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J. Anim. Sci. 2003. 81:232-238
© 2003 American Society of Animal Science

Evaluation of vaccination against methyllycaconitine toxicity in mice1

S. T. Lee2,{dagger}, B. L. Stegelmeier{dagger}, K. E. Panter{dagger}, J. A. Pfister{dagger}, D. R. Gardner{dagger}, T. K. Schoch{ddagger} and L. F. James{dagger}

{dagger} USDA, ARS, Poisonous Plant Research Laboratory, Logan, Utah 84341 and and {ddagger} Western Regional Research Center, Albany, California 94710

2 Correspondence:
1150 E. 1400 N. (phone: 435-752-2941; fax: 435-753-5681; E-mail:
stlee{at}cc.usu.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The purpose of this study was to determine whether larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin–protein conjugates or adjuvant alone to determine whether the resulting immunological response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations increased the calculated lethal dose 50% (LD50) for intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC)-vaccinated group, mouse survival was related (P = 0.001) to serum titers for methyllycaconitine doses up to 4.5 mg/kg of body weight. When mice with low antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50 estimates were 20% greater than the LD50 of the control group. However, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups. Overall, these results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Additional studies are warranted to continue development of potential larkspur vaccines for livestock.

Key Words: Delphinium • Lethal Dose • Mice • Toxicity • Vaccination


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Many larkspurs (Delphinium spp.) found on the rangelands of the western United States are poisonous to livestock, and annual losses attributed to larkspur poisoning are valued in millions of dollars (Williams and Cronin, 1966; Ralphs et al., 1988). Larkspurs contain over 40 norditerpenoid alkaloids that are toxic to mammals (Pelletier and Keith, 1970; Manners et al., 1992; 1993). These compounds act as potent neuromuscular blocking agents that block acetylcholine receptors in the muscle and brain resulting in muscle weakness, paralysis, respiratory failure, and death (Benn and Jacyno, 1983; Kukel and Jennings, 1994; Dobelis et al., 1999).

In general, the alkaloids possessing a N-(methylsuccinimido) anthranilic ester substituent at C18 (MSAL-type alkaloids), such as methyllycaconitine, are the most toxic of the larkspur alkaloids. The chemical structures of methyllycaconitine and related larkspur toxins are shown in Figure 1Go. Methyllycaconitine, with a reported i.v. LD50 of 4.8 mg/kg in mice (Panter et al., 2002), is the most prevalent of the highly toxic MSAL alkaloids in tall larkspurs of the western United States (Manners et al., 1995). Thus, the relatively high toxicity and prevalence of methyllycaconitine in tall larkspurs makes it the prime target for vaccination studies to alleviate larkspur toxicity.



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  		Figure 1. Chemical structures of lycoctonine, deltaline, methyllycaconitine, and 14-deacetylnudicauline.

 
Previously, we reported the chemical synthesis of lycoctonine, 14-deacetylnudicauline, and methyllycaconitine protein immunoconjugates (Lee et al., 2000). These alkaloid–protein immunoconjugates were injected into sheep and antibodies raised to the immunoconjugates. Antisera of sheep were obtained from blood samples and were found to bind the MSAL-type larkspur toxins in vitro. Using these antisera, sensitive ELISA were developed for the measurement of larkspur alkaloids in biological samples (Lee et al., 2000).

