Effects of concurrent infections on growth, development, distribution, and infectivity of adult Philophthalmus megalurus and Philophthalmus gralli

Chickens were infected concurrently with 10 and 20 metacercariae/eye of Philophthalmus megalurus and Philophthalmus gralli. After 14 and 34 days of growth, the adults were removed and worm lengths, return rates, stage of development, and distribution within the host recorded. In a second experiment, chickens infected on day 1 with 10 metacercariae/eye of P. megalurus were concurrently infected with a similar dose of P. gralli on day 14. At day 34, the worms were removed and evaluated for the same parameters as in the first study. The effect of concurrent infections on worm length of P. gralli but not P. megalurus was significant when compared to single species control groups. In all cases recovery rates of P. megalurus in concurrent infections were significantly lower than controls, whereas P. gralli adults were recovered at lower rates only in 20-metacercariae--14-day infections. Maturation of eggs was delayed in both species in concurrent infections at the higher infection levels. Normal distribution was disrupted more for P. gralli at the higher infection levels and longer growth periods. Philophthalmus megalurus adults rarely left their normal habitat in the conjunctival sac. A delayed infective dose of P. gralli affected both species by disrupting normal distribution patterns, delaying egg development, and, in the case of P. megalurus, reducing the recovery rate. The possible role of crowding and antagonistic effects is discussed.     

Growth, development and allometry of Philophthalmus nocturnus in the eyes of domestic chicks.

Fifty day-old chicks were each infected with 10 excysted metacercariae of Philophthalmus nocturnus Looss, 1907 around each orbit and growth, development and allometry were studied. The growth rate showed two phases over a period of 35 days, a limited lag phase lasting two days post-infection in which flukes did not exceed 440 microns in length, and a rapid phase during which growth was rapid and flukes reached a size of 3.008-3.504 mm on day 35. Five developmental stages were noticed during the course of development of the metacercaria to the egg-producing adult stage. Eggs appeared in the uterus on day 14 and oculate miracidia on day 25. The hindbody, testes and ovary showed positive allometric growth, the pharnyx less so, whereas negative allometric growth was shown by the forebody. Body width, oral sucker and ventral sucker were close to isometry, growing at the same rate as the body length.     

The effects of crowding on adults of Philophthalmus nocturnus grown in domestic chicks.

The effects of crowding on growth, reproduction, rate of recovery and site of infection of Philophthalmus nocturnus were investigated by infecting various groups of day-old chicks with 25, 50, 75, or 100 excysted metacercariae and examining them at 10 or 25 days post-infection. Considerable reduction in the body length and the size of the gonads was noticed in flukes grown under crowded conditions. Crowding also caused some change in the habitat of flukes, but had no effect on their rate of recovery. Very few flukes reached the ovigerous stage under overcrowded conditions and none reached the embryonated larval stage.     

Life cycle and identification of an eyefluke from Israel transmitted by Melanoides tuberculata (Müller, 1774)

A philophthalmid species from Israel using the freshwater snail Melanoides tuberculata as intermediate host was studied. The biological and morphological characteristics of all developmental stages of the life cycle of this philophthalmid were described, and compared to those of Philophthalmus lucipetus Rudolphi, 1819 from Israel, Philophthalmus gralli Mathis and Leger, 1910 from Jordan, Philophthalmus palpebrarum Looss, 1899, Philophthalmus nocturnus Looss, 1907, Cercaria distomatosa Looss 1896 from Egypt, and Philophthalmus lucknowensis Baugh, 1962 from India. The possible identity with 1 of these species is discussed. On the basis of comparative analysis of the data for all parasite stages in the life cycle, geographical distribution, snail hosts, and snail host specificity, we propose to designate the Israeli Melanoides tuberculata-transmitted eye fluke to Philophthalmus distomatosa n. comb. (Looss, 1896), (Digenea: Philophthalmidae).     

