IV- Identification of isolated parasites

1- Protozoa:

Trichodina: Trichodina species were observed in gills and skin scrape (Fig.1.a.b).

 

 

Fig. 1: Trichodina ciliated protozoa in wet mount (a) and stained with Geimsa stain (b) isolated from O. niloticus.

 

2- Trematodes:

2- a-Monogenia: the Microscopical examination revealed the presence of 

Cichlidogyrus liberianus (Fig.2). and Cichlidogyrus arthracanthus (Fig.3).

 

 

 

 

Fig. 2:Cichlidogyrus Liberianus showing attached to gill filaments on O.niloticus.

 

 

Fig. 3: Cichlidogyrus arthracanthus showing attached to gill filaments on O.niloticus.

 

2- d- Digenea

The results revealed the presence of Centrocestus sp., Clinostomum and Euclinostomum Encysted metacercaria sp(Fig 4-5-6)The external examination of some infected O. niloticus revealed the presence of scattered visible yellow to orange pea like encysted metacercariae on skin and embedded in the musculature with irritated scales Metacercariae of Clinostomum tilpaiae In spite of the large size (3–7 mm) of the clinostomatid cysts,The encysted metacercariae were detected in both skin and internal organs (branchial cavity, pharynageal regions and gills). Metacercariae of Euclinostomum Encysted metacercariae were large pea sized. They were spherical in shape, dark yellowish in color and embedded in the tissues of either anterior or posterior portions of the kidneys.

                                                    

Fig. 4: Encysted Centrocestus metacercariae in the gills of infected       O. niloticus.

 

  

 

 

Fig. 5: Stained excysted Clinostomum sp larvae.

 

 

 

 

 

Fig. 6: Stained excysted Euclinostomum sp. larvae.

 

3- Nematodes:

3- a- Acanthocephala: The parasitological examination revealed the presence of acanthocephalan tilapiae. (Fig.7-10).

3-b-Contracecum: The parasitological examination revealed the presence of acanthocephalan tilapiae. (Fig.11-13).

 

                                        

 

 

 

Fig. 7: Male Acanthocephala tilapae in O.niloticus

 

 

 

 

Fig. 8: Anterior end of Acanthocephala tilapae.

 

 

                       Fig. 9: Posterior end of male        Fig. 10: Posterior end of female

                            Acanthocephala tilapae                Acanthocephala tilapae

 

 

 

                                             Fig. 11: Contracaecum sp. Larvae

 

 

 

                                              Fig. 12: Anterior end of Contracaecum sp. larvae        

 

 

 

                                              Fig. 13: Posterior end of Contracaecum sp. larvae  

 

4- Crustacia: Isolation of Ergasilus from O. niloticus was obtained from gills (Fig.14-15).

                                 

 

 

 

                          Fig. 14: Ergasilus sp. attached to the gills of O.niloticus.

 

 

 

          Fig. 15: Ergasilus sp. in wet mount

 

VI- Identification of isolates of fungal organisms

The results of mycological examination allowed the isolation of 80 isolates from infected O. niloticus sampled from skin, gills, eye, and mouth lesions of infected O.niloticus (Fig.16-17) showed masses of mature and immature sporangia filled with large number of sporangiospores, the hyphae appeared profusely branched and were non septated, these morphological findings were characteristic of the saprolegnia species. The characteristic appearance of Saprolegnia sp. on Sabauraud's Dextrose agar (Fig. 18).

 

 

 

Fig. 16: Naturally infected O.niloticus showing cotton-like material on the skin.

 

 

 

Fig. 17: Saprolegnia sp. showing cotton-like material on the skin surface, eye, fins, gills and mouth

 

 

 

Fig. 18: Saprolegnia sp. on Sabauraud's Dextrose agar

Discussion

 

The present investigation was planned to detect the seasonal critical times for the occurrence of important parasitic and mycotic diseases that affecting O. niloticus under semi-intensive culture system in Sharkia Province. The detection of those critical times will help for lowering the negative impacts of such diseases.  

The results showed that parasitic infestations were found to be the major problems and the most prevalent diseases infecting cultured tilapia in semi-intensive farms. The percentages of parasitic and fungal fish pathogens represent 72.5% and 8% of examined diseased fish respectively. Some of these are obligate pathogens while others are opportunistic facultative organisms producing disease when fish health is compromised by injury or environmental stressors (Plumb, 1999). The results agree with El-Nobi (1998) and this could be attributed to the pond management, including poor water quality, improper nutrition and other stressors affecting O. niloticus.

