STUDIES ON SOME PROBLEMS OF PROTOZOALscholar.cu.edu.eg/?q=nehal-younis/files/rsl_d._nhl_lnhyy...STUDIES ON SOME PROBLEMS OF PROTOZOAL INFECTION IN FRESHWATER FISHES Thesis presented

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. Prof. Dr.

2012

Nisreen Ezz El-dien

Mahmoud

Mai El-Desoky

Al-Said Ibrahim

prof. of parasitology


Cairo university

prof. of fish

diseases and management


Cairo University

To…

My father,

My mother,

My husband

and my children

Acknowledgment

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 1-4

List of Figures

Figure (1) alive Oreochromis niloticus post

transportation to the laboratory.

40

Figure (2) Oreochromis niloticus infected with

Trichodina.

41


Chilodonella.

42


Trichodina.

43

Figure (5) Clarias gariepinus infected with

Trichodina.

44


Myxobolus dermatobia.

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).

56

Figure (11) Chillodonella hexasticha stained with

giemsa stain (X 100).

57

Figure (12) Henneguya branchialis stained with giemsa

stain (X 100).

58

Figure (13) Sporulated oocyst of cryptosporidium sp.

Stained with Modified Zeilnelson stain (X

100).

59

Figure (14) Sporulated oocyst of cryptosporidium sp.

Stained with Modified Zeilnelson stain (X

100).

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).

63

Figure (18) Trypanosoma mukasi stained with giemsa

stain (X 100).

64

Figure (19) Blood film stained with giemsa stain (X

100).

65

Figure (20) Blood film stained with giemsa stain (X

100).

66

Figure (21) Gill of Oreochromis niloticus infected with

Ichthyophthirius mulltifiilus.

68

Figure (22) Skin of Oreochromis niloticus infected with

Trichodina truttae.

69

Figure (23) Gill of Clarias gariepinus infected with

Trichodina truttae.

70

Figure (24) Gills of Clarias gariepinus infected with

Trichodina truttae.

71

Figure (25) Gills of Oreochromis niloticus infected with

Trichodina truttae.

72

Figure (26) Gill of Oreochromis niloticus infected with

Chillodonella hexasticha.

73

Figure (27) Gills of Oreochromis niloticus infected with

chillodonella hexasticha.

74

Figure (28) Gill of oreochromis niloticus infected with

Chillodonella hexasticha.

75

Figure (29) Gill of the Clarias gariepinus infected with

Henneguya branchialis.

76

Figure (30) Gill of Clarias gariepinus infected with


77

Figure (31) Eye of Oreochromis niloticus infected with


78

Figure (32) Eye of Oreochromis niloticus infected with


79

Figure (33) Intestine of Oreochromis niloticus infected

with Cryptosporidium sp.

80


with Balantidium sp.

81


with Balantitium sp.

82


with Eimeria sp.

83

Figure (37) prevalence of protozoan parasites among

examined fish.

86

Figure (38) The detected parasitic protozoa and their

incidence in both Oreochromis niloticus and

Clarias gariepinus.

89

Figure (39) Prevalence and seasonal dynamics of the

detected Ecto parasitic protozoa.

94


detected enteric parasitic protozoa.

96


detected Blood parasitic protozoa.

98

List of Tables

Table (1) prevalence of protozoan parasites among

examined fish.

85

Table (2) The detected parasitic protozoa and their


Clarias gariepinus.

88

Table (3) Seasonal incidence of parasitic protozoan

infection among examined fishes.

91

Table (4) Prevalence and seasonal dynamics of the


93


detected Enteric parasitic protozoa.

95



97

Table (7) result of water sample analysis from

Maryotia Channel.

99

Introduction

Introduction

1

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

Introduction

2

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.

Review of

Literatures

Review of Literature

3

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


4

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


5

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.


6

- 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


7

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.


8

- 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.


9

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.


10

- 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.


11

- 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.


12

- 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.


13

2-1-3- 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%).


14

- 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


15

incidence of Chilodonella among examined Clarias

gariepinus was (13.3%), peak of infection was in winter.

2-1-4- 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 15.7 + 1.5 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


16

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.


17

- 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.


18

- 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.

