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PRACTICAL MANUAL

COLLECTION, FIXATION, PRESERVATION AND MOUNTING OF PARASITES

I.

HELMINTHS Collection: The helminths infect different visceral organs of domestic animals and poultry. Whether the collection is to be made on necropsy from the slaughtered animals or the parasites to be collected must be alive otherwise their identification becomes a difficult task even for an expert. The staining process of the parasite starts right from its collection because the putrefactive changes have a considerable handicap on subsequent staining. Take due precautions so that the postmortem of a dead animal is done without any delay. Alimentary Tract : Before severing one part of the viscera from the other, ligate it. The parasites are liable to migrate from one part to the other and thus give a wrong impression about their seat of predilection. The search for schistosomes is made in the intact mesentery. Oesophagus is opened and screened for the presence of embedded worms viz., Gongylonema pulchrum in ruminants, G. ingluvicola in poultry and Spirocerea lupi in dogs. The infected parts are fixed in 10% formalin. Sarcocystis spp. is examined by filling water and scanning it. Cut open the entire contents of rumen, abomasum, small intestine and large intestine in separate large trays with identity of the contents. Wash the viscera in the respective trays with tap water, look for the parasites on the tissues by unaided eyes and collect the parasites by camel hair brush in a petri dish containing normal saline. In case no parasites are found, discard the viscera after scanning it with the magnifying lens. Further the materials can be processed in two ways : (a) Simple decantation : Large parasites like ascarids offer no difficulty and are picked up as such from the visceral contents. The others can be collected by adding sufficeint quantity of water, stiring the contents and then to let them decanted. The clearer supernatant liquid is poured off after 15min. or so. The process is repeated quite a few times till the sediments are relatively free from pigment or mucus. Thereafter the sediment is spreaded and the parasites are picked up with the help of a camel hair brush. Transfer them to a petri dish containing tap water and make them free from the sticking debris with the help of a camel hair brush. Finally transfer the parasites into the Petri dish containing saline water. This procedure is simple but time consuming. It requires no elaborate arrangement except the trays and adequate quantity of water. (b) Test sieve shakers (Plate XXII) : The watery contents of the trays are filtered through various grades of test sieves to get rid off repeated cumbersome decantation process. The commonly available convenient sieves have jointless 20cm diameter frame. They have sieves of 10, 20, 30, 40, 60, 80 and 100 mesh/sq. inch. The mesh may consist of durable sieve cloth or brass wire. The jointless frame should either consist of stainless steel or brass. The entire assembly should have highly polished non-sticky coating so that the worms are easily identified. The test sieves are fixed one below the other in the increasing order of their fineness. Most of the worms get entrapped in 10, 20, 30 and 40 number test sieves. It is very rare that some of them pass through number 40 test sieve and enter the number 60 test sieve. The water is used liberally in number 10 test sieve for washing visceral contents. All the test sieves are examined individually at the end of the process and the worms are collected with the help of a camel hair brush as usual.

Liver
The liver is dissected along the bile duct and the blood vessels. The parasites start coming out and can be collected with the help of camel hair brush. Liver flukes are usually free from debris. Give a wash in tap water and then transfer the parasites to a petri dish having normal saline. The tapeworm cysts are collected intact. One should be very careful while collecting them. A ruptured hydatid cyst is a potential danger for the collector.

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PRACTICAL MANUAL The nematodes like Gnathostoma spinigerum are processed likewise.

Lungs
The lungworm occurs primarily in trachea, bronchi and alveoli of their hosts. The conical lesions like atelectasis and emphysema in lungs are pathognomonic lesions. Dissection along the bronchi and bronchioles will reveal L4 larva and adult stage of nematodes viz., Dictyocaulus filaria, Protostrongylus rufescens and Muellerius capillaris in sheep and goats; Dictyocaulus viviparus in cattle and Metastrongylus spp. in pigs.

Aorta
The associated aorta should be searched out for filarial worms which may either be found completely embedded in a zigzag fashion e.g. Onchocerca armillata or a portion of the worm hanging out free in the lumen (Elaeophora spp.). In such cases, the heart/associated aorta is preserved in 10% formalin.

Heart
The heart along with the associated blood vessels should be examined for evidence of parasites and pathological changes. The heart of dog and cat may harbour Dirofilaria immitis. The cardiac muscles of pig and cattle may contain Cysticercus cellulosae and Cysticercus bovis, respectively.

Kidneys
The kidneys are examined first superficially and then after slicing. The sliced parts are kept in normal saline solution so that the parasites may leave the tissues themselves. The kidneys may be found infected with Stephanurus dentatus in pigs and Dioctophyma renale in dogs. Subcutaneous tissues The skin of the live or dead animals is searched out for the presence of worms and nodules. Parafilaria bovicola in cattle and Parafilaria multipapillosa in horses are found in subcutaneous tissues as thin fragile threads. The subcutaneous nodules are found containing the filarial worms viz., Onchocerca gibsoni or larvae of Hypoderma fly. Body Cavity In cattle and buffaloes, Setaria spp. worms are seen crawling upon the visceral mass in the cavity. These are collected in normal saline solution. Muscles Muscles of jaws, tongue, neck, diaphragm, shoulder and thigh are searched for cysticerci, small whitish structures (Sarcocystrs) in herbivores and pigs and pin point white dots (Trichinella spiralis) in pigs, dogs and cats only. Eyes White thread like worms, Thelazia and Setaria spp. in cattle and Oxyspirura in poultry birds may be found in eyes. Nasal Passages Schistosoma nasalis are searched in nasal granuloma cases. On rare occasions, leeches are found attached to the nasal mucosa.

Fixation
It is killing of tissues with a view to keep them near to the living tissues and to prevent autolysis and putrefaction. Autolysis is the lysis or dissolution of the cells by enzymatic action, probably as a result of rupture of lysosome. The purpose of fixation is defeated if it is carried out a long time after the death of the animal. The ideal aims of fixation are as follows : (a) To prevent or to arrest autolysis, bacterial decomposition and putrefaction. (b) To coagulate tissues for minimising the loss of easily diffusible substances. (c) To fortify the tissues against the deleterious effects of the various stages in the preparation of sections (dehydration, cleaning and wax impregnation).

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PRACTICAL MANUAL (d) To leave the tissues in a state that facilitates differential staining with dyes and other agents. The fixatives are chemical agents. The protein precipitant fixatives cause shrinkage of the tissues regardless of the osmotic pressure, whereas non-precipitant fixatives do not cause shrinkage. In general, the lesser is the distortion, better is the fixation. Before fixing, the parasites are stirred vigorously in normal saline solution to remove the mucus or other foreign material adhering to them. The different fixatives are : 1. Formalin Commercial formaldehyde is available as a 40% solution and is called formalin. Its long storage in the cold weather makes the formalin turbid due to formation of paraformaldehyde. This is polymerization phenomenon and is stopped by the addition of methanol. Such solutions are used again after filtration. Commercial formalin is acidic due to the presence of formic acid in it. It should be neutralised with sodium carbonate or calcium carbonate before use. Formalin is bufferred with : Sodium dihydrogen phosphate = 3.5 gm. Sodium hydrogen phosphate = 6.5 gm. Add formalin = 1.0 liter. Formalin in 0.85% normal saline is soluble better than water. It fixes proteins and compound fats either as preservative or as fixative and this property is helpful in the demonstration of lipids in frozen sections. The worms fixed in 10% formalin remain either as it is or be transferred to 70% alcohol after washing away the whole of the formalin in running water. 2. Mercuric chloride It is a protein precipitant and its rate of precipitation decreases after a few millimetres. Thus, pieces of tissues exceeding 5mm in thickness remain over- fixed at the periphery and under-fixed at the centre. Due to these defects, it is not used as fixative. The combining of this fixative with other reagents e.g. acetic acid, formalin, potassium dichromate etc. can be helpful in combating its defects. It can be used with many good fixatives. As mercuric chloride corrodes metal, fixatives containing it should never be stored in metal cap containers. 3. Zenkers fluid Potassium dichromate = 2.5 gm Sodium sulphate = 1.0 gm Mercuric chloride = 5.0 gm Distilled water = 100 ml Just before use, add 1.0ml glacial acetic acid to 20ml of the above mixture. The specimens to be fixed with this fixative should be allowed to remain in it for 24 hrs. or more. Thereafter to remove mercuric chloride from the specimens, they are transferred to a very dilute solution of tincture of iodine for a couple of hrs. Then they are washed in 70% alcohol and preserved. 4. Manns fluid Picric acid = 10.0 gm Mercuric chloride = 25.0 gm Formalin (40%) = 300.0 ml Water = 1000.0 ml The specimens are kept in this fluid for at least 12 hrs. Thereafter, they are washed in running water for 24 hrs. or more and later transferred to a diluted solution of iodine as in case of Zenkers fluid. They are washed in 70% alcohol and preserved later in it. 5. Bouins fluid Formalin (40%) = 25 ml

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PRACTICAL MANUAL Acetic acid = 5 ml Water / 70% alcohol = 75 ml Picric acid = to saturation The specimens to be fixed in this fluid should remain in it for at least 16 hrs. Later they are transferred to 50% alcohol, then to 70% alcohol in which they may be preserved. The fixation of helminths is done as follows : (i) Flukes Flukes are never fixed in 10% formalin as such because it causes their contraction and distortion. Such defects are overcome by pressing the flukes between two microscopic slides, which are kept in position by a cotton thread. Too much pressure should be avoided to prevent bursting or distortion of the specimens. After 2 hrs. or even overnight keeping of the slides in the fixatives, the threads are untied. The flattened specimens are preserved in 10% formalin or 70% alcohol. (ii) Tapeworms The scolices should never be missed since they are of great importance in identification of the worms. The thick tapeworms like Moniezia, Taenia need to be pressed in between two microscopic slides. All the scolices and thin specimens like Avitellina, Stilesia need more gentle handling. The scolices should preferably be pressed under coverslip No. 2 and fixed by drops of fixatives poured from the side. A minimum of 4 hrs. fixation is must for all the tapeworms. (iii) Nematodes They are fixed in a steaming mixture of 70% alcohol and 5% glycerine and thereafter the specimens remain preserved for a longer period. Another fixative and subsequent preservative is simmering 10% formalin. The tissues intended for section cutting should be fixed in Zenkers, Bouins or Manns fluid as described above or in 10% formalin.

Preservation
This constitutes the keeping of cells and tissues in a life like manner as far as possible. The agents bringing about this effect are called as preservatives. There is a common practice that specimens are preserved in 70% alcohol in glass tubes with corks. If the cork comes in contact with alcohol then the tannin dissolves in it and stains the specimens black. In order to avoid it, it is better to store them in glass stoppered phials. However, the black specimens can be soaked in pure beech wood creosote for 3 to 4 days and then transferred for the same period to 5% hydrochloric acid in 70% alcohol. Formalin as 10% solution can also be used as preservative.

Permanent mounts
The trematodes and cestodes are stained for making the internal organs visible for proper identification of the species. Various stains like acitic alum carmine, paracarmine, lithium carmine haemalum with 2% acetic acid and borax carmine are employed for this purpose. The stain is usually diluted with distilled water and staining is done for a prolonged period. The specimens are over stained and later differentiated in 0.1% acid alcohol or alum solution. After proper dehydration with graded alcohol, they are mounted in Canada Balsam / D.P.X. mountant. Nematodes preserved in 70% alcohol are cleared in the lactophenol. Later they are mounted in Canada Balsam. Alternately, the nematodes stored in 70% alcohol, are transferred to 10% glycerine for mounting in gum Arabic medium (Gum Arabic - 60, Glycerin - 40, chloral hydrate - 100, distilled water - 100 and thymol - 1).

