Dept. of Animal Medicine,

Fac. of Vet. Med., Assiut University, Egypt

Myxobolus Infestation in Ovaries of Sharptooth Catfish, Clarias gariepinus

(With 2 Tables and 5 Figures)

By

A.A. Elkamel and A. Tantawy*

*Department of Pathology, Faculty of Veterinary Medicine,

 Moshtohor, Banha University, Egypt

(Received at 26/9/2005)

الإصابة بطفيل ميکزوبولاس فى مبايض الأسماک القطية النيلية (القراميط)

أحمد عبدالهادى الکامل ،  أحمد عبد الحافظ طنطاوى

الهدف من هذه الدراسة هو اجراء دراسة ميدانية وملاحظة الأعراض الإکلينيکية ونسب الإصابة وکذلک التغيرات المرضية نتيجة اصابة مبايض الأسماک القطية النيلية (القراميط) بطفيل الميکزوبولس. تم فحص عدد 120 سمکة على مدار عام 2004 بمعدل 10 سمکات شهرياً ووجد أن عدد 15 سمکة کانت تحمل حويصلات طفيل الميکزوبولس فى المبايض.  وکانت معدلات الإصابة فى آخر فصل الخريف قليلة ثم ازدادت تدريجيا فى فصل الشتاء حتى وصلت إلى أعلى معدلاتها فى بداية فصل الربيع، ولم تسجل أى حالة من حالات الإصابة فى فصل الصيف. کما لوحظ أن الإصابة کانت فى مبيض واحد فقط فى 6 أسماک  (40%) من الأسماک المصابة فى حين کانت الإصابة فى المبيضين معاً فى 9 أسماک       (60%) من الأسماک المصابة. اما حدة الإصابة فقد ازدادت ايضاً خلال فصل الشتاء حتى  وصلت اعلى مستوى لها فى بداية فصل الربيع ثم تضاءلت سريعا مع بداية فصل الصيف.  اظهر الفحص الميکروسکوبى لحويصلات طفيل الميکزوبولس عدد کبير جداً من أبواغ (جراثيم) الطفيل عند درجات مختلفة من التطور والنمو. کما اظهر الفحص الميکروسکوبى لمبيض الأسماک المصابة أن حويصلات طفيل الميکزوبولس قد قامت بالضغط على الويضات القريبة مما تسسب فى ضمورها و موتها فى بعض الأحيان، کما تسببت الحويصلات فى حدوث اضطرابات دموية فى الأنسجة المجاورة لها. ونظراً لأن هذا الطفيل وجد فى مبايض القراميط فقد يکون نوعاً جديدا من الطفيليات والذى يحتاج لمزيد من الدراسة مستقبلاً.

SUMMARY

The main aim of this study was to investigate the clinical and postmortem findings, seasonal prevalence, and histopathological alterations that are caused by probably a new species of Myxobolus in ovaries of sharptooth catfish, Clarias gariepinus, in Assiut, Egypt. Out of 120 fish examined over one year (2004), ovaries of only 15 (12.5 %) fish were infested with macroscopic Myxobolus cysts (plasmodia and host cyst) that were embedded in the connective tissue among ova.  Prevalence of infestation started low in late autumn and increased over winter and reached maximum in early spring. Infestation was not recorded in summer. Six (40%) out of the infested fish had Myxobolus cysts in only one ovary, meanwhile, the reminder (60%) of infested fish had both ovaries infested. Also, intensity of infestation gradually increased over winter and was maximal in early spring, but abruptly declined in summer. Microscopic examination of plasmodia showed numerous typical Myxobolus spores at various developmental stages.  Mature spores are oval in shape with two anteriorly located polar capsules that have 4-5 coils of polar filaments. Microscopic examination of infested ovaries revealed that Myxobolus plasmodia were encapsulated within a thin connective tissue layer of host reaction.  Myxobolus cysts compress neighboring tissues causing atrophy of ova and local circulatory disturbances. Based on the tissue location of plasmodia and morphological character of the mature spores, the parasite in the present study might be a new species.

Key words: Clarias gariepinus, myxobolus, ovaries

Introduction

Commercial farming of sharptooth catfish, Clarias gariepinus, is a rapidly growing aquaculture industry in Upper Egypt. C. gariepinus, has recently gained a consolidate position in the food fish market as it is widely accepted by consumers in Upper Egypt.