This paper reports the use of lycoctonine, 14-deacetylnudicauline, and methyllycaconitine protein immunoconjugates as vaccines against methyllycaconitine toxicity in mice. Mice vaccinated with immunoconjugates were challenged by i.v. injection with methyllycaconitine. The effectiveness of the different immunoconjugates in elevating the LD50 of methyllycaconitine is reported.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Chemicals/Preparation of Alkaloid–Protein Conjugates
Freund’s complete adjuvant, Freund’s incomplete adjuvant, fetuin from fetal calf serum, 3,3',5,5'-tetramethylbenzidine, thimerosal, and polyoxyethylene sorbitan monolaurate (Tween 20) were obtained from Sigma Chemical Co. (St. Louis, MO). Reagent-grade BSA and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were purchased from ICN Biomedicals Inc. (Costa Mesa, CA). The methyllycaconitine used for dosing the mice was extracted from Delphinium barbeyi plant material using methods previously described (Gardner et al., 2000). The extracted methyllycaconitine was determined to be 76% pure when analyzed by flow-injection electrospray ionization mass spectrometry against a methyllycaconitine standard (Latoxan, Valence, France). Electrospray mass spectral data were acquired on a Finnigan LCQ Mass Spectrometer (Finnigan Corp., San Jose, CA). Samples were loop-injected into the electrospray source using a 50:50 methanol:1% acetic acid solution at a flow rate of 0.5 mL/min. The greatest impurity detected in the extracted methyllycaconitine had a mass of 465, which is consistent with the alkaloids 14-dehydrobrowniine and deltamine, and was present at approximately 4%. 14-Dehydrobrowniine and deltamine have both been found in D. barbeyi plant material, and previously reported LD50 estimates are one and two orders of magnitude greater than methyllycaconitine, respectively (Manners et al., 1993). The same lot of extracted methyllycaconitine was used exclusively in both vaccination trials. The extracted methyllycaconitine was dissolved in buffered physiological saline using previously established protocols (Manners et al., 1993). The methyllycaconitine solution was sterile filtered through 0.22-µm syringe filters and stored in sterile injection vials at 4°C for toxicity testing. Methyllycaconitine solutions prepared in this manner and stored at 4°C are stable for >6 mo after preparation (Panter, unpublished data).

The preparation of lycoctonine, 14-deacetylnudicauline, and methyllycaconitine conjugates was previously described in detail (Lee et al., 2000), and structures are shown in Figure 2Go. The targeted conjugation ratios of the lycoctonine-N-alkyl carbamate-protein conjugates were approximately 20:1 hapten to fetuin and approximately 23:1 hapten to BSA; these conjugates are designated as LYC and LYC-BSA, respectively. The targeted conjugation ratios of the 14-deacetylnudicauline-14-succinate-protein conjugates were approximately 20:1 hapten to fetuin and approximately 6:1 hapten to BSA; these conjugates are designated as DAN and DAN-BSA, respectively. The targeted conjugation ratios of the methyllycaconitine succinate-protein conjugates reactions were approximately 20:1 hapten to fetuin and approximately 5:1 hapten to BSA; these conjugates are designated as MLA and MLA-BSA, respectively. All alkaloid–protein conjugates were diluted to a concentration of approximately 1 mg/mL in deionized distilled water and stored (-120°C) as 1-mL aliquots until use.



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  		Figure 2. Structural depictions of the immunoconjugates lycoctonine (LYC), methyllycaconitine (MLA), 14-deacetylnudicauline (DAN).

 
The primary immunogen injection solution was prepared by adding an aqueous hapten–fetuin conjugate solution (1 mg/mL, 2.25 mL) to sterile saline (2.25 mL). This solution was in turn emulsified with complete Freund’s adjuvant (4.5 mL) yielding a total volume of 9 mL. Booster injection solutions were prepared in the same manner as the primary injection solution with incomplete Freund’s adjuvant used in place of complete Freund’s adjuvant. The control primary injection solution and control booster solution consisted of equal parts sterile saline solution emulsified with complete Freund’s adjuvant or incomplete Freund’s adjuvant, respectively.

