The Egyptian Journal of Hospital Medicine (Jan. 2017) Vol. 66, Page 28- 39

A Molecular and Microscopically Studies of Calicophoron Microbothrium (Paramphistomatidae) Alaa Abdel-Aziz Mohamed Samn Zoology Department, Faculty of Science (Boys),Al-Azhar University, Cairo, Egypt [email protected], [email protected]

ABSTRACT Background: Paramphistomiasis is a parasitic of and , which causes heavy economic black lashes especially in countries with advanced industry Aim of Study: the current study aimed to add more information about Calicophoron microbothrium (C. microbothrium) and clarify its biological role and how its miracidia infecte the molluscan intermediate . In addition, a brief description to Bullins truncates; the morphological, structural and chronological characteristics of the various intermolluscan stages of the parasite are studied in detail. Moreover, the present work showed the effective role of physical parameters (light, temperature, salinity and gas-phase (aerobic versus anaerobic)) on egg development and hatching and the biological activities of cercaria and metacercaria. Beside these routine techniques, PCR also was used as more advanced and accurate diagnostic technique based on the detection of nucleic acid. Where, 34 larvae and adult worms of Calicophoron microbothrium were isolated from naturally infected buffaloes. The results of the present study will facilitate the identification of this despise secular group of digeneans although its bad effect not only affect animal industry but also health. Furthermore, the current research clears the weak points in its life cycle to aperient settling this parasite. Keywords: Calicophoron microbothrium, Bullins truncates, paramphistome, biology, histology, Mas– PCR technique.