The effects of crowding on adults of Philophthalmus gralli (Trematoda) grown in chickens

Chickens were infected with six, 10, 30, 50, or 100 metacercariae of Philophthalmus gralli per eye and the adults were harvested 9, 14, 21, 35, and 50 days later. The worms recovered were measured and assessed for maturity. Significantly more adults from infections of six and 10 metacercariae per eye were recovered than from infections of 100 per eye. When metacercariae were placed in one eye, over one-fourth of the resulting adults were found in the opposite eye. Worms recovered from groups of zero to 10 were significantly longer than those in groups of 41 to 50 and 51 to 60 at all ages sampled. The normal movement of worms from the conjunctival sac to the outside of the nictitating membrane was hindered by crowding; when more than 10 were present, some worms developed in the sac to mature adults. Worms in all groups produced eggs in which miracidia developed at the same rate.     

Chemosensitivity of Philophthalmus megalurus (Trematoda) miracidia

Miracidia of the eyefluke Philophthalmus megalurus were tested in phi-chambers to determine if they reacted similarly to chemicals found stimulative for miracidia of Philophthalmus gralli, a closely related species. Philophthalmus megalurus miracidia were less responsive than P. gralli to the dicarboxylic amino acids and showed a significantly positive response only to 10 mM glutamic acid. These P. megalurus miracidia were chemonegative to 10 mM HCl and H2SO4, chemicals to which P. gralli miracidia gave a significantly positive response. Ammonia and Mg2+ did not elicit any response from P. megalurus miracidia.     

Identification of Stictodora lari (Heterophyidae) metacercariae encysted in the brackish water fish, Acanthogobius flavimanus

Metacercariae of the genus Stictodora encysted in the head tissue of Acanthogobius flavimanus (the gobies) caught at Sachun-gun, Kyongnam Province, were identified to be Stictodora lari Yamaguti, 1939 (Trematoda: Heterophyidae), a new parasite fauna in Korea. The metacercariae were 0.39-0.43 mm by 0.32-0.35 mm in size, long elliptical, and with a thin and transparent cyst wall. Total 200 metacercariae were collected from 50 gobies. In order to obtain adult worms two kittens and a puppy were infected each with 34-100 metacercariae, and total 33 adults were recovered between the day 4 and day 8 post-infection. The S. lari adults measured 0.95-1.18 mm long and 0.26-0.32 mm wide and the eggs in uteri 0.028-0.033 mm by 0.017-0.020 mm. The most characteristic morphological feature of these flukes was the presence of a gonotyl and gonotyl spines arranged in two groups; densely crowded group of 30-40 spines and linearly-arranged one of 30-40 spines, together of which made a comma (or reversed comma) shape along the lateral margin of the gonotyl. It has been proved by this study that S. lari is distributed in southern coasts of Korea.     

A comparison of the surface features of Philophthalmus megalurus and Philophthalmus gralli rediae by scanning electron microscopy.

General features observed on the surface of Philophthalmus megalurus and Philophthalmus gralli rediae include 2 rounded ambulatory buds, a tapered tail, a slitlike birth pore, and an oral opening surrounded by uniciliate sensory receptors. The tegument is folded in a ribbed pattern and is covered with small microvilli. The sensory receptors are concentrated on the lip of the oral opening but are less dense inside the buccal cavity. Both the birth pore and the buccal cavity are lined with a honeycomblike surface that may support the structural integrity of these highly extensible orifices. Major differences between the 2 species were noted in the structure of the sensory receptors. In P. megalurus they have flexible cilia that originate from a bulbous tegumental base. Receptors of P. gralli rediae are less dense, appear to have rigid cilia, and lack a bulbous base. In general P. megalurus rediae resemble more closely rediae of the philophthalmid Parorchis acanthus than they do those of P. gralli.     