Regarding the isolated parasites, Protozoa are the most prevalent Parasitic diseases of O. niloticus, Ruth (1990). Tricodinids are the most commonly encountered symbiotic groups in the aquatic environment. They found mainly on the skin and gills, either as parasites or as commensals and show very little host specificity(Woo, 2006). The results of parasitological examination and identification revealed the presence of Trichodina sp. in a percentage (42.5%) of total parasitic disease in fish examined. The research showed that Trichodina sp. represented the highest rate of infection among detected parasites in     O. niloticus, the results agree with (El- Gawady et al., 1992) and Younis (1999).

Dealing with the seasonal dynamics of Trichodina sp. parasite in the examined fish, it was clear that the higher prevalence was observed in summer season (64.8%), followed by spring (48%), then autumn (32.4%) and the least percentage was recorded in winter (24.8%). These results were similar with that obtained by Brewster (1997); El-Nobi (1998); Morozinska (2002) and Attia (2004). The higher prevalence of Tichodina infection among Tilapia during summer and spring seasons could be attributed to the high organic matter and the high temperature which are more suitable for parasite reproduction, while in winter the water temperature decreased and the parasitic prevalence consequently decreased(Attia 2004; Awad, 2007). On the other hand, Ramadan (1991); Mesally (1994); Salah et al. (1995) and Woo (2006) mentioned that winter and spring was found to be the season for sever Trichodina infection in fishes which stressed by harsh winter conditions and the lowest percentage of infection was recorded in summer.The results also revealed that the main clinical signs observed in heavily infected          O. niloticus may exhibited a grayish-blue color, formed by excessive mucus secretion in skin, gills and peeled epithelia, sluggish swimming. The excessive epithelial growth is believed to be a protective reaction, from the fish against the parasite. The results of Trichodina infection in the examined fish proved the drastic damage on the skin and gill epithelium. Similar signs were coincided with the description given by Noga (1996); Paperna (1996); Attia (2004); Awad (2007) and Abd El-Ghany et al. (2009).

The results of parasitological examination and identification revealed the presence of gill monogeneans at percentage (28%) of total parasitic disease in examined O. niloticus. This result was almost in agreement with El-Gawady et al. (1992) and Dewra (1995).A higher percentage was recorded by El-Gohary (2000)which was (80%), (78.9%) respectively. The variation in prevalence may be attributed to season, locality, physiological status of examined fish and environmental factors. The results revealed that the seasonal prevalence of monogenetic trematodeswas highest in spring season (38%), followed by summer (33.6%), Autumn (27.2%) and then in Winter (13.2%). These results were similar with that recorded by El-Nobi (1998); El-Gohary (2000); Attia (2004) and Awad (2007) this result may be due to that during warm seasons the climate temperature is suitable for the life cycle of the parasites.

The clinical signs observed in heavily infected O. niloticus with ectoparasitic monogenea usually results in excessive mucus, loss of appetite, sluggish movements and the fish swam near the surface of water. The infested fishes showed increased breathing frequency, and the gill covers were stretched open widely. The gills were pale in some fishes, hyperemic in others and covered with mucus secretions forming cloudy film of slime. Infested fishes were anemic and the fins were ragged with partial detachment of scales. Microscopically, the monogeneans were found attached to the gill filaments. Such obtained results are nearly similar those of theseclinical signs may be attributed to the organs of attachment and mode of feeding of monogenia. The hamuli, marginal hooklets, suckers and clamps of monogeneans all have direct contact with host tissue and may afflict mechanical damage directly. These result supported those of Attia (2004); Woo (2006); Awad (2007) and Abd El-Ghany et al. (2009).  

The results noticed in this work proved that mixed infection of Trichodina and monogeneans were more prevalent. However, monogenea can serve as vectors for viral and bacterial pathogens of fish which provided a portal of entry for invasive bacteria due to damage of the fish epithelium. Also, because they propagate and transferred rapidly between fishes under farming conditions this may be leading much damage to high morbidity and mortality. These were proved previously by Cusack and Cone (1986).

The results of parasitological examination and identification revealed the presence of three types of digenean encysted metacercariae in examined O. niloticus. Centrocestus sp. represented the highest one at percentage (5.9%) of total parasitic disease in examined fish. The results proved seasonal fluctuations among the examined fish. It was especially high during the warm season of the year in fresh water due to active shedding of cercariae. Similar observation was reported by Paperna, (1996); El-Nobi, (1998); Attia, (2004) and Awad, (2007), the last author recorded that encysted metacercaria (Centrocystus formosanus) were found in gills of black carp at the prevalence of 100% along the year. This may be attributed to the high abundance of the intermediate snail host in the examined earthen ponds in which black carp were reared.

