MYXOSPORIOSIS IN SEA MULLET SPECIES AT PORT-SAID, EGYPT ABSTRACT

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1 SCVMJ, VIX (1) MYXOSPORIOSIS IN SEA MULLET SPECIES AT PORT-SAID, EGYPT Eissa, I. A. M., Badran, A. F., Amina A. Dessouki* and Engi, A. El- Raziky** Dept. of Fish Diseases and Management, Fac. of Vet Med. Suez Canal University * Dept. of Pathology, Fac. of Vet Med. Suez Canal University ** Authrity of Veterinary Service, Port Said branch ABSTRACT The present investigation deals with myxosporiosis in both wild sea mullet Mugil cephalus and M capito. They were collected from Suez Canal area at Port-Said. The clinical picture revealed the presence of pale, white to pink, raised lesions on the scales and fins with large whitish, flat nodules on the surface of the apical area of the scales. In M. capito no lesions observed externally. Such nodules were also isolated from M cephalus from gill arch, liver and intestinal mesentery while in M capito, it was only found in intestinal mesentery. Based on the morphological and parasitological examinations, such isolated metazoan parasites were belonged to Cnidarian group, genus Myxobolus. The total prevalence of both fishes was 14% where M cephalus was prevailed (21%) than M. capito (7%) and seasonal prevalence revealed that the peak was in spring and there was no infection in summer. Besides, the histopathological alterations of the gills of infected M cephalus showed ballooning degeneration of the epithelial cell lining with adhesion and leukocytic infiltration mainly lymphocytes. The liver showed multiple cysts, each formed of thin fibrous capsule and contained numerous myxosporidian spores. Hepatic tissue had necrosis, degeneration and atrophy of hepatocytes. INTRODUCTION Nowadays, marine fishes take the upper hand as a good animal protein for man, animal and birds. On the other hand, like other species, they considered as a culture for parasites which affect this wealth or transmit it to man, animal and birds. Parasitic infestations represent the majority of the known infectious diseases affecting fish. Marine parasites include various types causing mortality, deformity, weight loss and different clinical abnormalities among the affected fish (Eissa, 22). Mullets are one of the most important resources of the Egyptian lagoons and represent a high percentage of fishing catches. These fishes are appreciated not only for their flesh but also for their female gonads. In Egypt, they have been found to be infected with several diseases from

2 222 Eissa et al., which myxosporiosis where myxosporeans show several localizations internally or exte-rnally (Faye et al. 1999, Sihem Bahri et al. 23, Diamant et al. 25 and Eiras et al. 27). So, this work was planned to investigate the clinical picture, total and seasonal prevalence, parasitological and histopathological examinations in both sea mullets Mugil cephalus and M capito. MATERIALS & METHODS Fish: A total number of 2 alive fish represented as 1 Mugil cephalus and 1 M capito were randomly and seasonally collected from Suez Canal area in Port Said throughout the whole period of study (28). The body weight of the examined fishes ranged from 1 to 15 g. Clinical examination: Alive fish specimens in the wellprepared glass aquaria were clinically examined for detection of any macroscopic lesions according to Conroy and Herman (1981). Postmortem examination: The fish specimens were subjected to postmortem examination for detection of any internal abnormalities (Noga, 1996). Parasitological examination: Skin and scales of freshly sacrificed fishes were examined with naked eye and magnified lens for searching about nodules. The operculi were removed to expose the gills. Also, the musculature, gastrointestinal tract, liver, kidney, spleen, heart as well as gall bladder and intestinal m- esentery were examined. They were placed on dissecting dish, scraped and then the nodules transferred to slides with a drop of distilled water. Myxosporial nodules were collected from tissue, crushed gently and spread thinly on a slide, cover slide with a 2% silver nitrate solution for 8 minutes, then rinsed in distilled water, placing slide face up in a dish of distilled water and exposed to direct sun light for 2 hours and allowed to dry and mount with neutral Canada balsam (Lucky, 1977). Histopathological examination: Specimens from gills and liver were fixed in 1% formalin and prepared for histopathological examination using H&E stain according to Carleton (1976). RESULTS Clinical examination: The lesions examined of the naturally infested Mugil cephalus were represented as pale, white to pink, raised lesions on the scales and fins large whitish, flat cysts on the surface of the apical area of the scales (Photo.1). In M. capito no lesions observed on the fishes. Postmortem examination: The parasites formed round or slightly oval whitish cysts appearing with different size on the surface of the organ. The cysts were collected from M. cephalus from different locations [on the distal part of scales, at the base of gill arch (Photo.2), liver (Photo.3), intestinal mesentery] and

