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Oral and parenteral vaccination of mice with protein-ergotamine conjugates and evaluation of protection against fescue toxicosis
Veterinary Immunology and Immunopathology 61 Ž1998. 305–316
Oral and parenteral vaccination of mice with protein-ergotamine conjugates and evaluation of protection against fescue toxicosis R.L. Rice a
c
a,1,)
, D.J. Blodgett a , G.G. Schurig a , W.S. Swecker b, C.D. Thatcher b, D.E. Eversole c
Departments of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA b Large Animal Clinical Sciences, Virginia– Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Accepted 3 November 1997
Abstract Acremonium coenophialum produces ergopeptide alkaloids in tall fescue Ž Festuca arundinacea Schreb... These ergot alkaloids decrease serum alkaline phosphatase ŽALP. activity, serum cholesterol and prolactin concentrations, as well as average daily gains ŽADG. in cattle. The objective of this study was to evaluate the protection of anti-ergotamine antibodies induced by either oral or parenteral vaccination with protein-ergotamine conjugates or passive vaccination with anti-ergovaline, monoclonal antibodies in a murine model of fescue toxicosis. Ergotamine ŽEG. was conjugated to bovine serum albumin ŽBSA. and cholera toxin subunit B ŽCTB. by the Mannich reaction. Mice were blocked based on weight and randomly allocated into five groups of 10 mice each. Treatment groups were as follows: Ž1. group vaccinated intraperitoneally Žip. with a BSA–EG conjugate and fed an endophyte-infected ŽEI. fescue diet ŽBSA–EG group.; Ž2. group orally vaccinated with a CTB–EG conjugate mixed with free cholera toxin ŽCT. and fed an EI fescue diet ŽCTB–EG group.; Ž3. nonvaccinated group fed an EI fescue diet ŽEI group.; Ž4. group passively vaccinated with anti-ergovaline, monoclonal antibodies and fed an EI fescue diet ŽMoAB group.; and Ž5. nonvaccinated group fed an endophyte-free ŽEF. fescue diet ŽEF group.. The EI diet contained 1.5 ppm of Ergovaline ŽEV., whereas no EV was detected in the EF diet.
) Corresponding author. 1210 Crown Dr., Mansfield, TX 76063, USA. Tel.: q1 817 551 8706; fax: q1 817 551 4584; e-mail: [email protected]. 1 Present address: Alcon Laboratories, 6201 South Freeway, Fort Worth, TX 76134, USA.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 5 6 - 6
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Respective diets were similar upon nutritional analysis. Unvaccinated mice in the EI group exhibited features of fescue toxicosis as indicated by decreased serum ALP activity and cholesterol, and decreased weight gain as compared to mice in the EF group. Antibodies against EG and EV were present in sera of mice in the BSA–EG and MoAB groups, respectively. Mice orally vaccinated with the CTB–EG conjugate developed secretory IgA ŽsIgA. antibodies and short-lived, systemic IgG responses against EG. Weight gains were increased in the BSA–EG and CTB–EG groups and tended to be increased in the MoAB group vs. the unvaccinated EI group. Serum ALP activity was decreased in the BSA–EG and MoAB groups as compared to the EF group. Serum ALP activity was further decreased in the BSA–EG vaccinated group as compared to the EI group. Cholesterol concentrations were decreased in the EI, BSA–EG and MoAB groups as compared to the EF group. Prolactin concentrations were similar in all groups. q 1998 Elsevier Science B.V. Keywords: Fescue toxicosis; Vaccine; Mice; Ergotamine; Prolactin
1. Introduction Tall fescue Ž Festuca arundinacea Schreb.. is often infected by the endophytic fungus, Acremonium coenophialum ŽMorgan-Jones and Gams, 1982.. The fungus produces ergopeptide alkaloids, especially ergovaline ŽEV.. Serum cholesterol concentrations and alkaline phosphatase ŽALP. activity are decreased in cattle grazing endophyte-infected ŽEI. fescue for long periods of time ŽLipham et al., 1989; Bond et al., 1984; Stuedemann et al., 1985.. Consumption of EI fescue forage by cattle also decreases serum prolactin ŽHurley et al., 1981; Thompson et al., 1987., and average daily gains ŽADG. ŽHemken et al., 1984; Stuedemann and Hoveland, 1988., which results in economic losses for producers ŽHoveland, 1990.. An economically sound solution to the fescue problem has not been forthcoming. Preventative methods would be preferable to new therapeutic drugs, which may cause residue problems. Development of a vaccine to protect cattle against fescue toxicosis may save producers millions of dollars and avoid potential environmental damage associated with pasture renovation, while allowing their cattle to benefit from the nutritional value of EI fescue forage. The purpose of this study was to evaluate the protection of specific antibodies induced by either parenteral vaccination with a bovine serum albumin–ergotamine ŽBSA–EG. conjugate, oral vaccination with a cholera toxin subunit B ŽCTB. –EG conjugate, or passive vaccination with anti-EV, monoclonal antibodies in mice against fescue toxicosis. Weight gains, serum ALP activity, cholesterol and prolactin concentrations were the indices used to evaluate the ability of polyclonal, anti-EG and monoclonal, anti-EV antibodies to confer protection in the mouse model of fescue toxicosis.