INTRODUCTION host with its ability of widespread, surviving for Paramphistomes are spreading worldwide, several months and shedding numerous especially in the warmer regions such as: , and , and mainly infect cercariae, and then so dangerous threaten to the , goats and . In addition, there are domestic animals (3). certain species of paramphistomes that infect The paramphistomes are conical or human it has medical importance beside their cylindrical digenean with thick bodies. They are economic one. Calicophoron microbothrium distinguished from other flukes by the ( microbothrium) is one of possession of a posteriorly located acetabulum. two of these parasites in Egypt as reported by The most familiar species are parasites of Ashour (1); the other species was Calicophoron domesticated livestock. It has been considered, gregarius. They were the most important flukes for a long time, that paramphistomes are identified in domestic Egyptian . completely non-injurious to their vertebrate This C. microbothrium has a life cycle hosts, but this view has been challenged by similar to that of the gastrointestinal trematodes. many authors (4-7). Where, an acute infection of Where, the adult flukes reside in the host's calves and sheep had been caused by the rumen and reticulum; their eggs are passed immature conical flukes in the small intestine, through host's faeces. When eggs reach water particularly in advanced cattle raising regions the miracidia hatch within 12-16 days and causing elevation of mortality rate in sheep penetrate suitable intermediate host. The about 30% as reported by Chauhan et al. (8) and parasite has a number of generations of rediae 21%-37.4% in cattle by Pande (9). In addition, before the production of free swimming Katlyar and Varsheney (10) recorded an cercariae that are changed to metacercariae after average percentage morbidity and mortality encysting on water plants; these plants become 41.24% and 57.62% in sheep and 68.59% and the source of the food causing the 75.53% in goats respectively. Moreover, Horak infection and beginning a new cycle (2). (6) recorded a loss of 11.4 kg of sheep's weight Seriously, these parasites may survive up to within 52 days and 27.3 kg in bovines after 140 years, so that they become a virtually constant days, while an uninfected bovine gained 50.9 kg infestation source for numerous snail during the same period. generations. As a result to that, the intermediate The clinical signs of acute paramphistomiasis in sheep, goats and cattle have been represented by 28 Received:121/0/2016 DOI: 10.12816/0034630 Accepted:22/10/2016 A Molecular and Microscopically Studies of Calicophoron Microbothrium restless and progressive decrease in appetite to a number of mortality among children like developed to complete , small happened in Assam, India (22). quantities of water are taken and the animals might stand with their muzzles in water for a Although the dangerous effects of this parasite long time; a condition known as polydipsia. on humans and economic animals, there were Diarrhoea developed 2-4 weeks after infection, limited number of papers has been studied the with fetid faeces and rectal hemorrhage. paramphistomes in Egypt. Ezzat (23) described Submandibular oedema (bottle jaw) has been specimens from Gazella dorcas which were noted in a number of outbreaks. Oedema of identified as . Tadros (24) lungs, hydrothorax, hydropsyeasdium and reported the presence of paramphistomes in ascites were reported by [Baldrey (11), Walker cows, buffaloes, sheep and camels in Shebin El- (12), Chauhan et al. (8), Pande (9), Haji (13), Kanater district in the Nile Delta, but he did not Bawa (14), Boray (15), Varma (16), Horak and designate these to known genera or species. Clank (17), Katiyar and Vershney (18) and Abdel-Ghani (25&26) described specimens, Horak (6)]. which were identified as P. cervi but his About how the parasite causing its disorder description was not based on anatomical and effects in animals, Horak (7) stated that histological features which were considered immature paramphistomes might penetrate the necessary for the specific identification of intestinal wall to just below the serosa and can paramphistomes. Also, he described briefly the be seen from the peritoneal side of the intestine. eggs and miracidia of these flukes and On rare occasions, the parasites perforated the conducted some experiments to determine the intestine and were found in the abdominal susceptibility of small laboratory animals to cavity. While, Dinnik (4) and Horak (6) reported infection. Ashour (1) gave the first detailed that many cattle and sheep were infected with morphological, anatomical and histological adult paramphistomes and the infection was account of paramphistomes in Egypt. He acquired by of few numbers of recorded two species under the metacercariae on one or several occasions, Paramphistomum, namely, C. (= Calicophoron) didn’t cause an obvious harm to the host. The microbothrium and C. khalili as well as one ingested metacercarine mature rapidly and serve species in the genus Carmyerus, C. gregarius. as a source of infection for successive Moreover, he conducted comparative generations of snails. These paramphistomes morphological and experimental studies on the can survive for some years, where the source of eggs and miracadia of both P. microbothrium infection is virtually consistent. and P. gregarious. Elkabbany (27) gave more The average daily egg produced by a single C. information about scanning and transmission microbothrium in infected sheep was estimated electron microscope of two species of about 75 eggs (6). Although this was not a large paramphistomes, C. microbothrium and C. output but the large number of parasites was gregarious. usually found in host with large number In addition to the previous causes, the difficulty according to Boray (19) who found 60.000 of paramphistomes' identification worms or excess of C. ichikawai/ 3-8 naturally morphologically and histologically leads to find infected sheep and Dinnik (4) returned that to new method easier. Therefore, the current study long life span of C. microbothrium in cattle used the PCR based techniques beside the which maintains its egg production for many routine techniques which should be a suitable years, that appearing the severity of this tool because it was providing rDNA ITS2 parasite. sequences that have proven as a suitable marker Surprising, it was possible that some species of for identification of paramphistomes species (28). paramphistomes which are parasites of wild ruminants were occasionally encountered in MATERIAL AND METHODS domestic ruminants if wild ruminants 1 Field studies: occasionally infect domestic ruminants' places 1.1 Collection of paramphistomes: and stocks live closely together (20&21). Mature specimens of paramphistomes Notably, there was asymptomatic in were collected from cows, buffaloes, sheep, and paramphistomes' natural hosts such as and goats, which slaughtered at Sharkiya and Mit monkeys, while in human causes serious health Ghamre Abattoirs. The flukes were found problems like , , abdominal pain, attached by their strong acetabula to the inner colic, and an increased mucous production. In surfaces of the rumen and the reticulum of their addition, in extreme situations this disease leads hosts. They appear in groups as pinkish to red 29