3 SCVMJ, VIX (1) one were collected from M.capitus [intestinal mesentery (Photo.4)]. Parasitological examination: Microscopical examination revealed round spores in frontal view and oval in lateral one containing two polar capsules isolated from distal part of scales (Photo.5), from the base of gill arch (Photo.6), from the intestinal mesentery (Photo.7). Based on the morphological and parasitological examinations, such parasites were belonged to class Cnidaria, genus Myxobolus. Total prevalence of myxosporiosis: The total prevalence was (41%) of both examined fishes, represented as (14%) in M. cephalus and (7%) in M. capitus. (Table1, Fig.1). Seasonal prevalence of myxosporiosis: Table (2) and figure (2) shows that the seasonal prevalence of myxosporiosis among M. cephalus and M. capitus was recorded as 4 and 28% in autumn, 24 and % in winter, 56 and % in spring respectively while in summer there were no infected cases. Histopathological findings: Liver of M.cephalus infested with myxosporidian cyst showed multiple cysts, each cyst formed of thin fibrous capsule and contained numerous myxosporidian spores. Hepatic tissue had necrosis, fibrosis, degeneration and atrophy of hepatocytes. The majority of the inflammatory infiltrates consisted of macrophages and lymphocytes (Photo.8). Gills of M. cephalus infested with myxosporidian cyst showed ballooning degeneration of the epithelial cell lining along with adhesion and leukocytic infiltration mainly with lymphocytes and few macrophages (Photo. 9). Table (1): Prevalence of Myxosporiasis among the examined fishes. Fish species No. of No. of examined fish infested fish % Mugil cephalus Mugil capitus Total

4 no. of examined fish 224 Eissa et al., No. of examined fish No. of infected fish 5 M.cephalus M. capitus Total fish species Fig. (1): Prevalence of myxosporiosis among examined fishes Table (2): Seasonal prevalence of myxosporiosis among examined fishes. Season Autumn Winter Spring Summer Total Fish spp. N % n % n % n % n=2 % Mugil. cephalus (n = 25 ) Mugil. capitus (n = 25 ) Total (n = 5 )

5 SCVMJ, VIX (1) M.cephalus M.capitus 6% 5% 4% 3% 2% 1% % 56% 24% 4% % Autumn Winter Spring Summer Fig. (2): Seasonal prevalence of myxosporiosis among examined fishes. (1) (2) Photo.(1): Mugil cephalus showing white cysts on the scales. Photo.(2): Large white cyst on the gill arch of Mugil cephalus.

6 226 Eissa et al., (3) (4) Photo.(3): liver of Mugil cephalus with myxosporidian cyst. Photo.(4): Intestinal mesentry of M. cephalus with myxosporian cyst. (5) (6) Photo. (5): Myxosporia.spp isolated from scales of M. cephalus (X1) Photo. (6): Myxosporia. spp isolated from gill arch of M. cephalus (X4)

7 SCVMJ, VIX (1) Photo. (7): Myxosporia.spp isolated from intestinal mesentery of M.capitus (X1) (8) (9) Photo. (8): Liver of M.cephalus infested with myxosporidian cyst showing large cyst contained the parasite with two polar capsules along with degeneration of hepatocytes.h&e.x4. Photo.(9):Gills of M. cephalus infested with myxosporidian cysts showed a ruptured myxosporidian cysts containing the parasites with bipolar capsule, along with ballooning degeneration, necrosis of epithelial cell lining the lamellae. H&E.X 4.

