Page 154 - The Veterinary Laboratory and Field Manual 3rd Edition

Parasitology 123

of lukewarm water kept in a 250 ml glass the steps 4–6 as for extraction of lungworm
beaker. larvae.
4 Leave the setting overnight under light. 6 Add 4 drops of 10% formalin, to kill the lar-
5 Discard the envelope and decant the super- vae, and 3 drops of iodine to stain them.
natant without disturbing the sediment 7 Examine the sediment using the low or
(15–20 ml). medium power lens (4× or 10/20×) of the
6 Collect the sediment in a centrifuge tube and microscope.
centrifuge at low speed (1000–1500 rpm) for 8 Examine morphological features includ-
10 min. Carefully discard the supernatant and ing the tail shape and measure the length
examine under low or medium power objec- of the larvae using a calibrated eyepiece
tive for larvae present in 1 ml of the sediment. (see Chapter 2 – microscope section), make
detailed drawings so that these can be com-
LarvaL cuLturE pared with those in text books (or consult a
Owing to the fact that many important hel- local expert). See Figure 3.4.
minth eggs look similar (that is, the common
Trichostrongyles) it is often necessary to One drawback of the above technique is the
hatch out the larvae to enable identification failure to obtain a clean culture without fae-
of the genus and species of parasite. In cases cal debris. Recent advancements in the field of
of anthelmintic resistance and/or assessment of molecular testing for anthelmintic resistance
treatment regimens in experimental and field tri- requires relatively pure larval culture and the
als this becomes very important. The following following protocol may yield a better result.
simple method is easy to perform but requires
patience and some practice to allow accurate Modified nematode larval culture
measuring of larvae and recording of data. Some
of the features used to identify parasite larvae 1 Weigh 30 g (minimum 10 g) faeces and place
are given in Figure 3.4. it into a 250 ml wide mouthed glass/plastic
Procedure beaker containing up to 1.5 cm of vermiculite
at the bottom.
1 Weigh out 10 g of the faecal sample and mix 2 Add same amount of vermiculite to the top
with charcoal or a desiccant (calcium carbon- and spray with tap water.
ate) to form a mixture with a dry consistency. 3 Mix faecal sample with vermiculite and push
2 Place the faeces in a culture jar (wide necked the mixture down to the bottom of the bea-
jar, 0.5 to 1 l volume) and place a Petri dish ker and make a hole in the middle to increase
on top. aeration to the culture.
3 Incubate the jar for 7–8 days at 28°C and 4 A plastic Petri dish is used as a lid for the
check the jar regularly. If necessary, add extra beaker but a rolled-up paper strip is placed
water to prevent the culture drying out. between the rim of the beaker and the Petri
4 After 8 days fill the jar with water, and allow dish to facilitate aeration.
to stand for 2–3 h. The larvae will migrate 5 Leave at room temperature (25ºC) for
into the water which can then be poured into 10–14 days but check regularly every other
a cylinder for sedimentation. day to moisten the culture by spraying with
5 Allow the sediment to settle and pour off water.
the supernatant or pour the suspension 6 For harvest, gently add lukewarm water on
of larvae into several test tubes and follow top of the culture up to the rim of the beaker.

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