2-1-5- 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


19

(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


20

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 2008. 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


21

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 2010. 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


22

pathogenic species as the parasite showed high deformation

of the filaments structure and complete disappearance of gill

lamellae.

2-1-6- 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:

2-2-1- Enteric parasitic protozoa:

2-2-1-1- 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


23

Canal (Ismailia) and Fayoum (Lake Quarun) during the

period from June to November 1996. 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


24

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.

2-2-1-2- 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.


25

2-2-1-3- 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.


26

2-2-2 Blood parasitic protozoa:

2-2-2-1- 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.


27

2-2-2-2- 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).


28

(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.

Material and

Methods

Material and Methods

29

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 50 -400 gm

and 210 Claries gariepinus species with average weight

150 - 400 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 70 × 40× 30 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.


30

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


31

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.


32

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-2-2-1- 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 H2O.

- 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).


33

III-2-2-2- 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.


34

- 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 + 100 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).


35

- 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).

Results

Results

36

IV. Results

IV. 1. Clinical and post-mortem examination of Oreochromis

niloticus and Clarias gariepinus infected with parasitic

protozoa:

- 435 a live fishes were clinically examined according to

(Hoffman, 1970).

IV. 1.1. 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

Results

37

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.

Results

38

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. 1.2. 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.

Results

39

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.

Results

40

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.

Results

41

Figure (2): Oreochromis niloticus infected with Trichodina

showed:

- Dark colouration of the body.

- Detached scales.

Results

42

Figure (3): Oreochromis niloticus infected with Chilodonella

showed:

- Haemorrhage and eroision of the caudal fin.


- Haemorrhagic area near the anal fin.

Results

43

Figure (4): Oreochromis niloticus infected with Trichodina

showed:


- Detached scales.

- Haemorrhage at the root of dorsal and anal fin in

the upper fish.

Results

44

Figure (5): Clarias gariepinus infected with Trichodina show

ulceration at tail.

Results

45

Figure (6): Oreochromis niloticus infected with Myxobolus

dermatobia. Show whitish to yellowish cysts in

eye.

Results

46

Figure (7): Gills of Clarias gariepinus show whitish cysts of


Results

47

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 27.8-43.2 × 27.5-44 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 103.3 – 123.5 um.

- The adhesive disc is saucer-shaped.

- The denticular ring is 30.82 – 45.31 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.

Results

48

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 36.3 – 61.2 × 19.8 – 28.8 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 9.2 –

11.4 um long and 5.4 – 7.1 um wide. Polar capsule are

pryriform in shape and of nearly equal sizes measuring 3.7

– 4.2 um long and 2.3 – 3.0 um wide. They constitute less

than half of the total spore length. Round iodinophilus

vacuole is found in the sporoplasm.

Results

49

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 (1.2 – 3 × 2 – 3) 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 12.2 – 16.2 ×

3.7 – 5.9 um and provided anteriorly with two elongated,

banana-shaped equal polar capsules. The sporoplasm

contains oval to round-shaped iodinophilus vacuole

measuring 1.7 – 3.4 × 1.4 – 2.5 um. The posterior end of

the spore is prolonged into two extended caudal processes

of 20.0 – 3o.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.

Results

50

Endo protozoan:

A- Enteric protozoan:

1- Cryptosporidium sp.

Description:

Subspherical oocysts 3.7 – 5.03 um × 3.03 – 4.69 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 21.8-42.04 um

× 20.5- 41.0 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.

Results

51

3- Eimeria (Kalman Molnari 1973):

Description:

Oocysts is spherical, diameter 10.4-11 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

(3.6 – 4.7) 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 (13.8 – 18) u.

the undulating membrane was thrown into 7 – 9 folds.

Results

52

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 (10.8 – 12.2) 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 (12.7 – 17) u in

length and (4.0 – 5.2) u in width. The cytoplasm stained light

blue and also contained the scattered red granules. An indistinct

Results

53

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 9.42-9.57 in length & 5.40-5.70 micron in

breadth, it is ovoid in shape containing a single vacule and

irregulary faint red chromatin masses, the cytoplasm is

differentiated.

Results

54

Figure (8): Unstained wet preparation smear of Trichodina

truttae (x 40).

Results

55

Figure (9): Lateral view of wet preparation smear of

Trichodina truttae stained with giemsa stain

(x 40).

