development of a pcr-based method for detection of delphinium spp. in poisoned cattle
DESCRIPTION
Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned CattleTRANSCRIPT
![Page 1: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/1.jpg)
Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned CattleDaniel Cook
![Page 2: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/2.jpg)
Delphinium (Larkspur)
![Page 3: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/3.jpg)
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
![Page 4: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/4.jpg)
N
OH
OH
OCH 3
OCH3
CH3OO
CH3O
O
N
O
O
Methyllycaconitine (MLA)
Toxic Alkaloids
![Page 5: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/5.jpg)
Objective
To develop a diagnositc tool to identify poisonous plants ingested by animals
![Page 6: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/6.jpg)
Polymerase Chain Reaction (PCR)
A DNA Photocopier
![Page 7: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/7.jpg)
Primer Design
![Page 8: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/8.jpg)
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)
![Page 9: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/9.jpg)
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%
![Page 10: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/10.jpg)
Live Cow Digestion
![Page 11: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/11.jpg)
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%
![Page 12: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/12.jpg)
Dead Cow Digestion
![Page 13: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/13.jpg)
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%
![Page 14: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/14.jpg)
Poisoned Cow
![Page 15: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/15.jpg)
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
![Page 16: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/16.jpg)
Dilutions-(Plant Material only)
![Page 17: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/17.jpg)
Digested Dilutions
![Page 18: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/18.jpg)
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
![Page 19: Development of a PCR-Based Method for Detection of Delphinium spp. in Poisoned Cattle](https://reader035.vdocument.in/reader035/viewer/2022062707/55855badd8b42a54608b5248/html5/thumbnails/19.jpg)
Acknowledgements
Jim Pfister John Constantino Jessie Roper Kermit Price