Coproculture
Aims:
Coproculture allows for the development of gastrointestinal nematodes of ruminants and horses from eggs into third stage larvae for more specific identification. This is done by simulating the optimal environmental conditions to support larval maturation in the laboratory setting.
Material:
- Faecal sample
- 1 plastic container (volume: 1,000 mL)
- 1 petri dish with a diameter greater than that of the plastic container
- Spring water
- Nebulizer
- 1 50 mL conical tube
- 1 laboratory centrifuge
- Pasteur pipettes
- Lugol’s solution
- Glass microscope slides and coverslips
Procedure:
Coprocultures may be performed on a faecal sample from individual animals or a pooled samples from multiple animals. Coprocultures are commonly performed on positive samples for which the faecal egg count (FEC) has been determined to identify the distribution of nematode species within the sample. Place 100 g of faeces in the bottom of the 1,000 mL plastic container (ratio 1:10) without mixing the sample. Place the co-culture in an incubator (incubation cabinet) set to 24 °C±1 °C for a duration of 10 days, humidifying the sample daily with a nebulizer. After 10 days, remove the container from the incubator and fill it completely with warm spring water. Carefully overturn the filled container onto a Petri dish and add an additional 40 ml of spring water to the Petri dish. Allow this to sit for 24 hours in an area of direct sunlight. This will allow the L3 larvae to emerge from the faecal sample and actively move from the container into the Petri dish. After the 24 hours, collect the ~40 mL of water (and larvae) within the Petri dish using a transfer pipette. Place the collected solution into a 50 mL conical tube and centrifuge the tube at 2,500 rpm for 10 minutes. Remove the majority of the supernatant, leaving approximately 5 mL of suspension in the bottom of the tube. The remaining liquid will contain the larvae.
Results:
Place a small amount of the larval suspension (approximately 50 µL) on a glass slide. To immobilize the larvae, add 1 drop of Lugol's iodine and place a cover slip on the slide. Examine the slide under an optical microscope observing the larvae at high magnification (up to the 40x objective). Focus on the defining morphologic characteristics and take careful measurements of the larvae to compare to morphometric keys reported in the literature for species identification.
References:
Euzeby, 1981. Diagnostic expérimentale des helmintoses animales. Travaux pratiques d’helmintologie vétérinaire. Livre 1. Paris, France.
Cernea, M.; Madeira de Carvalho, L.M.; Cozma, V. Atlas de Diagnostic al Strongilidozelor la Ecvine; Academic Press: Cluj-Napoca, Romania, 2008.
MAFF, 1986. Manual of veterinary parasitological techniques. Technical Bulletin No.18. Third ed. Her Majesty's Stationery Office London, UK
Van Wyk, J.A., Mayhew, E. (2013) Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide. Onderstepoort Journal of Veterinary Research, 80 (1). DOI: 10.4102/ojvr.v80i1.539