Results

56

Figure (10): Fixed smear of Trichodina truttae stained with

giemsa stain (X 100).

Results

57

.

Figure (11): Chillodonella hexasticha stained with giemsa

stain (X 100).

Results

58

Figure (12): Henneguya branchialis stained with giemsa stain

(X 100).

Results

59

Figure (13): Sporulated oocyst of cryptosporidium sp. Stained

with Modified Zeilnelson stain (X 100).

Results

60

Figure (14): Sporulated oocyst of cryptosporidium sp. Stained

with Modified Zeilnelson stain (X 100).

Results

61

Figure (15): Balantidium sp. Trophozoite (A) and cyst (B).

stained with Phosphotungestic-acid Haematoxylin

stain (Schaudin's stain) (X 100).

B

A

Results

62

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

Results

63

Figure (17): Flagellated form of Ichthyobodo necator stained

with giemsa stain (X 100).

Results

64

Figure (18): Trypanosoma mukasi stained with giemsa stain

(X 100).

Results

65

Figure (19): Blood film stained with giemsa stain (X 100).

Gametocyte of Haemogregarina clariae (A)

Merozoite of Haemogregarina clariae (B)

A A

A

B

Results

66

Figure (20): Blood film stained with giemsa stain (X 100).

Schizont of Haemogregarina clariae (C).

C

Results

67

IV. 3. The histopathological finding in infected fishes:

IV. 3. 1. 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. 3. 2. 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. 3. 3. 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.

Results

68

Figure (21): Gill of Oreochromis niloticus infected with

Ichthyophthirius mulltifiilus showing necrosis in

the rakers (arrow) (H & Ex 40).

Results

69

Figure (22): Skin of Oreochromis niloticus infected with

Trichodina truttae showing focal inflammatory

necrosis in the outer surface (s)

(H & E X 40).

Results

70

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).

Results

71

Figure (24): Gills of Clarias gariepinus infected with

Trichodina truttae showing sever congestion (v)

and epithelial stratification in the rakers (s) (H

& Ex 40).

Results

72

Figure (25): Gills of Oreochromis niloticus infected with

Trichodina truttae showing sever congestion (v)

and diffuse haemohages in the arch (h) (H &

Ex40).

Results

73

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).

Results

74

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).

Results

75

Figure (28): Gill of oreochromis niloticus infected with

Chillodonella hexasticha showing inflammatory

cells infiltration (m) in the arch (H & Ex 40).

Results

76

Figure (29): Gill of the Clarias gariepinus infected with

Henneguya branchialis show parasitic cyst

(arrow) in gill lamellaa (H & Ex 64).

Results

77

Figure (30): Gill of Clarias gariepinus infected with

Henneguya branchialis showing parasitic cyst

(arrow) in the gill filament (H & Ex 80).

Results

78

Figure (31): Eye of Oreochromis niloticus infected with

Myxobolus dermatobia showing the

magnification of parasitic cyst (H & Ex 40).

Results

79

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).

Results

80

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).

Results

81


Balantidium sp. showing inflammatory cells

infiltration (m) in the lamina propria of villi (H

& Ex 40).

Results

82

[


Balantitium SP. showing congestion in blood

vessels (v) with inflammatory cells infiltration

(m) in lamina propria (H & Ex 40).

Results

83


Eimeria sp. showing degeneration in mucosal

epithelium (d) with inflammatory cells

infiltration (m) in lamina propria of the villi (H

& Ex 40).

Results

84

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).

Results

85

Fish species Exam. Infect. %

Oreochromis niloticus 225 149 66.2

Clarias gariepinus 210 104 49.52

Total 435 253 58.16

Table (1): prevalence of protozoan parasites among

examined fish.

Exam. : number of examined fishes.

Infect. : number of infected fishes.

% : percentage of infection.

Results

86

Figure (37): prevalence of protozoan parasites among

examined fish.

Exam. : number of examined fishes.

Infect. : number of infected fishes.

% : percentage of infection.

0

50

100

150

200

250

300

350

400

450

Exam. Infect. %

Oreochromis niloticus Clarias gariepinus Total examined fishes

Results

87

- 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).