The results revealed thatCentrocestus species encysted metacercariae were detected in the gills of O.niloticus without any clinical signs in low infection rate. While the main clinical signs observed in heavily infested fish were respiratory distress, decreased feed intake and increased mucus secretion. The infected area showed inflammatory reaction especially in early infestation or when the cyst, was ruptured. This condition might represent a response to prolonged irritation by the parasite and its toxic product or the hyperplasia of chondrocytes or may be due to decrease the oxygen tension in the affected area (Attia 2004; Awad 2007 and Abd El-Aal et al., 2008).

Low infection rates were recorded for Clinostomum and Euclinostomum spp. at percentage (5.2%) and (4.9%) respectively. Results of the previous related studies, Abd-Alkareem (2000) found the prevalence rate was (4.4%) and (0.5%)respectively.Yimer (2000) found (9.09%) of Clinostomum and only 1 Euclinostomum species from all investigated fish. who found that 34% and 29% of this fish species were infected with these types of metacercariae, respectively; and that of Abd El Rahman (2005), who found that 45% and 11.8% of O. niloticus were infected with Clinostomum species and Euclinostomum species metacercariae, respectively. This can be refered to the difference in sampling periods and the ponds from which the examined fishes were obtained. The results also revealed that the seasonal prevalence of Clinostomum species and Euclinostomum species were during winter and spring seasons respectively. This may be due to the suppressed immunity of the cultured fish during cold temperature. It is worth to mention that extrapolation of seasonal variations from direct counts of larvae in fish since such infections normally persist for over 12 months, these results are in line with several authors as El-Nobi (1998); Attia (2004) and Woo (2006).

The results also revealed that the main clinical signs of Clinostomum infection observed in moderate and higher intensity of infection which showed debility, retarded growth, excessive mucus secretion, increased in the opercula movement and protrusion of operculum. Yellowish to orange color, pea like cyst were found scattered on skin and in bronchial cavity. In case of heavy infection, cysts were arranged in agape-like cluster. In dead tilapias, larvae encysted and moved over the mesentery and on skin. On the other hand, pathological impact of infection with Euclinostomum was not often obvious and depended on their number. Few cases showed related growth, abdominal distention and reddish ascetic fluid in the abdominal cavity, which could be attributed to functional damage of kidney. Euclinostomum was found distributed along the kidneys of infected tilapia in the form of grayish cyst. Congestion of infected kidneys was also observed. The present data werein agreement with previous observations (Paperna 1996; Abd El Rahman 2005; Abd El-Aal et al., 2008).

             The results of parasitological examination and identification revealed the presence of Contraceacum nematode at percentage (3%), it was higher during autumn (Abu-El-Ezz, 1988)While, Varjabedian (1993)mentioned that the majority of nematodes, reached their peak of infection during spring and summer, Aloo (2002)mentioned that no seasonal changes was recorded in infection level. This phenomenon may be attributed to many factors interacted together such as water temperature, fish host length, sex and feeding habits and propagation of the intermediate hosts and each nematode parasites had its own season of prevalence that varied in different hosts.

              The results also revealed that the main Clinical signs of Clinostomum infection observed the located of Contracaecum species larvae in the abdominal cavity causing abdominal distention. Also, the effect results from mechanical damage to the internal organs and production of toxic substances. Larvae penetrate in to the intermediate host's body through the wall of digestive tract and lead to its death, inflammatory response of digestive tract and pressure on internal organs. Intestine was voided from food. In severe infections, low body gain is common sign. Nematodes may not even show signs of illness, but they often have reduced reproductive capacity. The present result nearly coincided with that recorded by Abu-El-Ezz (1988); Moravec (1994) and Barson (2004).

Acanthocephala tilapiae was observed in a high prevalence as parasitic infection that varied from the other parasites and another of all host species analyzed (Ayotunde 2007; Omar et al., 2008). The results of parasitological examination and identification revealed the presence of Acanthocephala tilapiae at percentage (7.5%) that was prevalent during autumn. WhileGihan (1999)recorded that the highest rate was in spring followed by autumn. (Omar et al., 2008) showed that high prevalence of Acanthocephala tilapiae was observed in all host species analyzed. They mentioned that worms appear to be recruited in the summer, develop and mature through the winter, and reproduce sexually in late winter and spring. In addition Differential exposure to parasites and host susceptibility may explain the marked variation in parasite abundance among hosts. There were no clinical signs in light infection with Acanthocephala tilapiae parasite. While, there were signs of emaciation and congested intestine in heavy infection. These findings associated with infection are in harmony with those recorded by Kabata (1985). In the final host the Pathogencity of acanthocephala is related to the mode of attachment of the parasite species and to the systematic affiliation of the host. Long necked acanthocephalans which deeply penetrate into or through the intestinal wall with their presoma are more pathogenic than species with a short neck and a shallow mode of anchorage (Kabata 1985; Woo 2006).