G E N E R A L V E T E R IN A R Y P A R A S IT O L O G Y & H E

PRACTICAL MANUAL LARVAL STAGES OF TREMATODE PARASITES Body is roughly triangular in shape. Ectoderm bears numerous cilia. A stylet like spine is present at the anterior end for boring into IH. Germ cells are present. Eye spots present. Sac like gut present. A pair of cephalic glands (penetration gland). Appears like a sac and contains numerous germ cells. An oral sucker present. Two lateral appendages in the posterior half in Fasciola spp. only Intestine is simple sac like. Numerous germ cells present. A thickening (collar) is present at the level of oesophagus, behind which there is a birth pore. Body is oval shaped. Oral sucker is at the anterior end of the body. Ventral sucker lies in the middle of the body. Intestine like that of adult, and tail is simple

Miracidium

Sporocyst Redia

Cercarla Fasciola spp

Amphlstomes Know as "Cercaria pigmentata" Dark brown in colour. Round or globular in shape. Oral sucker at the anterior end Ventral sucker immediately anterior to the base of the tail. Body pigmented. Tail is simple, short and slender. Two eye spots present in the anterior half of the body. Pigment glands are present. Schistosoma spp. Known as "Furcocercus cercaria" Body is spindle shaped. Pharynx absent. Eye spots absent. Ventral sucker in the middle of the body. Tail is bifurcate distally (brevifurcate). Metacercaria Round encysted forms. Dark brown / blackish in colour Oral and ventral suckers visible under microscope. MORPHOLOGICAL CHARACTERS OF SOME ADULT TREMATODES FAMILY: FASCIOLIDAE GENUS: Fesciola, Fasclolopsls Fasciola hepatlca : Liver fluke Common name : Liver fluke of ruminants Definitive host : Sheep, Goat, Cattle Buffalo, Pig, Elephant, Dog, Cat, Man. Location Bile duct, gall bladder and liver. Sometimes occur in lungs, kidney and uterus as a berrant parasite. Intermediate hosts : Amphibious snails of species like Lymnaea truncatula, and Lymnaea luteola. Morphological characters : 1. It is smaller than, F. gigantica and measures 25 to 75mm x 5 to 12mm. 2. Shape : Leaf like, broader anteriorly than posteriorly. 3. At the anterior end there is a cone shaped projection known as cephalic cone followed by a pair of broad shoulders. 4. The posterior portion is 'V shaped

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PRACTICAL MANUAL 5. 6. 7. Oral sucker is terminal and surrounds the mouth opening. The Cuticle bears large numbers of backwardly directed small spines. Ventral sucker present at the level of shoulder. Both oral sucker and ventral sucker are of same size. 8. Intestinal caeca are highly branched only on the lateral sides and reach upto posterior end. 9. Testes are two in number, highly branched, tandem in arrangement and occupy the second and third quarters of the body. 10. Ovary Is highly branched and present anterior to testes. 11. Vitelline glands consists of large number of fine follicles and are present on either side of entire lateral fields. 12. Uterus is dark in colour and placed anterior to testes and contains large number of yellowish-brown coloured eggs. Fasciola gigantica : Common name, location and host : Same as F. heapatica Intermediate host : Aquatic snails of spp. Lymnaea auricularia var reufescence and L. acuminata. Morphological characters : 1. It is larger than F. hepatica and measures 25 1o75nrni x 5 to 1 2rnro. 2. Cephalic cone is smaller than Fasciola hepatica and shoulder are not distinct. 3. Ventral sucker is larger than oral sucker. 4. The posterior portion is broadly rounded or- 'U' shaped in nature. 5. Microscopically the intestinal caeca are branched both laterally and medially. 6. Other morphological characters of Fasciola qigantica are more or less similar to Fasciola hepatica. Fasclolopsis buski Common name : Intestinal fluke of Pig. Definitive hosts : Man, Pig and occasionally dog, Location : Duodenum and Jejunum Intermediate hosts : Aquatic snail : Gyraulus and Planorbis spp. Morphological characters : 1. These are large and thick flukes, elongate oval, slightly broader posteriorly than anteriorly. Size 30-75 x 8-20mm. Largest trematode of animals. 2. Cephalic cone and shoulders absent. 3. Oral sucker much smaller than ventral sucker and both the sucker are near to each other in anterior 1/3rd of the body 4. Intestinal caeca simple, convoluted and reach almost to posterior end. 5. Testes are tandem, branched and posterior in position. 6. Ovary is branched and anterior to testes. 7. Vitelline glands occupy the entire lateral fields. FAMILY: DICROCOELIDAE GENUS : Dicrocoelium and Eurytrema Dicrocoelium dendriticum : Common name Definitive host Intermediate hosts Lancet fluke Sheep, Goat, Cattle, Deer and rarely man. 1st (a) Snails : Zebrina detrita and Cionella lubrica. 2nd (b) Ants : Formica the fusca

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PRACTICAL MANUAL Morphological characters : 1. Size : 6-10 x 1.5-2.5 mm. Body is elongated and lencet like, narrow anteriorly and widest behind the middle, delicate and translucent. 2. Oral sucker is smaller than ventral sucker and these are close to each other in anterior 1/3 of the body. 3. Intestinal caeca simple and do not reach upto the posterior end. 4. Testes are slightly lobed and lie in tanden just posterior to the ventral sucker. 5. Ovary, globular in shape and post testicular, just close to posterior testis. 6. Uterus branched and the transverse coils of the uterus occupy most of the space behind the gonads and usually filled with numerous small brownish eggs. 7. Vitelline glands occupy the middle third of the lateral fields. Eurytrema pancreaticum Common name : Pancreatic fluke. Definitive host : Sheep, Goat, Cattle, Buffalo and occasionally man Location : Pancreatic duct, rarely in bile ducts and duodenum Intermediate hosts : 1st I.H. - Snails : Bradybaena spp. 2nd I.H. - : Grass hoppers : Conocephalus maculatus. Morphological characters : 1. Size : 8-16 x 5-8.5mm 2. Oral sucker is larger than the ventral. Both are situated at anterior 1/3 of the body. 3. Intestinal caeca simple and run upto 4/5 of the body length. 4. Testes are weakly lobed, horizontal and lie slightly posterior to the level of the sucker. 5. Ovary weakly lobed, situated near the median line behind the testes. 6. Uterus fills the posterior part of the body. 7. Vitelline glands are follicular and are situated in middle third of the lateral fields. FAMILY: OPISTHORCHIIDAE GENUS : Opisthorchls, Pseudamphistomum, Clonorchis Oplsthorchls tenuicoilis : Common name : Dog & cat liver fluke, Definitive host : Dog, Cat, Fox, Pig and man. Location : Bile ducts and rarely in intestine and pancreatic ducts. Intermediate hosts : 1st Snails : Bithynia spp. 2nd Fishes : Cyprinid fishes at genera : Idus, Cyprinus, Tinca, Barbus, Abramis, Leuciscus and Scardinus. Morphological characters : 1. Size : 7 to 12x1.5 to 2.5mm. 2. Shape : small to medium sized flukes with flattened and translucent body which is narrow anteriorly. 3. Suckers weakly developed, situated in anterior third of the body. 4. Intestinal caeca are simple and reach near to the posterior extremity of the body. 5. Testes spherical/slightly lobed, diagonal and situated in the posterior part of the body. Ovary small, slightly lobed and anterior to the testes. 6. The space between the ovary and ventral sucker is filled with ccoiled uterus containing eggs. 7. Vitelline glands consists of series of transversely arranged follicles and occupy the middle third of the lateral fields. They are found as 8 groups on each side.

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PRACTICAL MANUAL Opisthorchis sinensis (Clonorchis sinensis) : Common name : Oriental or Chinese liver fluke Definitive host : Man, Dog, Cat, Pig, Location : Bile ducts, and sometimes in the pancreatic ducts and the duodenum. Intermediate hosts : 1st Snail : Bulinus spp. Vivipara spp. 2nd Fish : Cyprinid spp. Morphological characters : 1. Size : 25x5mm 2. Body is flat, transparent, wide posteriorly and tappering anteriorly. 3. Oral sucker slightly larger than the ventral one, which is situated at the posterior end of the anterior 3rd of the body. 4. Intestinal caeca simple, reach upto the posterior end. 5. Testes are deeply lobulated or branched, tandem in arrangement and present in the posterior 6. Ovary small, slightly lobed and antero-testicular. 7. Vitellaria are granular aggregations in the lateral fields and extend slightly beyond the middle third of the body. FAMILY: PROSTHOGONIMIDAE GENUS : Prosthogonimus Prosthogonimus pellucidus : Common name : Oviduct fluke of poultry Definitive host : Domestic fowls, duck, geese and wide variety of wild birds. Location : Bursa of fabricius, sometimes oviduct and rarely intestine. Intermediate hosts : 1st Snail : Gyraulus spp. and Bithynia spp. 2nd : Nymphal stage of dragon fly. Morphological characters : 1. Size : 8-9 x 4-5 mm 2. Shape : Narrow anteriorly and broad posteriorly or pear shaped. 3. Ventral sucker present in the anterior half of the body, and larger than oral sucker. 4. Intestinal caeca simple and reach upto the posterior end. 5. Testes are horizontal in position and are situated at the middle of the body. 6. Ovary is anterior to testes and highly lobed. 7. Vitelline glands occupy the lateral fields form the level of the ventral sucker to the posterior end of testes. 8. Genital pore opens lateral to oral sucker. FAMILY: ECHINOSTOMATIDAE GENUS : Echinostoma : : Fowls, goose, duck and other aquatic birds. : Rectum and Caecurn : 1st : Snail : Lymnaea spp. 2nd : Tadpoles of Rana esculenta

Echinostoma revolutum Definitive host Location Intermediate hosts

Morphological characters : 1. It measure 10 to 22 mm long and upto 2.25 mm broad. 2. The shape is long and narrow,

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PRACTICAL MANUAL Oral sucker is surrounded by a head collar which is provided with 37 spines. Ventral sucker is larger than oral sucker and present in the anterior part of the body. 4. Intestinal caeca simple and reach upto the posterior end. 5. Testes are tandem in arrangement, elongated and oval, slightly lobed, present behind the middle of the body. 6. Ovary is anterior to testes, oval in shape. Uterine coils with eggs is situated in between the ventral sucker and ovary. 7. Vitellaria consists of coarse follicles and lie in lateral fields extending from the posterior border of the ventral sucker to the posterior end of the body. FAMILY: NOTOCOTYYLIDAE GENUS : Notocotylus Species : Notocotylus attenuatus Definitive host : Fowl, duck, goose and wild aquatic birds Location : Caeca and rectum Intermediate hosts . Planorbis, Lymnaea and Bulinus spp. of snails. Morphological characters: 1. Small size 2. Ventral sucker is absent 3. Testes lie side by side at the posterior end. 4. Ovary is lobed and placed in between the testes 5. Cirrus sac is present and vagina is half the length of the cirrus sac. 6. Genital pore opens directly behind the oral sucker. 7. Uterus forms more or less regular transverse, coils and extends from the ovary to the posterior of the elongated cirrus sac.
3.

FAMILY: SCHISTOSOMATIDAE GENUS: 1. Schistosoma 2. Oxientobilharzia (Syn. Oxnithobilharzia) 3. Biharziella 4. Bivitellobilharzia 5. Heterobilharzia 6. Trichobliharzia Schistosoma spp. : Common name Definitive host Location : Blood fluke : Ruminants, Equines, Pigs and Man. . Different veins such as mesenteric vein, nasal vein, portal vein, haemorrhoidal vein etc. : Snails : Indoplanorbis spp., Lymnaea spp. :

Intermediate hosts Morphological characters Males : 1. It measures 8.3 to 19.0 x 0.26 to 0.6 mm. 2. Body is irregularly striated and dorsally covered with tubercles. 3. Suckers are weakly developed and situated at the anterior part of cylindrical body near to each other. 4. Testes are 5 to 7 in groups on each side, situated slightly posterior to genital pore and opposite to ventral sucker. 5. Intestinal caeca bifuracated at the level of ventral sucker, unite at the posterior end of the body and continue as a single caecum. 6. A groove like structure called Gynaecophoric canal present on ventral surface. Females :

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PRACTICAL MANUAL
1. It measures 10 to 28 x 0.09 to 0.26mm. 2. The shape is elongated and smooth. Spines present in suckers and on posterior end. 3. Ovary oval and situated in the middle of the body. 4. Uterus occupies about half the distance between ventral sucker and the posterior end,

containing numerous eggs arranged in a single row. 5. Vitellaria situated in the posterior part on either side of the intestinal caecum. Orientobilharzia spp. 1. Similar to Schistosoma spp. 2. Testes more than 60 in number FAMILY : PARAMPHISTOMATIDAE Common name : Ruman fluke Definitive host : Cattle, Buffalo, Sheep, Goat, Deer and equines Location . Rumen and Reticulum Intermediate hosts : Snails : Indoplanorbis exustus. Morphological characters : Immature amphostomes : 1. These are oval shaped or pear shaped, anterior end is narrow with the oral sucker at the tip, and posterior end is broad, with a large posterior sucker. 2. Intestinal caeca is simple and may or may not reach up to posterior end. 3. Genital organs are not well developed, only faint impressions of ovary, testes and uterus are seen. Mature amphistomes : 1. These are large thick and fleshy, with an oval or pear shaped body. 2. Vental sucker found at the posterior end of the body and known as posterior sucker. 3. These are blood red or brownish in colour when freshly collected. Paramphistomum cervl : 1. It measures about 5 to 13x2 to 5mm. 2. Pear shaped, slightly concave ventrally and convex dorsally with a large subterminal posterior sucker. 3. Intestinal caeca simple and does not reach upto the posterior end of the body. 4. Testes are slightly lobed and tandem in arrangement. 5. Ovary is posterior to testes. 6. The vetelline glands are in compact groups in between pharynax and posterior sucker. Cotylophoron cotylophrum: 1. It measures 5 to 13 x 25mm. 2. Ventral sucker is present in posterior part of the body. 3. Intestinal caeca are highly convoluted and reach upto posterior end. 4. Testes are tandem in position, slightly lobed. 5. Ovary present posterior to testes. 6. The vitelline glands are present in compact groups between the pharynx and posterior sucker. 7. The genital opening is surrounded by a sucker known as genital sucker, which is prominent and present just behind the bifurcation of intestinal caeca. Gastrothylax crumenifer : 1. It measures 9 to 18 x 5mm. 2. This fluke has a large ventral pouch, which opens anleriorly and extend over the ventral surface upto the posterior sucker. 3. The oral sucker is small and terminal.