Alaa Samn flesh-likes patches in case of Calicophoron; 2.1 Preparation of tissue sections for however, Few flukes were seen free in the histological studies: lumen of the stomach. Snail's tissues for histological studies, using After the removal of the flukes from the haematoxylin, eosin and toluidine blue as the stomach, the flukes being attached to each method of Krichesky (34). other, therefore, they were put in normal saline 2.2 Follow up the life cycle of C. solution (0.7% NaCl). Where, saline make the microbothrium: fluke’s exhibit slow movement, in which the Using both morphological and histological body becomes narrower, elongated and the examination to all stages of the parasite life anterior end of the body moves in different cycle in the intermediate host and adult flocks. directions, while the posterior end is usually fixed to another fluke. Then all these samples RESULTS were stored in 95% ethanol. Examination of C. microbothrium's eggs 1.2 Morphological identification: revealed they are oval operculated, smooth- Some fresh flukes were pressed between two surfaced and white-grayish in color; their length glass slides and fixed in 70% alcohol for two 130-170 m and width 70-100 m (Fig 1). In days. The relaxed flukes were stained for the central of the egg, embryo was seen in an identification according to Eduardo technique early stage and surrounded by yolk cells (Figs 2 (29). All specimens were examined & 3). While, Figure 4 was showed the escaping morphologically and confirmed as process of miracidium and showing operculum paramphistomes before they included in and egg opening. Notably, the hatching of C. molecular study. microbothrium's eggs occurred more on the 1.3 DNA extraction: same periods in variable water mediums at the Genomic DNA collected from a small part of same temperature. adult flukes was extracted by the alkaline-lysis C. microbothrium's miracidium (Hot-SHOT) method (30). Mass-PCR technique shape like a torpedo; it was covered with cilia for detection of C. microbothrium was carried and carried on eye spots (Fig 5). The average out at molecular unit of Microbiology size of miracidium was measured about 199- Department, Faculty of Medicine Zagazig 270 x 50-60 m. Figs 6 & 7 showed miracidium University. penetrated the truncatus snail through 1.4 Molecular analysis the mantle cavity. The ITS2 (internal transcribed spacer region 2) By following up the infection stages of region was amplified using the primers GA1 snail by miracidium, using dissection of snail at [5_-AGA ACA TCG ACA TCT TGA AC-3_] different periods to detect the stages of (31) and BD2 [5_-TAT GCT TAA ATT CAG Sporocyst, it was found that Sporocyst was (32) CGG GT- 3_] . Polymerase chain reaction sacculer, elongated and curved in shape and (PCR) cycles were performed on Eppendorf measured 200 m x 100 m (Fig. 8, 9, 10 & Mastercycler epigradient machines. After the 11). Interestingly, Sporocysts were detected thermocycler process was finished, PCR mainly around the intestines and in mantle products were purified using PCR Microcon tissue of snails. columns and both strands were sequenced using While, rediae were opaque, an Applied Biosystems 3100 automated slightly curved in shape and had a limited cavity sequencer. as observed in Fig 12. In the young rediae the 1.5 Agarose gel electrophoresis: pharynx and the intestine were visible and they Amplification products of C. microbothrium contain embryo balls from which cercariae or adult flukes were visualized by electrophoresis daughter rediae would develop (Figs 12 & 13). on 1.5% agarose agar and staining by 0.5 µg/ml Rediae were detected around the intestines, ethidium bromide in the running buffer hepato- and in the mantle tissue of (33) procedure of Viljoen et al. . . The developing rediae 2 Laboratory studies: showed great variations in size and the mature The other part of the current study was carried rediae measured 700-1170 m in length and out in the Labe. Amounts of Calicophoron's 130-260 m in width (Fig. 14). mature parasites and eggs were collected from Otherwise, immature cercariae were infected sheep. In addition, snails those were liberated from rediae before their complete controlled under the stereomicroscope for development. In the young cercariae the body determination of natural infection. was small, eye spots were prominent and

