Journal of the Egyptian Society of Parasitology, Vol.44, No.3, December 2014 J. Egypt. Soc. Parasitol. (JESP), 44(3), 2014: 547 - 558 ENCYSTED METACERCARIAE OF FAMILY HETEROPHYIDAE IN INFECTED FISH IN DAKAHLIA GOVERNORATE, AN ENDEMIC FOCUS IN EGYPT By MAI A. HEGAZI, AISHA T. HASSAN, THANAA M. AL-NASHAR, OMAIMA I. ABO-ELKHEIR* and FATMA M. EL-LESSI Departments of Parasitology and Community Medicine & Public Health*, Faculty of Medicine for Girls Al-Azhar University, Cairo Abstract The study re-evaluated the status of encysted metacercariae (EMC) of Family Hetero- phyidae in fresh and brackish water fish in an endemic focus in Egypt, as well as to study their morphological pattern. Seasonal variation of EMC was matched with their preva- lence and intensity in infected fish. The study covered a period of one year from first of April 2011 to end of March 2012. The total percent of infection in the examined fish was 71.9% and 42.6% for brackish and fresh water respectively. The highest prevalence of the recovered EMC in both sources of water was for Heterophyes heterophyes (57.9%), followed by Pygidiopsis genata (39.7%), other Heterophyes spp. (21.09%), Haplorchis pumilio (19.5%), Phagicola spp (19.5%) and Stictodora tridactyla (6.2%). The highest mean intensity of the recovered EMC in both sources of water was for Pygidiopsis gena- ta (10.27±2.83), followed H. heterophyes (9.45±7.13), Haplorchis pumilio (1.76±2.03), Phagicola spp. (1.59±1.71), other Heterophyes spp. (0.82±0.99) and Stictodora tridacty- la (0.48±0.92). By using ANOVA test, there was significant difference in sizes of EMC of Pyigidiopsis genata (P=0.04) and Phagicola spp. EMC (P=0.03) in comparison to cor- responding ones in all fish species. By using Student T-test, there was significant higher size of Haplorchis pumilio EMC in Tilapia species in comparison to the corresponding one in Mugil species. Monthly variation showed that summer was the season with the highest prevalence and highest mean intensity of EMC in both brackish and fresh water fish. Also, undifferentiated EMC most properly of genus Centrocestus was demonstrated. Key words: EMC, Heterophyidae, Heterophyes, aequalis, dispar, Pygidiopsis, genata, Haplorchis, pumilio, Phagicola, longicollis, ascolonga, Stictodora, tridactyla Introduction taichui, Ha. yo-kogawi, Stictidora tridactyla, Family Heterophyidae (Odhner, 1914) Phagicoal longicollis, Ph. ascolonga and includes a set of over 50 genera having simi- Ph. italica (El-Sheikha and El-Sahazly, lar morphological features (Waikagul and 2008b), H. dispar (Mürrell and Fried, 2007), Thaenkham, 2014). The main zoonotic gene- H. nocens, H. katsuradi (Shalaby et al, ra are Metagonimus, Heterophyes (H), Cen- 2006), Haplorchis pleurolophocerca (Berger trocestus, Pygidiopsis, Stellantchasmus, and Marr, 2006), Stictodora sawakinensis Haplorchis and Procerovum (Chai and Lee, (Witenberg, 1928), Centrocestus cuspidatus 1990), Stictodora, Heterophyopsis, Di- (Bowman et al, 2008), C. formosanus (El- ochitrema, Phagicola, Appophalus, Crypto- Tantawy and El-Naggar, 2002), C. armatus cotyle, Plagiorchis, Brachylaima, Cotylurus (Ibrahim et al, 2009), C. unequiorchalis and Carneophallus (Berger and Marr, (Saad, 1994). The first intermediate hosts of 2006). In Egypt, the recorded genera and family Heterophyidae are different species species of family Heterophyidae were Het- of snails while second one harboring EMC erophyes heterophyes, H. aequalis, Pygidi- are different species of fish (Witenberg, opsis genata, Haplorchis pumilio, Ha. 1929). Definitive hosts are fish eating ani- 547 mals or birds and human consuming raw or in an endemic focus (Dakahlia Governorate) incompletely cooked fish (Dzikowski et al, in Egypt 2003). Heterophyiasis is commonly found in Materials and Methods Far East, Palestine, Mediterranean Coun- Mugil (M) and Tilapia (T) fish species (spp), tries, Hawaii, Ukraine, Canada and Al-aska the common intermediate host of fam Mugil (Seo et al, 1984; Chai and Lee, 1990; Dixon, (M) and Tilapia (T) fish species (spp), the 1997) and a zoonotic problem in Egypt common intermediate host of fam ily Het- (Shams El-Din, 2011) with H. heterophyes erophyidae, were collected form the Nile high infection rates (Chai and Lee, 2002). In River at Al-Mansoura City as fresh water Egypt, coastal regions inhabitants showed source and Al-Manzala Lake as brackish highest incidence of heterophyiasis (33.8%) water source. The later one is heterophyiasis than other helminthes in El-Meaddeya an endemic focus with high transmission (Abou-Basha, 2000) and 13.3% in Northern rate. Fish samples were obtained from local Egypt suffered from heterophyiasis (Lobna fishermen twice monthly over one year from et al, 2010). They added that infection rate the beginning