2. Materials and methods 2.1. Preparation of CTB–EG and BSA–EG conjugates by the Mannich reaction Ergotamine Žtartrate salt: Sigma Chemical Co., St. Louis, MO. was conjugated to CTB or BSA ŽSigma Chemical. for vaccination. Proteins were linked to EG using the
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Mannich reaction by modification of the method previously described ŽKelley and Shelby, 1990.. Conjugates were evaluated on 250 m m thin-layer chromatographic ŽTLC. plates of silica gel developed in chloroformrmethanol Ž9:1, vrv. solvent system and observed under a long wave lamp. Protein content of each protein-EG conjugate was determined by bicinchoninic acid assay ŽBCA; BCAe Protein Assay; Pierce, Rockford, IL.. 2.2. Experimental design Fifty BALBrc, male mice Ž22.0 " 0.34 g; average BW " SEM., 6 weeks of age, were blocked by weight and randomly allocated from outcome groups into five groups of 10 mice each. Treatment groups were as follows: Ž1. group vaccinated intraperitoneally Žip. with a BSA–EG conjugate and fed an EI fescue diet ŽBSA–EG group.; Ž2. group orally vaccinated with a CTB–EG conjugate and fed an EI fescue diet ŽCTB–EG group.; Ž3. nonvaccinated group fed an EI fescue diet ŽEI group.; Ž4. group passively vaccinated with anti-EV, monoclonal antibodies and fed an EI fescue diet ŽMoAB group.; and Ž5. nonvaccinated group fed an endophyte-free ŽEF. fescue diet ŽEF group.. Mice were individually housed in shoebox cages of clear polystyrene and exposed to a 14:10 h light:dark cycle. Mice were acclimatized to ground rodent chow for 1 week prior to day 1 of the dietary trial. All procedures involving the use of animals were reviewed and approved by Virginia Polytechnic Institute and State University’s Animal Care and Use Committee. 2.3. Diet preparation Concentrations of EV were determined in an identical genotype of EI and EF ‘Kentucky 31’ tall fescue seed ŽInternational Seed, Halsey, OR. by HPLC analysis as described ŽHill et al., 1993.. Ground rodent chow ŽHarlan Teklad, Madison, WI. was mixed with equal parts of ground EI and EF fescue seed by weight. The seed:chow diets were analyzed for nutrient content ŽNorth East DHIA, Ithaca, NY.. Dietary intake was limited to 5 to 6 g per mouse daily. Water was provided ad libitum. 2.4. Vaccination of mice Mice in the BSA–EG group were injected ip with 250 m g of a BSA–EG conjugate in Freund’s complete adjuvant and revaccinated 14 days later with 100 m g of the BSA–EG conjugate in Freund’s incomplete adjuvant. Anti-EG, IgG titers were determined on days 14 and 34 post-vaccination by ELISA. Mice in the CTB–EG group were orally vaccinated twice with 15 m g of a CTB–EG conjugate mixed with 5 m g of cholera toxin ŽCT. at a 10 day interval. Microgram amounts of uncoupled CT were added to the CTB–EG conjugate prior to oral dosing because pure CTB is less effective as an adjuvant compared to CT ŽWilson et al., 1993. and also because the Mannich reaction decreased the ability of CTB present in the CTB–EG conjugate to bind the GM 1 receptor. Anti-EG Žmucosal secretory IgA ŽsIgA. and systemic IgG. and anti-EV ŽIgG. antibody levels were determined at the end of the acclimation period prior to the start of
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the dietary trial period on day 0. All vaccinations and revaccinations with the BSA–EG or CTB–EG conjugates were performed before mice were exposed to fescue diets. For passive vaccination of mice in the MoAB group, anti-EV, monoclonal IgG1 antibodies were isolated from hybridoma culture medium by affinity chromatography Žanti-mouse, IgG agarose column; Sigma Immunochemicals, St. Louis, MO.. The IgG concentration per ml of supernatant was determined by BCAe analysis ŽPierce. and the activity of purified monoclonal antibodies was determined by ELISA ŽShelby and Kelley, 1991.. Mice in the MoAB group were injected ip with 200 m g of the affinity-purified, monoclonal IgG antibodies specific for EV 1 day before initiation of the treatment diets. On day 7 of the trial, the mice in the MoAB group were injected ip with an additional 500 m g of the same monoclonal antibodies. After 13 days on trial, mice were anesthetized with halothane and blood was collected from the retro-orbital sinus. Sera obtained were analyzed for ALP, prolactin and cholesterol concentrations. Activity of serum ALP and cholesterol concentrations were determined by the EKTACHEM 700rVitros System ŽJohnson and Johnson, Rochester, NY.. Mice were weighed on day 0 and day 12 of the dietary trial period to evaluate weight gains. 2.5. Determination of anti-EG sIgA coproantibodies in mice orally Õaccinated with a CTB–EG conjugate by indirect ELISA Fecal pellets were collected from individual mice after oral vaccination every 3 to 5 days for 20 days and frozen immediately at y708C until analysis. Fecal samples were re-suspended in phosphate buffered saline ŽPBS.. The fecal suspensions were immediately tested for sIgA coproantibodies against EG by modification of an ELISA procedure ŽDeVos and Dick, 1991.. 2.6. Determination of systemic IgG antibodies against EG or EV by indirect ELISA Antibodies against EG or EV in vaccinated mice were determined by modification of standard ELISA ŽShelby and Kelley, 1990; Shelby and Kelley, 1991.. Negative controls included wells with all reagents except serum Žno serum blanks., wells with pre-vaccinated sera or wells with no antigen Žno antigen blanks.. Purified anti-EV antibodies served as the positive control since anti-EV, monoclonal antibodies are cross-reactive with EG in ELISA ŽKelley and Shelby, 1990.. 2.7. Determination of mouse prolactin by radioimmunoassay (RIA) Serum Ž40 m l. from each mouse was assayed in duplicate by RIA to determine serum prolactin concentrations. All samples were assayed in a single assay and the intra-assay coefficient of variation was less than 10%. Reagents were purchased from R.F. Parlow ŽResearch and Education Institute, Torrence, CA.. Mouse prolactin standard Ž25 ngrml in PBS with 1% BSA. was used to construct a standard curve. Mouse prolactin was iodinated with I 125 for use as a tracer by the chloramine T reaction ŽSinha et al., 1972..
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2.8. Statistical analysis Differences in weight gains, ALP activity, cholesterol, and serum prolactin concentrations among the treatment groups were determined to be significant at probability values - 0.05 by analysis of variance. Linear contrast was used for pairwise comparisons. Spearman rank correlation was used for correlation analysis ŽSAS w , SAS Institute, Cary, NC.. 2.9. Results The BSA–EG and CTB–EG conjugates spotted onto TLC plates exhibited intense fluorescence at the origin, whereas free EG fluoresced and migrated when developed. Unconjugated protein controls remained at the origin and did not fluoresce. The CTB–EG conjugate given orally to mice induced both mucosal sIgA ŽFig. 1. and some mucosal IgG Ždata not shown. antibodies against EG. The sIgA and IgG coproantibodies against EG had similar kinetics, but sIgA reached higher levels. The sIgA concentrations ŽFig. 1. in the feces of the CTB–EG group increased 7 days post-vaccination and were present before the start of the dietary trial on day 20 post-vaccination. The systemic, IgG titers against EG in mice of the CTB–EG group peaked ŽLog 2 titers s 1. 14 days after vaccination and declined to background levels by the start of the dietary trial. Active vaccination of mice with a BSA–EG conjugate also induced systemic anti-EG antibodies ŽLog 2 titers s 8.. Mice in the MoAB group had anti-EV titers ŽLog 2 titers s 3.1.
Fig. 1. Mean optical density ŽO.D.. readings Ž"SEM. representing mucosal sIgA antibodies against EG prior to the start of the feeding trial on day 20. Mice were orally vaccinated with 15 m g of the CTB–EG conjugate and 5 m g of CT on days 0 and 10.
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after administration of an additional 500 m g of monoclonal antibodies. Two mice from the CTB–EG group became moribund after 48 h on the feeding trial and were euthanized for necropsy. Gastrointestinal hemorrhage and normal histological findings of major organs were found at necropsy. The small intestines were normal, but the lumen contained only large, gram positive bacilli and no gram negative bacteria. Gram negative bacteria were present in the colon. 2.9.1. Nutritional and EV content of dietary treatments The EV content of the dietary treatments was 1.5 ppm for the EI diet, whereas no EV was detected in the EF diet. The respective diets were similar upon nutritional analysis Ždata not shown.. 2.9.2. Weight gains and serum prolactin concentrations Weight gains ŽFig. 2. in the unvaccinated EI group were lower Ž P - 0.05. than weight gains observed in the EF group. Weight gains were increased Ž P - 0.05. in the BSA–EG and CTB–EG groups and tended to be increased Ž P - 0.09. in the MoAB vaccinated group vs. the unvaccinated EI group. Final body weights were similar Ž P ) 0.05. among groups, although mice in the EI group tended to weigh less. Weight gains were moderately correlated Ž r s 0.39, P - 0.05. to anti-EG titers. Although serum
Fig. 2. Average weight gains Ž"SEM. in mice during the 13-day immunization study. Treatment groups were: BSA–EG: mice vaccinated ip with 250 m g of a BSA–EG conjugate in Freund’s complete adjuvant and revaccinated 14 days later with 100 m g of the BSA–EG conjugate in Freund’s incomplete adjuvant; CTB–EG: mice orally vaccinated with 15 m g of the CTB–EG conjugate mixed with 5 m g of CT and fed an EI fescue diet; EI: nonvaccinated mice fed an EI fescue diet; MoAB: passively vaccinated mice with anti-EV, monoclonal antibodies and fed an EI fescue diet; and EF: nonvaccinated mice fed an EF fescue diet. Groups with unlike letters are different Ž P - 0.05..