8 228 Eissa et al., DISCUSSION Fish myxosporidiosis has an economical impact as infested fish are considered unmarketable due to the presence of external or internal nodules. Moreover, some authors declared that myxosporidian cases cause many trouble in humans (Boreham, et al. 1998, Ligia et al.21 and Moncada, et al. 21). The present study revealed the main clinical signs in the infested Mugil species with myxosporiasis as pale, white to pink, raised lesions on the scales and fins large flat cysts developed on the surface of the apical area of the scales which are white to pink, raised lesions on the scales. These most common features of clinical signs agree with the finding of Egusa et al. (199) and Rothwell et al. (1997). In the present study, some mullet fishes showed small white cysts found in gill arch, liver and intestinal mesentery. Diamant et al. (25) found that Kudoaiwatai n. sp. established multiple site infections, most commonly in the muscles and intracranial adipose tissue of the brain and eye periphery. Other sites were subcutaneous adipose tissue, nerve axons, mouth, eye, mesenteries, peritoneum, swim bladder, intestinal musculature, heart, pericardium, kidney and ovary. Also, these results agree with the finding of Brickle et al. (26) and Eiras et al. (27). The examined spores were round in frontal view and oval in lateral view and contain two polar capsules with large sporoplasm. Based on the morphological and parasitological examinations, such isolated metazoan parasites were belonged to Cnidarian group, genus Myxobolus according to the recent taxonomy described by the phylogeny tree of Smothers et al. (1994), Kent et al.(21), Fiala (26), Woo (26) and Dykova et al. (27). The present study showed that the prevalence of Myxosporiasis was 14% (21% for M. cephalus and 7% for M. capitus). These results nearly agree with that reported by Abd El-Aal et al. (21) who reported a prevalence of 18.64% in marine and brackish fish species collected from Port Said and Gbankoto et al. (21) who reported a prevalence of 18.3% in Sarotherodon melanotheron melanotheron. While this result disagree with that recorded by Sitja-Bobadilla and Alvarez- Pellitero (1993) who found the prevalence of infestation was 62.5% for Mugil capito, 7% for Mugil cephalus from Mediterranean Sea, Kalavati and Anuradha (1995) who recorded that the prevalence of parasitic infestation rate in Mugil cephalus was 53.7% from brackish water, Faye et al. (1997) who found that the prevalence was 2.88% in M. cephalus and 17.64% in M. curema due to infection of heart by Myxozoa and Faye et al. (1999) who found that the prevalence was 22.2% in M. curema and 2% in M. cephalus due to infection of gills by Myxozoa. This variation may be attributed to the fish species, difference in locality from which fish sample obtained, as the infestation with parasites is the result of interplay between environment, parasite and fish. This means

9 SCVMJ, VIX (1) that the occurrence of parasite may depend on the presence of intermediate hosts, the degree of water pollution and the state of health or powers of resistance of the host (El- Matbouli and Hoffmann 1989 ). Regarding to the seasonal prevalence of Myxosporiosis, the peak of Myxosporial infection was in spring 28%, while there was no infection in summer. These results were in agreement with Rothwell et al. (1997) as they recorded the peak of Myxosporial infection in Sea mullet, M cephalus in spring. In case of M. cephalus the peak of infection was in spring 56% followed by winter 24% then autumn 4% and summer %. In case of M. capitus the infection was observed only in autumn 28%.This may be due to change in the resistance of the host in relation to water temperature. The histopathological examination of the gills of M. cephalus infested with myxosporidian cyst showed ballooning degeneration of the epithelial cell lining along with adhesion and leukocytic infiltration mainly with lymphocytes and few macrophages. The liver in infested M. cephalus showed multiple cysts, each cyst formed of thin fibrous capsule and contained numerous myxosporidian spores. Hepatic tissue had necrosis, degeneration and atrophy of hepatocytes. This result agreed also with that obtained by Bucher et al. (1992) and Yokoyama et al. (2) who found relatively large and often multiple plasmodia resulted severe distension and proliferation of the bile ducts, attenuation and flattening of their epithelium, slight fibrosis was present in the pericholangiolar area. REFERENCES Abd El-Aal, A.A; Badawy, G.A. and Gattas, M.W. (21): Light and electron microscopic studies on myxosporean protozoans of some marine fishes with description of two new species. Assiut- Vet. Med.J., 44(88): Boreham, R.E.; S. Hendrick, P.J. O'Donoghue, and D.J. Stenzel. (1998). Incidental finding of Myxobolus spores (Protozoa: Myxozoa) in stool samples from patients with gastrointestinal symptoms. Journal of Clinical Microbiology 36: Brickle, P.; Chaganti-Kalavati and Mac- Kenzie, K. (26): Henneguya shackletoni sp. nov. (Myxosporea, Bivalvulida, Myxobolidae) from the Falklands mullet, Eleginops maclovinus (Cuvier) (Teleostei, Eleginopidae) in the Falkland Islands. Acta- Parasitologica. 51(1): Carleton, E.A. (1976): Carleton s histological teqnique 4 th ed. Oxf. Univ. Press, New York, Tronto. Conroy, D.A. and Herman, L.R. (1981): Textbook of fish diseases. T.F.H. publ, West Sylvania. Diamant, A.; Ucko, M.; Paperna, I.; Colorni, A. and Lipshitz, A. (25): Kudoa iwatai (Myxosporea: Multivalvulida) in wild and cultured fish in the Red Sea :

