Atlas of Ovine Parasitology

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Atlas of Ovine Parasitology

Description of the main parasitic forms eliminated in the faeces of small ruminants

Protozoa

Genus

Measurements

Description

Giardia

14 x 8 µm

Vegetative forms (trophozoites).

Cryptosporidium

5-7 µm

These spherical oocysts are so small that they must be stained to observe them.

Eimeria

17-56 µm

Recently eliminated, the oocysts are ovoid or spherical, refractive, with a swelling at the minor pole (micropyle) and a spherical zygote in its interior. Once sporulated they have four sporocysts, each with two sporozoites in their interior.

Nematodes

Strongylida* Trichostrongylus

79-118 x 31-56 µm

Oval, with the poles slightly unequal, the shell is thin and segmented.

Ostertagia/ Teladorsagia

80-103 x 40-56 µm

From oval to ellipsoidal, not very wide, having a morula with many small blastomeres.

Haemonchus

70-85 x 41-48 µm

Oval with asymmetric poles, embryonated with 16 to 32 cells.

Cooperia

70-83 x 32-36 µm

Poles are the same and rounded, the walls parallel, with 16-32 cells.

Bunostomum

79-97 x 47-57 µm

Oval, with rounded ends, fewer than 16 embryonic cells with dark granulation.

Chabertia

83-105 x 47-59 µm

From oval to ellipsoidal, wide, with slightly flattened poles and 16-32 small blastomeres.

Oesophagostomum 70-76 x 36-40 µm Nematodirus

Oval, thin-shelled, wide, with similar poles, 8-16 large cells.

150-230 x 67-110 µm Large, elliptical eggs. The embryo found in their interior is divided into 4-8 blastomeres, leaving a large liquid-filled space between the walls of the egg.

Marshallagia

160-200 x 75-100 µm Large eggs with parallel sides and a rounded end. A morula of 16 to

Strongyloides

45-65 x 25 µm

32 blastomeres can be seen in the interior.

Eggs are similar to strongylida but smaller, with the poles flattened and the formed larva situated inside.

Skrjabinema

54-63 x 30-34 µm

They are asymmetrical, with pointed, slightly rounded poles, pale grey in colour and almost translucent, allowing the embryo inside to be seen. The shell is thick.

Trichuris

70-80 x 25-40 µm

Eggs have a characteristic lemon shape, and are brown with two

Capillaria

40-50 x 22-25 µm

Eggs are similar to those of Trichuris, but smaller, with less prominent

refractive terminal plugs clearly prominent at the poles. terminal plugs, almost parallel walls and more flattened poles.

* Eggs of the different genera are very similar. They are medium-sized, greyish, with a more or less elongated or elliptical shape; the embryo (which can occupy the whole egg or just the central portion) being visible in the interior.

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Diagnostic techniques

Description of the main parasitic forms eliminated in the faeces of small ruminants (continued)

Cestodes

Genus

Measurements

Description

Moniezia benedeni

80-90 µm

Medium-sized eggs, quadrangular, a thick refractive capsule, with the oncosphere in the interior surrounded by the pyriform (pear-shaped) apparatus.

Trematodes

Moniezia expansa

50-60 µm

The same as M. benedeni but triangular in shape.

Avitellina

45 x 20 µm

Eggs without pyriform apparatus.

Thysaniezia

25 µm

Eggs without pyriform apparatus.

Stilesia

25 µm

Eggs without pyriform apparatus.

Paramphistomum

125-180 x 75-103 µm

Very large elliptical eggs (somewhat bigger than Fasciola), with a transparent polar operculum. The contents are grey in colour and occupy the whole egg.

Fasciola

130-150 x 70-90 µm

Very large elliptical eggs with a transparent polar operculum.

Dicrocoelium

38-45 x 22-30 µm

Small eggs with an asymmetric elliptical shape and a barely visible

The content is yellowish in colour and occupies the entire egg. brown operculum. In the interior, two darker germinal masses can be observed close to one of the poles and arranged transversely.