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Table 1 Effects of vaccination on serum ALP, prolactin and cholesterol concentrations in mice fed EI vs. EF fescue diets Treatment group 1
BSA–EG CTB–EG PO 2 EI MoAB 3 EF
ALP ŽUrl.
Cholesterol Žmgrdl. a
86.0"2.9 106.7"6.7 b,d 104.4"3.0 b,c 96.3"2.4 c 114.9"3.5d
a,c
90.6"4.1 95.0"3.7 c,b 91.5"4.3 a,c 89.9"3.2 a,c 102.6"3.2 b
Prolactin Žngrml. 8.5"0.9 b 7.4"1.3 b 6.5"1.1b 8.2"1.3 b 7.3"1.8 b
a–d
Groups with unlike letters differ Ž P - 0.05.. BSA–EG group, anti-EG IgG titers ŽLog 2 titerss8.. 2 CTB–EG PO: mice orally vaccinated with 15 m g of the CTB–EG conjugate and 5 m g of CT on days 0 and 10 prior to dietary trial. 3 MoAB group, anti-EV titers ŽLog 2 titerss 3.1.. Values represent group mean"SEM. 1
prolactin levels ŽTable 1. were not different Ž P ) 0.05. among groups, prolactin levels were moderately correlated Ž r s 0.42, P - 0.05. with anti-EG titers. 2.9.3. ALP actiÕity and cholesterol concentrations The ALP activity ŽTable 1. of the unvaccinated EI group was lower Ž P - 0.05. than the ALP activity of the EF group. The ALP activity of the BSA–EG and MoAB groups fed the EI diet was lower Ž P - 0.05. than EF group. The ALP activity was decreased in the BSA–EG group Ž P - 0.05. and tended to be decreased Ž P s 0.10. in the MoAB group as compared to the EI group. Within the vaccinated groups, ALP activity was lower in the BSA–EG group than ALP activity in the CTB–EG group. The ALP activity in the CTB–EG group was similar to the EI group and EF groups. Serum ALP activity was negatively correlated Ž r s y0.64; P - 0.05. with anti-EG titers. Cholesterol concentrations ŽTable 1. were decreased Ž P - 0.05. in the BSA–EG, EI, and MoAB groups as compared to the EF group.
3. Discussion Previous parenteral vaccines, which induced specific IgG antibodies against plant or fungal toxins, have resulted in various degrees of animal protection ŽCox, 1985; Stewart et al., 1988; Payne et al., 1993; Jonas and Erasmuson, 1979.. To protect cattle against fescue toxicosis, repeated parenteral vaccination of cattle may be required to induce adequate systemic anti-EG titers, since the small molecular weights of dietary alkaloid haptens will not induce anamnestic immune responses in vaccinated animals. However, repeated parenteral vaccination is both expensive and labor intensive for cattle producers. Therefore, traditional parenteral vaccination may not be the optimal route for the prevention of fescue toxicosis. In this study, EG was the model antigen because of its availability and structural ŽShelby and Kelley, 1991., as well as functional similarities to EV ŽMarple et al., 1988;
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McCollough et al., 1994; Kerley et al., 1994.. Ergotamine also cross-reacts antigenically with EV ŽKelley and Shelby, 1990. and is readily available at a moderate cost. Ergotamine must be conjugated to a carrier protein to be immunogenic, because of its low molecular weight. An indole nitrogen in EG and EV enables conjugation to protein carriers containing lysine residues by the Mannich reaction ŽRanadive and Sehon, 1967.. The Mannich reaction successfully conjugated BSA and CTB to EG, thus, these proteins must contain an adequate number of lysine residues in the proper orientation for conjugation to the indole nitrogen of EG. In the construction of conjugate vaccines, immunogenicity of the hapten can be modified by the carrier protein used. Both cholera toxin B ŽCTB. and whole cholera toxin ŽCT. function as carrier proteins and as adjuvants as well. The adjuvanticity of CTB and CT is only observed when co-administered with an antigen via the same route ŽHolmgren et al., 1993; Czerkinsky et al., 1989.. Oral vaccination with CT in microgram amounts induces both specific sIgA and specific plasma IgG responses originating in the gut-associated lymphoid tissue ŽGALT. ŽElson and Ealding, 1984; McKenzie and Halsey, 1984; Chen and Quinnan, 1989.. Secretory IgA ŽsIgA. is the major immunoglobulin class found in exocrine secretions ŽRussell and Mestecky, 1988.. Specific sIgA in secretions can neutralize antigens in the absence of demonstrable serum antibodies, and often protection is correlated best with local sIgA secretion rather than circulating antibody. Oral vaccination can produce high concentrations of antigen-specific sIgA at mucosal sites. However, traditional parenteral vaccination induces systemic IgM and IgG isotypes, but rarely serum IgA or sIgA isotypes because of the compartmentalization of systemic and secretory immune systems. In this study, oral vaccination of mice with a CTB–EG conjugate mixed with CT as an adjuvant induced sIgA and IgG coproantibodies against EG. Oral vaccination of mice with a CTB–EG conjugate also induced systemic IgG titers against EG. However, CTB and free CT did not prolong systemic IgG immune responses against EG as previously reported for other antigens ŽRussell and Wu, 1991.. Active vaccination of mice with a BSA–EG conjugate induced specific IgG antibodies against EG. In cattle with fescue toxicosis, decreases in weight gains cause major economic losses for producers. Thus, in evaluating the efficacy of vaccination protocols for the prevention of fescue toxicosis, protection against weight loss is of paramount importance. Active vaccination with a BSA–EG conjugate or oral vaccination with a CTB–EG conjugate increased weight gains, whereas passive vaccination with anti-EV antibodies tended to increase weight gains in mice fed an EI diet for a 13-day period. This supports the use of EG in a protein–hapten conjugate vaccine against fescue toxicosis and the investigation of vaccination of cattle fed an EI fescue diet. However, the observed weight gains were modest in the vaccinated mouse groups and robust weight gains will be necessary in vaccinated cattle for a vaccine against fescue toxicosis to be economically viable to producers. Besides weight gains, perhaps the physiological response of greatest importance in evaluating the efficacy of a vaccine against fescue toxicosis is serum prolactin concentrations. Hypoprolactemia in cattle grazing EI fescue is a fairly consistent finding. Ergovaline acts as a dopamine agonist at the DA 2 dopamine receptor of lactotrophs in the pars distalis to inhibit release of prolactin in vitro ŽStrickland et al., 1994.. Increases
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in serum prolactin concentrations are associated with increases in ADG and grazing time in cattle with fescue toxicosis treated with metoclopramide, a dopamine D 2 receptor antagonist ŽLipham et al., 1989.. However, increases in body temperature associated with fescue toxicosis are not decreased by metoclopramide therapy in sheep ŽAldrich et al., 1993.. This suggests a dual mechanism of action for ergot alkaloids in fescue toxicosis and thus vaccination may not protect against all indices associated with fescue toxicosis. In this study, serum prolactin concentrations were not different among groups and passive vaccination of mice with specific antibodies did not increase prolactin concentrations as previously demonstrated in cattle ŽHill et al., 1994.. Thyrotropin releasing hormone ŽTRH., however, may be needed to detect prolactin differences ŽThompson et al., 1987.. Stress also stimulates prolactin release from the pituitary in both ruminants ŽLamming et al., 1974; Lefcourt et al., 1986. and rodents ŽGala, 1990. and could be used to better detect differences in prolactin concentrations. In this study, blood was collected quickly from the mice and stress-induced prolactin release from