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A Molecular and Microscopically Studies of Calicophoron Microbothrium pigment and the tail was short and wide rather temperatures did not show any variation; while, than long (Figs. 15, 16, 17 & 18). But the light was found to be the most effective mature cercariae were large, active, and dark influence factor for hatching. pigmented and their body was 500-550 m long On the other hand, it was noted that tap water is and 200-400 wide; while their tail was 550-560 a good medium than distilled water and saline long and 80-90 m wide (Figs 18, 19, 20, 21 & solution (0.9%) for several of Paramphistomum 22). miracidia and some miracidia of Notably, it was seen that light was a great Paramphistomum species kept their viability factors in shedding cercariae, infected snail kept longer in lakes and pools compared with other (46-48) in the dark started shedding cercariae again after media . The previous results supported our being exposed to light. Cercariae were collected finding that newly emerged miracidia swam from the snails only one day in a week in order more active in distilled water and spring water. to regulate metacercarial age conformity and to Otherwise, it was recorded that too many obtain then in high numbers. miracidia had a negative effect on the Under the light source and in the experimental infection of snails and only the presence of vegetation in the water most young or medium sized snails were suitable for (47&49) cercariae encyst on the vegetation. During the infection . During the current experiment, encystment cercariae attaches with its ventral it was observed that the infection rate increased surface, and the material for cystation being to with the number of miracidia used; but contrast secrete from the pores all over the body (Figs 23 to this, it was seen that the large proportions of & 24). Metacercaria was in the form of a half miracidia had a negative effect. Concerning sphere and was surrounded with a thick layer of snail size, it was seen that the infection rate a cyst wall. These metacercariae were measured among the small and middle sized Bulinus 240-260 m in diameter (Figs 25, 26 & 27). truncatus is elevated their mortality rate is The sensitivity of polymerase chain increased too. reaction (PCR) was measured using various Generally, it was accepted that the miracidia number of lysate cells of C. microbothrium. lost their cilia during the penetration and After 22 cycles of PCR as little as one copy of entered the snail host tissues as a young the target gene was detracted by specific primer sporocyst. On the observation of miracidial on agarose gel electrophoresis (Fig. 28). Each penetration into the snails, both mechanical and (46-47) primer (P1 and P2) amplified several DNA enzymatic effects together play a role . The fragments that were polymorphic the species. transformation of the miracidium into a The results which obtained with primer 1 and 2 sporocyst is a gradual process and no clear line produced characteristic intense band patterns of can be drawn between the invading miracidium (50) C. microbothrium (769 bp) (Fig. 28). and young sporocyst . In the present study persistence of cilia of the anterior part of the DISCUSSION miracidium was also observed, 1-2 hours after It is well known that the intermediate host of C. the infection of snails. microbothrium varies according to the Available information on the development of C. (46) geographical regions; however, many types of microbothrium in the snail is very limited . (51) snails play the main role as intermediate host (25 Kıselev reported that, in some & 35-41), Bulinus truncates were recorded as an Paramphistomum species, the occurrence of intermediate host for heavy infestation of the daughter rediae were recorded only in autumn snails with various paramphistomes is reported months. Moreover, the production of redia was mainly in the late summer and autumn months less in number compared to the regular and the incidence varies between 3 to 75% in production of cercariae and they were observed th the infected areas (36-38 & 42-44). In the present after the 40 days of infection yet. The current study naturally infected Bulinus truncatus were experiment showed the daughter rediae in found at each month between April and Bulinus truncatus not only in autumn months November too. but also in different months of the year. Otherwise, it was noted that many factors affect Interestingly, in the current research, it is the development of Paramphistome eggs such possible to detect the rediae which caring as temperature and light; but the effect of daughter rediae from the external lateral parts of temperature rather than that of light (45-47). the mantle tissue of Bulinus truncatus before its Similar results were obtained from the current dissection on the examination under the study, where the development of C. stereomicroscope. Notably, the location of microbothrium eggs in various mediums and rediae in the snail tissue was an important factor 31

