Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187

The involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.     

Excystation of five digenean metacercariae in vitro and in the peritoneal cavity of the mouse

Excystation of metacercariae of Parorchis acanthus, Posthodiplostomum nanum, Posthodiplostomum sp., Posthodiplostomoides leonensis and Clinostomum tilapiae in mammalian saline at 39 degrees C was similar to their excystation in mouse peritoneal cavity. Extrinsic enzymes were not needed for C. tilapiae excystation but were an absolute necessity for P. acanthus excystation. Extrinsic pepsin enhanced P. nanum excystation but was not obligatory. Acid pepsin was an absolute requirement for Posthodiplostomum sp. and Posthodiplostomoides leonensis excystation. It was noted that acid saline could not be substituted for acid pepsin and that there was diversity in the condition involved in digenean excystation.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

The distribution of Posthodiplostomum cuticola metacercariae in young-of-the-year cyprinid fishes

The lowest frequency of metacercariae of Posthodiplostomum cuticola on the bodies of six young-of-the-year cyprinid species was found on the head and ventral body. Metacercariae distribution differed among host species which may have been related to the behaviour of the fish host.     

Early cysteine protease activity in excretory bladder triggers metacercaria excystment of Paragonimus westermani

The cysteine proteases of Paragonimus westermani metacercaria are known to initiate metacercaria excystment. However, their secretory sites, such as the intestine, tegument, and excretory bladder are not well defined. In this study, the metacercariae were labeled with bromodeoxyuridine (BrdU) to distinguish the initial activation sites. BrdU was labeled mainly at the excretory bladder and the excretory granules of the metacercariae and the newly excysted juvenile worms. This result shows that early 'defecation' of the proteases from the excretory bladder may accelerate the excystment of P. westermani metacercariae.     

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.     

Comparative life histories of two diplostomid trematodes, Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum

A lack of comparative studies limits our understanding of interspecific variation in parasite life histories, especially for species that incorporate asexual and resting stages into their life cycles. Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum are sympatric diplostomid trematodes that share the same first (pond snail, Physa gyrina) and second (fathead minnow, Pimephales promelas) intermediate hosts within lakes in Alberta, Canada. Interspecific differences in the body sizes of adults, cercariae, and metacercariae were evaluated from parasites recovered from laboratory-raised chickens, snails, and minnows, respectively. Differences in adult worm fecundity and cercariae production were estimated from chickens and snails exposed to known numbers of parasite larvae. Posthodiplostomum minimum adults, metacercariae, and eggs were 97, 86, and 5% larger than those of 0. ptychocheilus, respectively. The average numbers of eggs produced per day by P. minimum was 3 times higher than for O. ptychocheilus, and P. minimum produced approximately twice as many cercariae per day. Although the larger of the 2 species had higher egg and cercariae production, conclusions regarding covariation between adult worm size and the reproductive rates of other life cycle stages require further studies on related species.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

In vivo development of Echinostoma malayanum Leiper, 1911 with notes on effects of population density, chemical composition and pathogenicity and in vitro excystment of the Metacercaria (Trematoda: Echinostomatidae)

In vivo development of Echinostoma malayanum Leiper, 1911 was studied in white rats and the developmental process was arbitrarily divided into four stages: organogeny, vitellogenesis, formation of Mehlis' gland complex and cirrus sac, and oviposition. The percentage of development was 86-94. Population density affected the prepatent period of flukes and the normal prepatent period of 13-16 days was altered to 18-23 days in infection with 500-800 flukes. The majority of flukes in heavy infection were undersized and in the immature stage of development at patency. Data from chemical analysis of flukes revealed that protein, lipids, calcium and ash decreased quantitatively in flukes from higher population densities but no such change was observed as regards glycogen. Pathological changes in the rat intestine included lysis and destruction of mucosa, increased activity of goblet cells, oedematous and reticulated appearance of lamina propria and slight to moderate hyperplasia of epithelial cells. The metacercariae excysted in the medium containing trypsin plus sodium cholate an pepsin, though not essential for a high percentage of excystment, affected the rate. The reductant sodium dithionite substantially enhanced the rate and percentage of excystment. Excystation was optimal at pH 8, and 42 degrees C was more effective than 39 degrees C.     

Clarification of Cercaria sevillana (Digenea: Microphallidae) life cycle using morphological and molecular data

Cercaria sevillana is the cercaria larval stage that infects the gonads and the digestive gland of its first intermediate host, Nassarius reticulatus. In this study the decapodous crustacean Carcinus maenas was used to determine if it would serve as second intermediate host in the parasite's life cycle. The latter hypothesis was based on the knowledge that C. maenas is the second intermediate host of several other digenean species. After dissection, it was possible to observe encysted metacercariae in the antennal glands of the green crab. After biochemical excystment, the metacercariae were processed for light and scanning electron microscopy. The morphological features observed led us to conclude that this species was a microphallid fluke, and it was identified as Gynaecotyla longiintestinata. To establish a possible relationship between C. sevillana and this metacercariae, the ITS1 region was analyzed. Thus, DNA was extracted from C. sevillana and from the cysts isolated from the antennal glands. The ITS1 region was amplified and sequenced, and the alignment clearly demonstrated that the cercaria and the metacercariae belonged to the same species, G. longiintestinata.     