Schistosoma

132-247 x 38-60 µm

They are fusiform and one of the poles terminates in a point, although the smallest are frequently oval.

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Atlas of Ovine Parasitology

The main parasitic forms that can be found in a coprological analysis of ovine faeces (flotation/sedimentation techniques)

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Egg of gastrointestinal strongylida.

Egg of Nematodirus.

Eggs of Nematodirus and gastrointestinal strongylida.

Egg of Strongyloides.

Egg of Skjrabinema.

Egg of Trichuris.

Egg of Moniezia.

Egg of Moniezia (bloated).

Egg of Fasciola.

Egg of Dicrocoelium.

Egg of Schistosoma.

Oocyst of Cryptosporidium.

Unsporulated oocyst of Eimeria.

Sporulated oocyst of Eimeria.

Artefact.

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Diagnostic techniques

Coproculture technique The objective of coproculture is to be able to identify the genus and species of the parasites belonging to the Order Strongylida, which, as mentioned earlier, have eggs that are morphologically similar (except Nematodirus). For this reason, the method involves providing the eggs with the conditions necessary for the exogenous development of these nematodes from the egg phase until third stage larvae or L3 are produced, which will then be collected using the Baermann-Wetzel method. The larvae thus obtained can be differentiated morphologically, focusing on aspects such as tail length, the number of intestinal cells or the size of the larva, amongst others.

1 Take a quantity of faeces (50-100 g) and partially break it down with a mortar and pestle.

2 Moisten the faeces with a spray or add an absorbent (sepiolite), depending on its consistency.

3 Maintain the mixture in an oven at 22-25 ºC for 7-10 days, or at room temperature for 20-30 days (15 days if you have observed Nematodirus sp. eggs in the McMaster).

4 Stir the mixture every 48 hours to maintain the correct oxygenation, and moisten if required.

5 Recover the third stage larvae that have formed using the Baermann apparatus.

6 Use the microscope to identify the larvae according to their morphology.

7 Add a few drops of Lugol to immobilise the larvae.

Classification criteria for the larvae of ovine gastrointestinal nematodes.

Posterior end (P.E.)

Body length (T.L.)

short (<40 µm)

very large (>1000 µm)

medium (40-110 µm)

large (700-820 µm)

long (>110 µm)

medium (640-700 µm) small (600-640 µm) very small (<600 µm)

oesophagus

I. C.

P. E.

sheath

W. T.L.S. anus T.L. T.L. = Total body length of the larva.

T.L.S. = Length of the tail of the sheath.

W. = Width.

I.C. = Intestinal cells (size, number and form are important).

P.E. = Posterior extremity.

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Atlas of Ovine Parasitology

Washing Small cestodes should be submerged in tap water that is renewed constantly for 24 hours (if this is insufficient, they can be treated with H2O2 at 2-10% until they have become white and relaxed). The medium to large ones are first washed under running tap water, in an Erlenmeyer (conical flask), then placed in pepsin and into an oven at 38 ºC for more than 10 minutes (almost 24 hours in the majority of cases) until the internal organs can be distinguished using a stereo microscope. They are then washed in cold distilled water and a progressive dehydration is performed in a series of alcohols: 20-30-50-65 and 70% (10 minutes in each one).

Fixation In both cases by immersion in ethanol 70%.

Staining I

Clarify in a Petri dish with acid alcohol (50% of glacial acetic acid at 1% and 50% of ethanol 70%) (small: 1 hour, 30 min; large: 24 hours or more), monitoring the rinsing process throughout with the stereo microscope.

I

Stain (still in acetic acid) in the Petri dish with Semichón’s acetic-carmine (5-30 minutes).

I

Destain the external layers by submerging the worms in a Petri dish containing a solution of glacial acetic acid (from 1%, up to 50%) and ethanol 70% (10-30 minutes), shaking and changing the solution if it becomes heavily coloured during the process of destaining the cestode. Once destained, the worms are submerged in ethanol 70% to neutralise the acid.

