4.1 Introduction
4.2 Equipment
4.3 Methods for post-mortem differential parasite counts
4.4 Interpreting adult nematode counts
4.5 Identifying gastro-intestinal parasites of sheep and goats
4.6 Post-mortem examination for trematodes
4.7 Post-mortem examination for cysticercosis
Post-mortem parasite counts provide a more precise assessment of parasite burdens than parasite egg counts. For parasite counts, the intestinal tract from abomasum to rectum is required. The adult and larval nematodes are carefully washed out, counted and identified. In addition, a complete postmortem examination of all organs should be done, bearing in mind alternative causes of ill health or death. It is important to record all abnormalities and lesions observed. A number of parasites will be found in almost every grazing animal, irrespective of the state of its health. To assess the significance of parasite infections in field mortalities, it is therefore necessary not only to determine the species present, but also to assess the number of each species.
Methods suitable for differential parasite counting under field or laboratory conditions using simple, easily obtainable and inexpensive equipment are described below.
(a) One or two deep trays of about 30 x 45 x 15 cm. The precise size is not important. Suitable plastic trays are easily procurable. A rectangular shape facilitates pouring from them.(b) One or two large, wide-mouthed plastic jars or buckets of about 3-5 litres capacity. These are used to collect the contents of each organ examined and hence are called the "total contents" jars. Calibrate the sides of the "total contents" jar in litres.
(c) A large kitchen ladle or similar utensil with about a 40 ml capacity and with a handle about 12 inches long.
(d) A smaller, wide-mouthed glass or plastic jar of about 500-1000 ml capacity. This jar must have a close-fitting screw-top lid. Make a hole in the top of the lid as large as possible, without interfering with proper sealing between the edge of the lid and the top of the jar.
Cut and fix a piece of brass wire or nylon mesh (40 mesh per linear inch) neatly inside the lid.
Calibrate the sides of the "wash jar" in 100 ml gradations.
This jar is used to wash the colouring matter out of the faeces and is called the "wash jar".
(e) Two glass petri dishes about 9 cm in diameter.(f) An aqueous solution of iodine.
(g) A saturated aqueous solution of sodium thiosulphate.
(h) A light box or some white background material. A large white tile is very suitable. Paper will suffice, or the bottom of a petri dish can be painted white.
(i) A mounted needle or fine forceps to handle the worms during counting.
(j) A jug and a bucket for handling water are useful additions to field equipment, although suitable utensils may be readily procurable from the farmer.
(k) An illuminated background. Much eye-strain may result from doing large numbers of worm counts indoors where lighting is poor or variable. An illuminated background overcomes this. Electric lamps, preferably fluorescent, are fitted inside a wide shallow box. The top of the box is made from translucent white plastic or ground glass. Samples in clear glass petri dishes are placed on top. The diffuse white light shining up through the petri dishes provides a strong contrast for the stained worms and no shadows are cast.
Counts of gastro-intestinal parasites are most conveniently done by examining the abomasum, small intestines and large intestines separately.
The following techniques are quantitative procedures for isolating, counting and identifying adult and larval nematodes in the abomasum and adult nematodes in the small and large intestines.
4.3.1 Differential parasite counts of the abomasum
4.3.2 Isolating inhibited/immature larvae from the abomasum
4.3.3 Differential parasite counts of the small intestines
4.3.4 Differential parasite counts of the large intestines
(c) Wash the empty abomasum thoroughly in the tray several times, paying particular attention to cleaning between the folds of the mucous membrane. Add the washings to the total contents jar.
(g) Fill the wash jar with water. Screw the lid on securely. Invert the jar and shake it till most of the fluid is shaken out. Repeat this process until all faecal culturing matter is removed.
(h) Add water to make the volume in the wash jar up to 50 ml (for convenience).
(i) Pour small volumes into petri dishes.