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PRACTICAL MANUAL 4. 5. 6. 7. Intestinal caeca simple and ends at about the level of anterior border of the testes. Testes are lobed and horizontal in position with the ovary behind them. The posterior sucker is large and terminal and has a raised border. Uterus crosses from right to left at about the middle of the body In a 'S' shaped manner. Carmyerius spatiosus : 1. It measures 9 to 12mm long. 2. Ventral pouch is present. 3. The intestinal caeca reach upto the end of the 2/3rd of the body. 4. Testes are horizontal in position. 5 The posterior sucker is relatively small and spherical. Flschoederius spp. : 1. It measures 10 to 20mm long and 3 to 5mm wide. 2. Ventral pouch is present. 3. The intestinal caeca are not widely separated and end at a short distance behind the middle of the body. 4. One testes lies dorsal to the other. 5. Uterus runs forward in the mid-line. Gigantocotyle explanatum : (Found in the bile duct, liver and gall-bladder of buffaloes and cattle). 1. Pear shaped, slightly concave ventrally and convex dorsally with a large posterior subterminal sucker. 2. Intestinal caeca simple, convoluted and does not reach upto the posterior end of the body. 3. Tastes are larger than Paramistomurn cervi and diagonal In arrangements. 4. Ovary is posterior to testes. Gastrodiscus spp. (aegyptiacus/secundus) : 1. It is present in the large and small intestine of equines, pigs and elephants, measures 9-17 x 8-11mm. 2. Body is divided into two parts, anterior part is more or less cylindrical which is upto 4mm long and 2.5mm wide. The posterior portion is saucer shaped. 3. Oral sucker has two diverticulae / postero-lateral pouches. 4. Posterior sucker is subterminal and smaller than other amphistomes. 5. The testes are lobed, slightly diagonal and lie behind the middle with ovary posterior to them. 6. The vitelline glands occupy the lateral fields. 7. The genital pore opens at the level of the intestinal bifurcation. Gastrodiscoides hominis (caecal fluke of man and colon fluke of pigs) 1. Medium size 2. Body divided into two parts. Anterior being smaller than pasterior one and without papillae. 3. Pyriform in outline and bright reddish in colour. 4. Oral pouches present 5. Pasterior sucker has prominent deep cleft. Pseudodiscus collinsi (Colone fluke of equines) 1. Medium in size. 2. Body tapering anteriorly and rounded, posteriorly. 3. Intestinal caeca wavy. 4. Oral pouch present 5. Deeply lobed testes placed side by side.

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PRACTICAL MANUAL

CESTODES (TAPEWORMS) General Characters : They are commonly called as tapeworms due to their resemblance to a tape or a ribbon. The size may vary from a few milimeters to several metres in length containing a few to a large number of segments. The life cycle of a cestode includes eggs, cysts, occurring In the intermediate hosts and the adult tapeworm in the definitive host. They lack alimentary canal and the food absorption takes place at the microvilli of the Integument. The body is metamerically segemented and consists of three parts viz. Head (scolex), Neck (growth region) and Body (stroblla). The individual segment is called proglottid. The entire chain of segments Is called strobila. Anterior segments without well developed genital organs are immature, middle segments with well developed genital organs are mature and posterior segments containing eggs are gravid segments. Each mature segment may contain one or two sets of genital organs. Life cycle Is Indirect, except in Hymenolepis nana, and invertebrates or vertebrates act as intermediate hosts. MORPHOLOGICAL CHARACTERS OF COMMON CESTODE PARASITES CLASS: EUCESTODA FAMILY: ANOPLOCEPHALIDAE GENUS: Moniezia Moniezia spp. : Common name : Double pored tape worm of ruminants. Location and host : Found in the small intestine of sheep, goat, cattle and other ruminants. Intermediate hosts : Oribatid mites / grass mites. Morphological characters : Large sized, measuring about 600cm x 1.6cm. Body is thick and broad. The segments are more broad than long. The gravid segments appear like the cooked rice grains. Scolex : Shape is globular and measure 0.36 0.80 mm wide 4 prominent unarmed suckers. The rostellum and hooks are absent. Mature segment of the Moniezia expansa : The segments are broader than long and each segment contains 2 set of genital organs and the genital pores are bilateral. Ovary and vitelline glands together form a ring like structure on either side, median to longitudinal excretory canal. Testes are large in number and are scattered throughout the meduallary parenchyma. At the posterior border of each segment, there is a row of interproglottidal glands which are circular in shape and occupies the entire posterior border. Mature segment of Moniezia benedeni : 1. Segments are broader (upto 2.6cm) than M. expansa. 2. Each segment has 2 sets of genital organs and genital pores are bilateral. 3. Ovary & vit. glands together form a ring like or fan like structure on either side of segments. 4. Testes are in large number, scattered throughout medullary parenchyma.

1. 2. 3.

1.
2. 3.

4.

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PRACTICAL MANUAL 5. At posterior border of each segment the interproglottidal glands are arranged In a continuous row and form a band like structure in the middle portion.

GENUS: Anoplocephala Anoplocephala perfoliata : Location and host : : Large and Small intestine of equines. Intermediate hosts : : Cysticeraoid develops in oxibatid mites Morphological characters : 1. Adults 5-8 cm in length and 1.2cm in width 2. Scolex 2-3 mm in diameter and is provided with a "Lappet" behind each sucker. 3. Segments are wider than long, each with a single set of reproductive organs. Anoplocephala magna : 1. Occurs in the small intestine and rearely in stomach of horses. 2. Upto 8Ocm in length and 2.5cm In width. 3. Scolex is large, 4-6mm wide, and without lappets. Paranoplocephala mamillana : 1. Occurs in the small intestine and occasionally in the stomach of horses. 2. Measures 6-50 by 4-6mm. 3. Scolex is narrow, the opening of the suckers are slit like. FAMILY : THYSANOSOMIDAE Genus : Stilesia Avitellina spp. : Location and host : Intermediate host : Morphological characters :

Small intestine of ruminants Oribatid mites Adults may reach 3m in length and 3mm wide. They are almost cylindrical at the posterior end. Proglottids are short and not distinctly segmented as a result of which they appear as thick white threads.

Scolex of Avitellina spp. : 1. The scolex measures upto 2mm in diameter, slightly larger than Moniezia scolex. 2. Shape is globular and has 4 unarmed suckers. 3. The rostellum and hooks are absent. Mature segments of Avitelllna spp. 1. Very short and not well defined segments 2. Single set of genitalia. Uterus lies transversely at the middle portion. 3. Excretory canals are prominent with fewer no. of testes on either side. 4. Genital pores irregularly alternate, lateral to the excretory canals. Gravid segment of Avitellina spp. 1. These sgement are narrow, more long than broad and are beaded in appearance. 2. Each segment has a single egg capsule/paruterine organ, which is oval in shape and contains the fertilized eggs. Stilesia spp. : Location and host : Slilesia globipunctata occurs in the small intestine and S. hepatica in the bile ducts of sheep, goat, cattle and other ruminants. Intermediate hosts: Oribatid mites Morphological characters : Adults are 45-60cm long and upto 2.5mm wide. Proglottides are short, thin and the body coils like a ribbon. Scolex of Stilesia spp.:

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PRACTICAL MANUAL The scolex is smaller than Moniezia scolex. Globular in shape, Rostellum and hooks are absent and has 4 unarmed suckers. Mature segment of Stilesia spp. 1. Body is weakly segmented. 2. Single set of genitalia with Irregularly alternating genital pores 3. Eleven testes on either side of the excretory canal. 4. Ovary is globular and median to excretory canal. 5. Uterus is dumbbell shaped with two portions united by a transverse duct. Gravid segment of Stilesia Spp. : 1. The segments are more broad than long. 2. In the gravid segments all the genital organs are degenerated. Only two paruterine organs are present which contain fertilized eggs. FAMILY: DIPYLIDIIDAE GENUS: Dipylidium Dipylidium caninum : Common name Location and host Intermediate hosts

: Double pored tapeworm of dogs : Small intestine of dog, cat, fox and occasionally man. : Fleas belonging to the species Ctenocephalides canis, C. felis and Pulex irritans and the dog louse, Trichodectes canis. Morphological characters : The adult parasites may be upto 50cm long. Segments are more long than broad and are barrel shaped or cucumber seed shaped. Scolex of D. caninum : 1) Scolex is globular with 4 unarmed suckers. 2) Rostelium is present and bears 3 or 4 rows of rose thorne shaped hooks. Dipylidium caninum (Mature segment) 1. The mature segments are barrel shaped or cucumber seed shaped. 2. Each segment has 2sets of genital organs and genital pores are bilateral. 3. Ovary and vitelline glands together form a structure on either side of the segments, which looks like bunch of grapes. 4. Testes are in large number and scattered throughout the medullary parenchyma. (In gravid segments the eggs are in egg capsules, each containing upto twenty eggs.) FAMILY: DAVAINEIDAE Genus : Davainea Species : Davainea proglottina (Smallest tapeworm of poultry) Location and host : Duodenum of fowl, pigeon and other birds. Intermediate hosts : Gastropod molluscs like Limex, Aripn, Agriolimax spp. Morphological characters 1. Adults are microscopic (0.5-mm) with only 4-9 proglottids. 2. Rostellum and suckers are armed. 3. Genital pores alternate regularly. GENUS : Raillietina Location and host : Small intestine of domestic poultry and other birds. Intermediate hosts : 1) Raillietina cesticilus : Beetles. 2) R. echinobothrida : Ants 3) R. tetragona : Ants

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PRACTICAL MANUAL Scolex of Raillietina cesticillus : 1. Neck is absent and the scolex is large, which bears a wide rostellum armed with 400 to 500 small hooks. 2. The suckers are inconspicuous and unarmed. In scolex of Raillietina echinobothrida rostellum is present, which bears 200 number of hooks, arranged in 2 rows. The suckers are circular in outline and are armed with 8 to 10 rows of hooks, all of which are about twice as large as those of R. tetragona. Scolex of Rallietina tetragona Scolex is small. Neck is thin. The rostellum has one row of minute hooks. The suckers are oval in shape and armed with 8 to 10 rows of small hooks. GENUS: Cotugnia Cotugnia digonopora (Double pored tapeworm of fowl) Location and host : Small intestine of fowls. Morphological characters : 1. It is upto 1.07mm long. 2. Rostellum has two rows of small hooks, suckers unarmed. 3. Mature segment has two sets of genital organs. 4. Gravid segment has egg capsules, each containing one egg. FAMILY : DILEPIDIDAE GENUS: Amoebotaenia Amoebotaenia sphenoides : Location and host : Small intestine of fowls. Intermediate host : Earthworms Morphological characters : 1. Small worms and roughly triangular in shape, measuring upto 4mm long. 2. Entire body possess about 20 segments and these segments are narrow. 3. Rostellum has 12-14 spiner shaped hooks arranged in a single row. 4. Testes are about 12 or more and are present in the posterior part of each segment. 5. Genital pores irregularly alternate. FAMILY: HYMENOLEPIDIDAE GENUS: Hymenolepis Location and host : Small intestine of duck and geese. Morphological characters : Very thin parasites measuring 13mm by 1mm, Rostellum is retractile, bearing a single circle of hooks. Suckers are unarmed. FAMILY: TAENIIDAE GENUS : Taenia, Echinococcus Taenia spp. :
1. 2. 3. 4. 5. 6. 7. 8. Species Tsolium T. saginata T. hydatigina T ovis T. pisilormis T. taeniaformis T. multiceps T. galgeri Host man man dogs and wild carnivores Cat Dogs and wild carnivores -do Location Small intestine Small intestine Intermediate host Pigs Cattle Ruminants Sheep and goats Rabbits and hares Mice and rats
Ruminants, ungulates and man

Larval Stage Cysticercus cellulosae C. bovis C. tenuicollis C. ovis C. pisiformis C. fasciolaris


Coenurus cerebralis

Goats

Coenurus

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PRACTICAL MANUAL
9. T. sorialis Dog and Fox Logomorphs like rabbits cerebralis Coenurus serialis

1. 2. 3. 4. 5. 6.

7.