Alaa Samn for the production of daughter rediae; where, purity and concentration of the DNA has been daughter rediae developed mainly from the measured by utilizing the spectral system that is rediae developed in the external circle of the used in measuring the ultraviolet rays ranging snail mantle tissue. from 300-350 Angstrom for measuring the It has been recorded that the light was the most DNA this agreed with Dubs and Dhlaminiz (57). important factor for cercarial shedding of Each target gene was amplified by PCR or infected snails and the snails could continue to Mass–nested PCR with serially diluted DNA shed about 1-10 months (46-47& 52). We obtained extracted from purified C. microbothrium. The not only similar results but also saw the infected target genes included C. microbothrium wall Bulinus truncatus continued to shed cercariae in protein, small subunit ribosomal RNA (SSU the laboratory for more than 13 months; some rRNA) and random amplified polymorphic snails became free from infection after 11 DNA. The present study agreed with Lotfy et months. al. (55) where the second-round amplification Paramphistomum cercariae are usually encysted using 769 bp-primer of the target gene showed on the vegetation and other subjects in the water that the nested primer set specific for the gene and, they can stay viable for about 5 months proved to be the most sensitive one compared to when they are persevered in the refrigerator (25, the other primer sets tested and would therefore 35 & 46). In present study encystations occurred on be useful for the detection of C. microbothrium. the plastic sheet which was placed in the Petri They added that the most sensitive Mass–PCR dish and especially around its folds. It was also target gene and the primers cowpnest-F1 and observed that one fifth of the metacercariae kept cowpnest-R2 could detect as low as a single C. among wet filter paper in the refrigerator at 4C microbothrium. This highly sensitive Mass– kept their viability for about 6 months, while PCR method will be helpful for facilitating the most of them started death after the second detection of Paramphistomes in specimens with month. a low number of worms and could be a valuable Furthermore, in current study it was determined tool for detection of worm in environmental and that the prepatent period of infection was 102- clinical samples. 142 days and the infection rate was 19.6-77% in After data from the present figures were lambs. There were finding of Abdel Ghani (26) comparing diagrammatic representation banding and Kraneburg and Boch (53) in the C. cervi patterns of the species C. microbothrium supported the current results, where they obtained with P1 (primer) and P2 (each of them reported that, infection prepatent period was yields clearly banding pattern for the species 129 days in goats, 103-115 days in cattle and investigated), it was agreed with the report of sheep and the infection rate was 45.82% in Lotfy et al. (55). Notably, Acuna-Soto et al. (58) goats, 40.6% in sheep and 44.8% in cattle. and Aguirre et al. (59) were used the differences In the recent years, PCR was established as in the highly repetitive sequences in the non- valuable tool for routine diagnosis of infectious coding region of rDNA whereas Novati et al. . It has been widely used as an in vitro (60) and Troll et al. (61) based their test on the 16s method for detection of specific DNA from rRNA gene of other paramphistomes species. minute amounts of starting material. The assay Finally, the results of DNA and PCR indicated did not require radioactivity, no laborious that the use of the genetic techniques and the hybridizations and the procedure can be molecular biology have a great importance in performed in one day (54&55). Samn (56) reported discovering and identifying the genetic finger that PCR could be used for identifying the prints of the C. microbothrium that will help genetic finger prints of species of those who the specialized in this field in future. paramphistome. Luton et al. (32) and Anderson In addition, the current study gives general and Barker (31) compared the sensitivity of information on C. microbothrium life cycle and many PCR primer sets for detection of C. cleared points of weakness and strength that microbothrium. facilitated methods of elimination of this In PCR test was carried out by the extracted parasite. DNA from 34 worms of C. microbothrium. This led to doubling and finding the genetic sequence REFERENCES by using P1 and P2 the partial weight of the gene 1. Ashour AA (1976): Studies on the is estimated by 0.90 k Dalton. The extracted paramphistomes of some ruminants from A.R.E. DNA from C. microbothrium was utilized by M.Sc. Thesis, University of Ain Shams. the DNA extraction techniques (31&32). The 2. Olsen OW (1974): Animal Parasites: Their Life Cycles and Ecology. Dover Publications, Inc., New 32