              The results of parasitological examination and identification revealed the presence of Ergasilus sp. at percentage (4.9%), a higher prevalence was recorded in spring. While Mostafa et al. (1991)andMolnar et al. (1997)recorded that high level of Ergasilus infestation in summer and autumn seasons. This could be related to the environmental conditions and the presence of the infective copepodids.

The results also revealed that  the main clinical signs of Ergasilus infection were pale gills in mild to moderate infestation, erosion, degeneration and necrosis in gill filaments in sever cases, gills damage that resulted in loss of surface area for respiration and lead to suffocation, particularly at high water temperature and Excessive accumulation of mucus in the gill, This in turn may result in reduced growth rates and greater mortality (Grabda 1991; Mostafa et al., 1991; Molnar et al., 1997; Abd El-Rahim 1998; Woo 2006).

The results of mycological examination and identification revealed the presence of mycotic infection at percentage 8% of total diseased examined fish. This may be due to the fact that most of the serious mycotic pathogens are mostly secondary in nature and need great stress on such cultured fishes in order to find the porta of entery for producing a disease. The stress factors usually include environmental fluctuations and improper management practices with poor water quality (Udomkusonsri, 2003). The wet mounts from skin and gill tissues of infected O.niloticus showed the presence of large non-septet tubular cells. Spores of the fungi attach to injured or unhealthy tissue and establish a colony (mycelium). Microscopically, the identified agent was Saprolegnia diclina which found among O. niloticus in the examined semi-intensive farms in the winter season specifically. This disease (Saprolegniosis) is a serious disease that affects all species and ages of freshwater fishes and can also affect many estuarine fish (Woo and Bruno 1999; Aly and El-Ashram 2000; Polacheck 2007; Woo 2007). The low temperature constitutes a great stress factor affecting the health condition of O.niloticus and renders them prey to Saprolegnia fungus.(Mesallhy 1994; Naguib 1994; Aly et al., 1996; Aly and El-Ashram 2000; Polacheck 2007; Osama et al., 2008; Zaki et al., 2008).

The results revealed that the signs of Saprolegnia infection including general external signs which appeared anywhere on the body surface, fins, eyes and gills. The infected fish often develop grayish-white cotton-like growths on skin, fins, gills and eyes. The diseased fish showed hyperactivity and obvious evidence of irritation such as rubbing against the sides or bottom of tank which may be attributed to the inflammatory reactions, erythema at the base of fins, ulceration of the skin, this may be due to toxin secreted by saprolegnia hyphae, oomycete or zoospores due to fungal cells (hyphae) invade the injured tissue from which they infect surrounding healthy tissue. Also, loss of equilibrium with nervous signs, blindness, and enlargement of abdominal were also recorded. Evidence of respiratory distress was recorded. The gills appeared pale in colour with profuse mucus and covered by tufts of fungi. The same findings were also recorded by (Ruth 1990; Aly and El-Ashram 2000; Udomkusonsri. 2003; Osama et al., 2008; Zaki et al., 2008). The observed pale color of gills could be due to the coating of the gills by profuse mucus, necrotic debris and secondary mycosis (Plump 1997).

In conclusion, the result of this work proved the wide spread of parasitic and to lesser extent fungal infections in the O. niloticus cultured under the semi-intensive system in the earthen ponds. The wide spread of such pathogens are due to the improper management practice. The results revealed that summer and late spring are critical times for the occurrence of parasitic diseases. At the same time the problem of saprolegniosis in winter season represents an important problem for     O. niloticus semi intensively cultured in earthen ponds, especially after the days of severe cold weather fronts. A sever economical impact could be waited from the heavy fish mortality and high fish loss, so proper managemntal practices should be applied during these critical times that render O. niloticus susceptible for the occurrence of such diseases.  Raising of water column, continuous flow of water in the days of sever cold weather as well as  incorporation of immune-stimulant in fish ration could aid in avoiding of such loss by raising the fish resistance against these parasites and fungal agents.