10 23 Eissa et al., redescription and molecular phylogeny. J. Parasitol. 91(5): Dykova, I;, T. Tyml, I. Fiala and J. Lom. (27): New data on Soricimyxum fegati (Myxozoa) including analysis of its phylogenetic position inferred from the SSU rrna gene sequence. Folia Parasitologica 54: Egusa, S.; Maeno, Y. and Sorimachi, M. (199): A new species of Myxozoa, Myxobolus episquamalis sp. nov. infecting the scales of the mullet, Mugil cephalus L. Gyobyo-Kenkyu Fish-Pathology. 25(2): Eiras, J.C.; Abreu, P.C.; Robaldo, R. and Pereira Júnior, P.J. (27): Myxobolus platanus n. sp. (Myxosporea, Myxobolidae), a parasite of Mugil platanus Günther, 188 (Osteichthyes, Mugilidae) from Lagoa dos Patos, RS, Brazil. 59(4): Eissa, I. A. M. (22): Diseases caused by Microsporidia. Parasitic fish diseases n Egypt. Dar El-Nahda El-Arabia. Publishing, First Edition El-Matbouli, M. and Hoffmann, R. (1989): Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitol.Res. 75: Faye, N.; Kpatcha, T.K.; Fall, M. and Toguebaye, B.S. (1997): Heart infections due to myxosporean (Myxozoa) parasites in marine and estuarine fishes from Senegal. Bulletin of the European Association of Fish Pathologists. 17(3/4): Faye, N.; Kpatcha, T.K.; Debakate, C.; Fall, M. and Toguebaye, B.S. (1999): Gill infections due to myxosporean (Myxozoa) parasites in fishes from Senegal with description of Myxobolus hani sp. n. Bulletin of the European Association of Fish Pathologist. 19(1): Fiala, I. (26): The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. International Journal for Parasitology, 36, Gbankoto, A.; Pampoulie, C.;, Marques, A. and Sakiti, G.N. (21): Myxobolus dahomeyensis infection in ovaries of Tilapia species from Benin. J.of Fish Biology. 58, Kalavati, C. and Anuradha, I. (1995): A new myxosporean, Bipteria indica sp.n. (Myxospora: Sinuolineidae) from the gall bladder of the striped mullet, Mugil cephalus Linnaeus. Acta-Protozoologica. 34(4): Kent, M. L., B. Andree, J. L. Bartholomew, M. El-Matbouli, S. S. Desser, R. H. Devlin, S. W. Feist, R. P. Hedrick, R. W. Hoffmann, J. Khattra, S. L. Hallett,. J. G. Lester, M. Longshaw, O. Palenzeula, M. E. Siddall, and C. Xiao. (21). Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48: Ligia I. Moncada, Myriam C. López, Martha I. Murcia, Santiago Nicholls, Frecia León, Olga Lucia Guío, and Augusto Corredor (21): Myxobolus sp., Another Opportunistic Parasite in Immun-