Results

88

Protozoan species

Oreochromis niloticus Clarias gariepinus

No. of

exam.

No. of

infect. %

No. of

exam.

No. of

infect. %

Ectoparasitic protozoa:

Ichthyophthirius multifillis 225 8 3.5 210 4 1.9

Trichodina truttae 225 120 53.3 210 100 47.6

Chilodonella hexasticha 225 32 14.2 210 49 23.3

Myxobolus dermatobia 225 50 22.2 210 - -

Henneguya branchialis 225 - - 210 13 6.1

Ichthyobodo necator 225 10 4.4 210 - -

Enteric parasitic protozoa:

Cryptosporidium sp. 225 140 62.2 210 - -

Blantidium sp. 225 153 68 210 - -

Eimeria sp. 225 30 13.3 210 - -

Blood parasitic protozoa:

Trypanosoma mukasi 225 - - 210 86 40.9

Haemogregarina clariae 225 - - 210 31 14.7

Table (2): The detected parasitic protozoa and their


Clarias gariepinus.

Results

89

Figure (38): The detected parasitic protozoa and their incidence in both Oreochromis niloticus and

Clarias gariepinus.

0

10

20

30

40

50

60

70

Ichthyophthirius

multifillis

Cryptosporidium

sp.

Oreochromis niloticus Clarias gariepinus

Ichth. Trich. Chilo. Myxob. Henne. Ichthyo. Crypt. Blant. Eimeria Trypano. Haemogreg.

Results

90

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).

Results

91

Season Exam. Infect. %

Winter 115 37 32.17

Spring 100 70 70

Summer 118 92 77.96

Autumn 102 54 52.94

Total 435 253 58.16

Table (3): Seasonal incidence of parasitic protozoan

infection among examined fishes.

Results

92

- 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)

Results

93

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 115 37 32.17 7 (6.09) 34 (29.6) 20 (17.39) - (0) - (0) 9 (7.8)

Spring 100 70 70 1 (1) 69 (69) 54 (54) 2 (2) - (0) 1 (1)

Summer 118 92 77.9 - (0) 67 (56.3) 2 (1.7) 39 (33) 9 (7.63) - (0)

Autumn 102 54 52.94 4 (3.9) 50 (49) 5 (4.9) 9 (8.8) 4 (3.9) - (0)

Total 435 253 58.16 12 (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.

Results

94

Figure (39): Prevalence and seasonal dynamics of the


Ichth

yophth

iriu

s

Tri

chodin

na

Chil

odonel

la

Myxobolu

s

Hen

neg

uya

Cost

ia

Results

95

Season

No. of

examined

fishes

No. of

infected

fishes

% of

infection

No.

infected

with

Crypto.

(%)

No.

infected

with

Blant.

(%)

No.

infected

with

Eimeria

(%)

Winter 115 37 32.17 31 (27) 5 (4.3) 23 (20)

Spring 100 70 70 54 (54) 64 (64) 7 (7)

Summer 118 92 77.9 23 (19.49) 80 (67.8) - (0)

Autumn 102 54 52.94 32 (31.4) 4 (3.9) - (0)

Total 435 253 58.16 140 (32.18) 153 (35.2) 30 (6.9)

Table (5): Prevalence and seasonal dynamics of the detected

Enteric parasitic protozoa.

Results

96


detected enteric parasitic protozoa.

Cry

pto

spori

diu

m

Bla

nti

diu

m

Eim

eria

Results

97

Season

No. of

examined

fishes

No. of

infected

fishes

% of

infection

No. infected

with

Trypanosoma

(%)

No. infected

with

Haemogregarina

(%)

Winter 115 37 32.17 34 (29.6) - (0)

Spring 100 70 70 21 (21) 2 (2)

Summer 118 92 77.9 1 (0.8) 29 (24.6)

Autumn 102 54 52.94 30 (29.4) - (0)

Total 435 253 58.16 86 (19.77) 31 (7.13)

Table (6): Prevalence and seasonal dynamics of the detected

Blood parasitic protozoa.

Results

98



Try

pan

oso

ma

Hae

mogre

gar

ina

Results

99

IV. 6. Result of water analysis from Maryotia Channel:

mg/ L

NH4 1.49

NO3 0.89

P 0.45

Fe 0.411

Mn 0.118

Zn 0.00

Cu 0.002

Cd 0.00

Pb 0.00

B 0.116

Table (7): result of water sample analysis from Maryotia

Channel.