 

References

 

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IV- Identification of isolated parasites

1- Protozoa:

Trichodina: Trichodina species were observed in gills and skin scrape (Fig.1.a.b).

 

 

Fig. 1: Trichodina ciliated protozoa in wet mount (a) and stained with Geimsa stain (b) isolated from O. niloticus.

 

2- Trematodes:

2- a-Monogenia: the Microscopical examination revealed the presence of 

Cichlidogyrus liberianus (Fig.2). and Cichlidogyrus arthracanthus (Fig.3).

 

 

 

 

Fig. 2:Cichlidogyrus Liberianus showing attached to gill filaments on O.niloticus.

 

 

Fig. 3: Cichlidogyrus arthracanthus showing attached to gill filaments on O.niloticus.

 

2- d- Digenea

The results revealed the presence of Centrocestus sp., Clinostomum and Euclinostomum Encysted metacercaria sp(Fig 4-5-6)The external examination of some infected O. niloticus revealed the presence of scattered visible yellow to orange pea like encysted metacercariae on skin and embedded in the musculature with irritated scales Metacercariae of Clinostomum tilpaiae In spite of the large size (3–7 mm) of the clinostomatid cysts,The encysted metacercariae were detected in both skin and internal organs (branchial cavity, pharynageal regions and gills). Metacercariae of Euclinostomum Encysted metacercariae were large pea sized. They were spherical in shape, dark yellowish in color and embedded in the tissues of either anterior or posterior portions of the kidneys.

                                                    

Fig. 4: Encysted Centrocestus metacercariae in the gills of infected       O. niloticus.

 

  

 

 

Fig. 5: Stained excysted Clinostomum sp larvae.

 

 

 

 

 

Fig. 6: Stained excysted Euclinostomum sp. larvae.

 

3- Nematodes:

3- a- Acanthocephala: The parasitological examination revealed the presence of acanthocephalan tilapiae. (Fig.7-10).

3-b-Contracecum: The parasitological examination revealed the presence of acanthocephalan tilapiae. (Fig.11-13).

 

                                        

 

 

 

Fig. 7: Male Acanthocephala tilapae in O.niloticus

 

 

 

 

Fig. 8: Anterior end of Acanthocephala tilapae.

 

 

                       Fig. 9: Posterior end of male        Fig. 10: Posterior end of female

                            Acanthocephala tilapae                Acanthocephala tilapae

 

 

 

                                             Fig. 11: Contracaecum sp. Larvae

 

 

 

                                              Fig. 12: Anterior end of Contracaecum sp. larvae        

 

 

 

                                              Fig. 13: Posterior end of Contracaecum sp. larvae  

 

4- Crustacia: Isolation of Ergasilus from O. niloticus was obtained from gills (Fig.14-15).

                                 

 

 

 

                          Fig. 14: Ergasilus sp. attached to the gills of O.niloticus.

 

 

 

          Fig. 15: Ergasilus sp. in wet mount

 

VI- Identification of isolates of fungal organisms

The results of mycological examination allowed the isolation of 80 isolates from infected O. niloticus sampled from skin, gills, eye, and mouth lesions of infected O.niloticus (Fig.16-17) showed masses of mature and immature sporangia filled with large number of sporangiospores, the hyphae appeared profusely branched and were non septated, these morphological findings were characteristic of the saprolegnia species. The characteristic appearance of Saprolegnia sp. on Sabauraud's Dextrose agar (Fig. 18).

 

 

 

Fig. 16: Naturally infected O.niloticus showing cotton-like material on the skin.

 

 

 

Fig. 17: Saprolegnia sp. showing cotton-like material on the skin surface, eye, fins, gills and mouth

 

 

 

Fig. 18: Saprolegnia sp. on Sabauraud's Dextrose agar

Discussion

 

The present investigation was planned to detect the seasonal critical times for the occurrence of important parasitic and mycotic diseases that affecting O. niloticus under semi-intensive culture system in Sharkia Province. The detection of those critical times will help for lowering the negative impacts of such diseases.  

The results showed that parasitic infestations were found to be the major problems and the most prevalent diseases infecting cultured tilapia in semi-intensive farms. The percentages of parasitic and fungal fish pathogens represent 72.5% and 8% of examined diseased fish respectively. Some of these are obligate pathogens while others are opportunistic facultative organisms producing disease when fish health is compromised by injury or environmental stressors (Plumb, 1999). The results agree with El-Nobi (1998) and this could be attributed to the pond management, including poor water quality, improper nutrition and other stressors affecting O. niloticus.

