STUDIES ON SOME PROBLEMS OF PROTOZOAL INFECTION IN FRESHWATER FISHES NEHAL ABOU EL-KARAMAT YOUNIS. Prof. Dr. Mohammed Abed El Aziz Ahmed

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1 Cairo University Faculty of Veterinary Medicine Department of Fish Diseases and Management STUDIES ON SOME PROBLEMS OF PROTOZOAL INFECTION IN FRESHWATER FISHES Thesis presented By NEHAL ABOU EL-KARAMAT YOUNIS (B. V. Sc, 2008, Cairo University) For the degree of M. V. Sc. (Fish Diseases and Management) Under the supervision of Prof. Dr. Mohammed Abed El Aziz Ahmed Prof. and head of fish diseases and management department Faculty of veterinary medicine Cairo university Prof. Dr. Mai El-Desoky Al-Said Ibrahim prof. of fish diseases and management Faculty of veterinary medicine Cairo University Prof. Dr. Nisreen Ezz El-dien Mahmoud prof. of parasitology Faculty of veterinary medicine Cairo university 2012

2 To My father, My mother, My husband and my children

3 Acknowledgment

4 Acknowledgement No word can express my deep sincerest deepest thanks to prof. Dr. Mohammed Abd El-Aziz Ahmed professor and head of fish diseases and management department, faculty of veterinary medicine, Cairo university, for his kind encouragement, great interest, valuable advice and help during the course of this research. I am very thankful to Prof. Dr. Mai El-Desoky El-Said, professor of fish diseases and management, faculty of veterinary medicine, Cairo University, for her valuable advice during the course of this work. I would like to express my deepest thanks to Prof. Dr. Nisreen Ezz El-Dien Mahmoud professor of parasitology, Faculty of veterinary medicine, Cairo University, for her kind help during the practical work of this thesis and valuable advice. Finally, it is a great pleasure to record my kind gratitude to all members of fish diseases and management department, faculty of veterinary medicine, Cairo University.

5 List of Contents Introduction 1 Review of literature 3 Material and Methods 29 Results 36 Discussion 100 Summary 108 References 113 Arabic summary 4-1

6 List of Figures Figure (1) alive Oreochromis niloticus post transportation to the laboratory. Figure (2) Oreochromis niloticus infected with Trichodina. Figure (3) Oreochromis niloticus infected with Chilodonella. Figure (4) Oreochromis niloticus infected with Trichodina. Figure (5) Clarias gariepinus infected with Trichodina. Figure (6) Oreochromis niloticus infected with Myxobolus dermatobia Figure (7) Gills of Clarias gariepinus show whitish cysts of Henneguya branchialis. 46 Figure (8) Unstained wet preparation smear of Trichodina truttae (x 40). 54 Figure (9) Lateral view of wet preparation smear of Trichodina truttae stained with giemsa stain (x 40). 55

7 Figure (10) Fixed smear of Trichodina truttae stained with giemsa stain (X 100). Figure (11) Chillodonella hexasticha stained with giemsa stain (X 100). Figure (12) Henneguya branchialis stained with giemsa stain (X 100). Figure (13) Sporulated oocyst of cryptosporidium sp. Stained with Modified Zeilnelson stain (X 100). Figure (14) Sporulated oocyst of cryptosporidium sp. Stained with Modified Zeilnelson stain (X 100) Figure (15) Balantidium sp. Trophozoite (A) and cyst (B). 61 Figure (16) Oocyst of Eimeria unstained (X 100). 62 Figure (17) Flagellated form of Ichthyobodo necator stained with giemsa stain (X 100). Figure (18) Trypanosoma mukasi stained with giemsa stain (X 100). Figure (19) Blood film stained with giemsa stain (X 100)

8 Figure (20) Blood film stained with giemsa stain (X 100). Figure (21) Gill of Oreochromis niloticus infected with Ichthyophthirius mulltifiilus. Figure (22) Skin of Oreochromis niloticus infected with Trichodina truttae. Figure (23) Gill of Clarias gariepinus infected with Trichodina truttae. Figure (24) Gills of Clarias gariepinus infected with Trichodina truttae. Figure (25) Gills of Oreochromis niloticus infected with Trichodina truttae. Figure (26) Gill of Oreochromis niloticus infected with Chillodonella hexasticha. Figure (27) Gills of Oreochromis niloticus infected with chillodonella hexasticha. Figure (28) Gill of oreochromis niloticus infected with Chillodonella hexasticha. Figure (29) Gill of the Clarias gariepinus infected with Henneguya branchialis

9 Figure (30) Gill of Clarias gariepinus infected with Henneguya branchialis. Figure (31) Eye of Oreochromis niloticus infected with Myxobolus dermatobia. Figure (32) Eye of Oreochromis niloticus infected with Myxobolus dermatobia. Figure (33) Intestine of Oreochromis niloticus infected with Cryptosporidium sp. Figure (34) Intestine of Oreochromis niloticus infected with Balantidium sp. Figure (35) Intestine of Oreochromis niloticus infected with Balantitium sp. Figure (36) Intestine of Oreochromis niloticus infected with Eimeria sp. Figure (37) prevalence of protozoan parasites among examined fish. Figure (38) The detected parasitic protozoa and their incidence in both Oreochromis niloticus and Clarias gariepinus

10 Figure (39) Prevalence and seasonal dynamics of the detected Ecto parasitic protozoa. Figure (40) Prevalence and seasonal dynamics of the detected enteric parasitic protozoa. Figure (41) Prevalence and seasonal dynamics of the detected Blood parasitic protozoa

11 List of Tables Table (1) prevalence of protozoan parasites among examined fish. Table (2) The detected parasitic protozoa and their incidence in both Oreochromis niloticus and Clarias gariepinus. Table (3) Seasonal incidence of parasitic protozoan infection among examined fishes. Table (4) Prevalence and seasonal dynamics of the detected Ecto parasitic protozoa. Table (5) Prevalence and seasonal dynamics of the detected Enteric parasitic protozoa. Table (6) Prevalence and seasonal dynamics of the detected Blood parasitic protozoa. Table (7) result of water sample analysis from Maryotia Channel

12 Introduction

13 Introduction I. Introduction - Aquatic species are considered one of the most important sources of animal proteins, The world can relay on it to compensate the shortage in high quality protein due to the rapid increase of human population (Abd El-Aziz, 2002). - Increasing intensification of fish and lack of health management measures lead to many disease problems in fish about 80% of fish diseases are parasitic (Eissa I., 2002). - Protozoa are one of the major sectors of fish parasites. That have been long neglected because of its inherent difficulty in studying compared to other larger parasites (Omeji et al. 2011). - Ecto and Endo parasitic protozoa occupy a very important sector as one of the hazardous threats to fish health in terms of low weight gain as the infection may cause the fish to stop feeding so moderate infection on small fish may prove a fatal disease (Enayat 2011), Excess mucous formation on the skin which make the skin to appear slimy and exhibited cloudiness (Nyaku et al. 2007). - In recent years high mortalities occurred among fishes in Maryotia Channel in Giza. Oreochromis niloticus was the most affected fishes, the examined fished revealed the 1

14 Introduction presence of external protozoa related to aquatic pollutant (Abd El-Aziz and Zaki, 2010). So in this study investigate the most common protozoal infections among wild fishes in Giza governorate in relation to water changes, this can be achieved through (Aim of work): 1- Surveying the most common protozoal infection in certain water resources in Giza governorate. 2- Identifying the detected protozoa. 3- Studying the clinical, post mortem and histopathological changes in the examined fishes in relation to the identified protozoa. 4- Monitoring the water quality of water resources from which the fish samples were collected. 2

