development of a pcr-based method for detection of delphinium spp. in poisoned cattle

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

Delphinium (Larkspur)

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

N

OH

OH

OCH 3

OCH3

CH3OO

CH3O

O

N

O

O

Methyllycaconitine (MLA)

Toxic Alkaloids

Objective

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

Polymerase Chain Reaction (PCR)

A DNA Photocopier

Primer Design

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)

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%

Live Cow Digestion

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%

Dead Cow Digestion

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%

Poisoned Cow

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

Dilutions-(Plant Material only)

Digested Dilutions

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

Acknowledgements

Jim Pfister John Constantino Jessie Roper Kermit Price