Morphological characters These are large tapeworms and the size may vary from 40mm to 8m. The mature segments are square shaped. The gravid segments are more long than broad. Taenia scolex : 1. Rostellum is present and it has two rows; of hooks. One row of small another row of large hooks arranged alternately. (Rostellum & hooks absent in T. saginata) 2. Suckers are unarmed and shape is globular. Taenia spp. (mature segments) : Segments are more or less square shaped. Each segment has one set of genital organs. Genital pores irregularly alternate. Ovary is single but bilobed and is present in the posterior part of each segment. Vitelline glands are found in a compact mass, they are placed transversely in the posterior border of the segment, just behind ovary. Testes are in large number and they are scattered throughout medullary parenchyma. The uterus is a blind sac like tube present longitudinally at the middle of the segment. Taenia spp. (Gravid segments) : 1. The gravid segments are more long than wide. 2. Gravid uterus has 5 to 10 branches on either side. Echinococcus granulosus Location and host: Small intestine of dogs and other carnivores and the larval stage, hydatid cyst, is found in various ungulates and man. Morphological characters : 1. It is a very small parasite. 2. The body has a scolex and 3 to 4 segments. The last segment is the gravide one and it is more than of the length of the body. 3. The mature segments has one set of genital organs. 4. The ovary is single and it is kidney shaped and is placed in the posterior part of segments. 5. Vitelline glands are in a compact mass. They are placed just behind the ovary in the posterior boarders of segments. 6. Testes are about 50 in number and they are scattered throughout medullary parenchyma. 7. The rostellum has two rows of hooks varying from 30 to 60 In number. FAMILY: MESOCESTOIDIDAE GENUS: Mesocestoides Location and host Intermediate host Small intestine of dogs and other carnivores. 1st : Oribatid mites, in which cysticercoid develops. 2nd : Amphibia, reptiles, birds and mammals in which tetrathyridium develops in the peritonial cavity. Special feature : Tetrathyridia multiplies asexually by longitudinal splitting of the scolex in the second intermediate host as well as definative host. Mesocestoides Iineatus (Gravid segments) 1. The gravid segments are more or less square shaped. In the segments all the genital organs are degenerated. : :

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PRACTICAL MANUAL 2. Gravid uterus is replaced by an oval shaped paruterine organ containing fertilized eggs.

CLASS: COTYLODA FAMILY: DIPHYLLOBOTHRIIDAE GENUS: Diphyllobothrium Diphyllobothrium latum (Broad fish tapeworm) Location and host : Small Intestine of man, dog, cat, pig and other fish eating animals. Intermediate host : 1st : Cyclops like Diaptomus gracilis, in which procercoid develops. 2nd : Fresh - water fish, in which pleurocercoid develops. Morphological characters: Scolex has no suckers but has weakly muscular grooves called bothria. Mature segments are quite different from class Eucestoda with a bilobed ovary in the centre, follicular vitalline glands distributed in the paranchyma along with testes, genital pore at mid ventral portion and a rosette shaped uterus. Eggs are operculated. MORPHOLOGICAL CHARACTERS OF SOME CESTODE EGGS Moniezia spp. (Cattle & Sheep) Egg shell capsule is roughly square shape with rounded corners or some what traingular in shape. (M. expansa - triangular. M. benedini - square) Embryophore is pear shaped and is called pyriform apparatus and has a cap,and contains a hexacanth embryo. Size : 60-80 micron in diameter Anoplocephala spp. (Horse) Similar to Moniezia egg. Taenla spp. (Cat, Dog, Man) Egg shell capsule absent Small egg, round shape, Embryophore thick, radially striated. Hexacanth embryo present. Size : 35-80 m Hymenolepls spp. (Birds, Rats, Man) The egg shell is thin enclosing the hexacanth embryo. Wide space present between egg shell and embryophore Comparatively larger than eggs of Taenla species. Size : 60-80 m diameter. Dipylidium caninum (Dogs and Cats) Large oval capsules containing 4 to 20 eggs which have thick but unstriated embryophore. Size : 25-40 fr m diameter. Dlphyllobothrlum latum (Dog, Cat and Man) Small oval egg with thin shell (Similar to trematode egg) Ends appear pointed than round Operculum present Germinal mass undifferentiated and uniformly distributed. COMMON LARVAL STAGES OF CESTODES Cysticercus tenuicollis 1. It is the bladder worm stage of Taenia hydatigena.

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PRACTICAL MANUAL 2. 3. It develops in the peritoneal cavity of sheep, goat and cattle. It has a single scolex, a long neck and a bladder like cavity which is filled with large amount of serous fluid. The cyst wall is thin and translucent. Coenurus cerebralis It is the bladder worm state of Multiceps multiceps or Taenia multiceps. It develops in 1W brain, spinal cord and sometimes In the subcutaneous tissue of sheep and goat. Shape is nearly circular and the cyst wall is very thin and transparent. Large number of scolices are formed in the inner surface of cyst wall which are very clearly visible from outside. 5. The cyst contains large amount of serous fluid. Hydatid cyst 1. It is the bladder worm stage of Echinococcus granulosus. 2. The size of the cyst may vary and may reach upto 50cm in diameter. 3. Shape is round or spherical. 4. Cyst wall has 2 layers. Outer layer is very thick and tough, but inner germinal layer is very thin. 5. Scolices are not attached to the inner germinal layer, and present inside the daughter cysts. These daughter cysts containing the scolices are known as brood capsules. Sometimes these brood capsules may be ruptured so that the scolices are found freely floating in the cyst fluid and these freely floating scolices are known as hydatid sand. 6. These cysts develop in liver and lungs of all animals, human beings and even birds. NEMATODES (ROUND WORMS) Nematodes belong to the Phylum NematheIminthes, commonly known as "Round worms" The Class Nematoda are "true rond worms", present in all domestic livestock, birds and human beings. Characters : 1. Body is unsegmented, cylindrical and elongate, tapering at both the ends, covered with a tough transparent or translucent cuticle. 2. Body cavity and alimentary canal present. 3. Length of the worms varies from few mm to severel centimetres. 4. Sexes are separate, male parasites are smaller than females. 5. Body wall consists of cuticle, subcuticle and musculature. 6. In the anterior end some specialized structures such as head bulbs, amphids and alae are presents, which are commonly sensory in function. 7. Life cycle may be direct or indirect. FAMILY: ASCARIDIDAE GENUS : Ascaris, Neoascaris, Toxocara, Toxascaris Ascaris suum (Gross): Common Name : Large roundworm of pigs Location and Host : Small intestine of pigs Morphological characters : 1. The males measures 15 to 25cm in length and 3 to 4mm in diameter. Female parasites measure upto 45cm in length and 5mm in diameter. 2. These are very large stout worms. Both ends taper. 3. The cuticle is thick and the worm is fairly rigid 4. The mouth opening has 3lips, one dorsal and 2 subventral. Each lip bears on its inner surface a row of minute denticles.

1. 2.

3. 4.

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PRACTICAL MANUAL Parascaris equorum (Ascaris megacephala) : Common Name : Large roundworm of equines Location and Host : Small intestine of equines and rarely cattle. Morphological characters : 1. Males 15-28 cm and females upto 50cm long. 2. These are rigid stout worms with a large, head. 3. Three lips are conspicuous and are separated by three small intermediate lips. 4. Male tail has small lateral alae. In addition to number of precloacal and post cloacal papillae, a single median papilla occur on the anterior border of cloaca. Toxocara vitulorum (Neoascaris vitulaxum) : Common Name : Large round worm of cattle. Location and Host : Small intestine of cattle and buffaloes. Morphological characters: 1. Anterior portion is narrower than body. 2. The lips are narrow anteriorly and broad at the base. The cuticle Is thin and translucent so that the internal organs are visible to outside. 3. Males measure upto 25cm by 5mm and females 30cm by 6mm. Toxocara canis (Gross) : Common Name : Large round worm of dogs or arrow headed worm. Location and Host : Small intestine of dog and fox. Morphological characters : 1. The male parasites measure upto 10cm and female parasites measure upto 18cm. 2. The anterior end has small lips and a large cervical alae. The anterior end is bent ventrally. 3. The posterior end of the male is slightly coiled and curved and this end has a small caudal alae and a terminal narrow appendage. FAMILY: HETERAKIDAE GENUS : Ascarldla, Heterakis Ascaridia galli (Gross) Common Name : Large round worm of poultry Location and Host : Small intestine of fowls, guinea fowls, turkey, goose and wild birds. Morphological characters : 1. Male parasites measure 5 to 7cm and female 7.2 to 11 cm. 2. Anterior end has 3 small lips. 3. Both the ends are pointed and cuticle is thick. They are whitish in colour. Ascaridia galli (Male posterior end) Morphological characters : 1. Tail of the male has a small caudal alae, ten pairs of short and thick papillae. 2. There is a circular preclocal sucker with thick cuticular rim. 3. Spicules are two in number and of equal size. Heterakis gallinarum : Common Name : Caecal worm of fowls.

: Location and Host Cacea of fowls, ducks, goose and other birds. Morphological characters : 1. Males measure 7-13 mm and females measure 10-15mm.

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PRACTICAL MANUAL 2. 3. 4. 5. 6. There are large lateral alae extending some distance down the sides cf the body. Oesophagus has a strong posterior bulb. Tail of male has a large alae, a prominent precloacal sucker and 12 pairs of papillae. Spicules are unequal, the right one is long and slender. The left one is short and broad. Vulva lies behind the middle of the body. FAMILY: OXYURIDAE GENUS: Oxyuris Oxyurls equi : Common Name : Pin-worm of equines Location and host : large intestine of equines Morphological characters : 1. Males measure 9-12 mm and females upto 150mm in length. 2. The male has one pin-shaped spicule. 3. Adult female is slatey-grey or brownish in colour with a long narrow tail which may be more than 3 times as loog as the rest of the body. FAMILY: STRONGYLOIDIDAE GENUS : Strongyloides Parasitic forms are parthenogenetic, Species : S. papillosus (ruminants), S. westeri (horse pig & zebra), S. stercoralis (man, dog, cat, fox), S.cati (cat), S. ransomi, (pig), S. avium (fowl) Location : Small intestine Morphological characters : 1. Small thin worms. Only females are parasitic. 2. Oesophagus is long and filariform. 3. Uterus contains a single row of eggs. FAMILY: STRONGYLIDAE GENUS: STRONGYLUS Common Name : Large strongyle of horses Location and host : Large intestine of equines Morphological characters : a) Strongylus vulgaris (Anterior end) : 1. Has a large, deep and oval buccal capsule. Mouth opening surrounded by external and internal leaf crowns. 2. Two ear-lobe shaped dorsal teeth present at the base of the buccai capsule. 3. The thickening of the internal wall of the buccal capsule forms the dorsal gutter. b) Strongylus edentatus (Anterior end) : 1. Head separated from body by a constriction. 2. Buccal capsule cup shaped and does not have teeth. Dorsal gutter present. c) Strongylus equinus (Anterior end) : 1. Has oval buccal capsule with external and internal leaf crowns. 2. A large dorsal tooth with bifid tip and 2 subventral teeth preset. FAMILY : TRICHONEMATIDAE GENUS : Oesophagostomum

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PRACTICAL MANUAL Type of species : O. columbianum Common Name : Nodular worm of sheep and goats Location and host : Colon of sheep, goat, camel, wild antelopes Morphological characters : 1. Males measures 12-16.5 mm and females 15.21.5mm. 2. At the anterior end cuticle forms a mouth colar which is fairly high. 3. On the ventral surface, the anterior end has a cervical groove. 4. In between mouth colar, and cervical groove, the expanded part is known cephalic vesicle. 5. A cervical alae is present at the anterior part below the cervical groove, which is pierced by cervical papillae. 6. External and internal leaf crowns present in the anterior margin of the buccal capsule. 7. In males, bursa is well developed. The ventroventral, lateroventral and mediolateral, posteriolateral rays are united except at the tips. There are two equial spicules. 8. In females, genital pore is anterior to cloaca and has a short vagina. The ovijector is kidney shaped, raised above the cuticle. Pimply gut : It is a disease condition caused by the 3rd stage larvae of Oesophagostomum spp. in the intestine of previously sensitized sheep, goat, cattle, buffaloes and pig. In this case nodules are formed in large numbers in the walls of both the small and large intestine. These nodules contain green to yellowish green pus or caseous material and the larvae. Some nodules may be calcified. FAMILY : SYNGAMIDAE GENUS : Syngamus Syngamus trachea : Common Name : Gapeworm of fowls. Location and host : Occurs throughout the world in the trachea of fowl, turkey, pheasant, goose and wild birds. Morphological characters : 1. These worms are bright red in colour when fresh and both sexes occur permanently in copulation. 2. Males 2-6mm long, Female 5-20mm long. 3. Buccal capsule is cup shaped, without leaf crowns. It bears 6-10 small teeth at the base. 4. Male bursa is short with stout rays, spicules are equal. 5. Vulva opens in the anterior third of the body. FAMILY : ANCYLOSTOMATIDAE Genus : Ancylostoma, Bunostomum, Gaigeria Ancylostoma caninum : Common Name : Hookworm of dog. Location and host : Small intestine of dog, cat, fox and rarely human beings. Morphological characters : 1. Males 10-12 mm and females 14-16mm long. 2. Freshly recovered worms are grey or reddish in colour depending upon the presence of blood in their alimentary canal. 3. The anterior end is bent dorsally, so the worm appears like a hook.