Myxosporea are economically important fish parasites which form an abundant and diverse group. They cause heavy infections, extensive lesions, and mortalities in cultured fish (Lom and Dykovâ, 1995). In Africa, about 100 species are currently known from the continent (Fomena and Bouix, 1997). In Egypt, myxosporean parasites were examined in River Nile fish by Aziza (1980), Imam et al. (1987), Abdel Ghaffar et al. (1998), and Ali (1999, 2000). Currently, study of myxosporean infections focuses on pathogenicity and significance of the parasite in both aquaculture and captured fish (Lom and Dykovâ, 1995).

Clarias gariepinus, a carnivorous bottom feeder, acts as a host for plenty of parasites that have tremendous effects on fish health and population throughout the River Nile.  In Egypt, C. gariepinus had been found to be infested with Myxobolus lazeri (Aziza, 1980), and Myxobolus clarii (Mandour et al., 1993). Histozoic Myxobolidae cause great destruction of the host tissues and are of serious concern to fish culture (Kabata, 1985). 

There are scanty data on Myxobolus infestations and the pathology they cause in fish ovaries (Lom and Dykovâ, 1995; Gbankoto et al., 1998; Reed et al., 2003). In the present study, prevalence and intensity of infestation of C. gariepinus ovaries with probably a new species of Myxobolus have been investigated over one year. In addition, clinical and postmortem findings and histopathological alterations of C. gariepinus infested ovaries were studied.

MATERIALS and METHODS

Fish:

A total of 120 live, apparently healthy, female specimens of sharptooth catfish, Clarias gariepinus, of 300-800 g were collected from January 2004 to December 2004 (10 fish /month) from El-Ibrahemia canal and its tributaries, Assiut city.

Parasitological examination of samples

A-Clinical examination:

Fish were externally examined after capture for any apparent clinical signs or lesions. Fish were incised according to (Stoskopf, 1993) to examine ovaries for macroscopic Myxobolus cysts to determine prevalence of infestation (number of infested fish divided by the number of examined fish per month). Longitudinal incision was made in both ovaries to determine total numbers of Myxobolus cysts to determine intensity (number of cysts per infested fish) of infestation. Cysts were examined for size, consistency, and contents.

B-Microscopic examination:

Impression smears were made from cysts, air dried, and then fixed in 40% ethanol for 10 min. Fixed smears were stained with Methylene blue or Lugol’s iodine solution.

Histopathological examination

Infested ovaries were excised from infested fish, fixed in 10% neutral buffered formalin for 48 hours, and then processed for microscopic examination. Thin paraffin sections were stained with Haematoxylin and Eosin (H&E), Toluidine blue, and Periodic Acid Schiff’s (PAS) stains.

Results

Parasitological examination of samples

Longitudinal incision of infested ovaries showed whitish round to oval cysts (Myxobolus plasmodia and host cyst) that were randomly scattered and embedded in connective tissue among ova (Fig. 1). Cysts were seen by naked eyes and were 1.2- 1.5 mm in diameter. Cysts were located in ovaries of immature females and mature females at off-spawning seasons, while ovaries of mature females at spawning season needed more careful examination because cysts are of average size of mature ova but of different color. Interestingly, wall of cysts collected in spring, the primary spawning season, were fragile and readily ruptured releasing mature spores; in contrast, wall of cysts collected during late autumn and winter were relatively firm and resistant to rupture if compared to those collected in spring.

Seasonal prevalence and intensity of infestation

Out of the 120 fish examined, ovaries of only 15 (12.5%) fish were found to be infested with Myxobolus cysts. Infestations were not seen during summer, but were recorded at a relatively low rate when temperature started to drop in late autumn (Table 1). Prevalence gradually increased over winter and reached maximum when temperature started to rise in early spring, and then declined again in late spring (Fig. 2).

Intensity of infestation was determined according to number of cysts per ovary and whether one or both ovaries were infested (Table 2).  Six (40%) out of the infested fish had Myxobolus cysts in only one ovary, meanwhile, the reminder (60%) of infested fish had both ovaries infested. Infestation was considered severe when 10 or more cysts were seen in one or both ovaries, while was considered moderate when 6-9 cysts were seen in one or both ovaries. Females were considered lightly infested when had 1-5 cysts in one or both ovaries.

Generally, during winter, when temperature is lowest in season, most cases of infestation were light. When temperature starts to rise, intensity of infestation gradually increases where moderate cases were recorded. Furthermore, Intensity of infestation continues to increase in spring when severe cases of infestation were seen and then rapidly declined and even disappeared in summer. In addition, during late autumn, when temperature starts to decline, infestations re-emerge when light cases of infestation were seen again.