Regarding the isolated parasites, Protozoa are the most prevalent Parasitic diseases of O. niloticus, Ruth (1990). Tricodinids are the most commonly encountered symbiotic groups in the aquatic environment. They found mainly on the skin and gills, either as parasites or as commensals and show very little host specificity(Woo, 2006). The results of parasitological examination and identification revealed the presence of Trichodina sp. in a percentage (42.5%) of total parasitic disease in fish examined. The research showed that Trichodina sp. represented the highest rate of infection among detected parasites in     O. niloticus, the results agree with (El- Gawady et al., 1992) and Younis (1999).

Dealing with the seasonal dynamics of Trichodina sp. parasite in the examined fish, it was clear that the higher prevalence was observed in summer season (64.8%), followed by spring (48%), then autumn (32.4%) and the least percentage was recorded in winter (24.8%). These results were similar with that obtained by Brewster (1997); El-Nobi (1998); Morozinska (2002) and Attia (2004). The higher prevalence of Tichodina infection among Tilapia during summer and spring seasons could be attributed to the high organic matter and the high temperature which are more suitable for parasite reproduction, while in winter the water temperature decreased and the parasitic prevalence consequently decreased(Attia 2004; Awad, 2007). On the other hand, Ramadan (1991); Mesally (1994); Salah et al. (1995) and Woo (2006) mentioned that winter and spring was found to be the season for sever Trichodina infection in fishes which stressed by harsh winter conditions and the lowest percentage of infection was recorded in summer.The results also revealed that the main clinical signs observed in heavily infected          O. niloticus may exhibited a grayish-blue color, formed by excessive mucus secretion in skin, gills and peeled epithelia, sluggish swimming. The excessive epithelial growth is believed to be a protective reaction, from the fish against the parasite. The results of Trichodina infection in the examined fish proved the drastic damage on the skin and gill epithelium. Similar signs were coincided with the description given by Noga (1996); Paperna (1996); Attia (2004); Awad (2007) and Abd El-Ghany et al. (2009).

The results of parasitological examination and identification revealed the presence of gill monogeneans at percentage (28%) of total parasitic disease in examined O. niloticus. This result was almost in agreement with El-Gawady et al. (1992) and Dewra (1995).A higher percentage was recorded by El-Gohary (2000)which was (80%), (78.9%) respectively. The variation in prevalence may be attributed to season, locality, physiological status of examined fish and environmental factors. The results revealed that the seasonal prevalence of monogenetic trematodeswas highest in spring season (38%), followed by summer (33.6%), Autumn (27.2%) and then in Winter (13.2%). These results were similar with that recorded by El-Nobi (1998); El-Gohary (2000); Attia (2004) and Awad (2007) this result may be due to that during warm seasons the climate temperature is suitable for the life cycle of the parasites.

The clinical signs observed in heavily infected O. niloticus with ectoparasitic monogenea usually results in excessive mucus, loss of appetite, sluggish movements and the fish swam near the surface of water. The infested fishes showed increased breathing frequency, and the gill covers were stretched open widely. The gills were pale in some fishes, hyperemic in others and covered with mucus secretions forming cloudy film of slime. Infested fishes were anemic and the fins were ragged with partial detachment of scales. Microscopically, the monogeneans were found attached to the gill filaments. Such obtained results are nearly similar those of theseclinical signs may be attributed to the organs of attachment and mode of feeding of monogenia. The hamuli, marginal hooklets, suckers and clamps of monogeneans all have direct contact with host tissue and may afflict mechanical damage directly. These result supported those of Attia (2004); Woo (2006); Awad (2007) and Abd El-Ghany et al. (2009).  

The results noticed in this work proved that mixed infection of Trichodina and monogeneans were more prevalent. However, monogenea can serve as vectors for viral and bacterial pathogens of fish which provided a portal of entry for invasive bacteria due to damage of the fish epithelium. Also, because they propagate and transferred rapidly between fishes under farming conditions this may be leading much damage to high morbidity and mortality. These were proved previously by Cusack and Cone (1986).

The results of parasitological examination and identification revealed the presence of three types of digenean encysted metacercariae in examined O. niloticus. Centrocestus sp. represented the highest one at percentage (5.9%) of total parasitic disease in examined fish. The results proved seasonal fluctuations among the examined fish. It was especially high during the warm season of the year in fresh water due to active shedding of cercariae. Similar observation was reported by Paperna, (1996); El-Nobi, (1998); Attia, (2004) and Awad, (2007), the last author recorded that encysted metacercaria (Centrocystus formosanus) were found in gills of black carp at the prevalence of 100% along the year. This may be attributed to the high abundance of the intermediate snail host in the examined earthen ponds in which black carp were reared.

