This document describes the development of a PCR-based method to detect Delphinium species (larkspur) in the rumen contents of cattle poisoned by grazing on these plants. Primers were designed that specifically target larkspur DNA. Testing showed larkspur DNA could be detected by PCR up to 72 hours after ingestion, even when diluted to concentrations lower than what is expected in the rumen during poisoning. This larkspur detection method using PCR shows potential as a diagnostic tool for cattle poisoned by grazing on larkspur plants.

1. Development of a PCR-Based
Method for
Detection of Delphinium
spp. in Poisoned Cattle
Daniel Cook

2. Delphinium (Larkspur)

3. Clinical Signs
-Staggering gait
-Muscular trembles
-Periodic sternal then lateral recumbency
(this can lead to death for various reasons)
-Difficulty breathing (rapid and shallow)
-Death occurs from respiratory paralysis and/or
bloat

4. CH3O
N
OH
OCH3
OH
OCH3
CH3O
O
O
N
O
O
Methyllycaconitine (MLA)
Toxic Alkaloids

5. Objective
To develop a diagnositc tool to identify poisonous
plants ingested by animals

6. Polymerase Chain Reaction (PCR)
A DNA Photocopier

7. Primer Design

8. Primer Specificity
1- D. occidentale
2 – D. nutallianum
3 - D. andersonii
4 – D. glaucescens
5 – D. scaposum
6 – D. ramosum
7 – D. barbeyi
8 – D. occidentale
9 – Aconitum
10 – M. sativa (alfalfa)

9. Methods
 In Vivo Rumen Digestion (Live Cow)
5 grams of larkspur incubated in digestion bags in rumen of cannulated cows
Time Course 4h-48h
 DNA extraction
 PCR
 Agarose gel electrophoresis – 1.5%

10. Live Cow Digestion

11. Methods
 In Vitro Rumen Digestion (Dead Cow)
5 grams of larkspur incubated in digestion bags in rumen fluid in incubator
Time Course 8h-48h
 DNA extraction
 PCR
 Agarose gel electrophoresis – 1.5%

12. Dead Cow Digestion

13. Methods
 In Vitro Rumen Digestion (Poisoned Cow that Died)
5 grams of larkspur incubated in digestion bags in rumen of cannulated cows
(8h) removed and incubated in rumen fluid in incubator
Time Course 8h-72h
 DNA extraction
 PCR
 Agarose gel electrophoresis – 1.5%

14. Poisoned Cow

15. Dilutions-sensitivity of detection
 Larkspur (g) : Alfalfa (g)
 8 hours of live digestion
 Sensitivity to PCR
 Mimic cow rumen content (larkspur likely would
represent 5-10% of rumen contents in poisoned animal

16. Dilutions-(Plant Material only)

17. Digested Dilutions

18. Summary
 Larkspur specific primers were developed
 Larkspur can be detected 48-72 after ingestion of
larkspur and death of animal.
 Larkspur can be detected in matrix more dilute than what
one would expect to find in rumen of poisoned animal
 Results suggest this has potential as a diagnostic tool

19. Acknowledgements
 Jim Pfister
 John Constantino
 Jessie Roper
 Kermit Price