Microscopic examination

Microscopically, plasmodia were encapsulated within a thin fibrous connective tissue capsule of host reaction and infiltrated with few lymphocytes with dilated blood capillaries (Fig.3). Plasmodia were filled with numerous typical Myxobolus spores where mature spores located centrally, while the developing ones were peripherally arranged.  Furthermore, plasmodia collected in early spring had mainly mature spores, while plasmodia collected during autumn and winter had mainly developing spores.

Mature spores are oval in shape with slightly pointed anterior end and more rounded posterior end, and measuring 10.6 X 9.4 μ (Fig.4).  Also, mature spores have at the anterior end two oval polar capsules with pointed anterior end and rounded posterior one. Polar capsules are of equal size, and measuring 4.8 X 3.5 μ. Each polar capsule has 4-5 coils of polar filament. Sporoplasm contains an iodinophilus vacuole that stain positively with lugol’s iodine solution. Furthermore, thin sections of infested ovaries stained with PAS showed positively stained dark red spores.

Histopathological examination

Toluidine blue and H&E stained sections showed that Myxobolus cysts exert pressure atrophy over the adjacent ovarian tissues and cause disturbances in local circulation. Adjacent ova show degenerative changes in nuclei and cytoplasm and separation of the squamous cell layer that covers ova (Fig.5).

Discussion

Present study revealed that Myxobolus infestation of ovaries of sharptooth catfish, C. gariepinus, is a mildly spread among wild population. Prevalence of infestation was 12.5% of all fish examined over one year. Water temperature has a great influence over seasonal prevalence of myxosporean infestations (Negm-Eldin et al., 1999). The prevalence of ovarian infestations with Myxobolus cysts increased in winter and early spring, while decreased in autumn. Interestingly, during summer, there was no record of Myxobolus cysts in the ovaries of fish examined. Similar annual cycles were reported with other myxosporean infestations (Negm-Eldin et al., 1999). In accordance with Clifton-Hadley et al., (1986) who concluded that water temperature influences maturation of spores and development of myxosporean infestations in fish, in the present study, mature spores were seen in plasmodia collected in early spring, while developing spores were seen in plasmodia collected during autumn and winter.

Intensity of infestation has a cycle similar to that of prevalence.  Intensity of infestation has gradually increased over winter and spring and then abruptly declined in late spring and summer. Sudden decline in prevalence and intensity of infestations during summer may be due to dispersing of intact cysts with eggs laid by infested mature females during spawning or, alternatively, rupture of cysts releasing mature spores in ovarian tissues. Ovarian contractions during egg lying might promote rupture of the cysts. This is supported by the fact that the cysts’ walls are fragile and easily to be ruptured during egg laying season, but relatively harder during off-spawning seasons. Furthermore, it is supported by the fact that sporogenesis is completed during winter, and by spring plasmodia contain fully developed mature spores.

Dispersing of Myxobolus cysts or spores with laid eggs might be the primary route of spreading of infection and completing of the parasite’s life cycle. It is not clear how this parasite reaches fish ovaries, its target organs. The exact mechanism of host invasion is unknown, but many freshwater myxosporeans have an alternate stage of development in oligochaetes (Oumouna et al., 2002) or ploychaetes (Bartholomew et al., 1997) which produces actinosporean spores that invade host. Oral route of transmission is also common route for myxosporean infestations (Lom and Dykovâ, 1995). In either case, the sporoplasms cross the epithelial barrier and are carried by the blood stream or lymphatic system to the target organ (Kabata, 1985).

Encapsulation of Myxobolus cysts within a thin connective tissue capsule of host reaction indicates that plasmodia severely irritate ovarian tissues stimulating a proliferative inflammatory response. This capsule is driven from the surrounding population of connective tissue cells and from compressed cells of the neighboring tissues (Lom and Dykovâ, 1995).

The extent of damage to tissues infested with Myxosporea depends on species of parasite and its life cycle stage, intensity of infestation and the host reaction (Lom and Dykovâ, 1995). Microscopic examination of infested ovaries revealed that the myxobolus cysts replaced original ovarian tissues, compressed neighboring ova, and caused disturbances in circulation in neighboring tissues. Henneguya oviperda causes similar lesions in ovaries of pike, Esox lucius, in Europe, where atrophy of large number of ova was observed with local circulartory disorders (Lom and Dykovâ, 1995). Myxobolus dahomeyensis has been reported to hinder the successful breeding of several species of tilapia and their hybrids in Benin. M. dahomeyensis is found in ovaries of brooder tilapia where it penetrates inside the mature ova and liquefies the content causing total destruction of mature ova. In severe cases of infestation, ovaries become like sacs full of whitish fluid with spores and damaged ova (Gbankoto et al., 1998).