Titer/Competitive Binding Assays
Titer and competitive binding assays were performed on 96-well NUNC F96 Maxisorp polystyrene microtiter plates (VWR Scientific Products, Denver, CO). Toxin-BSA coating conjugates were dissolved in carbonate buffer (250 ng/100 µL, 0.05 M, pH 9.6) and 100 µL added to each well of a microtiter plate. Microtiter plates were incubated (2 h, room temperature [RT] and then 16 h, 4°C), inverted to remove excess coating solution, covered with an adhesive plate sealer, and stored in a plastic bag (-20°C; up to 6 mo). The plates were washed (3x) with saline-Tween buffer (0.15 M NaCl, 0.5% Tween 20) and blotted dry. Blocking buffer (150 µL; 0.1 M Tris, pH 7.5, 0.1% Tween 20, 5% skim milk powder) was added, and then the plates were incubated (1 h, RT). The plates were then washed (3x) and blotted dry.

For the competitive binding assay, free methyllycaconitine (50 ng) diluted in the blocking buffer (50 µL) was added to the wells in triplicate followed by 50 µL of antiserum diluted (1:30,000 and 1:60,000 for the mice injected with the MLA and DAN immunoconjugates, respectively) in the blocking buffer. For titer measurements, made in the absence of free toxin, the serum was diluted serially (1:1000 to 1:512,000) in blocking buffer serial dilutions and added to the wells in singlet. The plates were incubated (2 h, RT), washed (4x), and HRP conjugated goat anti-mouse IgG (100 µL) diluted 1/10,000 in blocking buffer was added to all wells. The plates were incubated (1 h, RT), washed (4x), and 100 µL of tetramethylbenzidine/H2O2 substrate (pH 5.5; 30°C; Bos et al., 1981) was added to each well. After 10 min, the reaction was stopped by the addition of 50 µL of 0.5 M H2SO4 to each well and the UV absorbances were measured at 450 nm with a BIO-RAD model 3550-UV microplate reader (Bio Rad Laboratories, Hercules, CA).

Vaccinations/Mouse Bioassay
Two independent vaccination trials with subsequent toxicity challenge using methyllycaconitine were completed in mice. White Swiss-Webster male mice (Simonsen Labs, Gilroy, CA; weanlings 15 to 20 g) were initially injected s.c. with primary injection solution (0.2 mL) for a dose of 50 µg of hapten–fetuin conjugate. Booster injections (0.2 mL) with the same dose of hapten–fetuin conjugate in incomplete Freund’s adjuvant were given at 3-wk intervals. Control mice were initially injected s.c. with control primary injection solution. At the same time, the treated mice received booster injections the control mice were given control booster injections (0.2 mL).

In Trial 1, two groups of 45 mice each were vaccinated with either MLA or DAN conjugate and received one booster. A third group of 45 mice was treated with control vaccines. Ten days after the single booster, 10 mice from each group were killed and their sera collected to determine whether the mice had developed titers to the conjugates and to determine whether the antisera bound free methyllycaconitine in vitro before dosing the mice with methyllycaconitine. Sixteen days after the booster and a 12-h fast, vaccinated and control mice were weighed, separated into treatment groups of 35 mice each, and dosed with i.v. injections of methyllycaconitine. Methyllycaconitine injections were performed via the tail vein in mice restrained in a plastic mouse block. The mice were maintained under a heat lamp for 15 min to dilate the tail vein. The tail vein was cleaned with 70% ethanol and i.v. injections were accomplished with a tuberculin syringe equipped with a 1.27-cm-long 27-gauge needle. The volume injected varied depending on the dosage delivered. Methyllycaconitine-induced death occurred in <5 min after injection. Time of injection, clinical effects, and time of death were noted and recorded.

In Trial 2, three groups of 100 mice each were vaccinated with MLA, DAN, or LYC conjugates. A fourth group with 30 mice was treated with control vaccines. Ten days after the second booster and a 12-h fast, vaccinated and control mice were weighed, separated into treatment groups, and dosed with i.v. injections of methyllycaconitine using the procedure described in Trial 1. Time of injection, clinical effects, and time of death were noted and recorded. In this trial, mice that did not die within 5 min after injection were euthanized with carbon dioxide. Blood was drawn via heart puncture immediately after methyllycaconitine-induced death or euthanasia, sera were obtained, and titers were determined.