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PRACTICAL MANUAL Buccal capsule is deep. The anterior margin of the buccal capsule has three teeth on either side, of which the 1st pair smallest and the 3rd pair is largest. 5. The base of the buccal capsule has a pair of triangular dorsal teeth and the pair of centrol-lateral teeth 6. Male bursa is well developed and the spicules are of equal size. 7. In the female, the vulva is situated in the posterior 3rd of the body. Bunostomum trigonocephalum / phlebotomum ; Common Name : Hookworm of sheep, goat and cattle. Location and host : Small intestine (ileum and jejunum) Morphological characters : 1. Males 12-17 mm long. Females 19-26 mm long. 2. Anterior end is bent dorsally. 3. the anterior magin of the buccal capsule bears a pair of chitinous plates and a dorsal cone at the base. 4. Male bursa is well developed with large lateral lobes and an asymmetrical dorsal lobe. 5. Spicules are spoon shaped. 6. In the female, vulva opens near the middle of the body. Gaigeria pachyscelis : Common Name : Hookworm of sheep and goats. Location and host : Duodenum Morphological characters : 1. Males upto 20mm and females upto 30mm long. 2. Buccal capsule contains a large dorsal cone and a pair of subventral lancets having several cusps. 3. Male bursa has small lateral lobes and a large dorsal lobe. FAMILY : TRICHOSTRONGYLIDAE GENUS : Haemonchus Haemonchus contortus : Common Name : Stomach work of ruminants or Twisted wireworm or Barbers pole worm. Location and host : Abomasum of sheep, goat, cattle and other ruminants. Morphological characters : 1. Males 10-20mm long. Females 20-30 mm long. 2. Buccal capsule is small and it contains a dorsal lancet. 3. male bursa has elongate lateral lobes and a small dorsal lobe, which is asymmetrically situated against the left lateral lobe and is supported by an inverted Y shaped ray. 4. In the female, the vulva is present in the posterior third of the body and the vulva is usually covered by a triangular or tongue shaped vulval flap or the vulval flap may be reduced to a small knob-like structure. 5. In the male, the spicules are of equal in size, dark brown in colour with barbed ends.
4.

Mecistocirrus digitatus (Abomasum of ruminants) Male posterior end The bursa has well developed lateral lobes. Dorsal lobe is symetrical Spicules are equal, long and slender. They are united together throughout their whole length. Cooperia spp. (Small intestine and rarely abomasum of ruminants)

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PRACTICAL MANUAL Male posterior end The well developed bursa has a small dorsal lobe. Spicules are short, stout, brownish pigemented and have a wing like expansion at the middle. Paracooperia nodulosa (Small Intestine, caecum and colon of buffaloes) Head with a cephalic vesicle Spicules sub-equal and trifurcated In females, the genital openinig is covered with a flap. Trichostrongylus axei and other species. (Hair worm of ruminants) Location : Abomasum and small intestine Anterior end : Buccal capsule, teeth, cutting plates and leaf crowns absent. Excretory pore situated in a notch at the anterior end. Posterior end Well developed bursa, especially lateral lobes. Dorsal lobe very small, dorsal ray slender. Characteristic short and thin ventro ventral ray. Two unequal, dissimilar, twisted and spatula shaped spicules. Dictyocaulus filaria (Thread/lung worm of sheep and goats) Male posterior end. Spicules are equal, stout and dark brown in colour and are boot or sock shaped. FAMILY : THELAZIIDAE GENUS : Spirocerca Spirocerca lupi: Common Name Location and host : : Oesophageal worm of dog Occurs in the well of the oesophagus, stomach & aorta, rarely in the stomach of dog, fox, wolf, jackels. Coprophagus beetles.

Intermediate Hosts :

Morphological characters : 1. Males 30-54mm long. Females 54-80mm long. 2. Worms are usually coiled in a spiral manner, appear blood-red in colour, when freshly removed. 3. Lips are trilobed and pharynx is short. 4. Male tale bears a caudal alae, 4 pairs and one unpaired median pre-cloacal papillae two pairs of post-cloacal papillae and a group of minute papillae near the tip of the tail. 5. In the male, the left spicule is very large and ringt spicule is vary small. Gongylonema pupulchrum: Common Name : Oesophageal worm of sheep, goat and cattle. Location and Host : Occurs on the oesophageal mucosa, firmly embedded in a zigzag fashion. It may also occur in the rumen. Intermediate Hosts : Coprophagus beetles Morphological Characters : 1. Males upto 62mm long. Females upto 145mm long. 2. The cuticle at the anterior and has a cervical alae and a number of found or oval thickenings termed as cuticular bosses. FAMILY : TRICHURIDAE

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PRACTICAL MANUAL GENUS : Trichuris Trichuris ovis : Common name : Whip-worm Location and host : Caecum of sheep, goat, cattle and other ruminants. Morphological Characters : 1. Male 50-80mm and females 35-70mm long. 2. Anterior part of the body is long and slender, while the posterior part is much thicker. 3. In males the hind end is curled and there is one spicule surrounded by a protrusible sheath which is usually armed with fine cuticular spines. 4. In females the vulva is situated at the beginning of the wide part of the body. 5. The worms are white in colour. FAMILY : FILARIIDAE GENUS : Dirofilaria Dirofilaria immitis : Common name Location and host : : Heart-worm of dogs. Right ventricle and pulmonary artery of dog, cat, fox, wild carnivora and sometimes in man.

Intermediate host : Mosquitoes Morphological Characters : 1. Males 12-16cm and females 25-30cm long. 2. The worms are slender and white !n colour. 3. In males, the hind end is spirally coiled, and the tail bears small lateral alae and large number of papillae. Spicules are unequal, left is long and pointed and right is short and ends bluntly. 4. In females, vulva is situated just behind the end of the oesophagus. FAMILY: SETARIIDE GENUS: Setaria, Stephanofilaria Setaria digitata : Location and host : Peritoneal cavity of cattle, buffalo, zebra Intermediate host : Mosquitoes Morphological Characters : 1. Worms are several centimeters long, milk-white in colour and taper towards the hind end, which is spirally coiled. 2. Mouth is surrounded by a cuticular ring which bears dorsal and ventral arid frequently also lateral prominences. 3. In males spicules are Unequal and in females the tail has spines or conical projections Stephanofilaria assamensis : Location and host : Skin of catle, particularly at the hump region. Intermediate host : Musca conducens Morphological characters : 1. Small milk-white parasites, males 2.3-3.2mm and females 6.1-8.5mm. long. 2. The oral aperture is surrounded by a protruding cuticular rim which has a denticulate edge. 3. The cuticle is spiney and transversely striated. MORPHOLOGICAL CHARACTERS OF OTHER NEMATODES

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PRACTICAL MANUAL Tetrameres spp. Mature female Mature female is globular or spherical in shape and blood red in colour. (Male is slender and filiform and white in colour). Marked sexual dimorphism. Draschia megastoma (Stomach worm of equines) Anterior end Head is prominent and is separated from the body by a constriction forming the shoulders. Two trilobed lateral lips present around mouth. Pharynx is funnel shaped. Gnathostoma spinigerum (Stomach worm of cat) Anterior end Two large distinctly trilobed lips. Large head bulb armed with 6-11 rows of backwardly directed hooks. Anterior 2/3 of the body armed with large flat spines. Ascarops strongylina (Stomach worm of pigs) Anterior end 2 small lateral lips with a tooth on each side internally. Cervical alae on left side only. Pharyngeal wall with a number of spiral thickennings. Physocephlus sexalatus (Stomach worm of pigs) Anterior end Cervical cuticle inflated followed by 3 cevical alae on each side. Asymmetrical cervical papillae. Pharynx with annular thinkennings. Physaloptera praeputialis Anterior end The cuticle forms a collar like projectin at the anterior end. Two large lips with three internal denticles and one large external tooth. Buccal capsule absent. Cervicle papillae prominent. Posterior end A large prepuce like fold of cuticle completely encloses the caudal end of the body. Fertilized female A conspicuous ring of brown cement material around the vulvae opening. Thelazia rhodesi (Eye worm of cattle and buffalo) Anterior end Small buccal capsule with inconspicuous lips. Cuticle bears prominent transverse striations. Trichinella spiralis in muscle section Numerous cysts are found within the muscle fibres. Larvae are spirally coiled and encapsuled in the cysts. IDENTIFICATION OF ACANTHOCEPHALA Eggs Elongate Larva (Acanthor) present within the egg. Spines present on One end of the larva. Larval stages Acanthella, Cystacanth Occur in beetles (interrnediale hosts) Adult Stage Macracanthorhynchus hirudinaceus

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PRACTICAL MANUAL (Thorny headed worm of pig). The cuticle is transversely wrinkled. Proboscis is globular and is armed with 6 rows of hooks.

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PRACTICAL MANUAL

SOME COMMON EGGS OF NEMATODES


Ascaris spp. (Pigs, Man) Sub-globular shape. Brownis-yellow in colour Thick shell with prominent albuminous projections. The unsegmented yolk appears as a compact mass in the centre. Size :50-80 micron in diameter. Toxocara vltulorum (Cattle and Buffalo) Round or sub-globular In shape and the thick egg shell Is finely pitted. Size: 75-95 m x 60-75 m. Toxocara canis (Dog, Cat) Sub-globular in shape with thick finely pitted shell. Size : 90-75 m Toxascaris leonina (Dog, Cat) Oval with broadly rounded ends. Egg shell smooth. Size :75-85 m x 60-75 m Parascaris equorum (Horses, donkeys) Similar to Toxocara egg. but egg shell is thick and pitted. Size : 90-100 rn Ascaridla galli (Birds) Oval shape. Thick but smooth egg shell. Size : 73-92 x 45-57 m Oxyuris equi (Horses, donkeys) Elongate, oval shape, flattened at one side. A plug or operculum at one pole. Size : 73-92 m Strongyloides spp. (Cattle, horse, dog, bird) Comparatively small in size and oval in shape. Egg shell is thin with blunt poles. Well developed larva is present inside. Strongyle spp. (All domestic animals) Eggs of nematodes belonging to Superfamilies, Strongyloidea, Trichostrogyloidea, Ancylostomatoidea. Oval shape, size varies with species (small, medium, large eggs) Thin egg shell. Embroyonic mass has 2-64 cell divisions. Size : 60-70x36-45 m Spirocerca lupl (Dog) Thick shelled and gelatin capsule shaped. Comparatively very small in size. Contains fully developed 1st stage larva inside. Size : 30-37 x 11-15 m Trichuris spp. (Cattle, Sheep, Goat, Dog, Man & Pig) Barrel shaped with thick shell. Dark brown coloured. Transparent plugs present at either poles. Yolk is unsegmented. Size : 50-10x23-65 m

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PRACTICAL MANUAL LARVAL STAGES OF NEMATODES IN FAECAL SAMPLE/SMEAR First & Second Larval Stage (L1 & L2) of Strongyles (Bovine, Equine, Ovine, Canine, Feline, Porcine) Rahabditiform oesophagus. Larve of Strongyloides spp. Oesophagus is long, Rhabiditiform It is 1/3 of the length of the body. Third Larval Stage (L3) Sheath of previous larval stage present. Filariform oesophagus.

IN BLOOD SAMPLE/SMEAR Microfilaria Cattle, Equines Ex. Setaria spp. Elongate, worm like. Sheathed. Dirofilaria immitls : Dog Unsheathed. Anterior end tapering. Tail is straight. IN SKIN SCRAPINGS Microfilaria Cattle, Horse Ex. Stephanofilaria, Onchocerca, Parafilaria, Long and slender larvae

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PRACTICAL MANUAL DEMONSTRATION OF ORGANS AND TISSUES OF THE HOST AFFECTED BY ENDO AND ECTO PARASTTES Endoparasites like helminths and protozoa affect different systems, organs and tissues of the host. A large number of helminths and protozoa are found in different parts of the gastro-intestinal tract. However, some are found in the bile ducts, liver, respiratory tract, urino-genital tract, gall bladder, kidneys, pancreas, lymphnodes, muscles, eyes and body cavities. Some of the helminths like schistosomes and protozoa like Babesia, Theileria, Trypanosoma, Plasmodium species are found also in the blood vascular system. Ectoparasites are generally found on the body surface and in subcutaneous tissue, but few may occur in muscles, G.I. tract, urino-genital system, respiratory passages and ears of human beings, animals and birds. The following organs/tissues affected with parasites are demonstrated. 1. Liver affected with Fasciols gigantica : Immature stages are more pathogenic which migrate in the liver paranchyma and cause destruction of liver tissue, and production of haemorrhagic tracts resulting in fibrosis and cirrhosis of liver, particularly in harbivorous animals. Adult parasites are found in the bile ducts which cause inflammation, thickeninng, fibrosis and calcification of the bile duct wall. 2. Bile duct and liver affected with Gigantocotyle explanatum : This is an amphistome found in the bile ducts of cattle and buffaloes. The immature stages which occur in the duodenum are more pathpgenic, but the adults in the bile ducts are less pathogenic. However, in heavy infections they may block the bile ducts and cause digestive disturbances and jaundice. 3. Liver and peritoneal cavity affected with Cysticercus tenuicollis : C. tenuicollis, the larval or metacestode stage of canine tapeworm Taenia hydatigena, migrate in the liver and finally reach the peretonial cavity of harvivores and pigs. They are not much pathogenic, but in heavy infections may cause destruction of the liver tissue during their migration, resulting in digestive disturbances. 4. Liver and lungs affected with Hydatid cyst : Hydatid cyst, the larval or bladderworm stage of canine tapeworm Echinococcus granulosus, generally develops in the liver and/lungs of domesticated animals and human beings. The size of the cyst may vary from a pea-nut to a football. Large sized cysts produce pressure atrophy in the liver or lung tissues, resulting in digestive disturbances or respiratory problems, respective to their location. 5. Liver of calf affected with larvae of Toxocara vitulorum : During their development the larval stages of many nematodes migrate through the liver, including the larvae of T. vitulorum. They cause damage to the liver tissue. 6. Brain of goat affected with Coenurus cerebralis : Coenurus cerebralis cysts, the bladder worm stage of Taenia multiceps, develops in the brain and/spinal cord of sheep, goat and cattle.

