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
To My father, My mother, My husband and my children
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.
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
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. 40 41 42 43 44 45 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
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). 56 57 58 59 60 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). 63 64 65
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. 66 68 69 70 71 72 73 74 75 76
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. 77 78 79 80 81 82 83 86 89
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. 94 96 98
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 85 88 91 93 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. 95 97 99
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
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
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
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
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: 2-1-1- 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
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
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
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
Review of Literature 2-1-2- 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
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