.. “Grass sickness” investigation report. Vet. Rec. 3, 37–45, 75–89.
        78. Uzal F. A., Robles C. A.. “Clinical signs of mal seco, a grass sickness-like syndrome of horses,” in Proceedings of the 1st International Workshop on Grass Sickness, EMND and Related Disorders, Bern, Switzerland, 7–8. .
        79. Uzal F. A., Robles C. A., Olaechea F. V.. Histopathological changes in the celiacomesenteric ganglia of horses with ‘mal seco’, a grass sickness-like syndrome, in Argentina. Vet. Rec. 130, 244–246.
          doi: 10.1136/vr.130.12.244pubmed: 1285752google scholar: lookup
        80. Valko M., Morris H., Cronin M. T. D.. Metals, toxicity and oxidative Stress. Curr. Med. Chem. 12, 1161–1208.
          doi: 10.2174/0929867053764635pubmed: 15892631google scholar: lookup
        81. Walker E. M. Jr., Walker S. M.. Effects of iron overload on the immune system. Ann. Clin. Lab. Sci. 30, 354–365.
          pubmed: 11045759
        82. Weinberg E. D.. Iron loading and disease surveillance. Emerg. Infect. Dis. 5, 346–352.
          doi: 10.3201/eid0503.990305pmc: PMC2640766pubmed: 10341171google scholar: lookup
        83. Weinberg E. D.. Iron, infection and sudden infant death. Med. Hypotheses 56, 731–734.
          doi: 10.1054/mehy.2001.1328pubmed: 11399125google scholar: lookup
        84. Wood J. L. N., McGorum B. C.. Equine dysautonomia: has grass been blamed unfairly all this time?. Equine Vet. J. 31, 451–452.
        85. Wood J. L. N., Milne E. M., Doxey D. L.. A case–control study of grass sickness (equine dysautonomia) in the United Kingdom. Vet. J. 156, 7–14.
          doi: 10.1016/S1090-0233(98)80055-5pubmed: 9691846google scholar: lookup
        86. Wylie C. E., Proudman C. J.. Equine grass sickness: epidemiology, diagnosis, and global distribution. Vet. Clin. North Am. Equine Pract. 25, 381–399.
          doi: 10.1016/j.cveq.2009.04.006pubmed: 19580947google scholar: lookup

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        1. Harte T, Smith D, Moore J, Wells B. Review of published research on primary dysautonomia of domestic animals. Vet Rec 2026 Jan 3;198(1):e30-e40.
          doi: 10.1002/vetr.5499pubmed: 40482055google scholar: lookup
        2. McGorum BC, Pirie RS, Glendinning L, McLachlan G, Metcalf JS, Banack SA, Cox PA, Codd GA. Grazing livestock are exposed to terrestrial cyanobacteria. Vet Res 2015 Feb 25;46:16.
          doi: 10.1186/s13567-015-0143-xpubmed: 25828258google scholar: lookup