Myxosporea are host, organ and tissue specific (Molnar, 1994).  Myxosporean infestations had been reported in C. gariepinus in Egypt.  Aziza (1980) described Myxobolus lazeri from kidneys, while Mandour et al. (1993) reported Myxobolus clarii from testisof C. gariepinus.  Mature spores morphology is the key feature in identification of Myxobolus (Kabata, 1985).  Mature spores of M. lazeri (9.8 X 6.1 μ) are smaller than those of the parasite of the present study (10.6 X 9.4 μ). In addition, polar capsule of M. lazeri spores are smaller (5.1 X 2.3 μ) than those of the parasite spores of the present study (4.8 X 3.5 μ).  Morphological characters of the parasite’s spores in the present study are close, but not similar, to those of M. clarii that is found in testis of C. gariepinus (Mandour et al., 1993). Mature spores of the parasite in the present study are relatively larger but within the average size as mature spores of Myxobolus clarii. Size of the polar capsules of M. clarii spores (5.1 X 2.5 μ), however, is smaller than those of spores of the present study.

Plasmodia of Myxobolus gariepinus reported by Reed et al. (2003) in ovaries of C. gariepinus in Botswana were 2-3 mm in diameter, while fully mature plasmodia of the parasite in the present study was 1.2-1.5 mm. Mature spores of M. gariepinus (13.9 X 10.8 μ) are larger than those of the parasite the present study. Furthermore, polar capsules of M. gariepinus spores are measured (6.2 X 3.5 μ) and contain 5-6 coils of polar filaments, while polar capsules of the parasite of the present study were smaller and contain 4-5 coils of polar filament. 

Based on its host species, tissue location, and mature spores morphology and dimensions, the parasite in the present study might be a new species. Classification of the parasite in the present study, however, needs further investigations including comparative ultra structure study and molecular identification.

Sharptooth catfish is widely accepted by consumers in Upper Egypt as a relatively cheaper choice of fish protein. Commercial farming of sharptooth catfish has significantly increased in Upper Egypt over the past few years. With no obvious method of treatment or control, ovarian infestation of sharptooth catfish with Myxobolus may affect fecundity (Lom and Dykovâ, 1995) and thus populations of wild and cultured fish.

Acknowledgment

We would like to thank Dr. Shaban M. Ahmed, professor of fish diseases and management, Faculty of Veterinary Medicine, Assiut University for all the help and guidance he provided through this study.  We would like, also, to thank Dr. Gamal Abed, Professor of Zoology, Faculty of Science, Assiut University for his input and help in identifying of the parasite studied.

References

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Table 1: Seasonal prevalence of Myxobolus infestation in Clarias gariepinus ovaries.

Table 2: Intensity of Myxobolus infestation in Clarias gariepinus ovaries.

Fig. 1: A photograph of Clarias gariepinus ovary incised longitudinally and infested with Myxobolus plasmodia (P) that are embedded in connective tissue among ova.

Fig. 2: Seasonal prevalence of Myxobolus infestation in Clarias gariepinus ovaries.

Fig. 3: Light microscope photograph of Myxobolus plasmodium in Clarias gariepinus ovary. Plasmodia are enclosed in fibrous connective tissue capsule (CT) infiltrated with few lymphocytes. Germinating cells (GC) are located peripherally, while developing spores (DS) are toward the center. Toluidine stain (400X).

Fig. 4: Light microscope photograph of Myxobolus mature spores stained with methlyene blue. Spores are oval in shape with sporoplasm (SP) and two polar capsules (PC) (1000X).

Fig. 5: Light microscope photograph of a plasmodium of Myxobolus full of spores (SP) encapsulated within a connective tissue capsule (CT) and compressing neighboring ova (CO) of Clarias gariepinus ovary. H&E (400X).

Fig. (4) Light microscope photograph of Myxobolus mature spores stained with methlyene blue. Spores are oval in shape with sporoplasm (SP) and two polar capsules (PC) (1000X).

Fig. (5) Light microscope photograph of a plasmodium of Myxobolus full of spores (SP) encapsulated within a connective tissue capsule (CT) and compressing neighboring ova (CO) of Clarias gariepinus ovary. H&E (400X).

Myxobolus Infestation in Ovaries of Sharptooth Catfish, Clarias gariepinus

By

Elkamel A.A.¹ – Tantawy A.²

¹Departement of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Egypt

²Department of Pathology, Faculty of Veterinary Medicine, Moshtohor, Banha University, Egypt