15 Review of Literatures

16 Review of Literature II. Review of Literature (1) Impact of Protozoal infection and main predisposing factors. - Shalaby and Ibrahim (1988) Concluded that External protozoa are the most dangerous group that causes severe mortalities. - Stoskopf (1993) Mentioned that protozoan infections have been recorded as the most critical parasitic infections on the external body surface leading to severe destructions of gills accompained with economic losses and mortalities in freshwater fish. - Roberts (1995) reported that some macroscopical lesions of protozoa on fish may be observed by fishermen or consumers that may lead to rejection of fish as the white spots that caused by Ichthyophthirius multifiliis species. - Woo and Poynton (1995) Mentioned that protozoans undoubtedly represent one of the most important groups of pathogens which negatively affect the health state of cultured and feral fish. There are a number of protozoan parasites long recognized as causative agents of sever diseases. - Tomas (1999) Stated that parasites, causing little apparent damage in feral fish populations, may become causative agents of diseases of great importance in farmed fish 3

17 Review of Literature leading to pathological changes, decrease of fitness or reduction of the market value of fish. - Al-Rasheid et al. (2000) Recorded that the most identified protozoa are belonging to ciliates. They can easily spread among most of fish hosts. Un controllable or recurrent infection with ciliated protozoans is indicative of unhygienic husbandry problems. - Roberts et al. (2000) Studied that in cultured fishes, protozoa often cause serious outbreak of disease. So the presence of dense populations of fish kept in particular environmental conditions may favor certain parasites so that the protozoa population increases to a very high level. - Eissa I. A. (2002) stated that increasing intensification of fish and lack of health management measures lead to many disease problems in fish. About 80% of fish diseases are parasitic especially in warm water fish. - Omeji et al. (2011) reported that protozoa are one of the major sectors of fish parasites that attack fish causing massive destruction of skin and gill epithelium. Even moderate infection of these organisms on small fish may prove a fatal disease, since the infection may cause the fish to stop feeding. - Omeji et al. (2011) Fish protozoa result in economic losses not only mortality, but also from treatment 4

18 Review of Literature expenses, growth reduction during and after outbreak of disease and this militate against expansion of aquaculture. Protozoan parasites cause serious losses in fish ponds and wild and their lesions render the fish un marketable. (2) Prevalence of protozoa in fishes and main clinical signs: 2-1 Ectoparasitic protozoa: Ichthyophthirius multifiliis: - Dickerson and Clarck (1996) stated that Ichthyophthiriasis affects both cultured and aquarium fishes causing large losses in fish cultures. - Robert et al. (1998) denoted that Ichthyophthirius multifiliis could constitute significant economic losses in fish production. - Robert et al. (1998) mentioned that fish infected with Ichthyophthirius multifiliis may have white specks on their skin as though they were sprinkled with salt. Because of this appearance, Ichthyophthirius multifiliis is called White Spot Disease. - Bernad (2000) examined (692) Salmonid fishes in Olsztyn in Poland. The detected protozoa were Ichthyophthirius multifiliis. 5

19 Review of Literature - Manicini et al. (2000) denoted that Ichthyophthirius multifiliis infected many species of wild fish in the central south region Cordoba, Argentina. - Muzzall (2000) succeeded to isolate Ichthyophthirius multifiliis from Trout species (366 rainbow trout, 16 brook trout, 103 brown trout). - Popovic et al. (2001) mentioned that the parasitological examination of rudd (seardinius crythrophthalnus hesperidicus) in lake Vrana, Coroatia revealed the 27% of the examined fish were harbouring Ichthyophthirius species. - Davis et al. (2002) mentioned that crowding stress increases the susceptibility of fish to infectionwith Ichthyophthirius multifiliis due to suppression of innate defences. - Kenneth et al. (2002) performed study on stressed channel catfish. proved that increase the susceptibility of stressed channel catfish for Ichthyophthirius multifiliis. - Kin et al. (2002) reported 3 ciliates cause losses among (15) species of ornamented tropical fishes in Koreas, Ichthyophthirius multifiliis was the most common parasite. - Sigh et al. (2004) mentioned that Ichthyophthirius multifiliis cause erosion of the epithelium and thickening of the gills, this could be attributed to inflammatory 6

20 Review of Literature processes which occurred during infection with this parasitic ciliated protozoa. - Svobodova and Kolarova (2004) mentioned that Ichthyophthiriosis is one of the most serious parasitic diseases of fresh water fish. It can cause large losses of tench (one of the original European cyprinid species) as well. The Ichthyophthirius multifiliis parasites between epidermis and in gill epithelium. Higher temperature of water, dense stock of fish for several weeks and total weakening of the fish by malnutrition or starvation are important conditioning factors affecting the outbreak of Ichthyophthiriosis. - Nesreen Saad (2008) detected the prevalence of Ichthyophthirus multifillis was (58%) from Clarias gariepinus. Fish were collected from Abassa farmed aquaculture El-Sharkia governorate. - Garcia et al. (2009) performed study on swordtails fishes collected in ornamental fish farm in sao Paulo, Brazil. For detection of protozoan parasites in fishes. Fish were monthly collected from ponds and tanks for one year. The prevalence rates in fish from tanks and ponds were, respectively (34.2%) and (22.5%) for Ichthyophthirius multifiliis. 7

21 Review of Literature - Osman et al. (2009) mentioned that Ichthyophthiriasis usually occur due to stressful conditions as poor quality water or sudden chill. - Osman et al. (2009) concluded that the ciliate Ichthyophthirius multifiliis parasitizes the skin of fresh water teleosts and is considered to be one of the most pathogenic fish protozoans. - Malgorzata et al. (2010) concluded that Ichthyophthirius multifiliis is a wide spread ectoparasitic ciliate that occurs in temperate, subtropical, and tropical zones, and may cause considerable loss of fish, particularly under farm or hatchery conditions. - Omeji et al. (2011) examined (120) Clarias gariepinus fishes [comprising 30 dead and (30) live fishes, sixty each from the wild (River Benue) and a pond] for protozoan parasites infection. Ichthyophthirius multifiliis was the most common protozoan parasites found in Clarias gariepinus. These protozoan parasites constitute (37.08%) of the total parasites encountered for fishes in the pond and (42.51%) of fishes in the wild. - Omeji et al. (2011) observed several damages for the body parts of the Clarias gariepinus fishes. Erosion of the epithelium on the skin and thickening of the gills as well as excess mucous secretion on the gills was caused by Ichthyophthirius multifiliis. 8

22 Review of Literature Trichodina species: - Leong and Wong (1990) mentioned that heavy infections of potozoans in Seabass from Thailand were the primary cause of disease outbreaks in these fish, showing symptoms of haemorrhage on the body, fin rot and scale loss. - Khan (1991) demonstrated Trichodina Truttae in captive Atlantic salmon. - Ramadan (1991) investigated Oreochromis niloticus in lake Manzalah and found that they were infected with different types of parasites including Trichodina. The prevalence of infection was highest in T. zilli and lowest in Oreochromis niloticus. He added that female fishes were heavily parasitized than males and the large sized fishes were more subjected to parasitic infection than smaller ones. - Lumanlan et al., (1992) stated that fresh water fishes imported into Philippines were infected with one or more parasite species such as Trichodina. - Novoa et al., (1992) listed the results of a microbiological survey in an on growing turbot farm. The parasites which were isolated occurred in low prevalence as the ciliates Trichodina sp. 9

23 Review of Literature - Radheyshyam et al., (1993) mentioned that fish health can be affected by environmental stressors as malnutrition, coverage of water surface by lotus plants, aggrevated hypoxic and hypercarbic conditions, all these also can induce proliferation of the fish pathogens, it was recorded in such a case that the main protozoa encountered in fish were Rrichodina sp. These infections caused severe losses n weight gain. - Bunkley and Williams (1994) listed that very heavy infections of Trichhodina spheroideri killed wild specimens of orange file fish Alutenus schoepfi and white spotted file fish Cantherhines macrocerus in Puerto Rico. - Gosper (1995) demonstrated parasitic disease of perch that including Trichodina infection and white spot diseases. - Loubser et al., (1995) performed study on some fishes from the Bay of Dakar, Sengal and found that four of these fish species were infected by three known Trichodinid species, i.e. Trichodina jadranica, Trichodinid lepsii and Trichodinid rectuncinata. - Ekanem and Obiekezie (1996) recorded the effect of Trichodina maritinkae on the growth of Heterobranchus longifilis fry, significant reduction in weight was observed due to the extensive histopathological changes associated with the infection. 10