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NOTE: In the case of a field post-mortem, procedures (f)-(k) can conveniently be carried out on return to the laboratory. |
For cattle, multiply the total count for each species by 20 to arrive at the total burden in the animal examined (assuming that an original volume of 4 litres was used). For sheep and goats, multiply the count for each species by 10 or 15 to arrive at the total burden (assuming that an original volume of 2 or 3 litres was used).
For Haemonchus, small differences in worm burdens may cause significant differences in their pathogenic effect. For this reason, a more accurate assessment of the burden should be obtained by carrying out a total abomasal count of Haemonchus as opposed to the sub-sampling procedure described above.
4.3.2.1 Principle
4.3.2.2 Application
4.3.2.3 Equipment
4.3.2.4 Procedure
This is a quantitative procedure for isolating, counting and identifying larvae from the abomasal mucous membrane.
This technique is carried out in conjunction with the isolation of adult abomasal parasites. It can be used to determine:
· the number of abomasal nematodes present as immature larvae, and hence the ratio of immature larvae to adult nematodes;· the number and seasonal occurrence of inhibited larvae
To prevent immature larvae from being counted as inhibited larvae, the number of inhibited larvae should be determined only in animals kept isolated from reinfection for at least 21 days. This allows non-inhibited larvae to complete development.
· A tray or bucket· Normal physiological saline, 0.9% (see the Appendix at the end of this handbook for the formulation)
· Sieve or nylon net, 32-m m mesh
· Baker
· Petri dishes
· Wash bottle
· Pasteur pipette
· Microslides/coverslips
· Microscope/dissecting microscope
(b) Leave the abomasum to soak overnight.
(c) Remove the abomasum, rinse well with saline solution and discard.
(f) Using a dissecting microscope, examine an aliquot of 10 ml in a petri dish and count the larvae.
(g) To identify the parasite species, transfer further sub-samples by Pasteur pipette to microslides for examination under the microscope.(h) The total number of larvae is calculated as follows: number in 10 ml sub-sample x 20 = total abomasal larval count.
The principle and application of the differential parasite counts of the small intestine are the same as those for parasite counts of the abomasum.
(a) The procedure used for the small intestines is similar to that for the abomasum.(b) When examining the small intestines it is convenient to "run" the intestines out, free from the mesentery, into one tray.
(c) Initially, the gut is washed by pouring water into one end of the gut and flushing it out into the total volume jar. For further washing and scraping, the intestine has to be opened.
(d) It is important to scrape the mucous membrane in some manner to recover the smaller parasites, especially Trichostrongylus.
(e) Opening and scraping can be done quickly, efficiently and easily in one operation using a simple instrument that can be made by any skilled metal fitter. The instrument is called a "gut-runner".
The gut-runner
(f) When the small intestines have been opened, scraped and washed, place all of the contents plus all of the washings in the total contents jar.(g) The procedure for sampling, washing, sub-sampling, staining and counting is the same as previously described for parasites of the abomasum.
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NOTE: Even very large numbers of the smaller nematodes can be very easily overlooked unless some kind of washing procedure is used. They are very difficult to detect when mixed with faecal material. |
The principle and application of the differential parasite counts of the large intestine are the same as those for parasite counts of the abomasum.
(a) Uncoil the large intestines into one tray. Open them with scissors, placing the opened portion into the second tray.(b) The nematodes of the large intestines are easily seen. There are relatively few of them and they can be picked off with forceps as the gut is opened and can be placed in a petri dish containing water. Few parasites will be overlooked using this procedure.
(c) When the contents are fluid because of diarrhoea, or when a more precise count is required, the contents should be processed as described for the abomasum and small intestine. A large open sieve of 40 mesh/inch brass wire can be used.
Table 4.1 provides a guideline for interpreting adult nematode counts.
