The Life History of Calicophoron ijimai (stomach fluke of cattle) in New Zealand
[Read before the Canterbury Branch, December 5, 1950; received by the Editor, February 12, 1951.]
A total of 45.6% of the cattle examined was infested with stomach flukes.
Planorbis kahuika (Finlay and Laws) is shown to be an intermediate host for Calicophoron ijimai.
All stages in the life history of C. ijimai are described.
A note on the rearing of snails under laboratory conditions is included.
A stomach fluke of cattle has been recorded in New Zealand, under the names of Amphistomum conicum and Paramphistomum cervi, but the vector has not been previously determined, nor has any significance been attached to the pathogenicity of this organism. The question of pathogenicity is not dealt with here, but details of structure and life history are given. The life history of the fluke is described from laboratory infestations which produced larval stages identical with those collected from Lake Wairarapa. A brief note on the New Zealand intermediate host is included. The adult has been described in general from specimens collected over a ten-month period from widely separated localities. Histological details which enabled the material to be placed as Calicophoron ijimai (Nasmark, 1937) came from longitudinal serial setions cut from eight specimens.
Technique and Method.
In the culture of snails, petri dishes 5in by lin were used. Tap water in Wellington was toxic, so filtered rain or creek water was used. Elodea suspended in the water provided the necessary vegetation, and calcium carbonate, in a fine suspension, was added. Egg masses were placed in freshly prepared dishes, pure cultures of Planorbis kahuika 1.5 mm. in diameter being available three weeks from the laying of the eggs. Boiled spinach was a useful food supplement.
Description of the Adult (Plate 100)
Diagnosis.—Length, 9.06 mm. (mean). Breadth, 4.33 mm. (mean). Dorsal line evenly curved. Acetabulum, 2.4–4.6 mm., Calicophoron type (described by Nasmark, 1937). Pharynx, 1–1.8 mm. in diameter, ijimai type. Oesophagus, short, .92 mm. long. Testes, .79–1.71 mm. in length, oval, strongly lobed, situated side by side rather than behind one another. Genital atrium, Calicophoron type. Egg size, 109.7 × 61.7 μ
Description.—Live contracted specimens measured at 18° C., from 4 × 2 mm. to 15 × 7 mm., increasing in length at least one and
[Footnote] * Present address, c/o Zoology Department, Otago University, Dunedin.
a-half times as the temperature approached 35° C. The body is sub-cylindrical, and is covered by an unmarked cuticle which extends into the acetabulum and into the pharynx. The inner border of the pharynx is uneven. In life the prominent acetabulum is terminal, but, when killed, uneven contraction draws the acetabulum ventrad. The genital atrium lies one-sixth to one-third of the total length from the anterior end. It is surrounded by muscular tissue which is subject to great diversity in extent and form. In 58 out of 77 specimens, each more than 10 mm., the genital papilla protruded. In the majority of cases, the papilla is surrounded by an area bounded by a delicate ridge. The pharynx has a posterior sphincter and leads into a short, wide oesophagus. The gut bifurcates anteriorly to the genital atrium, the two limbs extending in large folds laterally, terminating level at the commencement of the acetabulum. Vitellaria extend in wide bands on either side, from the anterior to the posterior regions of the body, emptying into longitudinal ducts which are connected by transverse ducts with a triangular shaped vitellarium situated medially in the posterior third of the body adjacent to the ovary. Lobed testes lie obliquely tandem, anterior to the vitellarium. The anterior testis is the larger. Vasa efferentia empty into a prominent convoluted vas deferens which opens into the common genital duct in the genital papilla.
The small oval ovary is situated to the left of the median line, behind the testes and anterior to the acetabulum. A much convoluted wide oviduct passes forward from the ovary between the testes, broadening anteriorly to the testes into a widely coiled uterus which is usually distended with eggs. Male and female ducts open through the genital papilla into the genital atrium. Crossing between the excretory canal and Laurer's canal was observed.
No flame cells were observed in adult C. ijimai.
Occurrence and Incidence of C. ijimai.
Juvenile flukes of average size, 5 × 2 mm. were found attached to the mucosa of the rumen in groups of from three or four to two hundred closely packed individuals of the same size, while the size of individuals of one group differed from the size of individuals of another group. This grouping indicates the possibility that juvenile flukes reach maturity together and migrate together to the rumen. Having spent some time in the rumen, it would seem that C. ijimai migrates to the reticulum along the strip of smooth epithelium between the rumen and the reticulum. Here half-grown flukes 10 × 4 mm. were found, particularly where the cattle were more heavily infested with the young stages. Only large flukes were found in the reticulum, very few in the rumen. Heavily infested cattle carried up to twelve large specimens in a reticulum “cell” one square inch in area. Such heavy infestations are explained by the way the animals feed. At Boggy Creek, Featherston, cattle grazed ankle to knee deep over swampy areas and along drains, preference being shown in summer months to a well-watered pasture. As the summer progressed, the water decreased, the concentration of intermediate stages increased, and heavy infestations resulted.