24 Review of Literature - Mohamed A. H. H. (1999) performed study that aimed for screening of the presence of Trichodina sp. among Oreochromis niloticus and Oreochromis aureus collected from various fish farms in the eastern province of Sauudi Arabia during January. Clinical signs of Trichodiniasis only appear on fish with heavy infection including sluggish movement, loss of appetite, black colouration, necrosis and ulcer on different parts of the body, detached scales and excessive accumulation of mucous in gill pouches. - Schisler et al., (1999) listed that gill ecctoprotozoa on juvenile Rainbow trout and Brown trout in the upper Colorado river. They found Trichodina On fish examined. They mentioned that peaks of infection intensity and ectoprotozoa richness occurred in August and September, because of high mean water temperatures and low flows during that time. - Robert (2003) Stated that Trichodina is one of the most common ecto-parasitic protozoa of wild and cultured fishes. - Svobodova and Kolarova (2004) mentioned that representatives of the genera Trichodina are frequent protozoa occurring in weakened Tench. Their rapid development and direct lifecycle can be problematic in both recirculating systems and in systems with poor water quality. 11

25 Review of Literature - Nesreen Saad I. (2008) recorded the incidence of Trichodina among examined Clarias gariepinus which was (58.7%). The peak of infection was in Autumn. while in Oreochromis niloticus the incidence of Trichodina was (42.7%) and peak infection was in winter. - Mahmoud et al., (2011) performed study on (330) Oreochromis niloticus and (140) Claries gariepinus were collected from three different ecosystems at Kafr El-sheikh province to detect prevalence and seasonal variation of external protozoa affecting fishwater fishes. Parasitic protozoa recorded an incidence of (55.5%) among total examined Oreochromis niloticus. The incidence of parasitic protozoa among total examined Clarias gariepinus reached (29%). Recorded highest infection during spring followed by summer then winter and autumn. - Omeji et al., (2011) performed study on a total number of (120) Clarias gariepinus were examined for protozoan parasites infection, sixty each from the wild and a pond (cultured) over a period of six months. The Prevalence of Trichodina sp. was (20.93%) among the protozoa found on the parts of the sampled fishes. 12

26 Review of Literature Chilodonella - Ali et al., (1988) recorded the protozoa infecting three species of carp raised in ponds in Iraq, one of the three protozoa was Chilodonella. - Lucky et al., (1989) examined (14) fish species from masco pool, Borno and recorded that fish species infected with (13) species of protozoa including chilodonella. They added that the prevalence of chilodonella cyprinid in examined carp from masco pool was (95%). - Hahlweg (1990) stated that chilodonella species caused great losses among reared tench larva. - Hossin (1992) isolated chilodonella species from gills of Oreochromus niloticus and Clarias gariepinus. - Urawa (1992) listed the occurance of chilodonella piscicola on juvenile pacific salmon reared at (204) hatcheries in northern Japan. The percentage of positive hatcheries was (8.8%) from chilodonella piscicola. - El-Khatib (1993) isted higher prevalence of external protozoa in Claries gariepinus fish about (63%). The recorded percentage for chilodonella was 58% but in Oreochromus niloticus the percentage was (55%). 13

27 Review of Literature - Kuo et al., (1994) examined many diseased ornamental fishes. They found several protozoan parasites including Chilodonella species. - Al-Shaikh et al., (1995) listed some fish parasites as Chilodonella cyprinid from the lower reaches of Diyala River, Mid Iraq. - Dickerson and Clark (1996) mentioned that Chilodonella Brooklyn Ella one of the most common ciliates parasitic to fish. They added that some of the ciliate protozoa are highly pathogenic to fishes but fish have evolved an immune system which can perform a protective response against parasite challenge. - Noble and Summer felt (1996) listed the diseases encountered in Rainbow trout cultured in recirculating systems, those caused by different parasites, one of them was Chilodonella. - Schisler et al., (1999) examined (112) rainbow trout and 204 brown trout in upper Colorado river that demonstrated Chilodonella in examined fish gills. They mentioned that peaks of infection occurred in August and September, because of high temperature and distended low flow. - Nesreen Saad I. (2008) recorded the incidence of Chilodonella among examined Oreochromis niloticus was (17.9%), peak of infection was in autumn. While the 14

28 Review of Literature incidence of Chilodonella among examined Clarias gariepinus was (13.3%), peak of infection was in winter Myxobolus dermatobia: - Abu El-Waffa et al (1999) previously isolated Myxobolus dermatobia from rounded, whitish cysts in the eye of Oreochromis niloticus without mentioning the infection rate or histopathological finding in their report. - Eduardo et al., (2002) described a new myxoporean species called myxobolus absonus species was found infecting freshwater fish in the river at state of Säo Paulo, Brazil. The cysts were found free in the opercular cavity. The spores are large (length mm) and oval in shape with anterior end slightly pointed. - Eissa et al., (2006) has indicated that Myxobolus species is one of the most endemic parasites at the earthen pond facilities in Nile Delta, Egypt. They also concluded that the reason behind the endemic nature of the parasite, could be the endemic presence of the tubificid intermediate host, Tubifex tubifex at the mud of the earthen pond bottom. - Eissa el at., (2010) has concluded that the endemic existence of the Myxxobolus tilapiae in the earthen pond facilities at Abassa, Sharkiya could played a critical role in the recurrent columnaris outbreaks in such facilities by concurrent infection routes. The concurrent infections 15

29 Review of Literature could be a multifactorial problem that jeopardize the entire existence of aquaculture fishes at the earthen pond facilities Egypt's wide. - Mohammed el al (2002) described six Myxobolous species from Nile fish, five of which are new and one is redescribed. - Molnar et al., (2002) studied Myxobolus species that recorded in skeletal muscle of six cyprinid fish species using morphological and molecular biological methods. intracellular developing Myxobolus spores identified as Myxobolus cyprinid from the Common carp and Myxobolus musculi. - Mahmoud A. M. et al (2004) described Myxobolus dermatobia affections from eye of Tilapia zillii from the River Nile at El-Kanater El-Khayeria, northen Cairo, Egypt. They mentioned that individual eyes showed up to 8 white cysts or nodules (1-2 mm in diameter) distributed peripherally around the corneal margin of the eye. Some fish showed two-eye affection. The histopathological examination of the infected eyes showed sever pathological lesions including lymphocytic and eosinophilic granular cells infiltration with hemorrhages and congested blood vessels. 16

30 Review of Literature - El-Matbouli et al., (2005) used light and electron microscopic method for studying the route of the sporoplasm of Myxobolus cerebralis from epidermis to Rainbow trout cartilage resulting in whirling disease. They concluded that after penetration of triactinomyxon sporoplasms of Myxobolus cerebralis through skin, fins, gills and buccal cavity. During the first hour following penetration, the sporoplasm migrates between the epidermal cells. Then, it enters the epithelia and multiplies intracellularly. These stages migrate deeper into the subcutis, then through the peripheral nerves and CNS, after (21) days the parasite reach the head cartilages, also during their migration occur multiplication of parasites so increase in number. - Probir et al., (2006) described a new Myxosporean species, called Myxobolus dhanachandi species from a freshwater fish from the state of Manipur, India. - Rebecca et al., (2006) studied the risk that caused by Myxobolus cerebralis infection to rainbow trout in Madison River which located in Montana in USA. - Nesreen Saad I. (2008) recorded that, the infection rate of Myxobolus spp. in Oreochromis niloticus was (8.8%). Fish were collected from aquaculture at abbasa fish farm and private fish farms at Kalubia and Giza governorates during different seasons. 17

