Oxibendazole

CASE STUDY: Effects of Non-Specific Immunostimulation of Prepartum Mares on Colostral Quality and Foal Immune Function1

Abstract

Only a few reports exist in the literature concerning the types of leukocytes present in the colostrum and milk of mares. Recent studies in other farm animals suggest a role for colostral leukocytes in the transfer of immunity from dam to neonate. This report investigated the influence of EqStim(R) (Propionobacterium aches; Immunovet, Tampa, FL) and levamisole, two immunomodulators, on colostral quality of mares and immune function of their foals in the immediate postpartum period. Although no enhancement in colostral quality or the immune function of foals by either immunomodulator was detected, no negative side effects to either treatment were observed. In addition, we believe this to be the first report to characterize the population of leukocytes present in colostrum obtained from

clinically healthy mares. The mean percentage (+/- SE) of neutrophils, macrophages, epithelial cells, and lymphocytes was 13.2 (+/- 4.99), 22.4 (+/- 8.57), 1.3 (+/- 0.52), and 63.2 (+/- 9.23), respectively. This profile of cell types present in the colostrum of the mare provides a reference point for future studies investigating the role of colostral leukocytes in the transfer of immunity from mare to foal.

(Key Words: EqStim(R), Propionobacterium aches, Immunity, Levamisole, Mare Colostrum.)

Introduction

There are significant numbers of leukocytes present in colostrum and, to a lesser extent, in milk of various farm animal species. In sheep, swine, and cattle, these leukocytes are functional and show increased expression of cell surface markers indicative of activation. Furthermore, these cells are absorbed intact by the neonatal gut. It has been suggested that colostral lymphocytes preferentially migrate to the lymph nodes and mucosal surfaces where they may contribute to the immune defenses of the neonate (Le Jan, 1996). To our knowledge, there are no reports that characterize the leukocyte population in the colostrum of healthy mares. The immunostimulant EqStim(R) (Propionobacterium acnes; Immunovet, Tampa, FL) has been shown to nonspecifically activate cells of the immune system and enhance disease resistance. Flaminio et al. (1998) reported an increase in peripheral cluster of differentiation (CD) 4+ Tcells within 7 d after EqStim(R) administration. Another study reported that administration of levamisole to unvaccinated mares 4 to 6 wk prior to foaling enhanced colostral concentrations of immunoglobulin G (IgG) and immune function of foals (Krakowski et al., 1999). The objective of Experiment 1 was to determine the effects of EqStim(R) administration to prepartum mares on somatic cell count (SCC) in colostrum and milk and the peripheral leukocyte population of foals at birth and 7 d of age. The objective of Experiment 2 was to determine whether the administration of levamisole at the time of vaccination of prepartum mares altered colostral quality as determined by concentration of IgG and leukocyte profile.

Materials and Methods

Experiment 1. Eight Quarter Horse mares were assigned randomly to either the control (n = 3) or EqStim(R) (n = 5) treatment. EqStimg(R)-- treated mares were injected intravenously with 5 mL of EqStim(R) on d 10, 8, and 6 prior to their expected foaling date; controls received a similar volume of saline. A 30-mL sample of colostrum and a 6-mL sample of the foal's blood were collected immediately after foaling. At 7 d after foaling, another 30-mL sample of milk and 6-mL sample of the foal's blood were collected. Colostrum and milk samples were submitted to the Kansas Dairy Herd Improvement Association laboratory (Manhattan, KS) for determination of fat, protein, lactose, non-fat solids, and SCC. Peripheral blood leukocytes were counted and isolated from the foal's blood, and cell phenotype was determined using monoclonal antibodies (VMRD, Pullman, WA) against CD4 (#HB61A), CD8 (#HT14A), B cell antigen (#B29A), granulocyte-monocyte (GM1; #DH59B), and major histocompatibility complex-2 (MHC-2; #H42A) antigen as previously described (Flaminio et al., 1998). Cell phenotype was determined using a FACScan(TM) flow cytometer (Becton Dickinson, San Jose, CA), and cell phenotype was recorded as the percentage of cells staining positive for a given marker. All data were analyzed with the PROC MIXED procedure of SAS (SAS System for Windows, Release 8.02, SAS Institute, Inc., Cary, NC) as a repeated measures design. The model included fixed effects for treatment and time and their interaction; mare was considered a random effect. Unless otherwise indicated, comparisons between treatments and/or time were made only when a significant F test (P