The results revealed thatCentrocestus species encysted metacercariae were detected in the gills of O.niloticus without any clinical signs in low infection rate. While the main clinical signs observed in heavily infested fish were respiratory distress, decreased feed intake and increased mucus secretion. The infected area showed inflammatory reaction especially in early infestation or when the cyst, was ruptured. This condition might represent a response to prolonged irritation by the parasite and its toxic product or the hyperplasia of chondrocytes or may be due to decrease the oxygen tension in the affected area (Attia 2004; Awad 2007 and Abd El-Aal et al., 2008).

Low infection rates were recorded for Clinostomum and Euclinostomum spp. at percentage (5.2%) and (4.9%) respectively. Results of the previous related studies, Abd-Alkareem (2000) found the prevalence rate was (4.4%) and (0.5%)respectively.Yimer (2000) found (9.09%) of Clinostomum and only 1 Euclinostomum species from all investigated fish. who found that 34% and 29% of this fish species were infected with these types of metacercariae, respectively; and that of Abd El Rahman (2005), who found that 45% and 11.8% of O. niloticus were infected with Clinostomum species and Euclinostomum species metacercariae, respectively. This can be refered to the difference in sampling periods and the ponds from which the examined fishes were obtained. The results also revealed that the seasonal prevalence of Clinostomum species and Euclinostomum species were during winter and spring seasons respectively. This may be due to the suppressed immunity of the cultured fish during cold temperature. It is worth to mention that extrapolation of seasonal variations from direct counts of larvae in fish since such infections normally persist for over 12 months, these results are in line with several authors as El-Nobi (1998); Attia (2004) and Woo (2006).

The results also revealed that the main clinical signs of Clinostomum infection observed in moderate and higher intensity of infection which showed debility, retarded growth, excessive mucus secretion, increased in the opercula movement and protrusion of operculum. Yellowish to orange color, pea like cyst were found scattered on skin and in bronchial cavity. In case of heavy infection, cysts were arranged in agape-like cluster. In dead tilapias, larvae encysted and moved over the mesentery and on skin. On the other hand, pathological impact of infection with Euclinostomum was not often obvious and depended on their number. Few cases showed related growth, abdominal distention and reddish ascetic fluid in the abdominal cavity, which could be attributed to functional damage of kidney. Euclinostomum was found distributed along the kidneys of infected tilapia in the form of grayish cyst. Congestion of infected kidneys was also observed. The present data werein agreement with previous observations (Paperna 1996; Abd El Rahman 2005; Abd El-Aal et al., 2008).

             The results of parasitological examination and identification revealed the presence of Contraceacum nematode at percentage (3%), it was higher during autumn (Abu-El-Ezz, 1988)While, Varjabedian (1993)mentioned that the majority of nematodes, reached their peak of infection during spring and summer, Aloo (2002)mentioned that no seasonal changes was recorded in infection level. This phenomenon may be attributed to many factors interacted together such as water temperature, fish host length, sex and feeding habits and propagation of the intermediate hosts and each nematode parasites had its own season of prevalence that varied in different hosts.

              The results also revealed that the main Clinical signs of Clinostomum infection observed the located of Contracaecum species larvae in the abdominal cavity causing abdominal distention. Also, the effect results from mechanical damage to the internal organs and production of toxic substances. Larvae penetrate in to the intermediate host's body through the wall of digestive tract and lead to its death, inflammatory response of digestive tract and pressure on internal organs. Intestine was voided from food. In severe infections, low body gain is common sign. Nematodes may not even show signs of illness, but they often have reduced reproductive capacity. The present result nearly coincided with that recorded by Abu-El-Ezz (1988); Moravec (1994) and Barson (2004).

Acanthocephala tilapiae was observed in a high prevalence as parasitic infection that varied from the other parasites and another of all host species analyzed (Ayotunde 2007; Omar et al., 2008). The results of parasitological examination and identification revealed the presence of Acanthocephala tilapiae at percentage (7.5%) that was prevalent during autumn. WhileGihan (1999)recorded that the highest rate was in spring followed by autumn. (Omar et al., 2008) showed that high prevalence of Acanthocephala tilapiae was observed in all host species analyzed. They mentioned that worms appear to be recruited in the summer, develop and mature through the winter, and reproduce sexually in late winter and spring. In addition Differential exposure to parasites and host susceptibility may explain the marked variation in parasite abundance among hosts. There were no clinical signs in light infection with Acanthocephala tilapiae parasite. While, there were signs of emaciation and congested intestine in heavy infection. These findings associated with infection are in harmony with those recorded by Kabata (1985). In the final host the Pathogencity of acanthocephala is related to the mode of attachment of the parasite species and to the systematic affiliation of the host. Long necked acanthocephalans which deeply penetrate into or through the intestinal wall with their presoma are more pathogenic than species with a short neck and a shallow mode of anchorage (Kabata 1985; Woo 2006).