31 Review of Literature - Fantham et al (2009) described some Myxosporidia found in freshwater fishes in Quebe province, Canada. - Carlos et al., (2010) performed study on Amyxosporean parasite in the gill lamellae of the freshwater teleost fish (sciades herzbergii) from the Poti River (Northeast of Brazil) called Myxobolus sciades species. Histological analysis revealed the close contact of the cyst-like plasmodia with the basal portion of the epithelial gill layer. - Zhang et al (2010) recorded the infection of Myxobolus turpisrotundus sp. N. in allogynogenetic Gibel carp, Carassius auratus gibelio (Bloch). - Carlos et al (2012) examined Amyxosporean infecting the gill filaments of the freshwater telosts collected from Amazonian river, Brazil, using light and electron microscopy Henneguya species: - Mohamed (1999) used light microscopical description for one Myxozoan species called Henneguya ghaffari, which infects the Nile Perch (Lates niloticus) in lake Wadi El- Raiyan in Egypt. The spores is characterized by a triangular thickening at the base of the caudal processes which adherent to each other for two-thirds of their length, then bifurcate to very fine processes. Prevalence of infection was 18

32 Review of Literature (34.6%) and peaked during winter and early spring. Infection was concentrated along the intestinal tract. - Boguyana et al., (2001) mentioned that Myxosporidea are frequently described in fish and have an importance in Ichthyopathology. In Africa, about (135) species of these parasites are currently known to infect freshwater, brackish and marine fishes. - Amina (2002) made redescription, supported by light microscopy photographs, for various immature stages and for mature Henneguya suprabranchiae. spores infecting the intestine of the Nile catfish (Clarias garipinus), Large cysts containing immature and mature stages were present in outer layer of the intestine. They caused sever damage to smooth muscle layer and atrophy due to increased size and resultant pressure of the plasmodial mass. - Bianca et al., (2003) described a new species of Myxosporea, called Henneguya chydadea, is parasitizing the gills of fish collected from a lake in state of Säo Paulo, Brazil. They recorded that about (88.3%) of examined fish had gills parasitized by Myxosporeans. The prevalence of the parasite ranged from (80%) in the spring and fall, (93%) in the summer and (100%) in the winter. - Edson et al., (2005) made survey of Myxosporean parasites of fish species cultivated in Brazil. The main fish 19

33 Review of Literature used in survey was Prochilodus lineatus hich considered the most consumed fish species in Brazil. Myxobolus porofilus was the only Myxosporean species reported to parasitize Prochilodus lineatus. However, during the survey, they found a new species of Henneguya called Henneguya caudalongula parasitizing the gills of Prochilodus lineatus. - Abraham et al (2008) described three new species of Hennegaya parasites of freshwater fishes in Cameroon (Central Africa). - Dalia et al (2009) examined (140) Clarias gariepinus for studying the impact of the infection with Henneguya branchialis. - Soheir et al., (2009) made general survey of protozoan parasites that infect freshwater fishes in Qena province, which considered the first time to do general survey in Qena province. Several species of freshwater fishes in Qena city were captured during the period from January 2006 to May Fishes were brought a live to the laboratory and examined for Henneguya species by a light microscope. A total number of fishes examined were (581) belonging to (14) species. Three species of Henneguya were recorded from (2) species of fishes. The first species is suggested to be Henneguya mandouri, the second 20

34 Review of Literature species is Henneguya assuiti and the third one is Henneguya nilotica. - Dalia et al., (2010) performed a study aimed to isolation and identification of respiratory form of Henneguyosis in naturally infected catfish Clarias gariepinus. (400) alive catfish Clarias gariepinus were randomly collected from different water ecosystems and private fish farms in Ismalia governorate, Egypt. The clinical signs, postmortem examination revealed respiratory manifestation, sluggish swimming, loss of appetite, congestion on gills and presence of cyst like structure on the gill filaments. Parasitological examination revealed great number of spores in the milky fluid inside the cysts, which were identified as Henneguya branchialis. The prevalence of infection was (17.5%) and highest rate of infection was found in spring season and in female specimens more than males. - Abdel -Baki et al., (2011) studied the validity, impacts and seasonal prevalence of Henneguya suprabranchiae infecting Clarias gariepinus from the River Nile. For this study catfish were collected from March 2009 to March a total of (240) freshly caught catfish Clarias gariepinus were collected from the boat landing site at Beni-Suef governorate. The overall prevalence was (35%) with maximum rate of infection in spring and minimum rate of infection in summer. Henneguya suprabranchiae is a 21

35 Review of Literature pathogenic species as the parasite showed high deformation of the filaments structure and complete disappearance of gill lamellae Ichthyobodo necator: - Urawa and Kusakari (1990) stated that Ichthyobodo necator could survive and reproduce in seawater after transferring of infected Chum salmon fry from freshwater to seawater. - Kuo et al (1994) Collected many ornamental fishes, they commonly found protozoan parasites including Ichthyobodo necator. - Mailto Olya (2008) mentioned that Costia is an invasive disease that affect aquarium and free-living fish. It is caused by the flagellate Ichthyobodo necatrix. At the anterior end of the protozoan has two flagella by means of which it swims. 2-2 Endoparasitic protozoa: Enteric parasitic protozoa: Cryptosporidium: - Barbara et al., (1990) listed that Cryptosporidium is an important intestinal pathogen causing economic and pathological problems in immuno compressed fish. - Nisreen et al (1998) collected (213) fish (Mugil cephalus and Tilapia zillii) from miditerranean sea (Port Said), Suez 22

36 Review of Literature Canal (Ismailia) and Fayoum (Lake Quarun) during the period from June to November Cryptosporidium nasorum was recorded from (27.23%) of them. - Alvarez et al., (2004) examined the turbot Scophthalmus maximus for description of Cryptosporidium scophthalmin species. Samples were collected from different farms on the coast of Spain. The parasite was found mainly in the intestinal epithelium and very seldom in the stomach. Oocysts were almost spherical with (4) naked sporozoites. Sporulation was endogenous, as fully sporulated oocysts were found within the intestinal epithelium, lumen and faces. Infection prevalence was very variable and juvenile fish were most frequently observed and intensively parasitized. - Thaddeus et al (2007) performed study to determine mechanical passage of Cryptosporidium parvum Oocysts via handling of fish caught. - Mloslav et al (2008) detected Cryptosporidium fragile sp. n. (Apicomplexa) from Black-spined toads, from Malay. - Brian et al., (2009) examined a freshwater Angel fish hatchery where variable levels of emaciation, poor growth rates, anorexia, restlessness and increased mortality within their fish. Multiple chemotherapy trials had been attempted without success. The youngest fish was the most severly affected and demonstrated the greatest morbidity and 23

37 Review of Literature mortality in infected fish large numbers of protozoa were identified both histologicallly and ultrastructurally associated with the gastric mucosa. The protozoa were morphologically most consistent with Cryptosporidium. - Rona et al., (2011) mentioned that cryptosporidiosis is protozoal disease caused by protozoan parasites of the genus Cryptosporidium (Apicomplexa). Cryptosporidiosis is an emerging disease in both wild and farmed fish in numerous countries world wide Blantidium species: - Grim JN (2006) Collected Balantidium jocularum from intestine of its fish host from lizard island, Australia. Used electron microscope to study the contents of food vacuoles. - Zhokhov and Molodozhnikova (2006) Recorded number of protozoa species parasitizing fishes in the Volga basin. One of them was Balantidium ctenopharyngodoni. - Lim et al. (2007) Redescriped the Balantidium ctenopharyngodoni which collected from the hindgut of Grass carp in China from Honghu lake using light and electron microscope. - Mingli et al., (2009) examined Xenocypris davidi fish from Niushan lake fishery in China and make redescription of Balantidium polyvacuolum which detected in hindgut of this fish species. 24