Of the 425 cattle examined, the majority were station cattle, two-year-old steers, reared for beef, in which the incidence was lower than that found in farm cattle. Of the cattle examined, 45.6% was infested. Half the total number of cattle infested contained from 70–229 flukes; fifteen animals from 220–750, and sixteen had 750 or over.
Adults were collected at the Gear Meat Works, Petone, and were transported in cold saline (.85g NaCl to 100 ccs.) to the laboratory where they were placed in petri dishes, 3½in × 1½in, containing cold Tyrode's solution. As the temperature was raised, streams of spermatozoa exuded from the protruding genital papilla accompanied by mucilaginous strands which coagulated and obscured the eggs, which were extruded later. Some flukes exhibited looping movements, others attached themselves by their acetabula to one another, forming a chain similar to chains found in the reticula of infested cattle.
At 35° C., 9 out of 10 adults remained alive and active for 30 days, the Tyrode's solution being renewed daily.
Life History (Plate 101)
The Egg.—The eggs measured, ranged in size from 55–143 μ × 44–78 μ compared with 133 μ × 55 μ given by Nasmark.
The egg is sub-ovoid with a strong transparent outer shell, the operculum visible as a jagged line. The egg is commonly found attached to the glass vessel by a short pedicel.
The embryo in progenetic eggs contains from two to eight cells. It lies in a central position on the outer surface of the nutrient cells which are separated from the shell by a transparent layer. Division is rapid, extending in all directions. After four days at 27° C., the embryo fills the posterior two-thirds of the shell, and by the eighth day the miracidium becomes defined. At ten days, flame cells are active, and contractions pass along the body of the miracidium. Cilia set up currents which violently agitate the excretory granules within the shell. The trebratorium contracts and expands as it pushes through the gelatinous cap immediately under the operculum, which opens suddenly. In two minutes the miracidium is free.
To obtain eggs, adult flukes were kept overnight in Tyrode's solution in petri dishes. In the morning, the flukes were removed and the eggs washed in tap water. Tap water was poured over the eggs, which were held at 27° C. Ten days later, 82% of the eggs hatched. Hatching could be precipitated on the tenth day by a slight drop in temperature, brought about by changing the water. However, by maintaining the temperature, hatching could be delayed until the eleventh day.
The Miracidium.—The newly hatched miracidium measure 215–235 μ × 40–42μ. It is covered with a transparent cuticle and is clothed with cilia 13 μ long. The retractile trebratorium extends forward when the miracidium is moving. From the anterior tip of the trebratorium the primitive gut, 88μ × 13μ, extends posteriorly. Four deeply staining cells, two on either side of the primitive gut are visible. These correspond in position to penetrative glands described by Srivastava (1938). The germ mass occupies at least one third
of the body cavity. It usually consists of two germ balls, one of which develops into a redia, the other breaking up into its component cells to form separate embryos in the sporocyst. The excretory system consists of two longitudinal ducts joined by a median transverse duct. Two flame cells are connected with the longitudinal ducts by short tubules entering anteriorly to the transverse duct.
The speed with which the miracidium moves through the water varies with the temperature. At 30° C., it is most active, activity decreasing up to 40° C and down to 8° C. Activity also varies in the presence of different species of snails. Snails were placed in clean filtered rain water uncontaminated by contact with molluses. There was no change in the steady rotatory movement in the presence of Potamopyrgus antipodum, P. corolla, and Myxas ampulla, but when placed with Planorbis kahuika, the miracidium increased its speed, bending, twisting, and turning until it crossed the path along which Planorbis kahuika had travelled. The miracidium then turned into the path and steadily overtook the snail, attaching itself to the fleshy portion of the body posterior to the eye stalks where it penetrates. Once within, the miracidium moves to the digestive tubules, adjacent to the most anterior portion of the gut.
Under laboratory conditions, the life of the miracidium varies from 4–24 hours.
The Sporocyst.—The four sporocysts measured, ranged in size from 430–855 μ × 115–170 μ. Surrounding the anterior end of the narrow primitive gut is a ring-shaped group of cells resembling a pharynx. The sporocyst itself is large and sluggish, bounded by a body wall one cell thick. The excretory system of the sporocyst consists of two flame cells, longitudinal and transverse trunks situated in positions corresponding to their positions in the miracidium. From twelve to fifteen embryos are inside the sporocyst, one to three of the rediae moving freely in the body cavity and often appearing ready to escape with cerariae embryos already in evidence. No birth pore is visible. The sporocyst is the most sensitive of the parthenitae, and once removed from the digestive tubules of the snail, it rarely survives more than one hour.