Helminth parasites of Chapalichthys encaustus (Pisces: Goodeidae), an endemic freshwater fish from Lake Chapala, Jalisco, Mexico

A total of 6 helminth species were recorded during helminthological examination of 50 Chapalichthys encaustus from Lake Chapala, Jalisco, Mexico. Helminth species identified included: Salsuginus sp. (an undescribed monogenea); Posthodiplostomum minimum (metacercariae); Cyclustera ralli (metacestode); Polymorphus brevis (cystacanth); Contracaecum sp. (nematode larvae); and Rhabdochona lichtenfelsi (adult nematode). Of these, 2 (Salsuginus sp. and R. lichtenfelsi) are specialist species. The observed species richness, individual parasite abundance, and diversity were low. Data suggest that host specificity is an important factor contributing to observed community composition and richness. Host feeding habits and helminth species availability seem to determine the characteristics of these helminth assemblages.     

Infectivity of Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum (Trematoda: Diplostomidae) cercariae following exposure to cadmium

The effect of cadmium (Cd) exposure on infectivity of cercariae of 2 trematode species (Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum) to their second intermediate fish host was evaluated. Individual fathead minnows (Pimephales promelas) were exposed to cercariae that had been treated with Cd solutions at concentrations of 0, 2, 20, or 200 microg/L for 2 hr. Two weeks later, the numbers of encysted metacercariae in the brain (O. ptychocheilus) and body cavity (P. minimum) of the fish were evaluated. ANOVA analyses indicated a strong negative effect of Cd concentration on cercaria infectivity. The species x Cd concentration interaction was not significant, indicating that the magnitude of Cd-induced reduction in infectivity was similar between O. ptychocheilus and P. minimum. The results show that short-term exposure to Cd, even at low concentrations, interferes with transmission processes that affect the recognition and penetration of cercariae, the migration and survival of metacercariae within the second intermediate host, or both.     

Estimation of the endohelminth parasite species richness in freshwater fishes from La Mintzita Reservoir, Michoacán, Mexico

In total, 9 endohelminth species were found to parasitize 7 fish species (2 cyprinids, 4 goodeids, and 1 poeciilid) from La Mintzita Reservoir, Michoacán, in central Mexico; 5 were larvae, including 3 allogenic species (Clinostomum complanatum, Tylodelphys sp., Posthodiplostomum minimum) and 2 autogenic species (Serpinema trispinosum, Spiroxys sp.). Four were enteric autogenic adults, i.e., Margotrema bravoae, Bothriocephalus acheilognathi, Proteocephalus longicollis, and Rhabdochona lichtenfelsi. The metacercariae of P. minimum reached the highest levels of prevalence and mean abundance among host species. Our results confirm the depauperate nature of the helminth communities of freshwater fishes from central Mexico. On the basis of this data set, we estimated the total endohelminth species richness for each component community by using 7 nonparametric estimators whose performance was evaluated with the unscaled measures of bias, precision, and accuracy. We found that Chaol and Bootstrap are the most precise and least biased methods for the 7 component communities: however, species richness was consistently underestimated. The underestimation was an unavoidable consequence of the patchy distribution of helminth species among different component communities, particularly at the small sample size used in our study.     

Unusual pathogenicity of a common metacercaria of fish

The metacercaria of Posthodiplostomum minimum centrarchi Hoffman, 1958, is reported from an unusual host, Roccus saxatilis, from North Carolina. Although few dead or moribund fish were observed, there were many deformed fish, few of which survived. The metacercariae were in the musculature and orbit of the eye instead of in the usual location in the centrarchid hosts.     

Excystment of Didinium nasutum

SYNOPSIS. Excystment of Didinium nasutum is solely dependent upon dense bacterial populations and is independent of the type of medium in which the bacteria have been grown. Beers' hypothesis of the need for a factor from proteose-peptone is thereby not confirmed, but the need for living, intact bacteria as postulated by him is fully shown. The process of excystment is initiated by bacteria, but their presence is not required throughout the excystment. Axenically grown paramecia, shown to be adequate as food organisms for didinia, will not induce excystment. Five types of bacteria including Gram-positive, Gram-negative, aerobic and anaerobic all suffice to induce excystment, suggesting that the initiation of this process is caused by some general metabolic process or product of bacteria.     

Metacercarial aggregation in Digenea (Fasciola hepatica and Paramphistomum daubneyi): environmental or species determinism?

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.     

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.     

A new species of Paragonimus (Trematoda: Troglotrematidae) from a cat infected with metacercariae from mountain crabs Larnaudia larnaudii

The identification of Paragonimus spp. lung flukes is based on the morphology of both the metacercaria and the adult. A very small Paragonimus sp. metacercaria was found in a freshwater crab caught in Kanchanaburi Province, West Thailand, an area where metacercariae of Paragonimus heterotremus had not been found. The metacercariae cysts were 180-204 microm in diameter, which was smaller than metacercariae of P. heterotremus. The coefficient of difference in body size between this metacercaria and P. heterotremus was 1.69, which was greater than a subspecific difference. Adults recovered from the lungs of a cat fed with the metacercariae were morphologically similar to, but smaller than, P. heterotremus dissected from the lungs of a feline experimental host. The tegumental spines of the worm in this study were singly spaced in arrangement, which is similar to, but larger than, the spines of P. heterotremus. Therefore, Paragonimus pseudoheterotremus is proposed as a new species.     

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.