38 Review of Literature Eimeria: - Molnar and Oqawa (2000) Collected thirty-two specimens of 11 freshwater fish species from lake Biwa, Japan and surveyed coccidian infections. Four fish species proved to be infected with Apicomplexans belonging to the genus Goussia. - Molnar et al (2003) Surveyed (14) freshwater fish species from Lake Kenyir, Malaysia for coccidian infections. Six fish species proved to be infected with Apicomplexans belonging to the genus Goussia. - Molnar K. et al (2005) performed a study to confirm the host specificity of Goussia spp. By oral infection by Oocyst which introduced to many species including Cyprinid fish. - Iva Dykova and Lom (2006) Stated that Eimeria infecting fishes is different compared with typical Eimeria species from higher vertebrates in endogenous sporulation and thin wall of Oocysts. - El-Mansy A. (2008) Described Goussina molnarica n. sp. From the gut of the African catfish Clarias gariepinus. Elliptical sporulated Oocysts were found in the feces and in the mucous covering the epithelium in the examined catfish specimens. Infection prevalence was highest in April and Lowest in November. 25

39 Review of Literature Blood parasitic protozoa: Trypanosoma: - Negm El-Din (1998) Successfully performed experiment on transmission of Trypanosoma mukasi from the blood of Clarias gariepinus to eight freshwater fish species using Batracobdelloides tricarinata as vector, such cross transmission showed that the Trypanosoma was not host specific. - Negm El-Din (1999) Stated that Trypanosoma mukasi was concurrently transmitted from Oreochromis niloticus to Clarias gariepinus using the leech vector Batracobdelloides tricarinata. Transmission was more successful in immature Clarias gariepinus. - Overath et al (1999) Stated that the haemoflagellates of the genus Trypanosoma are prevalent in freshwater fishes and are transmitted by leeches as vectors. - Gibson et al. (2005) Collected (22) cloned Trypanosoma isolates from (14) species of European freshwater fish and (1) species of African freshwater fish and examined by molecular phylogenetic analysis. 26

40 Review of Literature Haemogregarina: - Negm El-Din M. M. (1999) recorded morphological changes of Haemogregarina nili in fishes using the leech vector for the first time. - Davies A. J. and Johnston (2000) mentioned that fishes are hosts for a variety of intraerythrocytic parasites. - Davies A. J. et al (2004) reviewed past and current researches on the Haemogregarina bigemina, recorded from (96) species of fishes at Mesnil. The parasite undergoes intraerythrocytic binary fission finally forming mature paired gamonts. - Adam et al (2009) examined groups of freshwater fishes including Oreochromis niloticus and Clarias gariepienus. Trypanosoma and Haemogregarina spp. were diagnosed in the blood of Clarias gariepienus. - Polly M. et al (2011) demonstrated the morphology of the Haemogregarina stages, their spatial layout. In its invertebrate vector (Leech). 27

41 Review of Literature (3) Impact of water quality on parasitic protozoa infection in fish: - Eissa I. A. (2002) Mentioned that external protozoan parasites, especially Trichodina are commensal in health fish, stress factor as aquatic environment pollution predispose for more progration of it on its host and convert such commensal into parasites. - Osman et al., (2009) Stated that pollution due to exposure to benzo-a-pyrene adversely affects the physiological and immunological status of catfish leading to increased susceptibility to infection with Trichodina species. In conclusion, infection with Trichodina in fish had the highest drastic effect on the health of fish. - Anssi et al. (2010) Mentioned that higher temperature may boost the rate of disease spread through positive effects on parasite fitness in a weakened host. This pattern was observed in several protozoal infections of fish as Chillodonella infections. - Papadimitriou et al. (2010) monitoring the protozoan's abundance and diversity in fish. They used protozoan as indicator of wastewater treatment efficiency. 28

42 Material and Methods

43 Material and Methods III. Material and Methods III-1 Material: III-1-1- Fish: - A total number of 435 alive fish samples including 225 Oreochromous niloticus with Average weight gm and 210 Claries gariepinus species with average weight gm fishes, were collected from natural Nile resources in Giza governorate. - Fishes were transported alive to the laboratory in plastic containers provided with oxygen supply. - Some fish samples were immediately examined and others were kept into glass aquaria sized Cm. containing dechlorinated tap water and fed on pilletted ration till examination according to (Stoskopf, 1993). III-1-2- Chemicals: A- Methyl alcohol: - Used for fixation of smears before staining with Giemsa stain. B- Canada balsam: - Used for fixation of cover slip to facilitate examination of protozoa. 29

44 Material and Methods c- Cider oil: - Used for examination of protozoa under oil immersion lens. D- Formalin soln. 10%: - Used for preservation of fish tissues till send to histopathology examination. E- Stains: (1) Giemsa stain: - Used for staining of some smears after fixation with methyl alcohol according to (Mary, 1982) (2) Modified Zeihl-Neelsen technique: according to (Henriken and Pohlenz, 1981) used for detection of cryptosporidium infection in the examined fish samples. (3) Phosphotungestic-acid Haematoxyline stain for staining of intestinal scraping smear after fixation with Schaudin's solution. (4) H & E (Haematoxyline and Eosin) used for histopathological examination according to (Banchroft et al., 1996). III-1-3 Water sample: Were collected from Maryotia Channel at the same time of fish collection the water sample were collected in clean full glass bottles of one liter capacity to be used in the analysis of 30

45 Material and Methods different water physical and chemical parameters as well as the checking of the heavy metals pollution. III-1-4- Atomic absorption spectrophotometer: For both qualitative and quantitative analysis of water sample as well as fish tissues for heavy metals pollution, atomic absorption spectrophotometer (Buck Model 210, VGP) was used. The different heavy metals absorbance were recorded directly from the digital scale and the quantitative titrations was calculated from the following equation: C1 = (A1/ A2) c (D/W) Where: C1 = concentration of heavy metals (μg/ ml). A1 = absorbance reading of sample solution. A2 = absorbance reading of standard solution C = concentration of heavy metals (μg/ ml) of the standard solution. D = dilution of the fish samples W = weight of muscle in each sample. 31

46 Material and Methods III-2 Methods: III-2-1 Clinical examination: - Live fish samples were put under observation in aquaria for investigation of any abnormal behaviour pattern. according to (Hoffman, 1970). III-2-2- Parasitological examination: III Macroscopical examination (external examination): - The fish under investigation were grossly examined for detection of any external lesions for visible cysts through examination of body surface, fins and gills. - fish have been sacrified (Killed) by severing the spinal cord by inserting of needle or scalpel just behind to head, the operculum was removed to expose gill arches then transfer gills to petridish containing distilled H 2 O. - Evasceration of fish for examination of internal organs (liver, spleen, kidney, stomach, intestine) which transported to petridishs. - Samples preferably from the gross lesions were taken from skin, gills and other internal organs preserved in formalin soln 10% for histopathological examination according to (Lucky, 1977). 32

47 Material and Methods III Microscopical examination: Smear preparation, Staining and Examination: - Blood films were prepared from caudal vessels of a live fish, spread on a dry clean slide, air dried, fixed with methanol and stained with Giemsa according to (Mary, 1982). - Smears were also taken from the body surface, fins and gills. - Some smears were fixed with methanol and stained with Giemsa stain. Staining Method: 1- Air dry smears at room temperature. 2- Fix in absolute methanol (1 minute), shake off liquid, but keep wet. 3- Place smears in stain for 10 minutes or place some stain on the slide. 4- Gently rinse in small stream of tap water. 5- Air-dry and observe (add a thin layer of immersion oil). 6- May add cover glass for long-term storage. 33