Experiment 2. Eleven Quarter Horse mares were assigned randomly to either the control (n = 5) or levamisole (n = 6) treatment. At 30 d prior to their expected foaling date, all mares were vaccinated for equine influenza, equine herpes virus (EHV)-- 1a, EHV-1b, EHV-4, tetanus, and equine encephalomyelitis (Fort Dodge Animal Health, Fort Dodge, IA). At this time, controls were dewormed with oxibendazole (Anthelcide(R) EQ; Pfizer Animal Health, Exton, PA) at 10 mg/kg of BW. Alternatively, mares treated with levamisole received 7.5 mg/kg of BW of levamisole-hydrochloride (Levasole(R); Schering-Plough Animal Health, Union, NJ) in 30 mL of corn syrup. Immediately after foaling, a 50-mL sample of colostrum was collected. A 30-mL aliquot was submitted to the Kansas Dairy Herd Improvement Association for determination of fat, protein, and SCC. A 10-mL aliquot was frozen until assayed for IgG content using a commercially available single radial immunodiffusion kit (#444-30; VMRD). Another 3-mL aliquot was diluted 1:5 with PBS, and 10 mL of this suspension was prepared (Hema 3(R) Stain Set; Fisher Scientific, Pittsburgh, PA) for differential counting of the cells present in the colostrum. All data were analyzed with the PROC MIXED procedure of SAS. The model included the fixed effect of treatment and age of the mare. Initial data analysis showed that age had no significant effect or interaction with treatment; therefore, the term for mare age was removed from the final model. Mare was considered a random effect.

Results and Discussion

Experiment 1. There was no detectable effect of treatment on the percentage of fat, CP, lactose, or nonfat solids in mammary secretions on d 0 or 7 (data not shown). The percentages of CP (NO.001), non-fat solids (P

Treatment with EqStim(R) did not influence SCC or total WBC count of foals on d 0 or 7 (Figure 2). There was no effect of treatment or time (Figure 3) on the percentage of cells staining positive for CD4, CDB, GM-1, or MHC-2. In contrast, the percentage of cells expressing the B-cell phenotype declined (P

Although the manufacturer's suggested dose of 1 mL of EqStim(R)/ 113 kg of BW corresponds to the 5-- mL dose given to the mares in this study, in retrospect, a larger dose may be required to stimulate an immune response in prepartum mares. Blood samples were collected from one mare prior to the first EqStim(R) treatment and 2 d after the second treatment. No changes in total WBC count or in the percentage of cells staining positive for the CD4, CDB, B cell,

GM-1, or MHC-2 markers were observed. Whereas Flaminio et al. (1998) reported elevated rectal temperatures in foals after the second treatment with EqStim(R), rectal temperatures of mares receiving EqStim(R) in the present study were consistently in the range of 37.5 to 38.9 deg C and in some cases actually declined following treatment with EqStim(R). Again, this result suggests that the dose of EqStim(R) administered in the current study might have been too low to alter the number or proportions of peripheral blood leukocytes in healthy, prepartum mares.

If, in fact, there was no immunostimulatory effect of EqStim(R) on treated mares, it is not surprising that there were no differences in the response criteria of foals from control or treated mares. However, temporal changes in some of the response criteria of foals were observed. The increase in total WBC count from d 0 to 7 agrees with previously reported values (Chavatte and Rossdale, 1991). There was no influence of treatment or time on the percentages of CD4+, CD8+, GM-1+, or MHC-2+ leukocytes, but the small decline in the percentage of cells staining positive for the B cell antigen suggests there may be some modulation of the developing immune system of the foals in the early neonatal period. Little information is available concerning the leukocyte population of clinically healthy newborn foals, suggesting that further studies in this area are needed. Experiment 2. No effects of treatment with levamisole on the percentages of fat (P=0.49) and CP (P=0.92) in the colostrum of mares were detected (data not shown). In addition, administration of levamisole did not influence the SCC (P=0.33) or IgG concentration (P=0.85) in the colostrum of mares (Figure 4). Similarly, there was no apparent effect of levamisole on the percentages of neutrophils (P=0.25), macrophages (P=0.22), epithelial cells (P=0.66), or lymphocytes (P=0.65) in the colostrum of mares (Figure 5).

The concentration of IgG in the colostrum of mares in this study agrees with the range of values reported by others (Kohn et al., 1989;

Lavoie et al., 1989). A beneficial effect of levamisole administration on the concentration of IgG in the colostrum of mares was not observed; however, others have reported positive effects of levamisole (Krakowski et al., 1999). This may be due to differences in experimental procedures. In the present study, a single dose of levamisole was orally administered at the time of vaccination, 30 d prior to expected foaling. Krakowski et al. (1999) gave three subcutaneous injections of levamisole at weekly intervals during the last 4 to 6 wk prior to foaling to mares that had not been vaccinated previously. Those researchers suggested that levamisole may stimulate the immune system of unvaccinated prepartum mares and subsequently their foals. However, the present findings indicate that levamisole did not enhance colostral concentrations of IgG in mares vaccinated 30 d prior to foaling.