the pituitary gland may not have occurred. Although specific antibodies against EG preserved weight gains in vaccinated mice fed an EI fescue diet, specific antibodies against EG and EV were not protective against decreases in ALP and cholesterol concentrations and specific antibodies actually exacerbated decreases in serum ALP concentrations. Although lower serum ALP values may not have overt clinical significance, reduced ALP values in vaccinated mice fed EI fescue diets demonstrate exacerbation of indices induced by fescue toxicosis. Exacerbation of indices associated with fescue toxicosis by vaccination has not been previously reported, but has been reported for other parenteral vaccines against selected plant or fungal toxins ŽMacDonald et al., 1990; Smith et al., 1992; Culvenor, 1978.. Decreases in ALP activity were likely an effect of systemic IgG antibodies binding antigen or influencing receptors, since ALP activity was decreased in the BSA–EG group and tended to be decreased in mice of the MoAB group ŽIgG., but not the CTB–EG group ŽsIgA.. Additional studies are needed to examine long term protection against fescue toxicosis by specific antibodies, because amounts of antibodies produced over time may not be sufficient to neutralize all the dietary EV absorbed from the gastrointestinal tract. Additionally, the low molecular weights of dietary alkaloid toxins will not induce anamnestic immune responses in vaccinated animals. Furthermore, protection of immunization against toxins in laboratory species does not always guarantee protection in the target species ŽFairclough et al., 1984.. Thus, long term testing of conjugate vaccines against fescue toxicosis is warranted in cattle and the use of novel vaccination protocols to increase and prolong immune responses will be needed.
4. Conclusions The Mannich reaction was used to conjugate EG to CTB or BSA as the protein carrier in a conjugate vaccine for fescue toxicosis. Oral vaccination of mice with the
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CTB–EG conjugate induced sIgA coproantibodies and a short-lived systemic IgG response against EG. Unvaccinated mice fed an EI fescue diet experienced decreased ALP activity, cholesterol concentrations and weight gains as compared to mice fed an EF fescue diet, which further validates the murine model of fescue toxicosis. Parenteral vaccination with the BSA–EG conjugate and oral vaccination with the CTB–EG conjugate increased weight gains in mice fed a EI fescue diet for 13 days. Passive vaccination with anti-EV, monoclonal antibodies tended to increase short-term weight gains in mice. Prolactin concentrations were similar in vaccinated mice fed an EI diet and those fed an EI or EF fescue diet. Vaccination did not protect against decreases in ALP activity and cholesterol concentrations in mice fed an EI fescue diet. Systemic, anti-EV antibodies tended to further decrease ALP activity when compared to the EI group.
Acknowledgements The authors gratefully acknowledge the John Lee Pratt Animal Nutrition Foundation for financial support of this research, Dr. Warnick for his statistical advice, Dr. Kelley at Auburn University for supplying the hybridoma, International Seed for generously supplying the fescue seed and Dr. Akers for analyzing serum prolactin by RIA.
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Hemken, R.W., Jackson, J.A., Boling, J.A., 1984. Toxic factors in tall fescue. J. Anim. Sci. 58, 1011–1016. Hill, N.S., Rottinghaus, G.E., Agee, C.S., Schultz, L.M., 1993. Simplified sample preparation for HPLC analysis of ergovaline in tall fescue. Crop Sci. 33, 331–333. Hill, N.S., Thompson, F.N., Dawe, D.L., Stuedemann, J.A., 1994. Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue. Am. J. Vet. Res. 55, 419–424. Holmgren, J., Lycke, N., Czerkinsky, C., 1993. Cholera toxin and cholera toxin B subunit as an oral mucosal adjuvant and antigen vector systems. Vaccine 11, 1179–1184. Hoveland, C.S., 1990. Importance and economic significance of the Acremonium endophytes to performance of animals and grass plant. In: Quisenberry, S.S., Joost, R.E. ŽEds.., Proc. Int. Symp. on AcremoniumrGrass Interactions: Program and Abstracts. Louisiana Agricultural Experiment Station, Baton Rouge, LA, p. 7. Hurley, W.L., Convey, E.M., Leung, K., Edgerton, L.A., Hemken, R.W., 1981. Bovine prolactin, TSH, T4 and T3 concentrations as affected by tall fescue summer toxicosis and temperature. J. Anim. Sci. 51, 374–379. Jonas, W.E., Erasmuson, A.F., 1979. The effect of immunizing mice with a derivative of 2-amino-5-chloro-3, 4-dimethoxybenzyl alcohol coupled to some bacteria on sporidesmin induced bilirubinaemia. New Zealand Vet. J. 27, 61–63. Kelley, V.C., Shelby, R.A., 1990. Production and characterization of monoclonal antibody to ergovaline. In: Quisenberry, S.S., Joost, R.E. ŽEds.., Proc. Int. Symp. on AcremoniumrGrass Interactions. Louisiana Agricultural Experiment Station, Baton Rouge, LA, pp. 83–88. Kerley, M.S., Larson, B. T,. Samford, M.D., Turner, J.E., 1994. Involvement of alpha 2-adrenergic receptors in ergovaline response of cattle. Proc. Tall Fescue Toxicosis Workshop, SERAIEG-8. Atlanta, GA, Oct. 23–25, p. 39. Lamming, G.E., Moseley, S.R., McNeilly, J.R., 1974. Prolactin release in the sheep. J. Reprod. Fertil. 40, 151–168. Lefcourt, A.M., Kahl, S., Akers, R.M., 1986. Correlation of indices of stress with intensity of electrical shock for cows. J. Dairy Sci. 69, 833–842. Lipham, L.B., Thompson, F.N., Stuedemann, J.A., Sartin, J.L., 1989. Effects of metoclopramide on steers grazing endophyte-infected fescue. J. Anim. Sci. 67, 1090–1097. MacDonald, O.A., Thulin, A.J., Weldon, W.C., Pestka, J.J., Fogwell, R.L., 1990. Effects of immunizing gilts against zearalenone on height of vaginal epithelium and urinary excretion of zearalenone. J. Anim. Sci. 68, 3713–3718. Marple, D.N., Osburn, T.G., Schmidt, S.P., Rahe, C.H., Sartin, J.L., 1988. Hypothalamic catecholamines in calves consuming fungus-infected, fungus-free or fungus-free tall fescue plus ergotamine tartrate. J. Anim. Sci. 66 Ž1., 373. McCollough, S., Piper, E., Moubarak, A., Johnson, Z., Flieger, M., 1994. Effects of fescue alkaloids on peripheral blood flow and prolactin secretion in calves. Proc. Tall Fescue Toxicosis Workshop, SERAIEG-8. Atlanta, GA, Oct. 23–25, p. 9. McKenzie, S.J., Halsey, J.F., 1984. Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J. Immunol. 133, 1818–1824. Morgan-Jones, G., Gams, W., 1982. An endophyte of Festuca arundinacea and the anamorph of Epichloe typhina, new taxa in one of two sections of Acremonium. Mycotaxon 15, 311–318. Payne, A.L., Than, K.A., Stewart, P.L., Edgar, J.A., 1993. Vaccination against lupinosis. Fourth International Symp. Poisonous Plants. Fremantle, W. Aust., p. 234. Ranadive, N.S., Sehon, A.H., 1967. Antibodies to serotonin. Can. J. Biochem. 45, 1701–1710. Russell, Mestecky, 1988. Russell, M.W., Wu, H.Y., 1991. Distribution, persistence, and recall of serum and salivary antibody responses to preoral immunization with protein antigen IrII of Streptococcus mutants coupled to the cholera toxin B subunit. Infect. Immun. 59, 4061–4070. Shelby, R.A., Kelley, V.C., 1990. An immunoassay for ergotamine and related alkaloids. J. Agric. Food Chem. 38, 1130–1134. Shelby, R.A., Kelley, V.C., 1991. Detection of ergot alkaloids in tall fescue by competitive immunoassay with a monoclonal antibody. Food Agric. Immunol. 3, 169–177. Sinha, Y.N., Selby, F.W., Lewis, U.J., Vanderlaan, W.P., 1972. Studies of prolactin secretion in mice by a homologous radioimmunoassay. Endocrinology 91, 1045–1053.