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PRACTICAL MANUAL The clinical entity varies according to the location of the cyst, which includes staggering gait, incoordination in movement, turning round and round, high steping, blindness and paralysis. 7. Heart of dog affected with Dirofilaria immitis : The heart worm, a filarial nematode, occurs in the right ventricle of dogs. These are white elongate parasites. They cause enlargement of the right ventricle, cardic insufficiency, respiratory distress. 8. Rumen affected with amphistomes : Large number of adult amphistomes are usually found attached to the wall of the rumen and reticulum of sheep, goat and cattle. When fresh they appear red in colour. Adult amphistomes are not much pathegenic and they only cause denudation of the rumen papillae. The immature amphistomes attached to the wall of the anterior part of the small intestine are highly pathogenic. 9. Intestine of dog affected with tapeworms : Two types of tapeworms, Dipylidium canium and Taenia spp. infect the small intestine of dogs. The mature segments of D. caninum are cocumber-seed shaped or barrel shaped and those of Taenia species are rectanugular or square shaped. These are not much pathogenic, but in heavy infections may cause cattrahal enteritis. 10. Small intestine of fowl affected with tapeworms : Tapeworms belonging to various genera such as Davainia, Raillietina, Amoebotaenia, Choanotaenia, Hymenolepis, etc. infect the small intestine fowls. They may cause catarrhal enteritis, decreased gain in body weight and reduced egg production. 11. Oesophagus of dog with tumor caused by Spirocerca lupi : The nematode S. lupi is known as oesophagial worm of dog and produce nodules or tumors in the wall of the oesophagus, stomach and aorta. The paraite remains in a coiled manner in the tumor and redish brown in colour. Large sized tumors cause difficulty in deglutition, vomition, difficulty in respiration, coughing and change in voice. 12. Small intestine of calf, pup, foal and fowls packed with ascaris worms : Ascaris parasites such as Toxocara vitulorum of cow and buffalo calves, Toxocara canis and Toxascaris leonina of dogs, Parascaris equorum of equines, Ascaris suum of pigs and Ascaridia galli of fowls infect the small intestine. They cause catarrhal enteritis, collic pain, irregular bowl, nervous symptoms, anaemia, pot bellied condition and stunted growth. 13. Intestine of harvivores showing pimply-gut lesions : Large number of nodules are seen on the serous surface of the intestinal wall and the affected gut is known as pimplygut. The nodules are formed due to the migration of third stage larvae of Oesophagostomum spp. into the intestinal wall of sensitized animals. Sensitized or previously exposed animals develop some kind of immunity, as a result the larvae cannot return back to the intestinal lumen to complete their development and are traped in the intestinal wall.

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PRACTICAL MANUAL There is infiltration of inflammatory cells around the the larvae, resulting in the formation of nodules. The larvae may remain alive for three months in the nodule and later they die and become calcified. The nodules contain a greenish pus like material. 14. Small intestine of dog with hookworms : Ancylostoma species of hookworms infect the small intestine of dogs. The parasites are voracious blood suckers and cause haemorrhagic enteritits and severe anaemia. 15. Large intestine of goat infected with Trichuris spp. : Trichuris species attach to the wall of the caecum and colone of sheep, goat, cattle and other animals. They are commonly known as whip worm because they look like a whip due to their long thread like anterior end and short broad posterior end. In heavy infection they may cause catarrhal enterities. 16. Skin affected with sarcoptic mange : Sarcoptic mange in domesticated animals and man is caused by Sarcoptes scabiei, a microscopic mite. They burrow into the skin and cuase itching, irritation, exudation, crust formation, keratinites, thickening and wrinkling of the skin and alopesia. 17. Oesophagus of buffalo affected with Sarcocystis fusiformis : The cysts of this sepcies are macroscopic and visible to the necked eye. They appear like cooked rice grains and lies parallel to the muscle fibres. They infect skeletal muscles like tongue, oesophagus, diaphragm, heart, etc., They are usually nonpathogenic and rarely cause muscular weakness. 18. Caecal and intestinal cocidiosis in poultry : Caecal coccidiosis is caused by Eimeria tenella and intestinal coccidiosis is by different Eimeria species like E. necatrix, E. acervulina, E. brunetti, etc. They cause severe haemorrhagic lesions on the caecal and intestinal wall with disquamation of the epithelium and inflammation of the intestinal wall. The caeca and intestine are swollen and filled with partially or fully clotted blood. PATHOLOGICAL LESIONS : ENDO- AND ECTO PARASITES Sr. Pathological lesion Endo or Animals affected No. ectoparasite involved 1. Pimply gut Oesophagostomum Sheep, goats, spp. larvae cattle 2. Oesophageal granuloma & Spirocerca lupi Dogs tumour in aorta 3. Tumour in stomach Habronema Equines megastoma 4. Nasal granuloma Schistosoma nasale Cattle 5. Punched necrotic abomasal Theileria annulata Cattle ulcers 6. Splenomegaly Haemoprotozoan Cattle, buffaloes, infections dogs & equines 7. Summer sores (cutaneous H. muscae Equines habronemosis) H. microstoma

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PRACTICAL MANUAL 8. 9. 10. 11. 12. White raised lesions on intestinal Eimeria spp. mucosa Greyish white pinhead to millet Schistosoma sized nodules on the liver surface indicum Red marks on intestinal mucosa Hook worms Intestinal ulcers Triodontophorus Thrombi and emboli in anterior Stronglus vulgaris mesenteric artery and its branches (Verminous arteritis) Nodules in connective tissue S. edentatus under peritoneum Nodules in the wall of small intestine Small strongyles Enlarged liver, haemorrhagic and Fasciola gigantica honeycombed with tracts of migration Caecal distension with Eimeria tenella haemorrhages Pneumonic lesions of parasitic Dictyocaulus spp. bronchitis Ringworm like lesions in T. axei abomasum / stomach, inflammation and corrugation of mucosa Abomasal mucosa is swollen and H. contortus covered with patechial haemorrhages, shallow ulcers Milk spots on liver Ascaris suum Cysts in striated muscles Sarcocystis spp. Sheep & goats Sheep Ruminants Equines Equines

13. 14. 15. 16. 17. 18.

Equines Equines Sheep, goat, cattle and buffaloes Poultry birds (3 to 7 weeks old) Cattle and sheep Sheep, goats and equines Sheep, goats

19.

20. 21. 22.

Depressed or sunken unclers Histomonas on liver meleagridis 23. Warble holes on the back of cattle Hypoderma bovis 24. Mucous membrane of horse stomachGastrophilus spp. infected with horse bots; circular pits develop where the larvae have been attached 25. Wet eczema on lumbo-sacral area, Fleas abdomen, inside the hind legs and the neck 26. Wool-less crusty patches around Psoroptes ovis shoulder, along the sides of the body 27. Chronic dermatitis in Stephanofilaria region assamensis

Pigs Cattle, sheep, goat buffaloes and pigs Poultry birds Cattle Equines

Dogs, cats and equines Sheep the hump Cattle

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PRACTICAL MANUAL

LABORATORY DIAGNOSIS OF HELMINTH INFECTIONS


FAECAL EXAMINATION Collection and Examination of Faeces Faeces intended for parasitological examination should be collected from the rectum unless the animal observed in the act of defaccation when the sample may be collected from the ground. With large animals, collection of faeces from the rectum can be accomplished by hand without difficulty. Smaller animals such as young lambs and dogs can usually be induced to defaecate by inserting a moistened finger into the rectum and gently massaging with a rotary motion until the external sphincter relaxes. For dogs, moist cotton swabs introduced into the rectum and rotated well to collect the maximum possible faecal matter and dipped in saline in a test tube are useful for examination of eggs. Suitable containers for the dispatch of samples to the laboratory are: 30ml wide mouthed screw capped bottles of glass or preferably of plastic, which should be filled to the top if possible so as to exclude air as much as possible and so diminish the rate of development and hatching of the eggs. Samples should be collected from several animals in an affected herd, some of which should be collected from the most seriously affected animals and a few from the less seriously diseased in order to observe the contrast in the counts. Faecal samples collected from the ground in a field in which the animals have been running, are less useful for diagnostic purposes, but where rectal samples cannot be obtained, these should be examined. Such samples should be selected from the most recently dropped faeces, and several samples should be collected. Since eggs embryonate rapidly, the faeces should be stored in the refrigerator unless examination is carried out within a day. For samples sent through the post, the faeces are fixed and preserved in 10% formalin to minimise development and hatching. The detection of parasite stages in faeces from animals suffering from diarrhoea may be markedly impaired. The diagnosis of parasitism by microscopic techniques is more reliable when repeated examinations are made. For example, negative results are sometimes obtained from faecal examinations if the parasites are not mature enough to deposit eggs. This might also occur when they are enclosed in tissue nodules in case of certain parasites such as strongyles or oesophagostomes. Tapeworm segments often leave the digestive tract without disintegrating, and negative results may be obtained unless segments are detected. Despatch of samples The samples should be despatched by the available means to the nearest well equipped laboratory by adopting the following procedure : i) Use container of 10-20 ml capacity and fill it up to 1/9th to 1/8th with the faecal sample. The remaining portion is filled with the preservative (10% formal saline). ii) For Eimeria oocysts, 2.5% potassium dichromate solution is used as a preservative. iii) For culturing nematode larvae, fresh faecal samples should be carried in thermos bottles with ice. iv) Stopper the bottle tightly and seal it with dark red resin or wax. v) Apply the following label on the container Host : Material : Preservative used : Time of despatch : Address of owner : vi) Pad the container in a stong cardboard or wooden box by filling the empty space with strips of papers, wooden saw dust or cotton.

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PRACTICAL MANUAL Macroscopic examination All the available information regarding quantity, form, consistency, colour, reaction, presence of blood/mucus in faeces should be noted. i) For this, 15-20 gm of faeces is spread in a large Petri dish. ii) Tapeworm segments/nematodes/immature trematodes are searched with the camel hairbrush. The recovered parasitic stages are identified with or without staining as described later. Microscopic examination It is of two types : 1. Qualitative examination (a) Direct Smear Method It is possible to demonstrate the presence of parasite stages (eggs or larvae of helminths, oocysts of coccidia) by the examination of a thin smear of emulsified faeces. A small quantity of faeces is placed on a slide and mixed with some droplets of water / normal saline, and a cover slip is placed on the fluid. The slide is investigated using a magnification of 10x and 40x. This method, however, suffers from the following drawbacks : i. It is effective only where the concentration of parasite stages is high, ii. it is frequently difficult to identify them since they are partially covered by debris iii. Quantitative results cannot be obtained. Parasites found with the direct smear method. Coccidia and helminth eggs (Only when high numbers are present); Cestode and trematode eggs (mainly in birds). Concentration methods: The principle of these methods is to concentrate all the eggs in a given amount of faeces into a small quantity of emulsion as possible and then examining a drop of it. (b) Sedimentation technique : Procedure 1. Place a lump of faeces ( -l tsp. / 5-10g or a few faecal pellets (5-10) of sheep or goats) in a cup or glass pestle. 2. Add approximately 50-100ml of tap water and mix thoroughly with a spatula/rod/mortar until all the faecal material are broken down. 3. The mixture is poured through a wire mesh seive with an aperture of 500-800mm to remove large lumps. The strained fluid is caught in a bowl. The seive is rinsed with water and the debris left on the screen is discarded. 4. Transfer the suspension to centrifuge tubes and centrifuge at 2000 rpm for 2 min. 5. Discard the supematent. 6. Mix the sediment well and take a small quantity of it and mix it with a drop of water on the slide. 7. Apply a cover slip and examine under low power objective of the microscope. 8. Thickness of the smear should be such that if the slide is placed on a newspaper, you should be able to read the fine print through the smear. Parasite stages detected with the sedimentation method : Eggs of trematodes. Larvae of lung worms; Oocysts of Coccidia.