Escape of rediae from miracidia of Philophthalmus megalurus and Philophthalmus gralli during in vitro culture

Miracidia of the eyeflukes Philophthalmus megalurus and Philophthalmus gralli contain a preformed larval stage that was identified as a redia by electron microscopy and, on living forms, by the presence of ambulatory buds and an oral opening. This redial stage escaped and actively moved about when the miracidium stopped swimming in pond water. No redial escape was observed in NaCl solutions even though miracidia became immobile, but escape was noted in Hanks' balanced salt solution (HBSS) after 7 hr of exposure. When miracidia were placed in RPMI-1640 and Eagle's minimum essential medium, rediae escaped much earlier than in pond water and HBSS. Redial escape in vitro will provide a good source of material to initiate cultures of this larval stage.     

A comparison of Texan and Hawaiian strains of the avian eyefluke, philophthalmus gralli, with a cautionary note on the importation of exotic animals

Philophthalmus gralli (Mathis and Leger, 1910) was introduced into the San Antonio, Texas area within the last 25-30 years from an unknown foreign source. Strains of P. gralli originally from Texas and Hawaii were compared for differences in morphology, growth patterns, mating compatibility, and isozyme mobilities. Metacercarial cysts of the 2 strains were compared for viability after storage at room temperature. Adult stages of the 2 strains were indistinguishable based on sucker ratios, vitellaria, and egg sizes. In single- and multiple-worm infections, both strains exhibited similar growth patterns when reared in chickens. Adults of the 2 strains, when transplanted in concurrent infections, readily cross-inseminated. Isozyme mobilities of 5 enzymes were identical in all adult worms examined from both strains. After 3 days, metacercarial cysts from both strains rapidly lost their ability to excyst. The cysts of the Texan strain remained viable for 4 days longer than the Hawaiian strain, although excystment rates were low during that period. The evolutionary implications of this imported species are discussed.     

A comparative study of the regenerative processes in a trematode, Philophthalmus gralli, and a planarian, Dugesia dorotocephala

Adults of Philophthalmus gralli, an eyefluke of birds, were laterally amputated mid-way between the anterior testis and the ventral sucker. Worms were sampled at various short time intervals (30 min-6 h) after in vitro culture and long time intervals (1-8 days) after transplantation back to the host. Specimens were fixed for scanning electron microscopy and light microscopy and compared to the planarian, Dugesia dorotocephala, which was laterally transected in the pharyngeal region and maintained in springwater. It was found that wound closure took place by 2 days in the planaria; however, at the end of 8 days wound closure had been initiated but not yet completed in P. gralli. Replacement of major tissues was observed only in D. dorotocephala. Because calcium had been reported to be critical for planarian regeneration, a histochemical stain for calcium distribution was carried out. At the end of the 2-day study, no differences could be found in calcium distribution between the two organisms. No areas of calcium concentration were noted in any tissue important for the regenerative process.     

Development of the large American liver fluke, Fascioloides magna, in white-tailed deer, cattle, and sheep.

The comparative development of Fascioloides magna in white-tailed deer, cattle and sheep has been studied. Flukes were recovered from 72% of 32 deer administered 40 to 500 metacercariae, from 82% of 11 cattle administered 10 to 500 metacercariae, and from 53% of 15 sheep administered 8 to 200 metacercariae. The percentage recovery of the flukes administered as metacercariae was 4.1% of 6,130 in deer, 5.7% of 2,510 in cattle, and 4.7% of 1,213 in sheep. Flukes were recovered only from livers of infected deer, while in cattle, 1 fluke was also found in the lungs of each of 2 animals. In sheep, all but 10 flukes were recovered from the livers; 6 were found in the lungs and 4 in the abdominal cavities. The black iron porphyrin pigment associated with F. magna infection was found to be most widespread in cattle and sheep, but was also a pathognomonic feature in deer. Growth of the fluke was similar in all 3 host species tested, but eggs were passed only from deer, the normal definitive host. In cattle, the eggs were retained in the liver, and F. magna was lethal to sheep before its own maturity was attained. In cattle and deer, flukes matured approximately 7 months after exposure, but immature migrating flukes were found 12 months after infection and apparently can remain in this retarded state for an undetermined period of time.     