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Smith, J.F., di Menna, M.E., Towers, N.R., 1992. Zearalenone and its effects on sheep. Proc. 12th Int. Cong. Anim. Reprod. The Hague, The Netherlands, Vol. 3, p. 1219. Stewart, C., Lamberton, J.A., Fairclough, R.J., Pass, M.A., 1988. Vaccination as a means of preventing lantana poisoning. Aust. Vet. J. 65, 349–352. Strickland, J.R., Cross, D.L., Birrenkott, Grimes, L.W., 1994. Effect of ergovaline, loline, and dopamine antagonists on rat pituitary cell prolactin release in vitro. Am. J. Vet. Res. 55, 716–721. Stuedemann, J.A., Hoveland, C.S., 1988. Fescue endophyte: history and impact on animal agriculture. J. Prod. Agric. 1, 39–44. Stuedemann, J.A., Rumsey, T.S., Bond, T., Wilkinson, S.R., Bush, L.P., Williams, D.J., Caudle, A.B., 1985. Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers. Am. J. Vet. Res. 46, 1990–1995. Thompson, F.N., Stuedemann, J.A., Sartin, J.L., Belesky, D.P., Devine, O.J., 1987. Selected hormonal changes with summer fescue toxicosis. J. Anim. Sci. 65, 727–733. Wilson, A.D., Robinson, A., Irons, L., Stokes, C.R., 1993. Adjuvant action of cholera toxin and pertussis toxin in the induction of IgA antibody responses to orally administered antigen. Vaccine 11, 113–118.
Oral and parenteral vaccination of mice with protein-ergotamine conjugates and evaluation of protection against fescue toxicosis R.L. Rice a
c
a,1,)
, D.J. Blodgett a , G.G. Schurig a , W.S. Swecker b, C.D. Thatcher b, D.E. Eversole c
Departments of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA b Large Animal Clinical Sciences, Virginia– Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Accepted 3 November 1997
Abstract Acremonium coenophialum produces ergopeptide alkaloids in tall fescue Ž Festuca arundinacea Schreb... These ergot alkaloids decrease serum alkaline phosphatase ŽALP. activity, serum cholesterol and prolactin concentrations, as well as average daily gains ŽADG. in cattle. The objective of this study was to evaluate the protection of anti-ergotamine antibodies induced by either oral or parenteral vaccination with protein-ergotamine conjugates or passive vaccination with anti-ergovaline, monoclonal antibodies in a murine model of fescue toxicosis. Ergotamine ŽEG. was conjugated to bovine serum albumin ŽBSA. and cholera toxin subunit B ŽCTB. by the Mannich reaction. Mice were blocked based on weight and randomly allocated into five groups of 10 mice each. Treatment groups were as follows: Ž1. group vaccinated intraperitoneally Žip. with a BSA–EG conjugate and fed an endophyte-infected ŽEI. fescue diet ŽBSA–EG group.; Ž2. group orally vaccinated with a CTB–EG conjugate mixed with free cholera toxin ŽCT. and fed an EI fescue diet ŽCTB–EG group.; Ž3. nonvaccinated group fed an EI fescue diet ŽEI group.; Ž4. group passively vaccinated with anti-ergovaline, monoclonal antibodies and fed an EI fescue diet ŽMoAB group.; and Ž5. nonvaccinated group fed an endophyte-free ŽEF. fescue diet ŽEF group.. The EI diet contained 1.5 ppm of Ergovaline ŽEV., whereas no EV was detected in the EF diet.
) Corresponding author. 1210 Crown Dr., Mansfield, TX 76063, USA. Tel.: q1 817 551 8706; fax: q1 817 551 4584; e-mail: [email protected]. 1 Present address: Alcon Laboratories, 6201 South Freeway, Fort Worth, TX 76134, USA.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 5 6 - 6
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Respective diets were similar upon nutritional analysis. Unvaccinated mice in the EI group exhibited features of fescue toxicosis as indicated by decreased serum ALP activity and cholesterol, and decreased weight gain as compared to mice in the EF group. Antibodies against EG and EV were present in sera of mice in the BSA–EG and MoAB groups, respectively. Mice orally vaccinated with the CTB–EG conjugate developed secretory IgA ŽsIgA. antibodies and short-lived, systemic IgG responses against EG. Weight gains were increased in the BSA–EG and CTB–EG groups and tended to be increased in the MoAB group vs. the unvaccinated EI group. Serum ALP activity was decreased in the BSA–EG and MoAB groups as compared to the EF group. Serum ALP activity was further decreased in the BSA–EG vaccinated group as compared to the EI group. Cholesterol concentrations were decreased in the EI, BSA–EG and MoAB groups as compared to the EF group. Prolactin concentrations were similar in all groups. q 1998 Elsevier Science B.V. Keywords: Fescue toxicosis; Vaccine; Mice; Ergotamine; Prolactin
1. Introduction Tall fescue Ž Festuca arundinacea Schreb.. is often infected by the endophytic fungus, Acremonium coenophialum ŽMorgan-Jones and Gams, 1982.. The fungus produces ergopeptide alkaloids, especially ergovaline ŽEV.. Serum cholesterol concentrations and alkaline phosphatase ŽALP. activity are decreased in cattle grazing endophyte-infected ŽEI. fescue for long periods of time ŽLipham et al., 1989; Bond et al., 1984; Stuedemann et al., 1985.. Consumption of EI fescue forage by cattle also decreases serum prolactin ŽHurley et al., 1981; Thompson et al., 1987., and average daily gains ŽADG. ŽHemken et al., 1984; Stuedemann and Hoveland, 1988., which results in economic losses for producers ŽHoveland, 1990.. An economically sound solution to the fescue problem has not been forthcoming. Preventative methods would be preferable to new therapeutic drugs, which may cause residue problems. Development of a vaccine to protect cattle against fescue toxicosis may save producers millions of dollars and avoid potential environmental damage associated with pasture renovation, while allowing their cattle to benefit from the nutritional value of EI fescue forage. The purpose of this study was to evaluate the protection of specific antibodies induced by either parenteral vaccination with a bovine serum albumin–ergotamine ŽBSA–EG. conjugate, oral vaccination with a cholera toxin subunit B ŽCTB. –EG conjugate, or passive vaccination with anti-EV, monoclonal antibodies in mice against fescue toxicosis. Weight gains, serum ALP activity, cholesterol and prolactin concentrations were the indices used to evaluate the ability of polyclonal, anti-EG and monoclonal, anti-EV antibodies to confer protection in the mouse model of fescue toxicosis.