PH was measured 7.4

It was noticed that increase prevalence rate of parasitic

protozoal infections in fishes collected from polluted water.

Discussion

Discussion

100

V. Discussion

The aim of this study was to investigate the most common

protozoal diseases among wild fishes in certain tributaries in

River Nile in Giza governorate to make a correlation between

some water pollutants & the evidence of some protozoa

infection among tributaries of River Nile in some localities in

Giza governorate.

The prevalence of parasitic protozoa among (435)

examined alive fish samples including (225) Oreochromis

niloticus & (210) Clarias gariepinus brought from certain areas

in Maryotia Channel and River Nile in Giza revealed that the

percentage of infection among the examined fishes from both

species was (58.6%). In examined Oreochromis niloticus the

prevalence was (66.2%) while Clarias gariepinus (49.52%), the

result which considerably higher than that mentioned by Abu

El-Waffa et al. (1999) and Nesreen (2008).

The results of examination of Oreochromis niloticus

revealed the presence of the following external protozoa at such

incidence levels (3.5%, 53.3%, 14.2%, 22.2% and 4.4%) for

Ichthyophthirius multifilis, Trichodina truttae, Chilodonela

hexxxasticha, Myxobolus dermatobia and Ichthyobodo necator

while Henneguya branchialis recorded 0% incidence among

examined Oreochromis niloticus, the result which indicate that

Trichodina trutttae (53.3%) represent the highest infection rate

Discussion

101

among examined Oreochromis niloticus followed by Myxobolus

dermatobia (22.2%) then, Chilodonela hexxxasticha (14.2%)

and later on Ichthyobodo necator and Ichthyophthirius multifilis

showing by infection rate of (4.4% and 3.5%) respectively. This

result come inagreement with that reported by Negm El-Din

(1999) and Nesreen (2008) except for Ichthyophthirius

multifilis which show lower rate of infection.

The results of examination of Oreochromis niloticus to

detect the prevalence of internal protozoa revealed the presence

of Eimeria sp., Cryptosporidim sp., Blantidium sp. At the

following prevalence rate (13.3%, 62.2% and 68%) respectively,

which means that the Blantidium sp. (68%) contributes the

highest infection rate among internal protozoa in examined

Oreochromis niloticus followed by Cryptosporidim sp. (62.2%)

then Eimeria sp. (13.3%).

The results of examination of Oreochromis niloticus for

the presence of blood protozoa revealed negative results on the

contrary Negm El-Din (1999) and Abu El-Waffa et al. (1999)

reported many species of blood parasites among this host.

Examination of Clarias gariepinus (210) sample revealed

the presence of external protozoa such as Ichthyophthirius

multifilis, Trichodina trutttae, Chilodonela hexasticha and

Henneguya branchialis in infection rate (1.9%, 47.6%, 23.3%

and 6.1%) which means that Trichodina trutttae (47.6%)

Discussion

102

contributes the highest incidence rate among external protozoa

in examined Clarias gariepinus followed by Chilodonela

hexxxasticha (23.3%) while Henneguya branchialis the

incidence rate was (6.1% ) and Ichthyophthirius multifilis only

(1.9%) the result which come lower than that recorded by

Nesreen (2008) and which may be attributed to the locality

difference.

The results also revealed that the highest prevalence rate

was in summer (77.9%) followed by spring (70%) and autumn

(52.94%) while the last one was in winter (32.17%).

The results also revealed no enteric protozoa were detected

in examined Clarias gariepinus while the examination toward

the blood protozoa revealed the prevalence of Trypanosoma

mukasi and Haemogregarina clariae at (40.9% and 14.7%)

incidence rates. It was important to mention that, according to

the available literature the previous works on protozoan

infection among Oreochromis niloticus and Clarias gariepinus

did not deal with the comparative study between external and

internal protozoan infection rate so, the present study is the first

which deal with that point of view.

Many previous recorded data agree with our finding

regarding the higher prevalence and importance of external

protozoa (Shalaby and Ibrahim, 1988) (Woo and Poynton

1995) (Ramadan, 1991).