Statistics and Calculations
Titer values were determined by the method of Raghava et al. (2001). The titer value in this study is defined as the sera dilution at which the optical density at 450 nm was 0.5. Serum from one mouse in each of the three groups was chosen as a standard. The standard sera titer curve was run on the same plate as titer curves from all of the other sera collected for the mice within the same group. All titer curves within each group were then normalized with respect to the standard titer curve. A mouse was determined to have a titer if the absorbance at a sera dilution of 1:1000 was greater than the absorbance of the control-immunized mice sera by three times the absorbance of the control mice sera at the same sera dilution. The competitive binding assay was positive when the absorbance readings were less than the blank by three times the standard deviation of the blank.

The LD50 for methyllycaconitine toxicity in each vaccination group was determined by a modified up-and-down method (Bruce, 1985) and was calculated using the PROC PROBIT procedures of SAS (SAS Inst., Inc., Cary, NC) on a logistic distribution of the survival data. Confidence (fiducial) intervals (95%) were also calculated using the same program.

In Trial 2, the probability of survival within each vaccination group was compared using titer and dose in a logistic regression model. We used the PROC PROBIT procedure with logistic distribution of SAS (SAS Inst., Inc.) to fit these models. Dose, titer, and their interaction were factors used in the model. Binary data (i.e., lived or died) were not transformed prior to analysis in SAS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Trial 1
All 10 mice in the MLA- and DAN-vaccinated groups killed previous to dosing with methyllycaconitine had titers. The sera from these mice also bound methyllycaconitine in vitro. None of the 10 mice in the control group had titers. Lethal dose 50% values and the 95% confidence intervals for control and MLA- and DAN-vaccinated groups are shown in Table 1Go. Overlapping 95% confidence intervals did not provide evidence of differences, although an increase of 8.6 and 34.7% was observed in the LD50 of the MLA and DAN groups, respectively, compared to the control group.


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  		Table 1. Calculated lethal dose 50% values for mice vaccinated in Trials 1 and 2 against methyllycaconitine with methyllycaconitine (MLA), 14-deacetylnudicauline (DAN), and lycoctonine (LYC) protein conjugates
 
Trial 2
Lethal dose 50% values and the 95% confidence intervals determined for the control, MLA, DAN, and LYC groups are shown in Table 1Go. Similar to Trial 1, the overlapping confidence intervals did not provide evidence of differences between the LD50 of the MLA, DAN, LYC, and control groups; however, an increase in the LD50 of the MLA, DAN, and LYC groups relative to the control group was again observed.

Variations in previously reported LD50 for methyllycaconitine of 7.5 (Manners et al., 1993) and 4.8 mg/kg (Panter et al., 2002), and those reported in this study of 2.91 and 3.86 mg/kg for Trials 1 and 2, respectively, were noted and may be explained in three ways. First, the relative purity of earlier isolations of methyllycaconitine from plant material is now known to vary significantly. Second, the mice used in Trials 1 and 2 in this study and in previously reported studies were from the same strain and from the same supplier, but were from different lots. Third, the mice in Trial 2 received an additional booster and were 2 wk older and an average of 8 g larger than the mice in Trial 1. All three reasons stated above may account for differences between previously reported LD50 values and the values reported in this study, whereas the second and third reasons may be explanations for the difference in the LD50 between mice in Trials 1 and 2 in this study. For these reasons a control group was included with each trial. Each treatment was compared to the control group run within the trial.

Relationship Between Mice Survival and Antibody Titer Levels: Trial 2
Sera collected from all mice vaccinated with MLA, DAN, and LYC immunogens in Trial 2 had titers, but none was observed in the composite sera of the control mice. The median titers were 41,989 for mice vaccinated with MLA, 90,187 for mice vaccinated with DAN, and 45,401 for mice vaccinated with LYC.

