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Recovery of Larval Parasites (RLP
- 1. Recovery of Larval Parasites from Soil, Tissues and Pastures Sabahat Amin M.Phil Parasitology
- 2. Larval parasites • “Parasite having larval stage in their life cycle known as larval parasite”. • Almost all nematodes, insects and arachnids are larval parasites. • Ex: – soil transmitted helminthes – flies maggots, etc.
- 3. • Larval parasites on pasture ( All GIT parasites) – Haemonchus contortus – Trichostrongylid • Larval parasites in soil – Whip worm – Ascaris – Pin worm • Larval parasite in tissues – Ascaris lubricoides
- 5. Recovery of larvae from soil • Livestock with access to pasture – exposed to many parasitic nematode species. • Uptake of infective 3rd stage larvae • L3 can survive on pasture, particularly in soil • For months or sometimes even longer • Depending on the temperature and humidity • For health and productivity of animals • It is necessary to check soil as well as pasture samples
- 6. • To determine the intensity and specie of nematode larvae. • For the recovery of plant and soil nematode several methods have been developed. – Reverse water flow and sieving – Centrifugal-flotation technique – Sugar-flotation-sieving. • Rapid method for the recovery of L3 is Centrifugal- flotation technique.
- 7. Centrifugal flotation technique • Sample range 50-500g. • Procedure: • For recovery of L3 larvae subsequent sieving process was performed. • A sieve of 200µm placed on 25µm . • Stack of sieves placed on rim of bucket. • Soil sample washed using jet of tap water
- 8. • Large particles remain on the 200µm sieve • Small particles were flushed through in both sieves and discarded. • Larvae and other particles between 25-200 µm • Remain on the 25µm sieve carefully collected into a 50 ml centrifuge tube • by either rinsing or removing with a disposable plastic pipette.
- 9. 50-500g Sieving process 200µm on the 25µm Wash with tap water Stop when no particle Larvae on 25µm sieve 50ml centrifuge tube Vigorous shaking Centrifuge 1800rpm/5min
- 10. Supernatant discarded To separate larvae Tube filled with sugar solution centrifuged 1800rpm Supernatant removed having L3 Place on sieve Wash to remove sugar Rinsed in chamber
- 11. Advantages • Easy • Affordable • Eco friendly • Simple • Fast • 20 sample/day
- 12. Pasture larval count • The concentration of larvae is higher in the lower portion • In ryegrass the 80% larvae were in the first 5cm of pasture height. • The supply of L3 larvae is not equally distributed. • Samples should be collected covering large area • by using walking transects.
- 13. Basic protocol 10cm cut 400-500g Place in plastic bag Put in plastic bucket Having 10- 20ml water 2ml detergent
- 14. Kept for 8- 10h agitation Pasture removed Wash-series of sieve L3 will be harvest Store at room temp.
- 15. Interpretation • Below 1000 L3/kg of DM trichostrongyloids are safe • Between 1000 and 5000 L3 are moderately infective. • Above 5000 L3/kg of DM indicate high infectivity.
- 16. Bearmann method • Used for both pasture and soil larval parasites.
- 17. Recovery of larvae from tissue • Different methods have been used – Spin method – Tissue digest method – Bearmann method – Sedimentation technique – The disadvantage of last 3 is time consuming and lower recovery of larvae.
- 18. Spin method • Spin method is rapid, quantitative, allowed maximum recovery of larvae. • Protocol : • Mice killed by cervical dislocation • Lungs and liver removed • Rinsed in saline • Homogenized at 17000rpm for 20 sec in 40ml sodium citrate • Suspension strained through triple layer gauze
- 19. • Into 50ml centrifuge • Wash 3 times • The first wash involved centrifugation at 1,000 g for 60 sec • second and third washes involved centrifugation at 500 g for 20 sec. • Larval counts of total sediment (0.3 ml) were performed.