Discussion

103

At the same time (Ali et al., 2003) mentioned that fishes

are hosts for many protozoan parasites and the infestation rates

by protozoa especially external ones which reported to be high.

Regarding the high prevalence of internal protozoa in

examined Oreochromis niloticus which revealed high rates of

infection toward Blantidium sp. followed by Cryptosporidim sp.

and Eimeria sp. agree with many previous finding (Barbara et

al., 1990), (Alvarez et al., 2004) (Brian et al, 2009) (Mingli et

al., 2009).

The results of water examination which revealed the

prevalence of certain pollutions such as high Ammonia, Nitrates

and Iron may explain the high levels of infection toward such

internal protozoa because such infections mostley occurred in

immuno compressed fishes (Barbara et al., 1990) and fishes in

such examined localities living in polluted water which have

immuno suppressive effects on such fishes which render them

susceptible to such infections.

Regarding the examination for the presence of blood

protozoa, no protozoa were detected in examined Oreochromis

niloticus while Clarias gariepinus showed the presence of

Trypanosoma mukasi at prevalence level (40.9%) and

Haemogregarina clariae at levels (14.7%). The difference in

prevalence of infection between the two fish species may be

attributed to the physiological and immunological difference

Discussion

104

which render Clarias gariepinus more susceptible to infection

by blood protozoa than Oreochromis niloticus.

Regarding the results of clinical and post mortem finding

among infected Oreochromis niloticus and Clarias gariepinus,

the results revealed that Oreochromis niloticus and Clarias

gariepinus infected with external protozoa (Ichthyophthirius

multifilis, Trichodina trutttae, Chilodonela hexxxasticha and

Henneguya branchialis) showed slimmy, pale skin with

detached scales, also the presence of certain haemorrhage

especially at the base of fins.

Also some abnormal finding were observed in infected

fishes in comparing with normal fishes including restlessness

and accumulation at the bottom of the aquarium.

In case of fishes infected with Ichthyobodo necator showed

excessive grey dirty mucous covering the skin of fish as well as

detached scales.

Our finding agree with (Shalaby and Ibrahim, 1988)

(Leong and Wong, 1990) (Stoskopf, 1993) (Roberts, 1995)

(Woo and Poynton, 1995) (Tomas, 1999) (Eissa, 2002) (Ali et

al., 1988) (Omeji et al, 2011).

The signs recorded in examined fishes infected with

internal protozoa including variable levels of emaciation,

anorexia, restlessness and increase mortalities. Such finding

agree with (Barbara et al., 1990) (Eduardo et al., 2002)

Discussion

105

(Brian et al, 2009) (Alvarez et al, 2004) (Mingli et al., 2009)

(Rona et al., 2011).

The clinical finding in Clarias gariepinus infected with

blood protozoa (Trypanosoma mukasi and Haemogregarina

clariae) including only emaciation.

The finding agree with (Negm El-Din, 1998h) (Negm El-

Din, 1999) (Gibson et al., 2005).

Regarding the seasonal dynamics variation the highest

prevalence was in summer followed by spring and autumn agree

with many finding (Nesreen Saad I., 2008) (Mohamed A. H.,

1999). This may be due to the availability of suitable

temperature for the growth and multiplication of such protozoa.

Regarding the examination of water samples from such

important Tributaries of River Nile. The results revealed the

presence of NH4 1.49, NO3 0.89, P 0.45, Fe 0.411, Mn 0.118, Zn

0.00, Cu 0.002, Cd 0.00, Pb 0.00 and B 0.116 (mg/L).

The presence of such pollutants as Ammonia, Nitrate and

Iron at such levels may be a potent predisposing factor for the

occurrence of infection by such protozoa. Also such pollutant

render the examined fishes immune suppressed and susceptible

to protozoal infection.

The results agree with (Eissa I. A., 2002) (Osman et al.,

2009) that correlated the protozoal infection to environmental

pollution, Also (Osman et al., 2009) that stressful conditions

Discussion

106

especially poor water quality may render fish susceptible to

infection by external protozoa.

Also the finding meeting the finding of (Barbara et al.,

1990) that immuno compressed fish susceptible to protozoal

infection.