No interactions between dose and titer were detected (P > 0.10) for any conjugate. No relationship was found (P = 0.365) between survival and titer with the MLA group; however, dose was related (P = 0.001) to survival. Both titer (P = 0.030) and dose (P = 0.0001) were related to survival of the mice in the DAN (7) group. Both titer (P = 0.001) and dose (P = 0.044) were also related to survival of the mice in the LYC (5) group. As the challenge dose of methyllycaconitine increased, fewer mice survived; conversely, more mice with higher titer levels survived than those with lower titer levels. However, at the very highest methyllycaconitine doses, the toxin dose was so great that all animals died regardless of titer level. Because both titer and dose were related to survival of the mice in the DAN and LYC groups, we recalculated the LD50 for mice in the DAN and LYC groups using only mice that had titers > 120,000 and > 47,000, respectively. This resulted in a 20% increase in LD50 values to 4.67 mg/kg for the DAN mice and 4.60 mg/kg with the LYC mice compared to the LD50 value for the control group. In both cases, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups.

To determine the significance of titer without the overwhelming effects from the highest methyllycaconitine doses, the logistic regression was run after removing methyllycaconitine dose levels of 4.5 mg/kg or greater. A methyllycaconitine dose of 4.5 mg/kg is approximately the LD75 for DAN and LYC mice. Using this "dose-corrected" model, titers alone were related (P = 0.001) to survival with the LYC group, whereas dose was not related (P = 0.828). In the DAN group using the "dose-corrected" model, only dose remained significant (P = 0.003), whereas titers were not related to survival (P = 0.253). The relationship between titer levels alone and the probability of mouse survival for all three conjugates is shown in Figure 3Go. The sigmoid-shaped curve for the LYC group in Figure 3Go shows that only this group fits a logistic regression.



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  		Figure 3. The relationship between titers (x-axis) and the probability of mouse survival (0 to 1 on the y-axis) for mice vaccinated with LYC ({square}), MLA ({triangleup}), or DAN ({circ}) in Trial 2.

 
When a strong relationship between titer and survival exists, and if titer is known, we can predict the probability of survival of an animal at different toxin doses. Figure 4Go shows the predicted relationship between methyllycaconitine challenge doses (up to 4.5 mg/kg BW), serum titer level, and the probability of mice survival for the LYC conjugate. As methyllycaconitine doses increase, higher titer levels are required to maintain protection (i.e., probability of survival), whereas lower titer levels provide a reduced protective effect even at lower methyllycaconitine doses.



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  		Figure 4. The predicted relationship between titers and the probability of mice survival for mice vaccinated with the LYC conjugate and dosed with methyllycaconitine at levels of 3.5 mg/kg BW ({blacksquare}), 3.7 mg/kg BW ({diamondsuit}), 3.9 mg/kg BW ({blacktriangleup}), 4.1 mg/kg BW (x), 4.3 mg/kg BW (•), and 4.5 mg/kg BW (+).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Several active immunization studies with animals have demonstrated protection against poisonous plant-induced toxicoses. Mice and rabbits vaccinated with ergot alkaloid protein conjugates displayed protection against endophyte-infected fescue toxicosis (Rice et al., 1996; Filipov et al., 1998). A field vaccine against phomopsin mycotoxicosis (lupinosis) in sheep has been successful in Australia (Payne et al. 1992; Allen et al., 1994; Than et al., 1994; Edgar et al., 1998). A similar vaccine provided protection in sheep against annual ryegrass toxicosis and flood plain staggers (Than et al., 1998). Sheep and cattle vaccinated with lantadene–protein conjugates resulted in mild protection against the hepatotoxic effects of lantana poisoning (Stewart et al., 1988; Pass and Stewart, 1992). Cox (1985) reported the vaccination of sheep with a protein conjugate that provided partial protection against the toxic metabolites of phytoestrogens.