11 SCVMJ, VIX (1) osuppressed Patients? Journal of Clinical Microbiology, May 21, p , Vol. 39, No. 5. Moncada, L.I., M.C.Lopez, M.I.Murcia, S. Nicholls, F. Leon, O.L. Guio and A. Corredor. (21). Myxobolus sp., another opportunistic parasite in immuno-suppressed patients? Journal of Clinical Microbiology 39: Noga, E.J. (1996): Textbook of "Fish disease" Diagnosis and treatment. Walworth Publishing Co., U S A. Pickering, A. D. and Pottinger, T. G. (1989): Stress responses and disease resistance in salmonid fish: effects of chronic elevation of plasma cortisol levels. Fish Physiol. Biochem. 7: Rothwell, J.T.; Virgona, J.L.; Callinan, R.B.; Nicholls, P.J. and Langdon, J.S. (1997): Occurrence of cutaneous infections of Myxobolus episquamalis (Myxozoa: Myxobolidae) in sea mullet, Mugil cephalus L, in Australia. Aust Vet J.;75(5): Sihem Bahri, Karl B., Andreeh and Ronald, P. Hedrick (23): Morphological and Phylogenetic Studies of Marine Myxobolus spp. from Mullet in Ichkeul Lake, Tunisia. J. Eukrriyot. Micmbiol. 5(6): Sitja-Bobadilla, A. and Alvarez-Pellitero, P. (1993): Zschokkella mugilis n. sp. (Myxosporea: Bivalvulida) from mullets (Teleostei: Mugilidae) of Mediterranean waters: light and electron microscopic description. Journal of Eukaryotic Microbiology. 4(6): Smothers, J. F.; C. D. von Dohlen, L. H. Smith, Jr. and R. D. Spall. (1994). Molecular evidence that the myxozoan protists are metazoans. Science 265: Woo, P.T. (26). Fish diseases and disorders, (volume 1), CABI Publishing. Yokoyama, H.; Inoue, D.; Sugiyama, A. & Wakabayashi, H. (2). Polymerase chanin reaction and indirect fluorescent antbody technique for the detection of Kudoa amamiensis (Multivalvulida: Myxozoa) in yellowtail Seriola quinqueradiata. Fish Pathol., 35:

12 232 Eissa et al., الملخص العربى إسماعيل عبد المنعم عيسى أحمد فكرى بدران أمينة دسوقى* إنجى الرازقى** قسم أمراض ورعاية األسماك كلية الطب البيطرى جامعة قناة السويس *قسم الباثولوجيا كلية الطب البيطرى جامعة قناة السويس **هيئة الخدمات البيطرية فرع بورسعيد تم تجميع 411 سمكة بورى و 411 سمكة طوبار تجميعا عشوائيا من المياه المالحة لمدينة بورسعيد )قناة السويس عند مدخل بحيرة المنزلة( فى المواسم المختلفة. أسفر الفحص اإلكلينيكى لألسماك المصابة بالميكزوسبوريا عن وجود حويصالت بيضاء موجودة على القشرة لبعض االسماك. بينما أظهر فحص بعض األسماك بعد نفوقها عن ظهور حويصالت بيضاء موجودة على غشاء الميزنترى الكبد التجويف الخيشومى. الطفيليات المسببة وكانت من نوع الميكزوسبوريا. وقد تم التعرف على كانت النسبة الكلية لإلصابة %41 و سجلت أسماك البورى النسبة األعلى %14 و أسماك الطوبار نسبة %7. سجلت النسبة الكلية الموسمية لإلصابة بالميكروسبوريديا أعلى نسبة إصابة فى فصل الربيع لم يتم و %12 %65 فى الصيف رصدها. و كانت أعلى نسبة فى سمك البورى فى فصل الربيع و قد سجلت االصابة لسمكة الطوبار فى فصل الخريف فقط بنسبة %12. أوضحت الصورة الهستوباثولوجية نتيجة اإلصابة بحويصالت الميكزوسبوريا تليف فى الكبد و وجود بعض االلتهابات بينما اصيبت الخياشيم بضمور فى الخاليا المبطنة للتجويف الخيشومى.