The histopathological finding of infected fishes showed skin

necrosis and focal inflammatory necrosis in the outer surface,

sloughing of epithelial layer and haemorhage.

In case of gills infection showing necrosis in the rakers

inflammatory cells infiltration, sever congestion.

In case of eye infection showing haemorrahage and

yellowish to whitish cyst, these findings agree with finding of

(Leong and Wong, 1990) (Omeji et al., 2011) (Ekanem and

Obiekezie, 1996) (Mohamed et al., 1999) (Eduardo et al., 2002)

(Mahmoud el al., 2004).

The histopathological examination of fishes 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.

This finding agree with finding of (Barbare et al., 1990)

(Alvarez et al., 2004) (Zhokhov and Molodozhnikova, 2006)

(Brian et al., 2009).

Discussion

107

From this study it could be concluded that:

- Maryotia Channel which is a tributary of River Nile in Giza

governorate suffering from pollutants by Iron, Nitrates and

increase in Ammonia levels.

- Oreochromis niloticus obtained from such tributary

suffering from infection by external and enteric protozoa

Ichthyophthirius multifilis, Trichodina trutttae,

Chilodonela hexxxasticha, Myxobolus dermatobia,

Ichthyobodo necator, Eimeria sp., Cryptosporidium sp, and

Blantidium sp. With incidence rate (3.5%, 53.3%, 14.2%,

22.2%, 4.4%, 13.3%, 62.2% and 68%) respectively. It was

of great importance to mention that, this is the first record of

Balantidium species among Oreochromis niloticus in Egypt,

also for studying their incidence, clinical signs of the

affected fish and histopathological lesions.

- Clarias gariepinus suffered from infection by external as

well as blood protozoa Ichthyophthirius multifilis,

Trichodina trutttae, Chilodonela hexxxasticha, Henneguya

branchialis, Tryanosoma mukasi and Haemogregarina

clariae (1.9%, 47.6%, 23.3%, 6.1%, 40.9%, 14.7%)

respectively.

- Polluted and abnormal water parameters in Maryotia

Channel may playing the role as a predisposing factor for

occurrence of protozoal infection and through

immunodepression of such fishes and rendering them

susceptible to infection.

Cairo University

Faculty of Veterinary Medicine

Department of Fish Diseases and Management

C. V.

Name: Nehal Aboulkaramat Younis

Date of birth: 5/8/1986

Site of birth: Libya

Nationality: Egyptian

Thesis for M. V. Sc. Entitled: Studies on some Problems of Protozoal Infection in Freshwater

Fishes

Under supervision of:

Prof. Dr. Mohammed Abd El-Aziz Ahmed Prof. and head of fish diseases and management department

Faculty of veterinary medicine, Cairo University

Prof. Dr. Mai El-Desoky El-Said Ibrahim prof. of fish diseases and management


Nisreen Ezz El-Dien Mahmoud Prof. of parasitology


ABSTRACT

In this study, investigated the relation between the parasitic protozoal

infection rate and water pollution in locations from which fishes were

collected.

In this study, the infestation rate of different protozoal diseases in wild

freshwater fishes namely: Oreochromis niloticus and Clarias gariepinus was

66.2% and 49.52% respectively. These fishes were collected from different

areas in Nile River in Giza governrate. The results proved the occurrence of

many protozoa including internal and external protozoa. The infestation rate

with these parasites was varied with seasons and species of fish. It was noticed

that highest rate of infestation was in summer season followed by spring,

autumn and finally winter. Histopathological studies revealed inflammatory

changes in the infected tissues.

Water samples were collected from Maryotia Channel for analysis. Key

words: (Protozoal infection – fresh water – fish).

Summary

Summary

108

VI. Summary

In this study, we investigate the parasitic protozoal

infection among wild freshwater fishes in Giza governorate,

effect of water quality on parasitic protozoa prevalence. A total

number of (435) alive fish were collected from Nile river in

Giza governorate in different seasons include (225 Oreochromis

niloticus and 210 Clarias gariepinus) and subjected for Clinical,

Parasitological and histopathological examination.

Water samples were collected from the same location from

which fishes were collected.

The results of this work could be summarized as

follows:

1- The prevalence of parasitic protozoa in Oreochromis

niloticus was 66.2% while in Clarias gariepinus was

49.52%.