of April 2011 till the end of was 22% in brackish water and 42% in March 2012. The total number of fish was fresh water fish Besides, between 79.2% and 887; 385 Mugil spp. (M. cephalus, M. capi- 100.0 % in all fish in Manzala Lake (Mugil to, and M. auratus) and 502 Tilapia spp. (T. spp. and Tilapia spp.) were infected with H. nilotica and T. zilli). Fish samples were heterophyes EMC (El-Sheikha and El- transferred in ice-box to the laboratory and Shazly, 2008a). Also, in fresh water canal in screened for the presence of any EMC by Ismailia Governorate T. zilli (95.37%) were the naked eye or added by a hand lens infected with heterophyid EMC (Ibra- (Mahdy et al, 1995). About 30 snips of each him and Soliman, 2010). The rate of infec- fish were taken from different parts mainly tion with heterophyid metacercariae was from the head, dorsum and tail regions as 37.9% of Mullet, Liza auratus fish in Lake well as visceral organs, fins and gills. The Manzala (Ghobashy et al, 2010). In hetero- EMC were identified microscopically phyiasis, noticeable symptoms of diarrhea, (Elsheikha and Elshazly, 2008a). Different abdominal pain, loss of appetite and nausea types of metacercariae were separated into 6 occur in the final host as result of inflamma- groups according to their morphology (di- tion due to worm attachment to the mucosa ameters, shape, thickness of cyst wall, and of the intestine and releasing of eggs. Sus- pigments content). Separation of EMC from ceptibility of children may be more (Parija et infected fish by artificial digestion method al, 2003). was done (Yokogawa and Sano, 1968). The Eggs released in the intestine can be digested material was filtered through a trapped into the blood stream and embolize sieve and washed several times with 0.85% in various regions of the body, particularly physiological saline. Different types of EMC with tissue reactions in the heart valves and were collected from the sediment and segre- myocardium causing cardiac failure as well gated using a binocular dissecting stereomi- in the central nervous system (Schmidt and croscope. Each group of 50 EMC was given Roberts, 2009). Eosinophilia is often present orally to 5 puppies (healthy, aging two in heterophyiasis infections (Parija et al, months and weighing about 3kg) in 10 ml of 2003). phosphate buffered saline solution (pH 7.6) The aim of the present work was to review by using stomach tube and mouth gage. One the current status of encysted metacercariae week later, infection in puppies was con- (EMC) of family Heterophyidae in brackish firmed by stool examination. Infected pup- and fresh water fish as the intermediate host pies were anaesthetized and sacrificed after 3 weeks post-infection (PI). The intestine 548 was taken in one piece and opened. The mu- alum carmine then identified microscopical- cosa was scraped gently and the contents ly on morphological basis (Witenberg, were collected and transferred to clean dish 1929). The prevalence and mean intensity of containing warm saline to prevent the con- different EMC in fish tissues from different traction of the worms. The contents were sources of water were done (Margolis et al, transferred to a conical flask. The superna- 1982). tant fluid was removed and the deposit was Statistical analysis: Data were computed washed several times in warm saline to re- and analyzed by using SPSS for Windows move any debris. The parasites were collect- version 15.0. Student T-test and ANOVA- ed and preserved in 70% alcohol. The col- test (F-test) were used for comparing data. P lected worms were stained with acetic acid value ≤0.05 was considered significant. Results Table 1: Percent infection of total examined fish with heterophyid EMC over one year in different water of Tilapia and Mugil fish Water Sources Prevalence (%) Intensity (cyst/gm.) Manzala Mansoura Total L. N. Manzala Mansoura (N=887) Total (N=636) (N=251) L. N. (N`=564) EMC spp. (N`=457) (N`=107) 71.6% 0.0% 57.9% 1 H. heterophyes 0±0 9.45±7.13 (n=327 ) (n=0) (n= 327 ) 3.23±4.56 Other Heterophyes 26.03% 0.0% 21.09% 1.15±1.005 0±0 0.82±0.99 spp. (n=119 ) (n= 0 ) (n=119 ) 38.9% 42.9% 39.7% Pygidiopsis genata 2.95±2.07 4.16±4.103 10.27±2.83 (n=178 ) (n=46 ) (n=224 ) 15.09% 38.3% 19.5% Haplorchis pumilio 1.41±1.60 2.62±2.69 1.76±2.03 (n=69) (n=41 ) (n=110 ) 19.7% 18.6% 19.5% Phagicola spp. 1.55±1.56 1.70±2.07 1.59±1.71 (n=90 ) (n=20 ) (n=110 ) Stictodora tridacty- 7.7% 0.0% 6.2% 0.68±1.03 0±0 0.48±0.92 la (n=35 ) (n=0 ) (n=35 ) Table 2: Grouping and morphological characters of recovered EMC Fish Fish spp. No. of examined No. of infected % of infection Water source Mugil 385 294 76.4 Manzala Lake Tilapia 251 163 64.9 Total Mugil & Tilapia 636 457 71.9 Mansoura Nile Tilapia 251 107 42.6 Both water sources All spp.