2. Materials and methods 2.1. Preparation of CTB–EG and BSA–EG conjugates by the Mannich reaction Ergotamine Žtartrate salt: Sigma Chemical Co., St. Louis, MO. was conjugated to CTB or BSA ŽSigma Chemical. for vaccination. Proteins were linked to EG using the
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Mannich reaction by modification of the method previously described ŽKelley and Shelby, 1990.. Conjugates were evaluated on 250 m m thin-layer chromatographic ŽTLC. plates of silica gel developed in chloroformrmethanol Ž9:1, vrv. solvent system and observed under a long wave lamp. Protein content of each protein-EG conjugate was determined by bicinchoninic acid assay ŽBCA; BCAe Protein Assay; Pierce, Rockford, IL.. 2.2. Experimental design Fifty BALBrc, male mice Ž22.0 " 0.34 g; average BW " SEM., 6 weeks of age, were blocked by weight and randomly allocated from outcome groups into five groups of 10 mice each. Treatment groups were as follows: Ž1. group vaccinated intraperitoneally Žip. with a BSA–EG conjugate and fed an EI fescue diet ŽBSA–EG group.; Ž2. group orally vaccinated with a CTB–EG conjugate and fed an EI fescue diet ŽCTB–EG group.; Ž3. nonvaccinated group fed an EI fescue diet ŽEI group.; Ž4. group passively vaccinated with anti-EV, monoclonal antibodies and fed an EI fescue diet ŽMoAB group.; and Ž5. nonvaccinated group fed an endophyte-free ŽEF. fescue diet ŽEF group.. Mice were individually housed in shoebox cages of clear polystyrene and exposed to a 14:10 h light:dark cycle. Mice were acclimatized to ground rodent chow for 1 week prior to day 1 of the dietary trial. All procedures involving the use of animals were reviewed and approved by Virginia Polytechnic Institute and State University’s Animal Care and Use Committee. 2.3. Diet preparation Concentrations of EV were determined in an identical genotype of EI and EF ‘Kentucky 31’ tall fescue seed ŽInternational Seed, Halsey, OR. by HPLC analysis as described ŽHill et al., 1993.. Ground rodent chow ŽHarlan Teklad, Madison, WI. was mixed with equal parts of ground EI and EF fescue seed by weight. The seed:chow diets were analyzed for nutrient content ŽNorth East DHIA, Ithaca, NY.. Dietary intake was limited to 5 to 6 g per mouse daily. Water was provided ad libitum. 2.4. Vaccination of mice Mice in the BSA–EG group were injected ip with 250 m g of a BSA–EG conjugate in Freund’s complete adjuvant and revaccinated 14 days later with 100 m g of the BSA–EG conjugate in Freund’s incomplete adjuvant. Anti-EG, IgG titers were determined on days 14 and 34 post-vaccination by ELISA. Mice in the CTB–EG group were orally vaccinated twice with 15 m g of a CTB–EG conjugate mixed with 5 m g of cholera toxin ŽCT. at a 10 day interval. Microgram amounts of uncoupled CT were added to the CTB–EG conjugate prior to oral dosing because pure CTB is less effective as an adjuvant compared to CT ŽWilson et al., 1993. and also because the Mannich reaction decreased the ability of CTB present in the CTB–EG conjugate to bind the GM 1 receptor. Anti-EG Žmucosal secretory IgA ŽsIgA. and systemic IgG. and anti-EV ŽIgG. antibody levels were determined at the end of the acclimation period prior to the start of
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the dietary trial period on day 0. All vaccinations and revaccinations with the BSA–EG or CTB–EG conjugates were performed before mice were exposed to fescue diets. For passive vaccination of mice in the MoAB group, anti-EV, monoclonal IgG1 antibodies were isolated from hybridoma culture medium by affinity chromatography Žanti-mouse, IgG agarose column; Sigma Immunochemicals, St. Louis, MO.. The IgG concentration per ml of supernatant was determined by BCAe analysis ŽPierce. and the activity of purified monoclonal antibodies was determined by ELISA ŽShelby and Kelley, 1991.. Mice in the MoAB group were injected ip with 200 m g of the affinity-purified, monoclonal IgG antibodies specific for EV 1 day before initiation of the treatment diets. On day 7 of the trial, the mice in the MoAB group were injected ip with an additional 500 m g of the same monoclonal antibodies. After 13 days on trial, mice were anesthetized with halothane and blood was collected from the retro-orbital sinus. Sera obtained were analyzed for ALP, prolactin and cholesterol concentrations. Activity of serum ALP and cholesterol concentrations were determined by the EKTACHEM 700rVitros System ŽJohnson and Johnson, Rochester, NY.. Mice were weighed on day 0 and day 12 of the dietary trial period to evaluate weight gains. 2.5. Determination of anti-EG sIgA coproantibodies in mice orally Õaccinated with a CTB–EG conjugate by indirect ELISA Fecal pellets were collected from individual mice after oral vaccination every 3 to 5 days for 20 days and frozen immediately at y708C until analysis. Fecal samples were re-suspended in phosphate buffered saline ŽPBS.. The fecal suspensions were immediately tested for sIgA coproantibodies against EG by modification of an ELISA procedure ŽDeVos and Dick, 1991.. 2.6. Determination of systemic IgG antibodies against EG or EV by indirect ELISA Antibodies against EG or EV in vaccinated mice were determined by modification of standard ELISA ŽShelby and Kelley, 1990; Shelby and Kelley, 1991.. Negative controls included wells with all reagents except serum Žno serum blanks., wells with pre-vaccinated sera or wells with no antigen Žno antigen blanks.. Purified anti-EV antibodies served as the positive control since anti-EV, monoclonal antibodies are cross-reactive with EG in ELISA ŽKelley and Shelby, 1990.. 2.7. Determination of mouse prolactin by radioimmunoassay (RIA) Serum Ž40 m l. from each mouse was assayed in duplicate by RIA to determine serum prolactin concentrations. All samples were assayed in a single assay and the intra-assay coefficient of variation was less than 10%. Reagents were purchased from R.F. Parlow ŽResearch and Education Institute, Torrence, CA.. Mouse prolactin standard Ž25 ngrml in PBS with 1% BSA. was used to construct a standard curve. Mouse prolactin was iodinated with I 125 for use as a tracer by the chloramine T reaction ŽSinha et al., 1972..