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PRACTICAL MANUAL (c) Flotation technique Separating the eggs from faecal debris by floating them on a variety of saturated solutions (1) common salt of specific gravity 1.18 - 1.19, (2) sugar solution sp. gr. 1.25, (3) sodium nitrate, (4) Glycerine, (5) Zinc sulphate solution (32.5%) of sp.gr. 1. 18, (6) A.E.X (Hcl of sp.gr. 1.03 + Ether + Xylol - equal parts). When faeces are emulsified in liquids of high specific gravity than that of eggs and either centrifuged or allowed to stand, the worm eggs and many protozoan cysts float to the top while the heavy coarse debris settles to the bottom. The top film can then be removed and examined. Many nematode and few cestode eggs float in a liquid with a specific gravity of between 1. 10 and 1. 20. Trematode eggs, which are much heavier, require a specific gravity of 1. 30-1.35. (d). Direct centrifugal floatation or D.C.F. method (Lanes method). Procedure Follow the steps 1,2 and 3 as mentioned in procedure for sedimentation technique. 4. Transfer the suspension to a conical measure and fill with tap water to the top and allow to settle for 30 min. 5. Discard the supernatant carefully upto a remaining sediment of approx. 10ml. 6. The sediment is stirred and a sample of 2ml is poured into a centrifuge tube. 7. The tube is placed in the centrifuge. With a pipette, saturated NaCI or any other floatation fluid is added until a convex meniscus stands above the top of the tube. 8. A thick 19 x 19mm square cover glass is placed on the tube, ensuring that no bubbe is trapped under it. The cover glass may additionally be coated with a solution of egg white and glycerol (1: 1) to improve adherence of the eggs. 9. The tube is centrifuged at 2000 rpm for 2-3 min. 10. Pick off the cover glass and place it on a slide and examine under a microscope by using higher magnification. (e). Wills technique (Levitation technique): Small floatation tube with emulsion of faeces in the above solutions upto its third is filled to its full capacity with the same saturated solution till a convex surface is formed and is allowed to stand for 30 to 60 minutes by which time eggs would have floated up. Apply a coverslip or slide to the surface, remove fast that drop of fluid, containing eggs, by means of capillary attraction and then examine that drop in the slide. Parasite stages found with the flotation method:Eggs of cestodes and most of nematodes; larvae of lung worms, oocysts of cooccidia. Trematode eggs are not satisfactorily detected with the flotation method. It is worthwhile to remember that almost all cestodes except the members of cotyloda do not discharge eggs, but gravid segments containing numerous eggs are shed and passed out. Hence it is not always possible to find cestode eggs in faecal samples. A sizable quanuty of the faeces is placed in a petridish and with the aid of two mounted needles, the whole mass is searched for tapeworm segments which will be whitish in colour showing active movements while fresh. 2. EGG COUNMNG METHODS OR QUANTITATIVE METHODS (PLATE - XVII) (a) Stolls Dilution Method - For counting nematode and trematode eggs. 1. Three grams of faeces is taken in a test tube (45 ml graduated) 2. Fill the tube upto 45ml with N/10 NaOH (4 gms NaOH, 1It water) and add 10-12 glass beads, close with a stopper and homogenize the faecal materials. 3. 0.15ml of the suspension is drawn with a pipette and placed on a slide and a cover slip is applied.

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PRACTICAL MANUAL 4. 5. (b) Count the total number of eggs. Multiply the number of eggs by 100 which gives the eggs per 1 gram of faeces. Mc Master Method This technique is used where the number of eggs or larvae per gram of faeces should be counted (EPG = Eggs per gram of faeces) Procedure 1. Weigh 3g of faeces and add 42ml tap water. 2. Homogenize and pour suspension through a 250mm aperture sieve, collecting the filtrate. 3. Collect the filtrate, agitate and fill a test tube of 15ml volume. 4. Centrifuge at 2000 rpm for 2 min. 5. Pour off the supernatant; agitate sediment and fill tube to previous level with flotation solution. 6. Invert tube 6 times and remove fluid with pipette to fill both chambers of McMaster slide quickly. 7. Examine on one chamber and multiply number of eggs or larvae under one etched area by 100, or two chambers and multiply by 50 to arrive at the number of eggs per gram (epg) of faeces.
MORPHOLOGICAL CHARACTERS OF HELMINTH EGGS (PLATE - XVIII & XIX)

Fasciola spp. i) Oval in shape and yellowish in colour ii) Operculum, embryonic mass and shell indistinct. Posterior knob absent. Paramphistomes i) Oval in shape and greyish white in colour. ii) Operculum, embryonic mass and shell distinct. iii) A small knob is present on the end opposite to operculum. Dicrocoelium dendriticum i) Flattened on one side and convex on other side. ii) Operculum is present at one end. iii) Ciliated miracidium is present inside the egg. Schistosoma indicum i) Oval or spindle shaped. ii) Ciliated miracidium is present inside it. iii) A terminal spine is present on one end of the egg. Opisthorchis spp. i) Shape resembles electric bulb. ii) Operculum at anterior end and small tubercular thickening at posterior end. iii) Contains a ciliated miracidium which is asymmetrical. Moniezia spp. i) Shape is triangular/quadrangular/globular. ii) Well developed pyriform apparatus is present. iii) Hexacanth embryo is present in this apparatus.

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PRACTICAL MANUAL Dipylidium caninum i) Egg capsule is globular in outline and contains 4-20 eggs, which are globular in shape. ii) The egg is having upper coat of albuminous layer, beneath which there is embryophore and oncosphere. iii) Hexacanth embryo is present within the oncosphere. Taenia spp. i) Globular in shape ii) Oncosphere has hexacanth embryo iii) Embryophore has radial striations. Hymenolepis spp. i) Globular/oval in shape. ii) Embryophore has thickenings at the poles and thickenings are provided with 4-8 polar filaments. iii) Hexacanth embryo is present within the oncosphere. Ascaris lumbricoides / A. Suum i) Oval or globular in outline and are thick shelled. ii) The outer albuminous layer has prominent projections or corrugations. iii) Unsegmented embryo is present inside the egg. Toxocara spp. / Parascaris equorum i) Subglobular in outline. ii) Thick shelled and outer albuminous layer is pitted. iii) Unsegmented embryo is present inside the egg. Ascaridia galli i) Oval in shape ii) Smooth shelled iii) Albuminous layer is absent. iv) Unsegmented embryo inside the egg. Oxyuris equi i) Elongated and slightly flattened on one side. ii) Provided with a plug at one pole. Strongyles i) Oval in shape ii) Thin shelled iii) Embryonic mass segmented when laid Strongyloides spp. i) Oval with blunt ends ii) Thin shelled iii) Contain a fully developed embryo when laid. Dictyocaulus filaria i) Eggs expelled in nasal discharge or sputum contain fully formed larvae when laid. ii) In faeces L1 is passed which has a small cuticular knob at anterior extremity and numerous brownish food granules in the intestinal cells. Trichuris spp. i) Eggs are brown in colour.

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PRACTICAL MANUAL ii) Barrel shaped with a transparent plug at either pole. iii) Contain unsegmented embryo when laid. Spirocerca lupi i) Eggs have thick shells, capsule shaped. ii) Contain larvae when laid. EXAMINATION OF SKIN SCRAPINGS, NASAL SCRAPINGS AND URINE Skin scrapings are examined for the recovery of parasitic mites. The scrapings must be obtained in a manner that takes into account both the nature of the lesion and location of the mites. These should be taken from moist part near the edge of the lesion avoiding the inclusion of large amount of dry crust, hair or wool. In some cases if the animal gets furious, it is advised to go for local anaesthesia. For collection, dip the scalpel blade in glycerine or liquid paraffin. Pick up a fold of animal skin at the edge of the suspected area pinching it firmly between thumb and forefinger. Now by holding the blade at right angles to the skin, scrub the crust of the fold several times till blood begins to seep through the abrasions. The scraping will adhere to the blade. Boil the material in 10% potassium hydroxide to dissolve the debris/hair etc. After cooling, pour the material into a centrifuge tube and centrifuge for 2min at 2000 r.p.m. Examine a drop of the sediment for mites under the microscope. Nasal scrapings, obtained with the help of the rotatory motion of spatula / scalpel in the nasal tract, and fresh nasal discharge are collected from the suspected animals in formal saline solution. It is centrifuged at 1500 r.p.m. for 5 min. and the sediment is examined under low power of microscope for schistosome eggs. Urine is sedimented with or without centrifuge machine and the resultant sediment is examined under low power of microscope for the presence of Stephanurus dentatus eggs in pigs and Dioctophyma renale eggs in dogs.

LABORATORY PROCEDURES IN HELMINTHOLOGY


Examination of Faeces (Coproscopy) A laboratory unit for routine diagnostic coproscopy can be Installed at a reasonable cost and effort. Equipment, material and tools necessary for microscopic excamination of faecal samples are as follows: Compound microscope Plastic specimen containers (objectives :10,40,100 X, eyplece:10X), with tight fitting lid Dissecting microscope and magnifying glass Centrifuge, Centrifuge tubes Slides and Coverslips (Plastic or glass) Pasteur pipettes Measuring cylinder Cotton wool, Flotation solutions etc. Wooden/plastic spatulas Sieves with different mesh sizes/ Tea strainer Dissection needles, forceps Points to Remember during Collection and Preservation of Faecal Samples 1. Faecal examination should be conducted on fresh faecal sample. 2. Faeces should be collected directly from the rectum of large animals where as owners of small animals ,should be instructed to collect at least 5 grams of faeces immediately after defecation. 3. The material obtained from the rectum on a thermometer or faecal loop should not be relied for routine examination.

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PRACTICAL MANUAL 4. 5. If rectal samples are not available owners should be asked to collect faeces immediately after defecation. 5-10 grams of faeces should be collected for routine faecal examination. the faeces are dispatched to the laboratory In plastic containers with a tight lid or closed plastic bags. Creation of anaerobic condition in the container by filling completely with the faeces can preserve the sample upto 7 days without refrigeration Faeces can be preserved by refrigeration upto 1-2 wks. Equal volume of 10% formalin or formal saline added to faeces can be used as preservative / fixative. Label the containers with description of owners name and address, date and place of collection, species, age and sex of animal and history sheet of the disease. Freezing of sample should be avoided as freezing will distort parasite eggs. It the faeces are dried, soak in small amount of water/flotation fluid till the faeces are soft. Macroscopic examination of faeces is made for consistency, colour, presence of blood or mucus, presence of adult or larval parasites and tape worm segments. Samples from 10% of herd/flock should be collected if the intensity or degree of infection of a group are to be assessed.

6. 7. 8. 9. 10. 11. 12. 13.

Macroscopic Examination Spontaneously discharged tapeworm segments and nematodes (roundworms, pinworms, trichurids etc.) can be recognized by direct macroscopic inspection of the faeces, by sedimentation and decantation of faecal suspension in water in a glass tray, or by pouring the faeces, mixed with tap water through a sieve,(aperture:0.3mm) which will retain the worms; nematodes, immature amphistomes and tapeworm proglottids, which are then transferred into a Petri dish containing saline and can be observed against a dark background by the naked eye or by using a magnifying lens. Interpretation of Microscopic Examination No egg in the whole sample negative (-) Few eggs in the whole sample low grade infection(+) Few eggs In each microscopic field moderate infection (+ +) Many eggs in each microscopic field heavy infection (+++) Examination of Urine Collect urine sample, spin at 1500 rpm for 2 min, transfer the sediment with a pipette on to a slide, add one or two drops of water, place a coverslip on the sample and examine microscopically. Schistosoma eggs in ruminants and man, Stephanurus eggs in pigs. Capillaria and Dioctophyma eggs in carnivores can be detected by this method. Examination of Blood for Detection of Microfilaria A variety of methods can be used to detect microfilaria In venous blood samples. Direct Blood Smear Procedure 1. Place one drop of heparinized or EDTA treated blood on a slide, add a droplet of physiological saline, mix and cover with a coverslip. 2. Examine directly under low power (10X) of a microscope for live microfilariae. Larvae can be immobilized by placing a drop of 10% formalin at the edge of the coverslip. For further confirmation examine under high power (40x).