Other active immunization studies with plant toxins have resulted in increased toxicosis after vaccination with protein–toxin immunoconjugates. Rats vaccinated against senecionine with a senecionine–protein conjugate demonstrated increased toxicosis when dosed with free senecionine toxin (Culvenor, 1978). Similarly, gilts vaccinated with zearalenone–protein conjugates showed increased toxicosis compared to nonvaccinated gilts fed zearalenone (MacDougald et al., 1990). Mice were reportedly protected against sporidesmin toxicosis after vaccination (Jonas and Erasmuson, 1979); however, later studies showed increased toxicosis resulted when sheep were vaccinated with the same toxin conjugate (Fairclough et al., 1984).

In the successful vaccination studies with lantana (Stewart et al., 1988; Pass and Stewart, 1992), phytoestrogen (Cox, 1985) and lupinosis poisoning (Payne et al., 1992; Allen et al. 1994; Than et al., 1994; Edgar et al., 1998), a positive relationship between antibody titers and vaccine effectiveness was observed. Other studies suggest that the generation of antibodies alone does not ensure protection against the toxin (Culvenor, 1978; Fairclough et al., 1984; Cox, 1985; Cadot et al., 1986). In our trials, antibodies that bound methyllycaconitine in vitro were raised in mice. Vaccinating mice with larkspur toxin–protein carrier conjugates appeared to provide a mild protective effect against methyllycaconitine toxicity. Nonetheless, in both trials, the 95% confidence limits for the LD50 between the vaccinated and control groups overlapped. In Trial 2, we found a strong relationship between antibody titer and survival with the mice vaccinated with the LYC immunogen. This allowed us to predict the probability of survival of LYC-vaccinated mice with a known titer. When mice with low antibody titers were removed from the DAN- and LYC-vaccinated groups and the LD50 recalculated, a 20% increase was observed in the LD50 values of these groups compared to the control group. Again, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups.

It has been suggested that the immune system has evolved to degrade macromolecules by breaking peptide bonds and carbohydrate linkages and that if the toxin cannot be degraded by the immune system, the presence of antibodies may act as a method to temporarily bind and redistribute the toxin (Edgar, 1994). This mechanism could act to protect the animal from acute toxicosis by effectively binding some of the toxin and reducing its distribution to receptor sites, or conversely, contribute to increased or delayed toxicoses by retaining the toxin in the animal’s circulatory system, preventing the toxin from being metabolized or eliminated from the animal. Interestingly, many of the successful plant toxin immunization studies have been with toxins that include peptide or carbohydrate linkages, such as the ergot alkaloids, phomopsin mycotoxins and corynetoxins implicated in fescue toxicosis, lupinosis, and annual ryegrass toxicosis, respectively. This information and our results suggest the toxin methyllycaconitine, which does not contain peptide or carbohydrate linkages, is bound by the antibodies, but not readily degraded by the immune system. The mild trend toward protection observed in these vaccination trials is likely due to the temporary binding of methyllycaconitine by the circulating antibodies reducing the amount available to bind to acetylcholine receptors.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Vaccinations may be a useful tool to reduce livestock losses to toxic plants. The results suggest that vaccination with the lycoctonine–protein conjugate alters methyllycaconitine toxicity in mice. Further studies will be required to evaluate the efficacy of vaccines targeted against larkspur toxins that kill livestock. Nonetheless, vaccination with lycoctonine or other similar conjugates may be useful in the future to decrease the effects of larkspur toxins in animals.


   Footnotes
 
1 The protocol for animal use in this research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Utah State University, Logan. We thank S. L. Durham, Utah State University, Logan, for statistical assistance. We thank R. A. Anderson, L. A. Buhler, M. J. Chambers, J. W. Hartle, E. L. Knoppel, and J. Marshall for technical assistance. Back

Received for publication April 4, 2002. Accepted for publication September 24, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Allen, J. G., K. A. Than, J. A. Edgar, G. H. Doncon, G. Dragicevic, and V. H. Kosmac. 1994. Field evaluations of vaccines against lupinosis. Page 427 in Plant-Associated Toxins: Agricultural, Phytochemical and Ecological Aspects. S. M. Colgate and P. R. Dorling, ed. CAB International, Wallingford, U.K.