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2.8. Statistical analysis Differences in weight gains, ALP activity, cholesterol, and serum prolactin concentrations among the treatment groups were determined to be significant at probability values - 0.05 by analysis of variance. Linear contrast was used for pairwise comparisons. Spearman rank correlation was used for correlation analysis ŽSAS w , SAS Institute, Cary, NC.. 2.9. Results The BSA–EG and CTB–EG conjugates spotted onto TLC plates exhibited intense fluorescence at the origin, whereas free EG fluoresced and migrated when developed. Unconjugated protein controls remained at the origin and did not fluoresce. The CTB–EG conjugate given orally to mice induced both mucosal sIgA ŽFig. 1. and some mucosal IgG Ždata not shown. antibodies against EG. The sIgA and IgG coproantibodies against EG had similar kinetics, but sIgA reached higher levels. The sIgA concentrations ŽFig. 1. in the feces of the CTB–EG group increased 7 days post-vaccination and were present before the start of the dietary trial on day 20 post-vaccination. The systemic, IgG titers against EG in mice of the CTB–EG group peaked ŽLog 2 titers s 1. 14 days after vaccination and declined to background levels by the start of the dietary trial. Active vaccination of mice with a BSA–EG conjugate also induced systemic anti-EG antibodies ŽLog 2 titers s 8.. Mice in the MoAB group had anti-EV titers ŽLog 2 titers s 3.1.
Fig. 1. Mean optical density ŽO.D.. readings Ž"SEM. representing mucosal sIgA antibodies against EG prior to the start of the feeding trial on day 20. Mice were orally vaccinated with 15 m g of the CTB–EG conjugate and 5 m g of CT on days 0 and 10.
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after administration of an additional 500 m g of monoclonal antibodies. Two mice from the CTB–EG group became moribund after 48 h on the feeding trial and were euthanized for necropsy. Gastrointestinal hemorrhage and normal histological findings of major organs were found at necropsy. The small intestines were normal, but the lumen contained only large, gram positive bacilli and no gram negative bacteria. Gram negative bacteria were present in the colon. 2.9.1. Nutritional and EV content of dietary treatments The EV content of the dietary treatments was 1.5 ppm for the EI diet, whereas no EV was detected in the EF diet. The respective diets were similar upon nutritional analysis Ždata not shown.. 2.9.2. Weight gains and serum prolactin concentrations Weight gains ŽFig. 2. in the unvaccinated EI group were lower Ž P - 0.05. than weight gains observed in the EF group. Weight gains were increased Ž P - 0.05. in the BSA–EG and CTB–EG groups and tended to be increased Ž P - 0.09. in the MoAB vaccinated group vs. the unvaccinated EI group. Final body weights were similar Ž P ) 0.05. among groups, although mice in the EI group tended to weigh less. Weight gains were moderately correlated Ž r s 0.39, P - 0.05. to anti-EG titers. Although serum
Fig. 2. Average weight gains Ž"SEM. in mice during the 13-day immunization study. Treatment groups were: BSA–EG: mice vaccinated ip with 250 m g of a BSA–EG conjugate in Freund’s complete adjuvant and revaccinated 14 days later with 100 m g of the BSA–EG conjugate in Freund’s incomplete adjuvant; CTB–EG: mice orally vaccinated with 15 m g of the CTB–EG conjugate mixed with 5 m g of CT and fed an EI fescue diet; EI: nonvaccinated mice fed an EI fescue diet; MoAB: passively vaccinated mice with anti-EV, monoclonal antibodies and fed an EI fescue diet; and EF: nonvaccinated mice fed an EF fescue diet. Groups with unlike letters are different Ž P - 0.05..
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Table 1 Effects of vaccination on serum ALP, prolactin and cholesterol concentrations in mice fed EI vs. EF fescue diets Treatment group 1
BSA–EG CTB–EG PO 2 EI MoAB 3 EF
ALP ŽUrl.
Cholesterol Žmgrdl. a
86.0"2.9 106.7"6.7 b,d 104.4"3.0 b,c 96.3"2.4 c 114.9"3.5d
a,c
90.6"4.1 95.0"3.7 c,b 91.5"4.3 a,c 89.9"3.2 a,c 102.6"3.2 b
Prolactin Žngrml. 8.5"0.9 b 7.4"1.3 b 6.5"1.1b 8.2"1.3 b 7.3"1.8 b
a–d
Groups with unlike letters differ Ž P - 0.05.. BSA–EG group, anti-EG IgG titers ŽLog 2 titerss8.. 2 CTB–EG PO: mice orally vaccinated with 15 m g of the CTB–EG conjugate and 5 m g of CT on days 0 and 10 prior to dietary trial. 3 MoAB group, anti-EV titers ŽLog 2 titerss 3.1.. Values represent group mean"SEM. 1
prolactin levels ŽTable 1. were not different Ž P ) 0.05. among groups, prolactin levels were moderately correlated Ž r s 0.42, P - 0.05. with anti-EG titers. 2.9.3. ALP actiÕity and cholesterol concentrations The ALP activity ŽTable 1. of the unvaccinated EI group was lower Ž P - 0.05. than the ALP activity of the EF group. The ALP activity of the BSA–EG and MoAB groups fed the EI diet was lower Ž P - 0.05. than EF group. The ALP activity was decreased in the BSA–EG group Ž P - 0.05. and tended to be decreased Ž P s 0.10. in the MoAB group as compared to the EI group. Within the vaccinated groups, ALP activity was lower in the BSA–EG group than ALP activity in the CTB–EG group. The ALP activity in the CTB–EG group was similar to the EI group and EF groups. Serum ALP activity was negatively correlated Ž r s y0.64; P - 0.05. with anti-EG titers. Cholesterol concentrations ŽTable 1. were decreased Ž P - 0.05. in the BSA–EG, EI, and MoAB groups as compared to the EF group.