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PRACTICAL MANUAL Knott's Concentration Method This is the standard test used to screen for microfilariae in blood which includes lysis of erythrocytes, fixation and staining of larvae. Procedure 1. Mix 1 ml heparinized or EDTA treated blood with 9ml of 2% formalin In a 15ml tube; mix by inverting the tube several times after closing the tube with your thumb. 2. Centrifuge the tuba for 5rnin at 1500rpm then discard the supernatant. 3. Add 1 or a few drops of 0.1 methylene blue or 0.2% methyl green solution to the sediment and mix well. 4. Remove a drop of stained sediment with a pipette, spread it on a slide and add a coverslip. 5. Examine all sediment, drop by drop, under the microscope. Note that heparinized (EDTA-treated) and formalinized blood samples are suitale for mailing to a laboratory for Haematocrit Method Procedure 1. Fill a heparinized microhaematocrit tube with blood and centrifuge for 1 min. 2. Place the tube under a dissecting microscope and examine the interface of the plasma and buffy coat for motility : microfilariae (and haemoftagellates) will be observed actively swimming in the plasma. 3. Break the tube at the buffy coat interface and dab the buffy coat and a small amount of plasma on a slide; add a coverslip and examine for live microfilariae (at 4OX) or allow it to air dry, fix for 1min with methyl alcohol and stain with Giemsa for 30rnin for identification (1.5ml Giemsa stain in 15ml distilled water buffered to pH 7.2). Note that heparinized and non-fixed (non-treated) blood specimens should be examined on the day of collection. Characteristics for Differentiation of Microfilaria Microfilariae of the pathogenic heartworm (Dirofilaria immitis) and of other, less pathogenic filaroid species can be discriminated on the basis of several criteria such as size, morphology, movement, numbers, staining characteristics. This needs experience, knowledge of regional prevalence, and thus it should best be performed by an expert diagnostic unit. Microfilaria of D. reconditum tend to move rapidly across the microscopic field with a snake like movement. D. immitis microfilaria tend to remain in the same area and coil and uncoil. Characteristic Dirofilaria Immitis Dipetalonema reconditum Body shape Usually straight Usually curved Body length 295-325 m 250-288 m Midbody width 5-7.5 m 4.5-5.5 m Shape of cranial end Tapered Blunt Shape of tall Straight Usually curved or hooked

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PRACTICAL MANUAL COLLECTION AND PROCESSING OF HELMINTH PARASITES Trematodes Fresh and live flukes are collected in normal saline and stored In 10% formalin or 70% alcohol. Cestodes Tapeworms including scolices (heads) should be placed in water at 37C for about one hour and then stored in mixture of 5% glycerine in 70% alcohol or 5-10% formalin. Nematodes Nematodes collected in saline are immediately transferred to 70-90% hot ethyl alcohol. Once the nematodes, are fixed they can be stored indefinitely in glycerin alcohol. Preparation and Preservation of Helminth Parasites involves the following steps. 1. Collection 6. Destalnlng 2. Stretching 7. Dehydration 3. Fixation 8. Clearing 4. Preservation 9. Mounting 5. Staining 10. Labeling and Storing of slides 1. Collection Once the worms are recovered from live animals or post mortem or from slaughter house are shaken vigorously in saline and later in 2 changes of saline to remove dirt and mucus. Worms are left in tap water for few minutes to few hours in refrigerated condition for killing. 2. Stretching Flukes and tapeworms to be stained should be pressed between two slides and held in place by tyinq with cotton thread or rubber band to keep them flat before fixing. Curled nematodes are relaxed in 70% hot ethyl alcohol. 3. Fixation It fixes the parasie in the present state and also preserves and hardens the specimen in natural condition. Trematodes, cestodes and larger nemadoes are fixed and mounted for generic or species identification. 10% formaline is used as fixative for flukes Bouins fluid / AFA for cestodes Hot 70% alcohol for nematodes 4. Preservation Trematodes and cestodes are best preserved in 5% formalin with 5% glycerine. For nematodes 70% alcohol with 5% glycerin is a good preservative. Nematodes can be stored in 10% formalin. 5. Staining All traces of fixing reagent should be removed from specimens prior to staining. Trematodes and cestodes are stained for Identification whereas nematodes are not stained as the Impermeable cuticle of nematodes does not allow penetration of stains easily. Trematodes may be stained with carmine or haematoxylin stains. Carmine has better penetrating power than haematoxylin stains. Borax Carmine stains should be used for alcohol fixed specimens and haematoxylin for formalin fixed specimens of parasites. Specimens fixed In Bouins/AFA solution should be washed In several changes of 70% alocohol where as specimen fixed In formalin be washed In water. Specimens are placed from 70% alcohol Into carmine stain for 24-48hrs. Borax Carmine stain can be diluted with equal amount of 70% alcohol for better

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PRACTICAL MANUAL penetration of the stain where as Haematoxylln stain should be diluted with 10 parts of distilled water. 6. Destalnlng Destaining is done to remove excess stain by using acidulated 70% alcohol. Carmine stained specimens are put into 70% alocohol before transfer into destaining solution. Haematoxylin stained specimens are rinsed in distilled water and passed through 35% to 50% alcohol (about 30 minutes in each grade) before transferring to the acidulated alcohol. While destaining, acid alcohol is changed occasionally and allowed to act until the worms become a light pink against a white back ground. The cortical layer should be free of stain whereas enough stain should remain to colour the internal organs. Note: Leaving the specimens in destaining solution for long period will result in dull coloured specimens. 7. Dehydration Specimens are dehydrated in different grades of alcohol starting from 35%,56%,70%,85%,95%, and 100% (absolute alcohol) leaving the specimen for 15-30 minutes in each grade of alcohol. 8. Clearing It Is done for improved visibility of Internal organs. Clove oil, oil winter green/methyl salicylate and xylene are used. Xylene is cheaper compared to other. Note : Specimen kept for long time in clearing solution will make the spacimen brittle. 9. Mounting While mounting correct amount of the mounting medium Is used to avoid air bubbles. Once the mountant shrinks after drying, the gap should be filled by adding some mountant at the edge. Once mountant is hardened the excess medium on the slide is scraped with a single edqed safety razor. Canada Balsam or DPX are commonly used mountants for trematodes and cestodes. 10. Labeling and storing of slides Label is placed on one end of the slide and should contain the scientific name of the specimen, host, locality, date of mounting and name of the individual who is submitting the specimen. The slides are stored in a slide box. Processing of Nematodes Nematodes are not stained. Formalin preserved nematodes are washed in running water and dehydrated before clearing. Large nematodes are cleared in beech wood creosote. Nematodes of medium to small size are cleared in lactophenol. Very small nematodes are cleared and mounted with glycerin. Clearing agents used for nematodes include lactophenol, beech wood creosote or 95% glycerin alcohol. After examination the nematodes from clearing agents are washed either with water or 70% alcohol before storing. Usually temporary mounts are made for nematodes. Glycerin is an excellent mounting medium for nematodes. In tropical climate the glycerine jelly used for mounting of nematodes become very thin, Rubin's mountant is a clearing agent as well as mountant for nematodes. Cotton Blue Lactophenol Technique For Nematodes 0.01 to 0.0025% aqueous solution of cotton blue in lactophenol is a good clearing agent for nematodes.

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PRACTICAL MANUAL

LIFE CYCLE OF PARASITES


It could be either direct or indirect. Could involve aquatic or terrestrial environment. Intermediate hosts could be invertebrates or vertebrates. Metamorphosis may or may not be present. A single egg may give rise to a single adult or numerous adults (Polyembryony / Paedogenesis). The stage infective to the host can enter the host actively or passively through : - food and water. - ingested with intermediate / paratanic host. - inoculated by blood sucking or biting arthropods. - Active skin penetration etc. The life cycle of a parasite from egg to adult worm is complete with or without the involvement of an intermediate host/vector. It is of two types : Direct : Indirect : No intermediate host/vector is involved e.g. gastrointestinal nematodes (Ascarids, trichostrongylids, strongylids, hookworms etc.) The life cycle of a parasite involves one or more intermediate hosts / vectors. (Trematodes, cestodes and some nematodes). It can happen in the following manners.

TREMATODE LIFE CYCLE (PLATE - IV & IX)


The general features of trematode life cycle are : 1. 2. 3. 4. 5. 6. Indirect life cycle. Both aquatic and terrestrial cycles observed. Eggs are composite. Snail is always the 1st intermediate host. Metamorphosis occurs. A typical life cycle involves the following stages : Egg - Miracidium - Sporocyst - Redia - Cercaria - Metacercaria - Immature fluke - Adult fluke. 7. 8. 9. 10. Miracidium is the infective stage to the snail. Multiplication of larval stage occurs in the snail (Paedogenesis). Sporocyst, redia and cercaria stages are seen in the snail. Cercaria or metacercaria is infective to the 2nd intermediate host or to definitive host. (Cercaria is the infective stage of schistosomes and Metacercaria is the infective stage of other trematodes). Each cercaria or metacercaria gives rise to a single adult worm. One miracidium released from one egg of a trematode gives rise to many infective stages (cercariae or metacercariae) - called polyembryony.

11. 12.

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PRACTICAL MANUAL 13. The life cycles of the following trematodes involve a second intermediate host in which metacercariae develop and infection of D.H. is by ingestion of 2nd I.H. (a) (b) Dicrocoelium dendriticum Eurytrema pancreaticum : : Ant (Formica spp.) i. ii. (c) (i) (ii) (d) (e) (f) (g) Prosthogonimus spp. Collyriculum faba Opisthorchis tenuicollis Clonorchis sinensis Nanophyetus salmincola Paragonimus spp. : : : : : Dragonfly naiads (nymphs). Cyprinid fish Cyprinid fish Salmon fish. Crab, Crayfish. Grasshopers(Conocephalus maculatus). Tree crickets (Oecanthus longicaudus). institution.

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PRACTICAL MANUAL

CESTODE LIFE CYCLE


Always indirect except in case of Hymenolepis nana. Either invertebrate or vertebrate intermediate hosts are involved. Life cycle could be terrestrial or aquatic. Definitive host gets infected by ingestion of metacestode stage. Metamorphosis occurs.

Eucestoda Life Cycle (Plate - VI & X) Life cycle is mostly terrestrial. Stages involved in the life cycle are : Egg (embryonated) - Cysticercoid - Adult tape worm OR Egg (embryonated) - Cysticercus / Strobilocercus / Coenurus / Hydatid / Tetrathyridium Adult tape worm. Embryonated egg develops either into cysticercoid when ingested by an invertebrate host or into Cysticercus / Strobilocercus / Coenurus / Hydatid / Tetrathyridium when ingested by a vertebrate host. Multiplication of larval stages do not occur in Cysticercoid, Cysticercus and Strobilocercus, whereas it occurs in Coenurus, Hydatid and Tetrathyridium. DH gets infected by ingesting the bladderworm stages or the IH along with the bladder worm larval stages. Cotyloda Life Cycle (Plate - VI) This life cycle is always aquatic, indirect with two intermediate hosts. It involves the following stages : Egg (unembryonated) - Coracidium - Procercoid - Plerocercoid - Adult. 1st IH is a copepod / isopod. 2nd IH is a fish. Coracidium is infective to 1st IH in which Procercoid develops. The 1st IH containing Procercoid is infective to 2nd IH in which Plerocercoid develops. The Plerocercoid undergoes multiplication and is infective to the D.H. The DH is infected by eating the 2nd IH containing the infective stage(s) in which the adult tape worm develops.

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PRACTICAL MANUAL

NEMATODE LIFE CYCLE


Can either be direct or indirect. Metamorphosis is not observed. Stages in the life cycle are : Egg - L1 - L2 - L3 - L4 - L5 - Adult. (i) Eggs hatch under optimal conditions (moisture and temperature) and larvae are free living for a time. Thereafter they develop into infective larvae L3, which are active and infect the definitive host via food and water (Strongylids and trichostrongylids) or by penetration of hosts skin (Ancylostoma and Bunostomum). (Plate - V) (ii) Eggs develop in the open under optimal conditions but do not hatch. The infective larvae L2 develop within the eggs and eggs containing L2 gain entry through oral route (Ascarids). Indirect : (Plate - XI) Stages infective to the IH is egg or L1, which develop upto L3 stage. L3 in the IH is infective to the DH. Infective stage enters DH actively or passively along with food / water / IH or inoculated by arthropods IH. It is of three sub types. (i) (ii) (iii) Eggs hatch or the worms are viviparous and larvae enter the intermediate host after a short free existence e.g. Habronema spp. Intermediate host is eaten by definitive host. Unhatched eggs are ingested by intermediate hosts (e.g. Spiruroidea), which are eaten by definitive host. Worms are viviparous and larvae enter the blood of the host from where they are eaten by blood sucking intermediate hosts, inside which infective larvae develop. When the intermediate host sucks blood of the definitive host, the infective larvae are released in the host through its skin e.g. filarids.

Direct : (Two sub types)

LIFE CYCLE OF ACANTHOCEPHALA


The life cycle is always indirect, requiring an arthropod intermediate host (Crustacean / insect). The life cycle stages are Egg - Acanthor - Acanthella - Cystacanth - Adult. Embryonated egg is laid. It has to be ingested by the IH for further development. In the IH, egg hatches liberating acanthor, develops into acanthella, which develop further and undergoes encystment and is called as cystacanth. DH gets infected by ingesting the IH.

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