              The results of parasitological examination and identification revealed the presence of Ergasilus sp. at percentage (4.9%), a higher prevalence was recorded in spring. While Mostafa et al. (1991)andMolnar et al. (1997)recorded that high level of Ergasilus infestation in summer and autumn seasons. This could be related to the environmental conditions and the presence of the infective copepodids.

The results also revealed that  the main clinical signs of Ergasilus infection were pale gills in mild to moderate infestation, erosion, degeneration and necrosis in gill filaments in sever cases, gills damage that resulted in loss of surface area for respiration and lead to suffocation, particularly at high water temperature and Excessive accumulation of mucus in the gill, This in turn may result in reduced growth rates and greater mortality (Grabda 1991; Mostafa et al., 1991; Molnar et al., 1997; Abd El-Rahim 1998; Woo 2006).

The results of mycological examination and identification revealed the presence of mycotic infection at percentage 8% of total diseased examined fish. This may be due to the fact that most of the serious mycotic pathogens are mostly secondary in nature and need great stress on such cultured fishes in order to find the porta of entery for producing a disease. The stress factors usually include environmental fluctuations and improper management practices with poor water quality (Udomkusonsri, 2003). The wet mounts from skin and gill tissues of infected O.niloticus showed the presence of large non-septet tubular cells. Spores of the fungi attach to injured or unhealthy tissue and establish a colony (mycelium). Microscopically, the identified agent was Saprolegnia diclina which found among O. niloticus in the examined semi-intensive farms in the winter season specifically. This disease (Saprolegniosis) is a serious disease that affects all species and ages of freshwater fishes and can also affect many estuarine fish (Woo and Bruno 1999; Aly and El-Ashram 2000; Polacheck 2007; Woo 2007). The low temperature constitutes a great stress factor affecting the health condition of O.niloticus and renders them prey to Saprolegnia fungus.(Mesallhy 1994; Naguib 1994; Aly et al., 1996; Aly and El-Ashram 2000; Polacheck 2007; Osama et al., 2008; Zaki et al., 2008).

The results revealed that the signs of Saprolegnia infection including general external signs which appeared anywhere on the body surface, fins, eyes and gills. The infected fish often develop grayish-white cotton-like growths on skin, fins, gills and eyes. The diseased fish showed hyperactivity and obvious evidence of irritation such as rubbing against the sides or bottom of tank which may be attributed to the inflammatory reactions, erythema at the base of fins, ulceration of the skin, this may be due to toxin secreted by saprolegnia hyphae, oomycete or zoospores due to fungal cells (hyphae) invade the injured tissue from which they infect surrounding healthy tissue. Also, loss of equilibrium with nervous signs, blindness, and enlargement of abdominal were also recorded. Evidence of respiratory distress was recorded. The gills appeared pale in colour with profuse mucus and covered by tufts of fungi. The same findings were also recorded by (Ruth 1990; Aly and El-Ashram 2000; Udomkusonsri. 2003; Osama et al., 2008; Zaki et al., 2008). The observed pale color of gills could be due to the coating of the gills by profuse mucus, necrotic debris and secondary mycosis (Plump 1997).

In conclusion, the result of this work proved the wide spread of parasitic and to lesser extent fungal infections in the O. niloticus cultured under the semi-intensive system in the earthen ponds. The wide spread of such pathogens are due to the improper management practice. The results revealed that summer and late spring are critical times for the occurrence of parasitic diseases. At the same time the problem of saprolegniosis in winter season represents an important problem for     O. niloticus semi intensively cultured in earthen ponds, especially after the days of severe cold weather fronts. A sever economical impact could be waited from the heavy fish mortality and high fish loss, so proper managemntal practices should be applied during these critical times that render O. niloticus susceptible for the occurrence of such diseases.  Raising of water column, continuous flow of water in the days of sever cold weather as well as  incorporation of immune-stimulant in fish ration could aid in avoiding of such loss by raising the fish resistance against these parasites and fungal agents.

 

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