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Alaa Samn

35. Altaif KI, Al-Abbassy N, Al-Saqur IM and maturity and its longevity in cattle. Bull. Epizoot. Jawad AK (1978): Experimental studies on the Dis. Afr., 10: 27-31. suitability of aquatic snails as intermediate hosts for 50. Dawes B (1960): The penetration of Fasciola Paramphistomum cervi in Iraq. Ann. Trop. Med. hepatica into Limnea truncatula and of F. gigantica Parasit., 72:151-155. into I. auricularia. Trans. Soc. Trop. Med. Hyg., 54: 36. Arru E and Deiana S (1962): Sull' andamento 9-10. stagianale della paramfistomosi intestinale 51. Kiselev NP (1967): The biology of (Paramphistomum cervi) dei caprini e degli ovini in Paramphistomum ichikawai Fukui, Veterinariya, Sardegna. Atti. Soc. Ital. Sci. Vet., 16: 231-232. Moscow 44: 51-53 (Ref: Helminth. Abst. (1969), 38: 37. Arru E and Deiana S (1969): Gli ospiti 1035). intermediti Paramphistomum (P. cervi) in Sardegna. 52. Varma, A.K. (1961): Observation on the biology Atti. Soc. Ital. Sci. Vet., 23: 909-912. and pathogenicity of Calicophoron microbothrium 38. Deiana S (1953): L`infestione del Bulinus (Flaschoeder, 1910) Journal of Helminthology, contortus da cercarie di bouis (Sonsino, 33:161-168. 1876) e del Paramphistomum cervi (Schrank, 1790) 53. Kraneburg W and Boch J (1978): Beinlge zer in diverse stagioni dell`anno. Biol. Sper., 5: 1939- Biologic und Pathogeitae des einheimischen 1940. Pansenegels Paramphistomum cervi. 3, Eutwicklung 39. Kamburov P, Vassilev I and Samnaliev P in Rind, Schaf und Reh. Berl. Munchtrachticarztl. (1976): The species composition of Wschr., 91: 71-75. Paramphistomum Fischoeder, 1901, in . 54. Haque R, Ali IK, Akther S and Petri AW Khelmintologiya, Sof., 1, 19-22 (Ref. : Helminth (1998): Comparison of PCR. Isoenzyme analysis and Abst. (1978), 47: 3497). antigen detection for diagnosis of Calicophoron 40. Sey O (1977): Examination of amphistomes microbothrium infection. J. Clini. Micro. 63(2):449- (Tremotoda: Paramphistomata) parasitizing in 452. Egyptian ruminant, 10: 47-50 (Ref.: Helminth, Abst. 55. Lotfy W M, Brant S V, Ashmawy K I, (1979), 48: 1635). Devkota R, Mkoji G M and Loker E S (2010): A 41. Sey O and Arru E (1977): A review of species molecular approach for identification of of Paramphistomum Fischoeder, 1901 occurring in paramphistomes from Africa and . Vet. Sardinian domestic ruminants. Riv. Parasit., 38: 295- Parasito., 174 :234–240. 301. 56. Samn A (2007): Molecular, Electrophoresis 42. Arfaa F (1962): A study on Paramphistomum patterns and microscopical studies for identification microbothrium in Khazistan S.W. Iran. Annals and diagnosis of some parasitic. Trematodes in Parasit. Hump. Comp., 37: 519-555. Egypt. Thesis submitted degree of Faculty, Science, 43. Katiyar RD and Varshney RT (1963): PhD Al–Azhar Univ. Amphistomiasis in sheep and goats in Uttar Pradesh. 57. Dubs I, Sibula M and Dhlaminiz S (2015): Indian J. Vest. Sci., 33: 94-98. Molecular analysis of selected paramphistome 44. Zharikov IS (1964): Ecological and isolates from cattle in South Africa. J. Helminthol., parasitological study of mollusks of the genus (2): 65-75. in the Minsk region, Sb. Mauch. Trud. 58. Acuna-Soto R, Samuelson J, GirulamI P and Belorussk. Nauchno-issled. Vet. Inst., 99-10. (Ref. Wirth d (1993): Application nof polymerase chain Helminth. Abst. (1965), 34: 1734). reaction to the epidemiology of Calicophoron 45. Dinnik JA (1958): Identification of liver fluke microbothrium. Am. J. Trop. Med. Hyg., 49(1):58- and stomach fluke eggs recovered from faeces of 70. infested animals. Bull. Epizoot. Dis. Afr., 6: 135- 59. Aguirre A, Warthurst DC, Guhi F and 138. Frame JA (1995): Polymerase chain reaction – 46. Kraneburg W (1977): Beitrige zur Biologie solution hybridization enzyme – linked and Pathogenitat des einheimischen Pansenegels immunoassay (PCR-SHELA) for the differential Paramphistomum cervi. I. Entwicklungsstadien in diagnosis of Calicophoron spp. Trans. Royal. Soc. der Aussenwelt und im Zwischenwirt, Berl-Munch, Trop. Med. and Hyg., 89:187-188. Tierarztl. Wschr., 90: 316-320. 60. Novati S, Sironi S, Granata A, Bruno S, 47. Lengy J (1960): Study of Paramphistomum Gatti, Seaglia M and Brandi C (1996): Direct microbothrium Fischoeder 1901, A. rumen parasite sequencing of the PCR amplified SSU rRNA gene of of cattle in Israel. Bull. Res. Coun. Israel, B (9): 71- Calicophoron microbothrium and the design of 130. primers for rapid differentiation from Calicophoron 48. Colvin Jr (1962): The longevity of mirecidia of microbothrium. Parasito., 112:363-369. Paramphistomum microbothrium in various media. 61. Troll H, Marti H and Weiss N (1997): Simple Proc. La Acad. Sci., 25: 109-116 (Ref: Helminth differential detection of Calicophoron Abst. (1966), 35: 1349). microbothrium in fresh stool specimens by sodium 49. Dinnik JA and Dinnik NN (1962): The growth acetate – acetic acid-formaline concentration and of Paramphistomum microbothrium Fischoeder to PCR J. of Clinic. Microbio., 35(7):1701-1705.

