The infection of freshwater fishes with three species of Henneguya in Qena, Upper Egypt

It is so for the first time to do general survey of protozoan parasites that infect freshwater fishes in Qena Province. The total number of fishes examined were 581 belonging to 10 families represented by 14 Species during the period from January 2006 to May 2008 . Three species of Henneguya were recorded from two species of fishes. The first species is suggested to be a new one called Henneguya mandouri sp.nov. The second species is Henneguya assuiti, Mandour et al., 1988 and the third one is Henneguya nilotica, Marwan, 1998.


INTRODUCTION
The genus Henneguya Thelohan, 1892 is one of the largest nine genera in the family Myxobolidae.The present parasites are placed in the genus Henneguya according to the morphological characteristics as described by Kudo (1966).In Africa, about 100 species of Myxosporean parasites were currently known (Fomena and Bouix, 1997).Examination of some freshwater fishes from Chari and Logone Rivers of Chad (Central Africa) revealed the presence of Myxosporean species of the genus Henneguya Thelohan, 1892.Henneguya ghaffari Ali, 1999 was recovered from gills and intestine of the Nile perch Lates nilotica from Chad in Senegal.Obiekezie and Schmahl, (1993) reported that Henneguya latero capsulate in the skin of cat fish (Clarias gariepinus) and Heterobranchus bidorsalis in Nigeria.In Egypt, the River Nile fish were first examined for Myxosporean parasites by Fahmy et al. (1975).then by Abed (1987), Iman et al, (1987), Abdel-Ghaffar et al. (1995a, b, 1998), Ali (1998Ali ( , 1999Ali ( , 2000)).Current and Janovy (1976) studied the ultrastructure of inter lamellar Hennguya exilis in the channel cat fish.Abdel Ghaffar et al, (1995) reported some Henneguya spp, from cat fishes.Mandour et al. (1988) described Henneguya assiuti collected from gills and respiratory trees of cat fish (Clarias lazera) .Ashmawy et al, (1989) reported the occurrence of Henneguya branchialis in the gills, intestine and secondary respiratory organ of Clarais lazera collected from Behera water bodies.Abdel Ghaffar and his colleges in 1995 described the same parasite in Giza Province.Abed, (1997)

MATERIALS AND METHODS
Several species of freshwater fishes in Qena City were captured during the period from January 2006 to May 2008.They were collected from different freshwater bodied in Qena, including Qus, Qeft, El-Tramssa, El-Maana, Dandara, Dishna and Nagh-Hamadi.
Fishes were brought alive to the laboratory and examined for Henneguya species by a light microscope.Impression smears were made from gills, fins, liver; kidney, gonads and intestine then were fixed and stained with Giemsa's stain.

Scanning electron microscopic studies
Cysts were fixed in phosphate buffer 3.5% gluteraldehyde at pH 7.4 for 3-4 hours, post fixed in 1% Osmium tetraoxide for 2 hours, washed in Na-phosphate buffer (pH 7.4).Fixed cysts were gently crushed in phosphate buffer to dislodge the spores that were transferred to glass cover slips which were mounted on copper sluds, gold coated and examined.

RESULTS
During the present study, 3 species of Henneguya were identified as following: 1-Henneguya mandouri sp nov.
The cysts of this parasite were collected from the middle and the base of the gill filaments of 32 out of 40 Lates niloticus fish (80%).With light microscopic level only large forms of spores were detected which have an ovoid body with a rounded anterior end Fig ( 1,2) which measured (11-13) x (6.0-7.5)µm, the tails were composed of 2 bifurcated filaments, measuring about (40-53) µm in length (Table 1).The polar capsules were equal in size and pyriform, situated at the anterior end, measuring about (3.1-4.3 x 1.5-2.2) µm.The ratio of polar capsule / spore length were (0.23-0.36) µm, the total length of spores (51-66) µm, length / posterior process ratio was (0.2-0.3) µm.Polar filament coils are (3-5) as shown in fresh spores (Fig. 3) the length of polar filament is (12.5-23)µm (Fig. 4).The sporoplasm granulated, occupied 75% of the spore cavity with one iodinophilous vacuole and 2 clearly visible sporoplasmic nuclei (Fig. 5).This species of Henneguya was studied by SEM which revealed that the plasmodia were whitish, ellipsoidal,\ and oval in shape, measuring (0.2-1x o.1-0.5)mm (Fig. 6), the spores were resolved more clearly by SEM, and revealed that the surface of spores were smooth (Figs.7-9) the two caudal appendages are equal, some spores are joined and appear as one appendage (Fig. 8), but in others were composed of 2 bifurcated filaments (Figs.9,10).Enlarged spore showed the site of the polar capsules and the polar filaments, the cyst wall (Cw), suture (S) the anterior groove (AG), the posterior groove (PG), and the edges of the spore, (E) as in (Fig. 11).

2-Henneguya assiuti Mandour et al. (1988):
This parasite was collected from gills of Clarias gariepinus, where 7 out 28 fish examined (25%) were infected with the parasite.Cysts are oval to round in shape, yellowish in colour, filled with fluid containing a suspension of mature spores which appear milky white by naked eye.Mature spores were pyriform in shape, measuring about (9.8-13.5 x 4.2-5.5)µm.The posterior end was prolonged into more or less extended process to form 2 often equal caudal appendages, measuring about (30.4-37.2) µm., the polar capsules appear equal in length (7.2-7.9 x 1.5-2) µm as in Fig (12) or The infection of freshwater fishes with three species of Henneguya in Qena 13 unequal especially in length (Fig. 13).Polar filaments are coiled (8-9 coils) inside the polar capsules and the iodinophilic inclusions are seen in fresh spores stained with iodine as in Fig. ( 14).