Table 4.1 A GUIDELINE TO THE INTERPRETATION OF ADULT NEMATODE COUNTS
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Nematode species |
Degree of infection |
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Light |
Moderate |
Heavy |
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CATTLE |
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Abomasal parasites |
1-5000 |
5000-10000 |
10000+ |
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Small intestinal parasites |
1-000 |
8000-20000 |
20000+ |
|
Haemonchus |
1-400 |
400-1500 |
1500+ |
|
Trichostrongylus |
1-10000 |
10000-25000 |
25000+ |
|
Cooperia |
1-5000 |
5000-10000 |
10000+ |
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SHEEP |
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Haemonchus |
1-500 |
500-1500 |
1500+ |
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Trichostrongylus |
1-1000 |
1000-10000 |
10000+ |
|
Nematodirus |
1-2500 |
2500-8000 |
8000+ |
|
Oesophagostomum |
1-50 |
50-150 |
150+ |
NOTE: These numbers should be considered only as a guideline in the interpretation of parasite burdens.
Tables 4.2 to 4.5 provide simple keys for identifying some common gastrointestinal parasites of sheep and goats, describing nematodes of the abomasum, small intestines and large intestines.
Table 4.2 A SIMPLE KEY FOR IDENTIFYING COMMON GASTRO INTESTINAL PARASITES OF SHEEP AND GOATS
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Organ location
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Head region
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Characteristics of Parasite |
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Anterior end |
Mature size |
Genus |
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Abomasum
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No cephalic swelling
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Cervical papilla present
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Large |
Haemonchus |
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Medium |
Ostertagia |
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Prominent excretory pore |
Small |
Trichostrongylus |
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Very long oesophagus |
Medium |
Strongyloides |
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Small intestines
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Cephalic swelling with striations
|
|
Small, coiled |
Cooperia |
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Medium, tangled |
Nematodirus |
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Distinct buccal cavity with teeth |
Head bent dorsally |
Large, stout |
Bunostomum |
|
|
Tapeworms
|
Scolex with suckers |
Large, 2 genital pores/seg. |
Moniezia |
|
|
|
Small, indistinct segments |
Avitellina |
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Caecum and large intestines
|
Small, indistinct |
Very long thin neck |
Large, whip-like |
Trichuris |
|
Leaf crowns present
|
Cervical papillae level with oesophagus |
Large, stout |
Oesophagostomum columbianum |
|
|
Cervical papillae behind oesophagus |
Large, stout |
Oes. venulosum |
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Further differential features are given in Tables 4.3-4.5.
Table 4.3 NEMATODES OF THE ABOMASUM
|
|
Haemonchus |
Ostertagia |
Trichostrongylus |
|
Mature size
|
Males 10 to 20 mm long. Females 18 to 30 mm. |
Males 7 to 8 mm long. Females 9 to 12 mm. |
Males 4 to 5 mm long. Females 5 to 7 mm. |
|
Large, easily seen, mostly in fundus region of abomasum
|
Mostly found at pyloric region of abomasum |
Very small, difficult to see without washing or staining. |
|
|
Much the same thickness throughout length of worm. |
Taper to very fine anterior end. |
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|
Head
|
Prominent large cervical papillae. |
Small cervical papillae set more posteriorly. |
No cervical papillae. Prominent excretory pore. |
|
Distance from anterior end about 3 times diameter between papillae. |
Distance from anterior end about 5 times diameter between papillae. |
|
|
|
Female
|
Vulva covered by large vulval flap. |
Small or no vulval flap. |
Simple genital opening without vulval flap. |
|
Red and white spiral striping visible in fresh specimens resembling "Barber's pole".