2- The isolated parasitic protozoa namely.

Ichthyophthirius multifillis.

Trichodina truttae.

Chilodonella hexasticha.


Summary

109

Myxobolus sp.

Eimeria sp.

Cryptosporidium sp.

Blantidium sp.

Ichthyobodo necator..

Trypanosoma mukasi.

Haemogregorina clariae.

3- Clarias gariepinus were infected with Ichthyophthirus

multifillis, Trichodina sp., Chilodonella sp., Henneguya

sp., Trypanosoma mukasi and Haemogregarina clarae,

with infection rate 1.9%, 47.6%, 23.3%, 6.1%, 40.9% and

14.7% respectively.

4- The Oreochromis niloticus was infected with

Ichthyophthirus multifillis, Trichodina truttae,

Chillodonella, Myxobolus sp., Eimeria, Crypto sporidium.,

Blantidium and Ichthyobodo necator with infection rate

3.5%, 53.3%, 14.2%, 22.2%, 13.3%, 62.2%, 68% and

4.4% respectively.

5- The clinical and postmortem examination of fishes

naturally infected with protozoan parasites revealed many

abnormal manifestation.

Summary

110

- In cases of Oreochromis niloticus and Clarias

gariepinus infected with external protozoa

"Ichthyophthirus, Chilodonella sp., Trichodina sp.

Henneguya branchialis, skin is slimy, pale with

detached scales blood spots especially at the base of

fins, signs of irritation is manifested.

- In cases of external flagellated protozoa as Ichthyobodo

necator, body covered with grey dirty mucous, detached

scales and signs of irritation.

- In case of Myxobolus infection in Oreochromis

niloticus fishes whitish to yellowish cysts ranged from 1

up to 8 in number was observed in the eye.

- In case of intestinal parasitic protozoa as Eimeria sp.,

Cryptosporidium sp. and Blantidium sp. Many signs is

recorded especially in young fishes as variable levels of

emaciation, anorexia, restlessness and increased

mortality within their fish.

- In case of blood parasites as Trypanosoma mukasi and

Haemogregarine sp. Emaciation in case of heavy

infection.

Summary

111

6- In this study the seasonal dynamics revealed that in total

examined fish species, the protozoal infection reached the

peak in summer 77.9% followed by spring 70% and

autumn 52.94% while the least one was in winter 32.17%.

- Seasonal dynamic of Ichthyophthirus multifillis reach

the peak in winter, while Trichodina sp. in spring.

- Seasonal dynamic of Chilodonella sp. reach the peak in

spring, Henneguya branchialis and Myxobolus sp. in

summer, while Eimeria sp. in winter.

- Seasonal dynamic of Cryptosporidium sp. in spring,

while Blantidium sp. and Haemogregarine sp. in

summer.

- Seasonal dynamic of Trypanosoma mukasi and

Ichthyobodo necator in winter.

Summary

112

- The analysis of water sample from Maryotia

Channel revealed:

element mg/ L

NH4 1.49

NO3 0.89

P 0.45

Fe 0.411

Mn 0.118

Zn 0.00

Cu 0.002

Cd 0.00

Pb 0.00

B 0.116

PH was measured 7.4

It was noticed that increase prevalence rate of parasitic

protozoal infections in fishes collected from polluted water.

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Arabic

Summary

1

امللخص العربي

2

3

4

العنصر )ملجم/ لتر(

NH4الىيتروجيه األموويومي 1441

NO3الىيتروجيه الىتراتي 0491

Pالفوسفور 04450

Feالحديد 04411

Mnالمىجىيز 04119

Znالزوك 0400

Cuالىحاس 04002

Cd الكادميوم 0400

Pbالرصاص 0400

Bالبورن 04116

السرية الذاتية

املستخلص العربي

ات عن بعض مشاكل العدوىــدراس

باألوليات يف أمساك املياه العذبة

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STUDIES ON SOME PROBLEMS OF PROTOZOALscholar.cu.edu.eg/?q=nehal-younis/files/rsl_d._nhl_lnhyy...STUDIES ON SOME PROBLEMS OF PROTOZOAL INFECTION IN FRESHWATER FISHES Thesis presented