The longevity and hatchability of Philophthalmus megalurus and P. gralli miracidia in different environmental conditions.

The effects of salinity, pH, and temperature on the longevity and hatchability of miracidia of Philophthalmus megalurus and P. gralli were determined. Miracidia of both species are able to hatch and survive at saline concentrations much above physiological levels, although these processes are reduced in 2.0--2.4% saline and completely inhibited at 2.6%. The greatest hatching rates for both species were found near neutrality (pH 6--8) but some miracidia hatched at the extreme pH levels of 3 and 12. Philophthalmus megalurus miracidia exhibited longer half-lives under acid conditions (pH 2--6) than P. gralli miracidia; conversely, P. gralli miracidia showed longer half-lives in alkaline conditions (pH 8--11). Hatching and longevity were much greater below room temperature (5--20 C) than above (30--50 C) for miracidia of both species. Temperatures above 50 C proved lethal for eyefluke eggs. Except in acid pH, P. gralli miracidia showed longer half-lives than miracidia of P. megalurus. Comparison to studies on schistosomes revealed that the inhibitory effects of physiological saline and host body temperature on the hatching process of schistosome eggs does not occur in these 2 species of eyeflukes.     

Early development of the laboratory-reared flounder,Pleuronichthys cornutus

Fertilized eggs ofPleuronichthys cornutus were obtained by both artificial fertilization and natural spawning of laboratory-reared fish. The present paper describes in detail the early development of the fish and the rearing methods employed to provide basic information for mass production of this species. Eggs and sperm for artificial fertilization were obtained from adult fish caught in the Ariake Sound, Kyushu in November and December of 1984. Their maturation was successfully induced by intermuscular injection of pituitary homogenate of the silver carp,Hypophthalmichthys molitrix. Fertilized eggs were also obtained in 1985 by natural spawning of a broodstock kept in a tank for a year. Hatched larvae were fed successively with rotifers,Artemia nauplii and the harpacticoid copepod,Tigriopus japonicus and reared for 80 days. Ten thousand young fish of about 33 mm TL were obtained in 1984 and 1985 with the survival rate of about 17%. Ten developmental stages were defined on the basis of the morphological characteristics: A) newly hatched to 4 day old larvae, 2.7 to 4.1 mm TL (2.6 to 3.9 mmNL), yolk sac present; B) 4 to 16 day old larvae, 3.8 to 5.9 mm (3.6 to 5.6 mm), yolk resorbed, actively feeding on rotifers; C) 15 to 30 day old larvae, 6.3 to 8.3 mm (6.0 to 7.9 mm), notochord straight, hypural fin ray visible; D) 24 to 40 day old larvae, 6.7 to 9.2 mm (6.4 to 8.8 mm), caudal notochord upturned (45°); E) 28 to 45 day old larvae, 7.9 to 10.8 mm (7.5 to 10.3 mm), caudal notochord upturned (45°–90°); F) 32 to 50 day old larvae, 10.8 to 15.7 mm (8.8 to 12.8 mm BL), eyes symmetrical; G) 35 to 66 day old larvae, 13.4 to 20.0 mm (10.9 to 16.3 mm), eyes asymmetrical, but left eye not visible from the right side; H) 40 to 75 day old larvae, 13.8 to 26.2 mm (11.3 to 21.4 mm), the upper edge of left eye visible over top of the head from the right side; I) 46 to 89 day old larvae, 20.1 to 27.4 mm (16.4 to 22.4mm), left eye on the edge of the head and pupil visible from the right side; and J) juveniles of 51 day old or over, 23.6 mm or more (19.3 mm or more), metamorphosis completed. One to three inflections were found for relative growth of total length, eye diameter, upper jaw length, preanal length, and distance between the base of the pectoral fin and the anus against the notochord length or body length. Two inflections were found for body length (or notochord length)-body weight relationship. Most inflections appeared at the stages of D, F and J, corresponding to the body length of 8, 9–12 and 18–22 mm respectively.     