I

Dehydrate (to prevent oxidation darkening the preparation): immerse the cestode in Petri dishes containing a series of alcohols: 80% ethanol (10 minutes), 90% (10 minutes), absolute (10 minutes) and finally, isopropylic alcohol (5 minutes) (because the absolute alcohol hydrates rapidly).

I

Clarify (to leave the cuticle transparent and the internal organs visible) by placing the worms in xylene for a maximum of 5 minutes.

Proglottids of Moniezia.

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Diagnostic techniques

Trematodes They can be processed in a similar way to the cestodes, by washing them various times in physiological saline solution (so that they discharge the ingested blood), though in this case many internal organs can be seen in their interior before fixation. As a fixative you can use formalin 10%, moving it constantly to prevent the specimens from contracting. Once fixed they can be preserved in formalin 3%.

In general, once the specimens have been processed, they are placed on a slide with mounting medium (Canada balsam, glycerogelatine, gum arabic, etc.) and can be examined under the microscope. Sometimes it is worth mounting the specimen directly in the clarifier, leaving them for a certain amount of time so the worms become transparent and the preparations will then be ready for study under the microscope. If the specimens are very thick or you want to observe the internal structures in more detail,they can be stained.

Of the available staining techniques for helminths, the following stand out: lactophenol cotton blue, Ehrlich’s haematoxylin and Horen’s trichrome stain.

Basic layout of the trematode.

Location of other ovine endoparasites (except adult helminths found in the viscera) Location

Digestive apparatus

Respiratory system

Circulatory apparatus

Nervous system Systemic parasitism

Parasites Oesophagus

Protozoa

Sarcocystis

Small intestine

Protozoa

Eimeria Cryptosporidium

Liver

Metacestodes

Hydatid cyst Cysticercus

Nasal fosase

Arthropods

Oestrus

Lung

Metacestodes

Hydatid cyst Cysticercus

Protozoa

Babesia Theileria Trypanosome

Trematodes

Schistosoma

Blood

Spinal medulla and brain

Metacestodes

Coenurus

Striated muscle, heart, oesophagus, diaphragm

Protozoa

Sarcocystis

Metacestodes

Cysticercus

Muscle and CNS

Protozoa

Toxoplasma

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Atlas of Ovine Parasitology

Gastrointestinal nematodes

Cooperia Location: small intestine.

Morphology: they have a slender anterior end without developed labia, a small buccal cavity and discrete cervical papillae that are hard to see, situated in the posterior third of the oesophagus.

Features a long cephalic vesicle that is normally divided into cephalic and cervical portions.

Anterior end.

Female Tail terminating in a point, relatively sharp but not spiny. The vulva is situated in the posterior half of the body and can have a vulvar flap.

Posterior end of female.

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Nematodes

Gastrointestinal nematodes

Cooperia oncophora Location: small intestine.

Male: the dorsal ray of the copulatory bursa measures 220-240 µm, its branches terminate in a horseshoe-shaped arch and close to the centre of each arch there is a small digitate. The spicules are large (228-300 µm), slender and smooth, without prominent edges, and terminate in a button.

Female: body markedly irregular in the region of the vulva, the tail is thin with annular striations. The anus is located 160 µm from the distal end. OviSpicules.

jectors 700 µm long.

Cooperia mcmasteri Location: small intestine.

Male: anterior end terminates in a spiral. It is similar to C. oncophora, but the copulatory bursa is bigger and the rays being longer and thinner, the spicules are more slender and slightly shorter. Long spicules (270 µm), with a boot-shaped termination, have an enlargement resembling a wing on the mid part.

Female: transverse vulva, the anterior labia dilates forming a shirttail.

Spicules.

Cooperia curticei Location: small intestine.

Slender anterior end without developed labia, small buccal cavity and cervical papillae not very evident and hard to see, located in the posterior third of the oesophagus. Features a long cephalic vesicle that is normally divided into cephalic and cervical portions.

Male: 4.6-6.8 mm x 0.075-0.08 mm.

