Nematodes

3.2.1. Collection of Fecal Samples (see Note 1)

1. With gloves on, take 6-10 g of fecal matter per animal by anal ring stimulation, avoiding contamination with free life nematodes or other elements that can lead to erroneous diagnosis.

2. When you have many animals, screening can take place in 8-10% of the same species.

3. Place the samples in the recipient and label (see Note 1)

4. Send to the laboratory under refrigerated conditions.

3.2.2. Microscopic Examination of Previous Enrichment: Willis' Technique (12) (see Note 2)

1. NaCl-satured solution: Dissolve by agitation and heat 360 g NaCl in 1000 mL of distilled water (see Note 3).

2. Place in the precipitation glass a sample of fecal matter about the size of a chickpea (or a teaspoonful) (see Note 4).

3. Fill the fourth part of the precipitation flask with saturated saline solution and mix with a glass rod until homogenization.

4. Strain to another glass, rinsing the first one with the saturated saline solution.

5. Fill the precipitation glass with the saline solution until the border forms a meniscus.

6. Place a clean slide on the liquid surface for 15 min. Then quickly invert the slide, add a drop of Lugol, put a cover on, and observe microscopically (see Note 5).

3.2.3. Fecal Nematode Egg Counts: Modified McMaster Method (13)

1. Homogenize in a mortar 5 g of fecal matter with the saturated solution, filter (through the strainer with wire mesh) in a precipitation glass, and add more saturated solution up to a volume of 100 mL (dilution 1:20).

2. Stir gently to avoid exaggerated formation of air bubbles and fill camera's cells with a Pasteur pipet.

3. Place on a microscope plate and after 2 or 3 min to begin the count using a magnification of 60-70x. The nematode eggs are added to the interior surface of the camera's cell, in the same plane that eventually bubbles.

4. Eggs from one, two, or four cells can be counted. If the eggs contained in one cell are counted, multiply by 40; if the eggs contained in two cells are counted, multiply by 20; and if eggs from four cells are counted, multiply by 10 (see Note 5).

5. Correction factor according to fecal consistency (for sheep and goats):

Normal x 1

Mash not formed x 2

Liquid thick x 3

Liquid diarrhea x 4

3.2.4. Coprocultures: Modified Roberts and O'Sullivan Technique (see Note 4)

1. Place 3 g of fecal matter in the mortar and crush.

2. Place the crushed feces in a 5-cm Petri dish and add the bacterial suspension under evaluation. Add some Telgopor pearls and chlorine-free water until a consistent mass is achieved.

3. Cover the Petri dish and incubate at 25-28°C during 10 d.

4. For the control coprocultures, proceed in the same way but without adding the bacterial suspension.

5. To collect the infecting larvae (L3), fill a Petri dish with chlorine-free water up to the edge; use another 10-cm Petri dish for a cover. Pour quickly to avoid spilling the water.

6. Place 5 mL of chlorine-free water in a 10-cm Petri dish and leave for 3-4 h.

7. Collect the water in the Petri dish containing the larvae and place them in an assay tube. Keep under refrigeration for 2-3 h and then remove the supernatants, placing 3-4 mL in the assay tube (see Notes 5 and 6).

3.2.5. Third-Stage Larvae (L3) Counts

1. After 3-4 h the larvae in the Petri dish will displace to the water of the dish. Using a Pasteur pipet, place a small volume of water containing larvae in the camera to count the number of larvae.

2. During microscopic observation, count the dead L3 larvae first. Then kill the remaining larvae by heat (placing the camera near the burner), and count the total L3 larvae.

3. Calculate viable total L3 larvae from the difference between total L3 and dead L3 larvae.

4. Continue in the same way for each coproculture (see Note 5).

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