The Redia.—The redia varies in size from 281 μ × 58 μ to 1013 μ × 172 μ. After escaping from the sporocyst, the redia develops in the digestive tubules in close proximity to the sporocyst. The redia is cylindrical, the mouth opens into a circular pharynx which leads to a prominent, triangular gut, frequently distended by orange-coloured food. About 48 large cells constitute the gut wall. The gut is supported laterally by parenchyme cells. It lies in the body cavity, which is filled with embryos in all stages of development. In one redia 15 separate embryos were counted, with, in addition, three to five less well developed embryos packed closely at the posterior end of the body cavity. The most highly developed cercariae lie transversely at the anterior end, immediately behind the gut. A prominent longitudinal trunk constitutes the main part of the excretory system. This trunk is supplemented by branches ending in flame cells. Five pairs of flame cells occur symmetrically. The system opens at the junction of the posterior third, with the anterior two-thirds, between two pairs of flame cells, the anterior pair facing posteriorly, the
posterior pair facing anteriorly. The excretory vesicle pulsates regularly. Cercariae escape from the birth pore situated a third of the length from the anterior end on the aspect away from the longitudinal trunk. The redia is able to move through the host tissues by strong undulatory contractions.
The Cercaria.—When extended, the body of the cercaria is one and a-half times as long as it is wide, but when contracted, the ratio is reversed. Dark brown granules are arranged irregularly along striations which are visible through the transparent cuticle. Light brown cystogenous cells completely obscure detail in mature cercariae and partially obscure it in immature cercariae.
When the cercaria escapes from the redia, diffuse eye-spots extend dorsally and laterally in long strands radiating from central concentrated eye-spots, and the tail is but a short stump. As the cercaria matures, the pigment becomes concentrated into two kidney shaped eye-spots situated antero-dorsally and the tail becomes long and motile. The tail is clear and transparent and, compared with a body length of 275 μ, is 344 μ long. The large muscular acetabulum occupies approximately one quarter of the body length of a normal resting cercaria. The mouth is surrounded by prominent cells forming a prepharynx. In the immature cercaria, this prepharynx is used as an anterior sucker, adhering very closely to objects. The prepharynx leads to a well developed pharynx which opens into a short oesophagus. The alimentary canal bifurcates a third of the body length from the anterior end, the two branches ending blindly, anteriorly to the acetabulum. The excretory system consists of a number of flame cells which empty into two prominent ducts distended with excretory granules. Commencing at the extreme anterior end, the ducts continue posteriorly to a point level with the middle of the acetabulum before turning at right angles to empty into the excretory vesicle. Excretory material from the vesicle flows into the main excretory duct which extends from the middle of and beneath the acetabulum (when viewed from the ventral aspect) to a point near the posterior end of the tail, where the duct bifurcates and empties through two openings, only one of which was seen to function at the one time. A transverse duct unites the two longitudinal ducts posteriorly to the bifurcation of the gut. A group of three or four darkly staining cells is sometimes visible between the transverse excretory duct and the acetabulum in the immature cercaria. This is probably genital anlagen. The movement of the cercaria is typical, sharp, twisting contractions accompanied by lashing of the tail. In addition, the cercaria employs a looping motion, using posterior acetabulum and anterior prepharynx alternately. As the cercaria develops, it moves vigorously, holding the oral opening wide, absorbing nutriment from the liver cells of planorbis kahuika. Mature cercariae were observed escaping from an exposed portion of the digestive gland near the mantle chamber; these remained active for two and a-half hours, although they did not encyst.
The Metacercaria.—The metacercaria is circular in outline, 233–239 μ in diameter. It is flat underneath and rounded, shallow crowned and when detached, narrow brimmed with the edges bent down. The cyst wall is clear. An opaque pigmented layer surrounding the
Stages in the Life History of Calicophoron ijimai
1–4 Development of miracidium; 5 Sporocyst; 6 Young redia; 7–10 Cercaria; 11 Metacercaria.
a., acetabulum; b.w., body wall; c., cyst; c.d., cuticle detail; e., embryo; ex.d., excretory duct; f.c., flame cell; g., gut; g.m., germ mass; n., yolk material; o., operculum; p., pedicel; p.g., primitive gut; r., redia.
metacercaria, obscures the anatomy of the cercaria. The anterior sucker and the acetabulum are visible and excretory tubules are evident. Metacercariae were found adhering to the sides of the glass containers in which infected Planorbis kahuika had been held. Two P. kahuika experimentally infected, yielded 37 and 21 escaped cercariae respectively during a 50-hour period. These were fed to a sheep, but no infection resulted.