48 Material and Methods - for detection of Cryptosporidium Oocyst in the examined fish samples, modified Zeihl-Neelsen technique is used according to (Henriken and Pohlenz, 1981). 1- Wet preparation of mucosal scraping as well as faecal samples were taken from ileum, jujenum and colon of tilapia fish. 2- Samples were spread on glass slide to prepare a thin transparent layer and left to dry at room temperature. 3- Then fixed with methanol 10% and stained with modified Ziehl- Neelsen technique for 10 minutes (Henrikson and Pohlenz, 1981). 4- The fixed slides were immersed in concentrated carbol fuchsin (1.0 gm. Fuchsin, 10 ml. ethanol and 90 ml. of 5% phenol) for 5 minutes. The slide were rinsed with tap water for 2 minutes and decolourized by 10% sulphoric acid for 30 second then rinsed in tap water for 2 minutes. 5- Application of counter-staining of 5% malachite green (5.0 gm. Of malachite green ml. of 10% ethanol) for one minute, then the slides were rinsed with tap water and left for air dried. - Squash preparations from liver, spleen and kidney as well as intestinal scrapings were made (preferably from gross lesions) on cover slips, fixed in Schaudin's solution and stained with Phosphotungestic-acid Haematoxylin stain (Drury and Wallington, 1980). 34

49 Material and Methods - Tilapia spp., specimens which showed eye opacity were examined where the eye was extirpated and dissected to reveal the nature of the opacity. - Fresh as well as stained smears were examined microscopically under x40 and x100 objectives using Cidar oil. Parasites were illustrated with microphotographs measured by the micrometer lens and were identified according to available literatures. Statistical tables and histograms were also made for clarifying the obtained data. III-2-3- Histopathological examination: - Autopsy samples were taken from the liver, gills, stomach, intestine, skin, eye ball and tail of fish in different groups and fixed in 10% formol saline for twenty four hour. Washing was done in tap water then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 degree in hot air oven for twenty four hours. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns by slidge microtome. The obtained tissue sections were collected on glass slides, deparaffinized and stained by hematoxylin and eosin stain then examined through the electric light microscope (Banchroft et al., 1996). 35

50 Results

51 Results IV. Results IV. 1. Clinical and post-mortem examination of Oreochromis niloticus and Clarias gariepinus infected with parasitic protozoa: a live fishes were clinically examined according to (Hoffman, 1970). IV In cases of Oreochromis niloticus and Clarias gariepinus infected with ecto-parasitic protozoa. 1-Ichthyophthirius multifiliis: Grey-white spots that give the fish's skin and fins the appearance of being sprinkled with salt. These granular white spots have "a bumpy" feel to the touch infection of the gills occurs before the skin and fins. In earlier stages, the fish may swim horizontally and rapidly rub or flash against solid objects in attempt to free themselves of the parasite. Fish also may appear sluggish and lie on the bottom of the pond or tank. In advanced cases bloody tins are common, with a thick mucous layer covering the body. 2- Trichodina truttae: The parasites don't cause distinctive lesions on the fish but do block the flow of oxygen when heavily loaded on the gills. As with most protozoa, environmental degeneration and 36

52 Results crowded conditions cause them to become more damaging, the trichodina attaches to hard, calcified surface such as scales and fin rays causing sever lesions and increasing mucous formation especially in stressed fishes. Also sluggish movements loss of appetike, black colouration, necrosis and ulcer on different parts of the body can be noticed in heavily infected fishes. 3- Chilodonella hexasticha: As most of protozoa stress conditions and environmental degeneration and crowded conditions cause them to become more damaging. In heavily infected fish increase mucous formation which accumulated on body surface and gill pouch. Also sluggish movement, loss of appetite, black colouration and necrosis, ulcer can be noticed on different parts of the body. 4- Myxobolus dermatobia: In wild Oreochromis niloticus fish, Myxobolus spp. Severly affected fish eye showing the presence of white cysts which were either unilateral or bilateral in the conea of affected fish. The size of cyst ranged from 1-3 mm with variable shape, being oval, rounded or irregular in shape. The cysts were very prominent and found usually at the prophecy of the eye. Number of cysts usually ranged from 1-8 cysts/eye. The shape of eye may cause fishes to be unmarketable. 37

53 Results 5- Henneguya branchialis: Cause respiratory form of Henneguyosis in naturally infected Clarias gariepinus. The clinical signs and postmortem examination revealed respiratory manifestation, sluggish swimming, loss of appetite, congestion on gills and presence of cyst like structure on the gill filaments. 6- Ichthyobodo necator: The observed clinical signs were thin grey film of dirty mucous on the body with detachment of scales. The fish rubbed their body against the wall of aquarium. IV In cases of Oreochromis niloticus and Clarias gariepinus infected with endo protozoan. A- Enteric protozoa: 1-Cryptosporidium spp.: Fish infected with Cryptosporidium showing variable levels of emaciation, anorexia, restlessness and increased mortality within fish. The youngest Oreochromis niloticus were the most severely affected and demonstrated the greatest morbidity and mortality in infected fish. The feces of severely infected young fish become watery with variable levels according to infection level with Abdominal distension was noticed. 38

54 Results 2- Blantidium spp.: The young Oreochromis niloticus fish was the most severely affected showing anorexia, emaciation due to histopathological lesion in fish intestine. 3- Eimeria spp.: The most affected fishes were the youngest Oreochromis niloticus which showed variable levels of emaciation and poor growth. The economic damage done by Coccidiosis has apparently been grossly underestimated because Coccidiosis in fish usually manifested itself as achronic infection. Mortality is gradual. Intestinal damage caused by rupture of epithelium by the escaping oocysts was noticed. B- Blood parasitic protozoa: 1- Trypanosoma mukasi: It is intercellular blood parasite. In case of heavy infection, fish showed emaciation. It is found in blood of Clarias gariepinus. 2- Haemogregarina clariae: It is intracellular blood parasite. In case of heavy infection, lead to emaciation. It is found in blood of Clarias gariepinus. 39

55 Results Figure (1): alive Oreochromis niloticus post transportation to the laboratory. Showed healthy fish move along the columen of water while the affected fish accumulated at the bottom. 40

56 Results Figure (2): Oreochromis niloticus infected with Trichodina showed: - Dark colouration of the body. - Detached scales. 41

57 Results Figure (3): Oreochromis niloticus infected with Chilodonella showed: - Haemorrhage and eroision of the caudal fin. - Dark colouration of the body. - Haemorrhagic area near the anal fin. 42

58 Results Figure (4): Oreochromis niloticus infected with Trichodina showed: - Dark colouration of the body. - Detached scales. - Haemorrhage at the root of dorsal and anal fin in the upper fish. 43

59 Results Figure (5): Clarias gariepinus infected with Trichodina show ulceration at tail. 44

60 Results Figure (6): Oreochromis niloticus infected with Myxobolus dermatobia. Show whitish to yellowish cysts in eye. 45

61 Results Figure (7): Gills of Clarias gariepinus show whitish cysts of Henneguya branchialis. 46

62 Results IV.2. Morphological identification of isolated parasites from the examined fishes: 1- Ichthyophthirius multifillis: Description: Ichthyophthirius multifillis was isolated from skin and gills of Oreochromis niloticus, it is oval or round holotrichus ciliat measures um, contain large horse shoe-shaped macronucleus and small micronucleus. The cytoplasm appears grossly granular containing many small vacules. 2- Trichodina truttae (Mueller, 1937): Description: - Trichodina truttae is reported from skin and gills of Oreochromis niloticus and Clarias gariepinus. It has a body of diameter measuring um. - The adhesive disc is saucer-shaped. - The denticular ring is um and carry (26 29) denticles. The ray of the denticle is thin, slightly curved posteriorly. The macronucleus is horse-shoe shaped while micronucleus is difficult to be detected. 47