Spicules of 111-165 µm.

Female: 5.8-8.05 mm x 0.075-0.1 mm.

Spicules.

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Atlas of Ovine Parasitology

Gastrointestinal nematodes

Marshallagia Marshallagia marshalli Location: small intestine.

The buccal capsule is small but well differentiated; they lack a cephalic vesicle and cervical wings.

The cervical papillae are highly developed and point backwards. The synlophe comprises 5056 longitudinal ridges perpendicular to the body surface.

Male: 10-13 mm. The genital pore is large with a long (280-400 µm) dorsal rib, bifurcated close to the distal end. Prebursal papillae are present.

The spicules (250-280 µm) are slender, yellowish buff in colour, trifurcated and with a ventral curvature close to the middle (lateral view). There is no gubernaculum.

Female: 12-20 mm. The vulva opens 2.5-5 mm from the tail, which is pointed with a terminal spine.

According to some authors, a vulvar flap is present, whilst others believe it is absent.

Spicules.

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Nematodes

Gastrointestinal nematodes

Nematodirus Location: small intestine.

The body is long, slender, whitish in colour and it usually appears rolled-up. The anterior region of the body is thicker than the posterior, with a cephalic vesicle; neither prebursal nor cervical papillae are present. The cuticle has transverse striations on the posterior portion.

Anterior end with cephalic expansion.

Male Size: 8-16 mm. The spicules are plain, filiform and very long (0.7-1.25 mm), jutting out from the body and fused at the end. There is no gubernaculum to guide them but they are united by a membrane (totally or just in the final part).

Long spicules fused at their ends.

Female Size: 19-25 mm. Body dilated toward the third or fourth part due to the fact that the uterus contains few eggs, but these are very large.

Vulva situated in the posterior third of the body.

The caudal end is short, generally conical and truncate, with a terminal spine.

Posterior end with terminal spine.

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Atlas of Ovine Parasitology

Gastrointestinal nematodes

Nematodirus filicollis Location: small intestine.

Male: 10-15 mm. Spicules of 680-950 µm, long and equal. They are slender and filiform and come to and end in a point, with a thin, lanceolate terminal tip of 16 µm, both of which are covered by a membrane.

Female: 12-20 mm. The body is thickened from the vulvar region almost until the distal end; this is due to uterus, which contains only a few eggs, but these are very large. The vulva is situated slightly in front of the border of the

Lanceolate termination of the spicules.

distal third. This measures 65-80 µm, is long and truncate, and ends in a short spine.

Nematodirus spathiger Location: small intestine.

Male: 10-19 mm. Each spicule terminates in a spoon-shaped piece with a blunt tip of 16 µm, and both are surrounded by a membrane.

Female: 15-29 mm. The vulva opens close to the posterior quarter of the body. The posterior end is blunt.

Spoon or brush-shaped tips of the spicules.

Nematodirus helvetianus Location: small intestine.

Male: 11-17 mm. Spicules of 0.9-1.25 mm, very close together, with a very long lanceolate end (35 µm), which is pointed and symmetrical and covered by a membrane.

Female: measures from 18 to 25 mm. The vulva opens transversally 1.2 mm from the blunt posterior end; the terminal spine is short.

Lanceolate termination of the spicules.

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Nematodes

Gastrointestinal nematodes

Trichuris Location: caecum and colon.

The anterior portion of the body and oesophagus is much longer and thinner than the posterior portion, with a simple buccal opening.

Anterior end (oesophageal portion).

Eggs in the interior of a female.

Female Size: 35-70 mm. The posterior end is only slightly curved, without forming a spiral. The vulva is situated at the beginning of the thick portion of the body.

Posterior end of female.

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It includes a chapter on the key diagnostic techniques used to identify the adult parasites, the eggs and the larval forms.

FĂŠlix ValcĂĄrcel Sancho

The main purpose of this Atlas of Ovine Parasitology is to provide a simple yet useful tool that will study the causes and help in the identification of the main parasites of sheep.

Atlas of Ovine Parasitology