Benn, M. H., and J. Jacyno. 1983. The toxicology and pharmacology of diterpenoid alkaloids. Page 153 in Alkaloids: Chemical and Biological Perspectives. Vol. 1. S. W. Pelletier, ed. Wiley, New York.

Bos, E. S., A. A. van der Doelen, N. van Rooy, and A. H. W. M. Schuurs. 1981. 3,3',5,5'-Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J. Immunoassay 2:187–204.[Medline]

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Cadot, R., J. Descotes, C. Y. Cuilleron, C. Grenot, and J. C. Evereux. 1986. Evaluation of active specific immunization against paraquat toxicity in rats. Vet. Hum. Toxicol. 28:226–229.[Medline]

Cox, R. I. 1985. Immunophysiological control of phyto-oestrogen toxicity. Page 98 in Plant Toxicology. A. A. Seawright, M. P. Hegarty, L. F. James, and R. F. Keeler, ed. Poisonous Plants Committee, Yeerongpilly, Queensland.

Culvenor, C. C. J. 1978. Prevention of pyrrolizidine alkaloid poisoning—animal adaption or plant control? Page 189 in Effects of Poisonous Plants on Livestock. R. F. Keeler, K. R. van Kampen, and L. F. James, ed. Academic Press, New York.

Dobelis, P., J. E. Madl, J. A. Pfister, G. D. Manners, and J. P. Walrond. 1999. Effects of Delphinium alkaloids on neuromuscular transmission. J. Pharmacol. Exp. Ther. 291:538–546.[Abstract/Free Full Text]

Edgar, J. A. 1994. Vaccination against poisoning diseases. Page 421 in Plant-Associated Toxins: Agricultural, Phytochemical and Ecological Aspects. S. M. Colgate and P. R. Dorling, ed. CAB International, Wallingford, Oxon.

Edgar, J. A., K. A. Than, A. L. Payne, N. Anderton, J. Baell, Y. Cao, P. A. Cockrum, A. Michalewicz, P. L. Stewart, and J. G. Allen. 1998. Towards a commercial vaccine against lupinosis. Page 196 in Toxic Plants and Other Natural Toxicants. T. Garland and A. C. Barr, ed. CAB International, Wallingford, UK.

Fairclough, R. J., J. W. Ronaldson, W. W. Jonas, P. H. Mortimer, and A. G. Erasmuson. 1984. Failure of immunisation against sporidesmin or a structurally related compound to protect ewes against facial eczema. N. Z. Vet. J. 32:101–104.[Medline]

Filipov, N. M., F. N. Thompson, N. S. Hill, D. L. Dawe, J. A. Stuedemann, J. C. Price, and C. K. Smith. 1998. Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed-based diets on rabbits. J. Anim. Sci. 76:2456–2463.[Abstract/Free Full Text]

Gardner, D. R., G. D. Manners, K. E. Panter, S. T. Lee, and J. A. Pfister. 2000. Three new toxic norditerpenoid alkaloids from the low larkspur Delphinium nuttallianum. J. Nat. Prod. 63:1127–1130.[Medline]

Jonas, W. E., and A. F. Erasmuson. 1979. The effect of immunizing mice with a derivative of 2-amino-5-chloro-3,4-dimethoxy benzyl alcohol coupled to some bacteria on sporidesmin-induced bilirubinaemia. N. Z. Vet. J. 27:61–63.[Medline]

Kukel, C. F., and K. R. Jennings. 1994. Delphinium alkaloids as inhibitors of {alpha}-bungarotoxin binding in rat and insect neural membranes. Can. J. Physiol. Pharmacol. 72:104–107.[Medline]

Lee, S. T., T. K. Schoch, D. R. Gardner, and B. L. Stegelmeier. 2000. Development of enzyme-linked immunosorbent assays for toxic larkspur (Delphinium spp.) alkaloids. J. Agric. Food Chem. 48:4520–4528.[Medline]

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