3. Discussion Previous parenteral vaccines, which induced specific IgG antibodies against plant or fungal toxins, have resulted in various degrees of animal protection ŽCox, 1985; Stewart et al., 1988; Payne et al., 1993; Jonas and Erasmuson, 1979.. To protect cattle against fescue toxicosis, repeated parenteral vaccination of cattle may be required to induce adequate systemic anti-EG titers, since the small molecular weights of dietary alkaloid haptens will not induce anamnestic immune responses in vaccinated animals. However, repeated parenteral vaccination is both expensive and labor intensive for cattle producers. Therefore, traditional parenteral vaccination may not be the optimal route for the prevention of fescue toxicosis. In this study, EG was the model antigen because of its availability and structural ŽShelby and Kelley, 1991., as well as functional similarities to EV ŽMarple et al., 1988;
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McCollough et al., 1994; Kerley et al., 1994.. Ergotamine also cross-reacts antigenically with EV ŽKelley and Shelby, 1990. and is readily available at a moderate cost. Ergotamine must be conjugated to a carrier protein to be immunogenic, because of its low molecular weight. An indole nitrogen in EG and EV enables conjugation to protein carriers containing lysine residues by the Mannich reaction ŽRanadive and Sehon, 1967.. The Mannich reaction successfully conjugated BSA and CTB to EG, thus, these proteins must contain an adequate number of lysine residues in the proper orientation for conjugation to the indole nitrogen of EG. In the construction of conjugate vaccines, immunogenicity of the hapten can be modified by the carrier protein used. Both cholera toxin B ŽCTB. and whole cholera toxin ŽCT. function as carrier proteins and as adjuvants as well. The adjuvanticity of CTB and CT is only observed when co-administered with an antigen via the same route ŽHolmgren et al., 1993; Czerkinsky et al., 1989.. Oral vaccination with CT in microgram amounts induces both specific sIgA and specific plasma IgG responses originating in the gut-associated lymphoid tissue ŽGALT. ŽElson and Ealding, 1984; McKenzie and Halsey, 1984; Chen and Quinnan, 1989.. Secretory IgA ŽsIgA. is the major immunoglobulin class found in exocrine secretions ŽRussell and Mestecky, 1988.. Specific sIgA in secretions can neutralize antigens in the absence of demonstrable serum antibodies, and often protection is correlated best with local sIgA secretion rather than circulating antibody. Oral vaccination can produce high concentrations of antigen-specific sIgA at mucosal sites. However, traditional parenteral vaccination induces systemic IgM and IgG isotypes, but rarely serum IgA or sIgA isotypes because of the compartmentalization of systemic and secretory immune systems. In this study, oral vaccination of mice with a CTB–EG conjugate mixed with CT as an adjuvant induced sIgA and IgG coproantibodies against EG. Oral vaccination of mice with a CTB–EG conjugate also induced systemic IgG titers against EG. However, CTB and free CT did not prolong systemic IgG immune responses against EG as previously reported for other antigens ŽRussell and Wu, 1991.. Active vaccination of mice with a BSA–EG conjugate induced specific IgG antibodies against EG. In cattle with fescue toxicosis, decreases in weight gains cause major economic losses for producers. Thus, in evaluating the efficacy of vaccination protocols for the prevention of fescue toxicosis, protection against weight loss is of paramount importance. Active vaccination with a BSA–EG conjugate or oral vaccination with a CTB–EG conjugate increased weight gains, whereas passive vaccination with anti-EV antibodies tended to increase weight gains in mice fed an EI diet for a 13-day period. This supports the use of EG in a protein–hapten conjugate vaccine against fescue toxicosis and the investigation of vaccination of cattle fed an EI fescue diet. However, the observed weight gains were modest in the vaccinated mouse groups and robust weight gains will be necessary in vaccinated cattle for a vaccine against fescue toxicosis to be economically viable to producers. Besides weight gains, perhaps the physiological response of greatest importance in evaluating the efficacy of a vaccine against fescue toxicosis is serum prolactin concentrations. Hypoprolactemia in cattle grazing EI fescue is a fairly consistent finding. Ergovaline acts as a dopamine agonist at the DA 2 dopamine receptor of lactotrophs in the pars distalis to inhibit release of prolactin in vitro ŽStrickland et al., 1994.. Increases
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in serum prolactin concentrations are associated with increases in ADG and grazing time in cattle with fescue toxicosis treated with metoclopramide, a dopamine D 2 receptor antagonist ŽLipham et al., 1989.. However, increases in body temperature associated with fescue toxicosis are not decreased by metoclopramide therapy in sheep ŽAldrich et al., 1993.. This suggests a dual mechanism of action for ergot alkaloids in fescue toxicosis and thus vaccination may not protect against all indices associated with fescue toxicosis. In this study, serum prolactin concentrations were not different among groups and passive vaccination of mice with specific antibodies did not increase prolactin concentrations as previously demonstrated in cattle ŽHill et al., 1994.. Thyrotropin releasing hormone ŽTRH., however, may be needed to detect prolactin differences ŽThompson et al., 1987.. Stress also stimulates prolactin release from the pituitary in both ruminants ŽLamming et al., 1974; Lefcourt et al., 1986. and rodents ŽGala, 1990. and could be used to better detect differences in prolactin concentrations. In this study, blood was collected quickly from the mice and stress-induced prolactin release from the pituitary gland may not have occurred. Although specific antibodies against EG preserved weight gains in vaccinated mice fed an EI fescue diet, specific antibodies against EG and EV were not protective against decreases in ALP and cholesterol concentrations and specific antibodies actually exacerbated decreases in serum ALP concentrations. Although lower serum ALP values may not have overt clinical significance, reduced ALP values in vaccinated mice fed EI fescue diets demonstrate exacerbation of indices induced by fescue toxicosis. Exacerbation of indices associated with fescue toxicosis by vaccination has not been previously reported, but has been reported for other parenteral vaccines against selected plant or fungal toxins ŽMacDonald et al., 1990; Smith et al., 1992; Culvenor, 1978.. Decreases in ALP activity were likely an effect of systemic IgG antibodies binding antigen or influencing receptors, since ALP activity was decreased in the BSA–EG group and tended to be decreased in mice of the MoAB group ŽIgG., but not the CTB–EG group ŽsIgA.. Additional studies are needed to examine long term protection against fescue toxicosis by specific antibodies, because amounts of antibodies produced over time may not be sufficient to neutralize all the dietary EV absorbed from the gastrointestinal tract. Additionally, the low molecular weights of dietary alkaloid toxins will not induce anamnestic immune responses in vaccinated animals. Furthermore, protection of immunization against toxins in laboratory species does not always guarantee protection in the target species ŽFairclough et al., 1984.. Thus, long term testing of conjugate vaccines against fescue toxicosis is warranted in cattle and the use of novel vaccination protocols to increase and prolong immune responses will be needed.
4. Conclusions The Mannich reaction was used to conjugate EG to CTB or BSA as the protein carrier in a conjugate vaccine for fescue toxicosis. Oral vaccination of mice with the
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CTB–EG conjugate induced sIgA coproantibodies and a short-lived systemic IgG response against EG. Unvaccinated mice fed an EI fescue diet experienced decreased ALP activity, cholesterol concentrations and weight gains as compared to mice fed an EF fescue diet, which further validates the murine model of fescue toxicosis. Parenteral vaccination with the BSA–EG conjugate and oral vaccination with the CTB–EG conjugate increased weight gains in mice fed a EI fescue diet for 13 days. Passive vaccination with anti-EV, monoclonal antibodies tended to increase short-term weight gains in mice. Prolactin concentrations were similar in vaccinated mice fed an EI diet and those fed an EI or EF fescue diet. Vaccination did not protect against decreases in ALP activity and cholesterol concentrations in mice fed an EI fescue diet. Systemic, anti-EV antibodies tended to further decrease ALP activity when compared to the EI group.
Acknowledgements The authors gratefully acknowledge the John Lee Pratt Animal Nutrition Foundation for financial support of this research, Dr. Warnick for his statistical advice, Dr. Kelley at Auburn University for supplying the hybridoma, International Seed for generously supplying the fescue seed and Dr. Akers for analyzing serum prolactin by RIA.
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