Observations on material from the field confirmed laboratory findings. Cattle infested with adult C. ijimai were traced to Boggy Creek, Featherston, where P. kahuika was collected in large numbers. From infected Planorbis, all stages from the sporocyst to the cercaria were found. Metacercaria at one time were found adhering to blades of grass which had been flooded with water from the lake. They were also collected from dishes containing infected Planorbis both from the field and from laboratory infestations. All stages observed in the laboratory were identical with those collected from Boggy Creek.
The Intermediate Host
Planorbis kahuika (Finlay and Laws) was found to be the intermediate host in New Zealand for Calicophoron ijimai.
Out of 477 adult P. kahuika collected from Boggy Creek, 7.33% were infected with various stages in trematode life histories. Of these, 20% of the infections were of an echinostome. The proportion, in which the various stages occurred, differed from month to month, but not sufficient information was obtained to enable any conclusion to be drawn. Parthenitae of C. ijimai were found only in the Featherston district, but the widespread occurrence of P. kahuika indicates the possibility of an increase in incidence of C. ijimai in the future.
Experimental infections of Isidora n.sp., Isidora lirata, Planorbis kahuika, Myxas ampulla, and Potamopyrgus corolla were attempted. A varying number of miracidia, from two per snail for two hours, to more than a hundred per snail, were placed in dishes. A total of 66 Isidora n.sp., laboratory bred; eight Isidora lirata from the Botanical Gardens; twenty-one Myxas ampulla, laboratory bred; twenty-two Potamopyrgus corolla, laboratory bred; and forty-four Planorbis kahuika, laboratory bred, were exposed to miracidia. All the tests proved negative except in the case of P. kahuika, of which a total of 25% was proven infected.
When on the 8th of February, twelve P. kahuika were placed in 50 cc. of water with more than two hundred miracidia, seven out of the twelve became infected. Two of the remaining five died overnight, and could not be examined as they were partially eaten by morning.
It was later found that C. ijimai provided ideal class material, no difficulty being experienced in obtaining a 100 per cent. infection of approximately 70 Planorbis, using miracidia hatched under the above conditions.
In the classification of the present material, difficulty was experienced owing to the different methods for separation of species. Different classifications of Paramphistomidae have been used by dif-
ferent writers, the one used in this paper being that of Nasmark (1937), in which species are separated by means of acetabulum, pharynx, and genital atrium types. Dawes (1936), also revised the family, basing his descriptions on general morphological details, many of the species described by Nasmark not being regarded as valid.
Nasmark described fourteen species of stomach fluke belonging to five different genera all inhabiting the stomach of Bos taurus. Five other species belonging to three genera are described from the stomach of Bas taurus indicus. Of the fourteen species, Dawes relegates eight to P. cervi, two to P. cotylophorum, and makes no reference to four others (Ceylonocotyle scoliocoelium, C. didranocoelium, Cotylophoron fulleborni, and Paramphistomum leydoni.)
On the one hand we see a drastic reduction in number of species, while on the other, in an effort to accommodate individual differences, the number of species has been increased. The question arises whether these differences are sufficiently constant in character throughout a species to be used as diagnostic features. The examination of a limited number of individuals does not give adequate information on the extent of variability within the species, hence in order to test the validity of the classification set out by Nasmark, large numbers of the different species described will have to be examined, and comparison made using life history studies.
The writer is indebted to Professor L. R. Richardson for helpful criticism during the presentation of the above work as a thesis for M.Sc. at Victoria University College. Thanks are also due to Mr. L. K. Whitten, of the Animal Research Station, Wallaceville, for his interest and assistance in the work. The assistance of Mr. A. W. B. Powell, of the Auckland Museum, who identified the snails, is gratefully acknowledged.
List of References.
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Daweb, Ben, 1936. On a Collection of Paramphistomidae from Malaya with a revision of the genera Paramphistomum and Gastrothylaw. Parasitology. 28 (3) 330–354.
Edgar, G., 1938. Paramphistomiasis of Young Cattle. Aust. Vet. Jour. 14 (1) 27–31.
Jonathan, S. R., 1950. The Life History of the Rumen Flukes of Cattle in New Zealand. Aust. Vet. Jour. 26 (7), 149–151.
Nasmark, K. E., 1937. A Revision of the Trematode Family Paramphistomidae. Zool. Bidr. Uppsala. 16, 301–565.
Simson, W. A., 1926. Amphistome Larvae in Intestine of Cattle and Sheep. Vet. Record. 6.
Srivastava, H. D., 1938. Life History and Pathogenicity of Cotylophoron cotylophorum Fischoeder 1901, Stiles and Goldberger 1910, of Indian ruminants, and biological control to check infestation. Ind. Jour. Vet. Sci. and Animal Husbandry. 8 (4), 381–385.
Stunkard, H. W., 1929. The Parasitic Worms Collected by the American Museum of Natural History Expedition to the Belgian Congo 1909, 1914. Bull, Amer. Mus, Nat. Hist. 58 (6) 233–289.