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails,Filopaludina martensi cambodjensis,in Pursat Province,Cambodia

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1–8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 μm in average diameter (163–190 μm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4–8.2)×1.4 (1.1–1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0–100% homology based on cox1 sequences and 98.9–99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.     

The early migratory behaviour of young Fasciola hepatica in sensitized mice

Earlier work has shown that when mice are sensitized with irradiated metacercariae the numbers of immature flukes that can be recovered from the peritoneal cavity 2 days after reinfection with normal metacercariae is significantly less than the numbers recovered from non-sensitized control mice. Experiments are now described which investigate the reason for this difference. An inflammatory cellular reaction, most marked in sensitized mice occurs in the intestinal wall but this does not delay the migration of challenge flukes into the peritoneal cavity. No effective protective mechanism operates at the intestinal wall because similar numbers of flukes are present in the livers of sensitized and non-sensitized mice at 12 and 14 days after infection. When livers of sensitized and non-sensitized mice were examined 2 days after infection significantly more flukes had already reached the liver in the sensitized group. This indicates that immature flukes migrate more quickly from the peritoneal cavity in mice previously sensitized with irradiated metacercariae and would account for the difference in the number of flukes recovered from the peritoneal cavity of sensitized and non-sensitized mice at 2 days after infection.     

Gynaecotyla squatarolae (Digenea: Microphallidae) from rats experimentally infected with metacercariae from the shore crab, Macrophthalmus dilatatus

Metacercariae of Gynaecotyla squatarolae (Digenea: Microphallidae) were discovered from the shore crab, Macrophthalmus dilatatus, purchased at a market in a coastal town of Taean-Eup, Chungcheongnam-do, Republic of Korea. Their adult flukes were confirmed by experimental infection of rats. The metacercariae of G. squatarolae were elliptical (391.1 x 362.5 micrometer), and the excysted metacercariae had progenetic genital organs, including the ovary and testes. To obtain adult flukes, 6 Sprague-Dawley rats were fed 500 metacercariae each, and killed at days 2, 4, and 6 post-infection. The adult flukes were identified as G. squatarolae (Yamaguti, 1934) Yamaguti, 1939, based on morphological characters, including 2 ventral suckers (1 large and 1 small), a large genital atrium equipped with the cirrus and the metraterm, separated male and female genital pores, a transversely long cirrus pouch, and extensive vitelline follicles. In the present study, it has been first proven that the shore crab M. dilatatus is a second intermediate host for G. squatarolae in the Republic of Korea.     

Echinostoma revolutum: metacercariae in Filopaludina snails from Nam Dinh Province, Vietnam, and adults from experimental hamsters

We detected metacercariae of Echinostoma revolutum in Filopaludina sp. snails purchased from a local market in Nam Dinh Province for the first time in Vietnam. Adult flukes were harvested from experimentally infected hamsters at days 14 and 17 post-infection. The metacercariae were round, 170-190 μm (n = 15) in diameter, with a cyst wall thickness of about 12 μm. A total of 37 collar spines were arranged around the head collar, and large excretory granules were seen in 2 canals of the excretory bladder. The 14-day old adult flukes were elongated, ventrally curved, and 5.0-7.2 × 0.8-1.3 mm (n = 20). The head collar had a total of 37 collar spines arranged in 2 alternating rows, including 5 corner spines on each side. The cirrus sac contained a saccular seminal vesicle, a prostatic gland, and an unarmed cirrus. Two tandem testes were smooth or slightly lobed. Eggs were ovoid to elliptical, 110-118 × 70-75 μm. These morphological characters were similar to those of E. revolutum and E. jurini. We tentatively identified it as E. revolutum because the validity of E. jurini remains to be elucidated. The taxonomic relationship of E. revolutum and E. jurini is discussed.