63 Results 3- Chilodonella hexasticha (Kiernik, 1909): Description: The parasite is recorded from the skin of Oreochromis niloticus. The body is oval to bear-shaped and flattened dorsoventrally, measuring um. The cytopolasm is coarsely granular containing oval to round-shaped macronucleus which constitutes about one third of the body and round micronucleus with variable size. Four to six ciliary bands are seen at the two sides of the ventral surface. The cytostome is cornucopia-like measuring 11.8 um in length. 4- Myxobolus dermatobia: Description: White and yellowish round cysts were observed in the eyes of Oreochromis niloticus. Uni-or bilaterally situated in the episclera causing slight exophthalmia and their size ranged between 1-2 mm in diameter. The detected cysts contain a great number of spores. The shape of the spores is being elongated, elliptical in shape. The spores measure um long and um wide. Polar capsule are pryriform in shape and of nearly equal sizes measuring um long and um wide. They constitute less than half of the total spore length. Round iodinophilus vacuole is found in the sporoplasm. 48

64 Results 5- Henneguya branchialis (Abu El-Wafa, 1988): Description: Macroscopic cysts were found firmly attached to the gill filaments especially at the base and suprabranchial organ of Clarias gariepinus. They are ovoid to round in shape, opaque white in colour, somewhat of firm consistency and with different sizes ( ) mm. when ruptured, the cysts ooze viscid whitish contents including a great number of mature Henneguya spores. The spore has elongated fusiform shaped body measuring um and provided anteriorly with two elongated, banana-shaped equal polar capsules. The sporoplasm contains oval to round-shaped iodinophilus vacuole measuring um. The posterior end of the spore is prolonged into two extended caudal processes of o.9 um length. 6- Ichthyobodo necator: Description: The parasite obtained from the body surface and fins of Oreochromis niloticus. The parasite found in two forms, one of them has two flagella and the other form without flagella. Ichthyobodo necator is oval or pyriform in shape and contain a single nucleus which located at one side, basophilic granules were seen in the cytoplasm. 49

65 Results Endo protozoan: A- Enteric protozoan: 1- Cryptosporidium sp. Description: Subspherical oocysts um um, with distinct colour. In each oocyst, there are elongated, vermiform and slightly curved sporozoites with nucleus at their wider ends. 2- Blantidium sp.(trophozoit and cyst) Description: A. Trophozoite: Ovoid in shape measuring um um in size. The body surface is covered with cilia. The cytostom is subterminal at the smaller end and the cytopyge is near the posterior end. The macronucleus is kidney shape and the micronucleus is in the notch of the macronucleus. There are many contractile and food vacule. B. Cyst: spherical in shape with size smaller than Trophozoit. The cyst wall consists of double membrane. It contains macronucleus in dense cytoplasme. 50

66 Results 3- Eimeria (Kalman Molnari 1973): Description: Oocysts is spherical, diameter um. Cyst wall measures 0.1 um thickness, single layered, colorless, and of uniform thichness. Sporocysts moderately compact with a relatively narrow space separating them from the cyst wall. B- Blood protozoan: 1- Trypanosoma mukasi: Description: This trypanosoma was detected in the blood of Clarias gariepinus as monomorphic trypanosome. It was diagnosed in fresh films by it s active motility among blood corpuscles. The body was long and narrow and usually curved. Both ends were tapering with a more slender anterior end. It measures (42 53) u in length and ( ) u in width at the nuclear level. The cytoplasm was lightly and finely granular and stained light blue. It contained a variable number of vacuoles. The nucleus was oval and usually located slightly in front of the middle or centrally. The free flagellum measured 15.9 ( ) u. the undulating membrane was thrown into 7 9 folds. 51

67 Results 2- Haemogregorina Clariae: Description: This parasite was scarcely noticeable in the erythrocytes of the fish Clarias gariepinus as three forms. Merozoites: Merozoites were elongated oval or slightly curved short broad bodies. One side was usually more convex than the other. It measured ( ) u in length and (4 6) u in breadth. The cytoplasm stained light blue and may contain one or two large vacuoles (one at each pole) and a variable number of deeply red staining granules scattered or arranged in a linear manner near the periphery of the body. The nucleus is a deeply red nearly compact chromatin mass. Gametocytes: The Gametocytes were longer and narrower bodies with one end more pointed than the other blunt end and partially encircling the host cell nucleus. It measured ( ) u in length and ( ) u in width. The cytoplasm stained light blue and also contained the scattered red granules. An indistinct 52

68 Results vacuole is rarely found near the rounded end. The nucleus appears to consist of a variable number of an irregular coarse deeply red chromatin masses and extended to the whole width of the body filling about 1/3 to 1/2 body length. Schizont: It measures in length & micron in breadth, it is ovoid in shape containing a single vacule and irregulary faint red chromatin masses, the cytoplasm is differentiated. 53

69 Results Figure (8): Unstained wet preparation smear of Trichodina truttae (x 40). 54

70 Results Figure (9): Lateral view of wet preparation smear of Trichodina truttae stained with giemsa stain (x 40). 55

71 Results Figure (10): Fixed smear of Trichodina truttae stained with giemsa stain (X 100). 56

72 Results. Figure (11): Chillodonella hexasticha stained with giemsa stain (X 100). 57

73 Results Figure (12): Henneguya branchialis stained with giemsa stain (X 100). 58

74 Results Figure (13): Sporulated oocyst of cryptosporidium sp. Stained with Modified Zeilnelson stain (X 100). 59

75 Results Figure (14): Sporulated oocyst of cryptosporidium sp. Stained with Modified Zeilnelson stain (X 100). 60

76 Results B A Figure (15): Balantidium sp. Trophozoite (A) and cyst (B). stained with Phosphotungestic-acid Haematoxylin stain (Schaudin's stain) (X 100). 61

77 Results Figure (16): Oocyst of Eimeria unstained (X 100). 62

78 Results Figure (17): Flagellated form of Ichthyobodo necator stained with giemsa stain (X 100). 63

79 Results Figure (18): Trypanosoma mukasi stained with giemsa stain (X 100). 64

80 Results A A B Figure (19): Blood film stained with giemsa stain (X 100). Gametocyte of Haemogregarina clariae (A) Merozoite of Haemogregarina clariae (B) 65

81 Results C Figure (20): Blood film stained with giemsa stain (X 100). Schizont of Haemogregarina clariae (C). 66

82 Results IV. 3. The histopathological finding in infected fishes: IV The histopathological examination of Oreochramis niloticus infected with external protozoa: Showed skin necrosis and focal inflammatory necrosis in the auter surface, sloughing of epithelial layer and subepidermal haemorrhage. In case of gills infection showing necrosis in the rakers, inflammatory cells infiltration, sever congestion and diffuse haemorrhage in the arch. In case of eye infection, showing haemorrahage and parasitic cyst in the champer in front of choroids and retina. IV The histopathological examination of Oreochranis niloticus infected with intestinal parasitic protozoa: Showed degeneration in mucosal epithelium with inflammatory cells infiltration in lamina propria of the villi, congestion in the blood vessels. Iv The histopathological examination of Clarias gariepinus infected with external protozoa: - Skin showed necrosis in epithelial layer with inflammatory cells inflaltration. - Gill examination show parasitic cyst. 67

83 Results Figure (21): Gill of Oreochromis niloticus infected with Ichthyophthirius mulltifiilus showing necrosis in the rakers (arrow) (H & Ex 40). 68

84 Results Figure (22): Skin of Oreochromis niloticus infected with Trichodina truttae showing focal inflammatory necrosis in the outer surface (s) (H & E X 40). 69

85 Results Figure (23): Gill of Clarias gariepinus infected with Trichodina truttae showing hyperplasia and stratification in the rakers (s) with sever congestion in the blood vessels (v) (H & Ex 40). 70

86 Results Figure (24): Gills of Clarias gariepinus infected with Trichodina truttae showing sever congestion (v) and epithelial stratification in the rakers (s) (H & Ex 40). 71

87 Results Figure (25): Gills of Oreochromis niloticus infected with Trichodina truttae showing sever congestion (v) and diffuse haemohages in the arch (h) (H & Ex40). 72

88 Results Figure (26): Gill of Oreochromis niloticus infected with Chillodonella hexasticha showing Sever congestion in the blood vessels of the filaments and lamellae (v) (H & Ex 40). 73

