Detection of Specific Antibodies to Clostridium Botulinum Type C in the Colostrum and Milk of Mares

Leonie C Hunter, Department of Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh.

Background

There is some evidence to suggest that an immune response occurs to the aetiological (causative) agent of grass sickness. Epidemiological studies have demonstrated that there is a lower incidence of the disease in older horses and horses that have been on a particular pasture for longer (Doxey et al, 1991; Gilmour and Jolly, 1974; Wood et al, 1998). Prior contact with a case of grass sickness is associated with a ten-fold decrease in likelihood of disease (Wood et al, 1998). This resistance that is seen to develop, may be in the form of an immune response. It has also been observed, that whilst there is a higher incidence of the disease in young horses, grass sickness is extremely rare in suckling foals. Although this could be due to differences in dietary or physiological factors, it could also be due to the presence of protective maternal antibodies against the aetiological agent of the disease.

Foals receive passive protection from antibodies in the mare’s colostrum. Unlike humans, there is no significant transplacental transfer of antibodies in the horse. Colostrum is formed by selective concentration of serum antibodies in the mammary gland. Immunoglobulin G (IgG) is the major antibody component of colostrum. The immunoglobulins in colostrum are absorbed into the foal’s circulation, immediately after birth through specialised intestinal cells. Therefore once the foal starts suckling it receives systemic passive protection from its mother. The specialised intestinal epithelial cells are rapidly replaced by mature cells, so that immunoglobulins cannot be absorbed after 24-36 hours post-birth.

The composition of the mare’s mammary gland secretions also changes at this time from colostrum to milk. Milk is produced locally in the mammary gland and the major antibody component is now IgA. Although the antibodies in milk cannot be absorbed into the foal’s circulation, they can provide the foal with local protection in the gastrointestinal tract.

We are currently investigating the hypothesis that grass sickness is caused by a toxicoinfection with Clostridium botulinum type C, where the organism grows and produces toxin in the gastrointestinal tract. C. botulinum type C can produce three toxins – C1, C2 and C3. The C1 toxin is neurotoxic, and this is the one we have looked for to date. We have detected the C1 toxin in the ileum and/or faeces by direct detection and/or after enrichment in 74% of acute grass sickness cases, 67% of subacute and chronic cases, compared to 10% of controls (Hunter et al, 1999; Hunter, 1999).

The study

The aim of this study was to look for the presence of specific antibodies to C. botulinum type C in the colostrum and milk of mares. We looked for antibodies to both the organism, using extracted surface antigens, and to the C1 toxin, by ELISA (enzyme-linked immunoabsorbent assay). Colostrum was collected up to 12 hours after birth, and milk was collected at 2 and 4 weeks after birth, and then at 4-weekly intervals until weaning. Samples were collected from 36 mares, 20 of which had been in contact with grass sickness, 2 had recovered from chronic grass sickness and 14 were not known to have been in prior contact with the disease. “In contact” was defined as either co-grazing with a horse that developed grass sickness, or grazing pasture where grass sickness had occurred previously.

Results

IgG antibody to both C. botulinum type C (surface antigens) and the C1 toxin were detected in the colostrum of all 36 mares (figure 1). IgA antibody was detected to C. botulinum type C and the C1 toxin in 58% of the colostrum samples (figure 1). With two exceptions, all the colostrum samples that were negative for IgA to the organism were also negative for IgA to the toxin. A positive correlation was observed between antibody levels to the organism and to the toxin, suggesting that the mares had been exposed to both C. botulinum type C and the C1 toxin.

IgG antibody was detected to C. botulinum type C in the milk samples of all 36 mares, and to the C1 toxin in 92% of the milk samples (figure 1). IgA antibody was detected to C. botulinum type C in 53% of the milk samples and to the C1 toxin in 39% of the milk samples (figure 1). The 17 milk samples that did not have detectable IgA to C. botulinum type C, also did not have IgA to the C1 toxin, and 15 of these samples were from mares that also did not have IgA to these antigens in the colostrum.

There were no significant differences in the antibody levels detected in the colostrum or milk between the mares that had been in contact with grass sickness and those that had not been in contact with the disease.

The antibody levels to the specific antigens were investigated over the course of the suckling period. There was a drop in specific antibody levels from the colostrum sample to the first milk sample collected at two weeks. This follows the same pattern as the total immunoglobulin levels, as milk has a lower total immunoglobulin content compared to colostrum. The specific antibody levels remained more or less constant in the milk samples from two weeks until weaning, as did the total immunoglobulin levels.

Discussion

IgG to both C. botulinum type C and the C1 toxin was detected in the colostrum of all 36 mares involved in this study, irrespective of whether the mares had previously been in contact with EGS. The presence of these specific antibodies in the colostrum, suggests that foals do receive systemic protection against the proposed aetiological agent of grass sickness. However, we do not know that the levels of antibodies detected are actually protective levels.

The detection of IgA to these antigens in the milk of some but not all mares demonstrated that some foals also received local passive protection from this organism and its toxin. The presence of IgA in the milk of some of the mares may be evidence of the recent exposure of these horses to C. botulinum type C. The levels of antibodies in the milk remained fairly constant until weaning, suggesting local passive protection against C. botulinum type C continues throughout the suckling period.

There were no significant differences in the specific antibody levels between horses that had previously been in contact with grass sickness and those not known to have been in contact with the disease. However, the majority of mares in the contact group were grazing land where grass sickness had not occurred for many years. We do not know whether there is a different distribution of C. botulinum type C between fields where grass sickness has occurred or not, it may be that the majority of horses are exposed to this organism, as these results suggest.

In summary, we have demonstrated the presence of specific antibodies to C. botulinum type C in the colostrum and milk of mares. These maternal derived antibodies may play a role in the protection of foals at both the systemic and local level from the proposed aetiological agent of grass sickness. Acknowledgements

I would like to thank both the many horse owners who provided samples from their mares for this study, and all those who offered their help. Your efforts were greatly appreciated. I would also like to thank Joyce McIntosh at the Equine Grass Sickness Fund for assistance in setting up this study, and the ‘Scottish Equestrian’ magazine for printing my article requesting volunteers! References

Doxey DL, Gilmour JS and Milne EM (1991). A comparative study of normal equine populations and those with grass sickness (dysautonomia) in eastern Scotland. Equine veterinary Journal, 23, 365-369.

Gilmour JS and Jolly GM (1974). Some aspects of the epidemiology of equine grass sickness. Veterinary Record, 95, 77-81.

Hunter LC, Miller JK, and Poxton IR (1999). The association of Clostridium botulinum type C with equine grass sickness: a toxicoinfection? Equine veterinary Journal, 31, 492-499.

Hunter LC (1999). The association of Clostridium botulinum type C and equine grass sickness. Equine News, Summer 1999.

Wood JLN, Milne EM, and Doxey DL (1998). A case-control study of grass sickness (equine dysautonomia) in the United Kingdom. Veterinary Journal, 156, 7-14.

milk_leonie_hunter

Figure 1: Percentage of mares with detectable IgG and IgA to C. botulinum type C surface antigens and to the C1 toxin in the colostrum and milk.