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

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