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A Molecular and Microscopically Studies of Calicophoron Microbothrium

Fig (1): Photomicrograph of Fig (2): Photomicrograph of Fig (3): Photomicrograph permanent preparation of egg Egg with fully formed of The mira-cidium (Mi) stained with aceto-carmine, miracidium (Mi) during leaving the shell. X:400 showing the operculum (O) and rupture of the vitelline vitelline cells (V.C.). X:600 membrane (V.M.). X:500

Fig (4): Photomicrograph Fig (5): Photomicrograph of Fig (6): Photomicrograph of of an empty egg after the perma-nent preparation of the histolo-gical section through miracidium has escaped miracidium stained by aceto- the mantle cavity of Bulinus showing operculum (O) carmine, showing the truncatus showing (Mi) 30- and egg opening (E.Op). terebratorium (Tr.), the shoulder 60 minutes after X:500 line (Sh.L), cilia (Ci), and nuclei penetration. X:270 of the epidermal cells (Nu.E) and germinal cells (G.C). X:400 35

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Fig (7): Photomicrograph of Fig (9): Photomicrograph of histo-logical section through Fig (8): Photomicrograph of section through the hemolymph Bulinus truncatus six hours section through the mantle space showing the young (Sp.) after penetra-tion, showing showing the young 72 hours post-infect ion, the (Mi) closely lodged within sporocyst (Sp.) 24 hours germinal cell in mitotic status. the tissue, (Ci), apical gland post-infection. X:400 X:400 (Ap. G.) and germ cells (G.C.). X:380

Fig (10): Photomicrograph of Fig (11): Photomicrograph of Fig (12): Photomicrograph section through the sec-tion through the of mature radia showing hemolymph space showing hemolymph space showing part the birth pore (B.P.) and the young (Sp.) 4 days post- of the mature (Sp.) 9-10 days the distribution of the infection. X:400 post-infection. X:250 germinal balls. X:325

36

A Molecular and Microscopically Studies of Calicophoron Microbothrium

Fig (13): Photomicrograph Fig (14): Photomicrograph of Fig (15): Photomicrograph of of section of the mature inter-radial stage of immature extra-radial stage of immature mother radia showing the cercaria (I.C.) representing cercaria representing stage III mouth (M), pharynx (P), stage II of the cercarial of cercarial development, gut (Gu) and (G.B.). X:450 development showing the showing the primordial of oral primordia of the eye spots sucker (O.Su.), eye spots (E.S.) (P.E.S.) and primordial of the and tail bud (T.Bu.). X:170 tail (P.T.). X:170

Fig (16): Photomicrograph of Fig (17): Photomicrograph of Fig (18): Photomicrograph of sec-tion through the infected extra-radial stage of immature extra-radial stage of immature snail showing the internal cercaria representing stage cercaria representing stage IX structure of immature VII of cercarial development, of cercarial development, cercaria (I.C.) cystogenous showing the oral sucker showing the branches of eye cells (C.C.) and T.Bu. X:150 (ACE), acetabulum (A) and spots-pigment (Br.E), the bud Tail (T). X:170 of the tail fin (B.F.). X:180

37

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Fig (19): Photomicrograph of Fig (20): Photomicrograph of Fig (21): Photomicrograph of section through infected snail section through the body of mature cercariae showing the showing the mature cercariae mature cercariae showing the tail (T) and mucoid tail fins with oral sucker (O.Su.) and superficial pigment (S.P.) of (F1, F2, and F3). X:120 ventral sucker (V.Su.), eye spots rods (RO.) and eye (E.S.). X:350 spots (E.S.). X:580

Fig (22): Photomicrograph Fig (23): Photomicrograph of Fig (24): Photomicrograph of mature cercariae showing un-stained whole-mounted of top view of metacercariae extrusion of cysto-genous metacercariae on the lettuce stained with aceta-carmine rods (R.O) through the leaves. X:45 showing the body, and the whole surface. X:120 eye spots. X:300 38

A Molecular and Microscopically Studies of Calicophoron Microbothrium

Fig (25): Photomicrograph Fig (26): Photomicrograph Fig. (27): Photomicrograph of of lateral view of of frozen section of Frozen section of the cyst metacercaria, stained with metacercaria stained with stained with toluidine blue acetocarmine showing the acetocarmine showing the showing the various layers of dome shape (Do.), the base cyst wall (W), (E.S.), the cyst including outer layer (Ba.) and the wall of the (O.Su.), (V.Su) and (O.L.), inner layer (I.L.) as cyst (W). X:350 pigmented tegument (Pi). well as pigmented wall (P.W.), X:320 the body, (V.Su.) and ventral blug (V.P.). X:300

Fig. (28): Photomicrograph of detection of Calicophoron microbothrium (paramphistomatidae) DNA using polymerase chain reaction Mass–PCR. Lane m = molecular size ladder; lane 1 : Control, Lane 6 and 14 [ positive samples by Mass–PCR at 796 bp.

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