|
Under high magnification tip of tail shows annular rings. |
Cuticle striations are annular. |
|
|
Cuticle striations are longitudinal. |
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|
Male tail
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Asymmetrical dorsal lobe in bursa. |
Bursal lobes are symmetrical. |
Bursal lobes are symmetrical. |
|
Spicules taper to barbed points. |
Spicules vary with species. |
Spicules vary with species. |
Table 4.4 NEMATODES OF THE SMALL INTESTINES
|
|
Strongyloides |
Cooperia |
Nematodirus |
|
Mature size
|
Usually only females are found, 3 to 6 mm long. |
Males 4 to 6 mm long. Females 5 to 7 mm |
Males 10 to 15 mm long. Females 15 to 20 mm. |
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These worms lose the iodine stain quickly when de-colourized with hypo solution. |
Usually coiled flat or in 1 or 2 tight coils. |
Usually tangled shape due to twisting of the "thin neck". |
|
|
Other features
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Very long oesophagus, one third to one half total length of worm. |
Body of female swollen at region of vulva. |
Female tail has prominent spine protruding from a blunt end. |
|
Eggs expressed from females have a fully developed larva in them, |
Male tail has short, stout spicules. |
Male tail has very long, slender spicules usually extending beyond the bursa. |
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|
Bunostomum |
|||
|
Mature size
|
Male 12 to 17 ram long. Female 19 to 26 mm. A stout worm much thicker than any other round worms of the small intestine. |
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Large buccal cavity has prominent teeth. B. trigonocephalum of sheep and goats has one large and 2 small teeth. |
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|
Head |
B. phlebotomum of cattle has 2 pairs of subventral teeth. |
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|
Other features |
B. trigonocephalum has short, twisted spicules. B. phlebotomum has long, slender spicules. |
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Table 4.5 NEMATODES OF THE LARGE INTESTINES
|
CAECUM |
Trichuris |
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|
Mature size |
Male 50 to 80 mm long. Female 35 to 70 mm long. The anterior end is very thin, the posterior end is thick. It is called the "whip-worm" because of its shape. |
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|
Other features |
Male has single spicule in spine-covered protrusible sheath. Female produces barrel-shaped eggs with a transparent plug at each end. |
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|
COLON |
Chabertia |
Oesophagostomum venulosum |
Oesophagostomum colombianum |
|
Mature size
|
Male 13 to 14 mm long. |
Male 11 to 16 mm long. |
Male 12 to 16 mm long. |
|
Female 17 to 20 mm. |
Female 13 to 24 mm. |
Female 15 to 21 mm. |
|
|
Other features |
Chabertia has a large globular buccal cavity that is visible to the naked eye in fresh specimens. There are no teeth in the buccal cavity. |
Small buccal cavity surrounded by leaf crown. Cervical papillae are situated posterior to the oesophagus. |
Small buccal cavity surrounded by leaf crown. Cervical papillae are situated opposite anterior region of oesophagus. |
For a more precise assessment of the liver fluke burden of an animal the liver can be examined post-mortem for its content of immature and adult flukes.
· A sharp knife
· A cutting board
· A medium size tray
· A wash bottle
· Petri dishes
· A laboratory counter
(a) Place the liver on a board and cut it into fine slices with a sharp knife.(b) After each cut is made apply pressure to the liver to squeeze out the flukes and wipe these off gently before making the next cut.
(c) When the whole organ has been sliced, place all of the material in a tray and cover with water.
(d) Remove the pieces of liver, again squeezing each piece as it is removed from the water.
(e) Pour water and flukes into a sieve and wash parasites until clean.
(f) Pour into petri dishes and count the flukes present.
(g) If very large number of immature flukes are present, count by a dilution technique.
The diagnosis of cysticercosis in carcasses is done during routine meat inspection according to rules and regulations which are specific for each country. The rules governing meat inspection attempt to accommodate the interest of the owner/butcher (carcasses should not be spoiled by too many incisions) and those of the veterinary public health service.
· A sharp knife
· A cutting board
· A laboratory counter (optional)
The routine inspection for Cysticercus bovis consists of the following:
(a) Two incisions parallel to the jaw of the external masseter muscles and one of the interior.(b) A longitudinal incision of the ventral side of the tongue.
(c) Incision of the heart septum.
(d) Two parallel incisions in the triceps muscle.
(e) Visual inspection of all exposed muscle surfaces.
As the judgement of carcasses is related to the number of cysts, a quantification of cysts may be required if the routine inspection only reveals 1-5 cysts. The masseter muscles, the heart, the tongue, the triceps muscle and the diaphragm should be removed from the carcass and examined by cutting the muscles in 5 mm thin slices, inspecting all exposed surfaces for live and dead cysts.