3-Henneguya nilotica Marwan (1998):
This parasite was observed in 2 out 28 Clarias lazera (7.14 %).The spherical cysts were observed on the tips of the secondary respiratory organ, measuring (1.0-3.0)mm., mature spores are ellipsoidal with a blunt anterior end and a tapering posterior end, measuring about (13.5-15 x 4.5-5.6)µm, the spore is surrounded by a homogenous dense sheath when the spores stained with Giemsa's stain.Sheathed spores have dissimilar staining reaction that may take deep violet, purple or blue colour Figs.(15,16).When the outer sheath was ruptured, the parasite was stained dark blue, while the sheath took a light violet color as in Figs.(17,18).Sheathed spores had the same body length as uncoated ones, but the later appeared broader reaching (4.5-5.6)µm, while their posterior tails were shorter reaching 19 in length, the two processes were seen adhering to each other as in Fig. (18).Each spore had two equal polar capsules which were elongated, thin and occupy approximately half the body length, they measured (6.5-7.9 x 1.5-2.5)µm.Polar capsules were seen adhering to each other at the anterior end of ‫ا‬the spore and separated a part in the lateral sides of the spore.Spores treated with Lugol's Iodine solution showed one or two iodinophilous vacuoles and 8-9 coils of polar filaments as in Fig. ( 14).These vacuoles were irregular in shape, situated in-between the two polar capsules, directly behind them or at the posterior third and adhering to the lateral side of the spore.

1-Henneguya mandouri sp. nov., present species from Bagrus bayad:
According to the available literature, 4 species of Henneguya have been reported namely Henneguya assiuti Mandour et al., 1988 Mandour et al., (1988) described Henneguya assiuti from Clarias lazera in Assiut Province by a prevalence of 21.02 % while in present study the same parasite was collected from the same host in Qena Province where the incidence of infection was is 25%.The spores described above are identified as Henneguya assiuti in account of the marked similarities between the spores of those described by Mandour et al., (1988) and Abed (1997).Mandour et al. (1988) reported that cysts occurred in gills and secondary respiratory organ; while Abed (1997) and Marwan (1998) and in the present work cysts were reported from the gills only Henneguya assiuti may be related to Henneguya sp.Abed, (1987) which was described from the gills of Lates niloticus due to the length of the spore and the long posterior processes, but differs in having bigger polar capsules reaching (7-9) µm in length, in comparison with the parasite of Lates niloticus which measures (3-4) µm.Also, the polar capsule of Henneguya assiuti has 8-9 polar filament coils while in Henneguya sp.there are 3-5 polar filaments coils.The total length of Henneguya sp. is larger than the present one.

3-Henneguya nilotica Marwan, 1998 from Clarias gariepinus:
Marwan (1998) described Henneguya nilotica as a new species from Clarias lazera in Assiut Province where the parasite was obtained from the secondary respiratory organ of 18.18 % of specimens collected from Assiut Province.During the present study the incidence of infection was 7.14 % in the same host.The spores that described above are identified as Henneguya nilotica on account of the marked similarities between the spores of the parasite with those described by Marwan (1998).
Table (2): shows a comparison between some previously described Henneguya species in the two fresh water fish species: Lates niloticus and Clarias lazera.
It is clear that the spores in the two descriptions are closely comparable both in their structure and measurements.Hennguya nilotica is similar to Henneguya testicularisas that is a sheathed myxozoan (Azevedo et al., 1997) which infects the testis of the Amazonian fish Moenkhausia oligolepis, but this parasite is unique in its site of infection, locality and host.Mature spores of this parasite have short caudal processes compared with the present sheathed species, being 13-14.5 µm.
Table (1): Showing the measurements by µm. of the three described species of Henneguya in the present work.

Table (2):
A comparison between some previously described Henneguya species in the two freshwater fish species: Lates niloticus and Clarias lazera, where: H-Henneguya, SBL-spore body length, PCL-polar capsule length, PFC-polar filament coils, PP-posterior process, TBL-total body length.And all measurements by µm.

Figs
Figs. (1-5): Light photographs of mature spores of Henneguya mandouri sp.nov. in Lates niloticus; Figs.1,2:Spores stained with Giemsa's stain, showed the ovoid body of the spores with rounded anterior end, polar capsules, and also showed the caudal process length, Fig.3: Fresh spore by using Lugol's iodine solution showed the sporoplasm, Fig.4: Spore stained with Giemsa's stain, showed the polar filament when it is extruded, Fig.5: Spores stained with Giemsa's stain showed the presence of two sporoplasmic nuclei.

Figs
Figs. (6-11): SEM micrograph of mature spores of Henneguya mandouri sp.nov; Fig. (6): SEM micrograph showing the site, size, and shape of Henneguya mandouri sp.nov.(Present parasite) cyst on gills of infected Lates niloticus fish, Fig. 7: SEM micrograph of Henneguya mandouri sp.nov.showing irregular masses of spores embedded in plasmodia, Fig. 8 : Showed that the two caudal appendages are joined with each other and appear as one appendage, Fig. 9: Showed that the caudal appendage composed of two bifurcated filaments, where the separation is near to its end, fig.10: The separation of caudal appendage is in the anterior part of it.Fig. 11: Is an enlarged spore showing the cyst wall (CW), suture (S), the anterior groove (AG), the posterior groove (PG), and the edges of spore (E).
studied the ultra-structure of the spores of Henneguya assiuti in the gill filaments and respiratory trees of the