89 Results Figure (27): Gills of Oreochromis niloticus infected with chillodonella hexasticha show vacuolization, necrosis, inflammatory cells infiltration (m) and sever congestion (v) of the blood vessels in the rakers and filaments (H & Ex 40). 74

90 Results Figure (28): Gill of oreochromis niloticus infected with Chillodonella hexasticha showing inflammatory cells infiltration (m) in the arch (H & Ex 40). 75

91 Results Figure (29): Gill of the Clarias gariepinus infected with Henneguya branchialis show parasitic cyst (arrow) in gill lamellaa (H & Ex 64). 76

92 Results Figure (30): Gill of Clarias gariepinus infected with Henneguya branchialis showing parasitic cyst (arrow) in the gill filament (H & Ex 80). 77

93 Results Figure (31): Eye of Oreochromis niloticus infected with Myxobolus dermatobia showing the magnification of parasitic cyst (H & Ex 40). 78

94 Results Figure (32): Eye of Oreochromis niloticus infected with Myxobolus dermatobia showing haemorrhage (h) and parasitic cyst (arrow) in the chamber in front of choroid and retina (H & EX 40). 79

95 Results Figure (33): Intestine of Oreochromis niloticus infected with Cryptosporidium sp. showing Degenerative change was observed in the lining mucosal epithelial cells (d) with inflammatory cells infiltration in the lamina propria (m) (H & Ex80). 80

96 Results Figure (34): Intestine of Oreochromis niloticus infected with Balantidium sp. showing inflammatory cells infiltration (m) in the lamina propria of villi (H & Ex 40). 81

97 Results [ Figure (35): Intestine of Oreochromis niloticus infected with Balantitium SP. showing congestion in blood vessels (v) with inflammatory cells infiltration (m) in lamina propria (H & Ex 40). 82

98 Results Figure (36): Intestine of Oreochromis niloticus infected with Eimeria sp. showing degeneration in mucosal epithelium (d) with inflammatory cells infiltration (m) in lamina propria of the villi (H & Ex 40). 83

99 Results IV. 4. Prevalence of parasitic protozoa in Oreochromis niloticus and Clarias gariepinus: - Out of the investigated (435) freshwater fishes (225 Oreochromis niloticus and 210 Clarias gariepinus) (253) fishes were found infected with protozoan parasites with an infection rate reaches (58.16%). - The Oreochromis nilooticus showed high infection rate with parasitic protozoa (66.2%) in Clarias gariepinus was (49.52) as shown in Table (1) & Figure (37). 84

100 Results Fish species Exam. Infect. % Oreochromis niloticus Clarias gariepinus Total Table (1): prevalence of protozoan parasites among examined fish. Exam. : Infect. : number of examined fishes. number of infected fishes. % : percentage of infection. 85

101 Results Oreochromis niloticus Clarias gariepinus Total examined fishes Exam. Infect. % Figure (37): prevalence of protozoan parasites among examined fish. Exam. : Infect. : number of examined fishes. number of infected fishes. % : percentage of infection. 86

102 Results - Regarding the incidence of parasitic protozoa in the examined fish species, it was found that, among Oreochromis niloticus, Balantidium species recorded the highest rate (68%) followed by Cryptosporidium species (62.2%). On the other hand, Trichodina species recorded the highest rate of infection (47.6%) among the examined Clarias species. It was noticed that Ichthyophthirius multifiliis showed the lowest rate (3.5% and 1.9%) among Oreochromis niloticus and Clarias gariepinus respectively. - During investigation, mixed infection with Trichodina species; Chilodonella species, Cryptosporidium species and Balantidium species in the examined Oreochromis niloticus was noticed. Also Clarias gariepinus showed mixed infection with Trichodina species, Chilodonella species, Trypanosoma species and Haemogregarina species. (Table 2 and Figure 38). 87

103 Results Oreochromis niloticus Clarias gariepinus Protozoan species No. of exam. No. of infect. % No. of exam. No. of infect. % Ectoparasitic protozoa: Ichthyophthirius multifillis Trichodina truttae Chilodonella hexasticha Myxobolus dermatobia Henneguya branchialis Ichthyobodo necator Enteric parasitic protozoa: Cryptosporidium sp Blantidium sp Eimeria sp Blood parasitic protozoa: Trypanosoma mukasi Haemogregarina clariae Table (2): The detected parasitic protozoa and their incidence in both Oreochromis niloticus and Clarias gariepinus. 88

104 Results Oreochromis niloticus Clarias gariepinus Ichthyophthirius multifillis Figure (38): The detected parasitic protozoa and their incidence in both Oreochromis niloticus and Clarias gariepinus. Cryptosporidium sp. Ichth. Trich. Chilo. Myxob. Henne. Ichthyo. Crypt. Blant. Eimeria Trypano. Haemogreg. 89

105 Results I.V. 5. Seasonal dynamics of the detected parasitic protozoa among examined fishes: - The peak of infection with protozoan parasites was recorded in summer (77.9%) followed by spring (70%) and autumn (52.94%) while the lowest rate was in winter (32.17%). (Table 3). 90

106 Results Season Exam. Infect. % Winter Spring Summer Autumn Total Table (3): Seasonal incidence of parasitic protozoan infection among examined fishes. 91

107 Results - Concerning the prevalence and seasonal dynamics of each parasite under investigation it was found that Trichodina species recorded the highest infection rate among the examined fishes (50.57%) with the peak of infection during spring season (69%). While the lowest rate was recorded by Ichthyobode necator (2.3%) with the peak of infection during winter season (7.8%). (Table 4) 92

108 Results Season No. of examined fishes No. of infected fishes % of infection No. infected with Ichyo. Multifiliis (%) No. infected with Tricho. (%) No. infected with Chilo. (%) No. infected with Myxo. (%) No. infected with Henne. (%) No. infected with Ichthyobodo necator (%) Winter (6.09) 34 (29.6) 20 (17.39) - (0) - (0) 9 (7.8) Spring (1) 69 (69) 54 (54) 2 (2) - (0) 1 (1) Summer (0) 67 (56.3) 2 (1.7) 39 (33) 9 (7.63) - (0) Autumn (3.9) 50 (49) 5 (4.9) 9 (8.8) 4 (3.9) - (0) Total (2.76) 210 (50.57) 81 (19.49) 50 (11.49) 13 (2.9) 10 (2.3) Table (4): Prevalence and seasonal dynamics of the detected Ecto parasitic protozoa. 93

109 Ichthyophthirius Trichodinna Chilodonella Myxobolus Henneguya Costia Results Figure (39): Prevalence and seasonal dynamics of the detected Ecto parasitic protozoa. 94

110 Results No. No. No. Season No. of examined fishes No. of infected fishes % of infection infected with Crypto. infected with Blant. infected with Eimeria (%) (%) (%) Winter (27) 5 (4.3) 23 (20) Spring (54) 64 (64) 7 (7) Summer (19.49) 80 (67.8) - (0) Autumn (31.4) 4 (3.9) - (0) Total (32.18) 153 (35.2) 30 (6.9) Table (5): Prevalence and seasonal dynamics of the detected Enteric parasitic protozoa. 95

111 Cryptosporidium Blantidium Eimeria Results Figure (40): Prevalence and seasonal dynamics of the detected enteric parasitic protozoa. 96

112 Results Season No. of examined fishes No. of infected fishes % of infection No. infected with Trypanosoma (%) No. infected with Haemogregarina (%) Winter (29.6) - (0) Spring (21) 2 (2) Summer (0.8) 29 (24.6) Autumn (29.4) - (0) Total (19.77) 31 (7.13) Table (6): Prevalence and seasonal dynamics of the detected Blood parasitic protozoa. 97

113 Trypanosoma Haemogregarina Results Figure (41): Prevalence and seasonal dynamics of the detected Blood parasitic protozoa. 98