We believe this to be the first report to characterize the somatic cell population present in the colostrum of healthy mares, although there are a wide range of reference values for the population of somatic cells present in the colostrum of humans and other farm animal species (Butler, 1999). When the cell population data were pooled across treatment, the mean percentages (and standard error) of neutrophils, macrophages, epithelial cells, and lymphocytes were 13.2 (+/- 4.99), 22.4 (+/- 8.57), 1.3 (+/- 0.52), and 63.2 (+/- 9.23), respectively. Although the percentages of macrophages and epithelial cells correspond very closely to values reported for the bovine, the percentage of neutrophils is quite low, and the percentage of lymphocytes is much greater. Most of the studies investigating cell types present in the colostrum or milk of mares have focused on the detection of mastitis (Freeman, 1993). Consequently, there is no reason to doubt the accuracy of the values reported herein. However, given the small number of mares sampled in this study, these results provide a reference point for further studies in this area. Further study using a greater number of mares is justified to corroborate the findings of this experiment.

Implications

Neither EqStim(R) nor levamisole, as administered to prepartum mares in this study, significantly influenced colostral quality or the immune function of foals in the immediate postpartum period. Also, no adverse side effects in response to treatment administration were observed. However, future study investigating the optimum dose or optimum time for administration as well as the potential benefits of these immunostimulants is warranted. Although there is ample evidence in the literature describing the role of colostral immunoglobulins in the passive transfer of immunity from dam to offspring, relatively little is known about the function of colostral leukocytes ingested by neonatal farm animals. Although this study provides a profile of the types of cells present in the colostrum of the mare, future studies should determine how these cells may modulate development of cell-mediated immunity and disease resistance in foals.

1 Contribution 02-460-1 from the Kansas Agricultural Experiment Station.

Literature Cited

Butler, J. E. 1999. Immunoglobulins and immunocytes in animal milks. In Mucosal Immunology. (2nd Ed.). P. L. Ogra, J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock, and J. R. McGhee (Eds.). p 1535. Academic Press, San Diego, CA.

Chavatte, P, and P. D. Rossdale. 1991. Red and white blood cell indices as an aid to the diagnosis of diseases of the newborn foal. Equine Vet. Educ. 3:28.

Flaminio, M.J.B.F., B. R. Rush, and W. Shuman. 1998. Immunologic function in horses after non-specific immunostimulant

administration. Vet. Immunol. Immunopathol. 63:303.

Freeman, K. P. 1993. Cytological evaluation of the equine mammary gland. Equine Vet. Educ. 5:212.

Gibbs, P. G., G. D. Potter, R. W. Blake, and W. C. McMullan. 1982. Milk production of Quarter Horse mares during 150 days of lactation. J. Anim. Sci. 54:496.

Hoffman, R. M., D. S. Kronfeld, J. H. Herbein, W. S. Swecker, W. L. Cooper, and P. A. Harris. 1998. Dietary carbohydrates and fat influence

milk composition and fatty acid profile of mare's milk. J. Nutr. 128:2708S.

Kohn, C. W., D. Knight, W. Hueston, R. Jacobs, and S. M. Reed. 1989. Colostral and serum IgG, IgA, and IgM concentrations in Standardbred mares and their foals at parturition. J. AVMA 195:64.

Krakowski, L., J. Krzyzanowski, Z. Wrona, and A. K. Siwicki. 1999. The effect of nonspecific immunostimulation of pregnant mares with 1,3/1,6 glucan and levamisole on the

immunoglobulin levels in colostrum, selected indices of nonspecific cellular and humoral

immunity in foals in neonatal and postnatal period. Vet. Immunol. Immunopathol. 68:1. Lavoie, J. P., M. S. Spensley, B. P Smith, and Mihalyi. 1989. Colostral volume and

immunoglobulin G and M determinations in mares. Am. J. Vet. Res. 50:466.

Le Jan, C. 1996. Cellular components of mammary secretions and neonatal immunity: A review. Vet. Res. 27:403.

Ullrey, D. E., R. D. Struthers, D. G. Hendricks, and B. E. Brent. 1966. Composition of mare's milk. J. Anim. Sci. 25:217.

J. L. TURNER2, PAS, M. J. ARNS3, PAS, AND J. E. MINTON

Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201

2Present address: Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003-8003. To whom correspondence should be sent: jlturner@nmsu.edu

3Present address: Department of Animal Sciences, University of Arizona, Tucson, AZ 85721-0038.

Copyright American Registry of Professional Animal Scientists Feb 2003
Provided by ProQuest Information and Learning Company. All rights Reserved