Mortada M.A. Hussein, Walid H. Hassan and Maha A. Mahmoud

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1 World Journal of Fish and Marine Sciences 5 (2): , 2013 ISSN IDOSI Publications, 2013 DOI: /idosi.wjfms Pathogenicity of Achlya proliferoides and Saprolegnia diclina (Saprolegniaceae) Associated with Saprolegniosis Outbreaks in Cultured Nile Tilapia (Oreochromis niloticus) Mortada M.A. Hussein, Walid H. Hassan and Maha A. Mahmoud 1 Department of Fish, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62512, Egypt 2 Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62512, Egypt 3 Advanced Center for the Diagnosis of Animal and Poultry Diseases, Fayoum 63517, Egypt Abstract: A study was conducted to evaluate the pathogenicity and pathology of Achlya proliferoides BSN-M005 and Saprolegnia diclina BSN-M003, isolated from saprolegniosis outbreaks against immature stages of Nile tilapia, Oreochromis niloticus. The cumulative mortality rates of the tested fish groups that exposed to high zoospore concentrations of A. proliferoides BSN-M005 and S. diclina BSN-M003 were 60 and 100% respectively. The histopathological changes associated with saprolegniosis lesions induced by S. diclina BSN-M003, were loss of the epidermis, edema of the hypodermis, different degrees of degenerative changes in the underling musculature and the fungal elements were observed penetrating the entire musculature. On the other hand, the histopathological changes associated with saprolegniosis lesions induced by A. proliferoides BSN-M005 showed mats of hyphae attached the surface of epidermis, necrosis, degenerative changes close to hyphae and sometimes fungal elements were observed penetrate into dermal layer but never reach the underling musculature. It is clear from our results that S. diclina BSN-M003 is highly pathogenic than A. proliferoides BSN-M005 to Nile tilapia. Key words: Saprolegniosis Achlya proliferoides Saprolegnia diclina Pathogenicity INTRODUCTION Pathogenic members of the order Saprolegniales, which includes two families, the Saprolegniaceae Saprolegniosis is an infectious fungal disease that is (e.g. Achlya, Brevilegnia, Dictyuchus, Saprolegnia and widespread in all stages of the life cycle of fish. The Thraustotheca) and the Leptolegniaceae (Aphanomyces, disease causes serious losses in fish farms and hatcheries Leptolegnia and Plectospira), totaling 132 species in and considered as one of aquatic disease implicated in about 20 genera [7] are considered the causative agents mass mortalities of cultured and wild fish in many that incriminated in such infections. The most important countries [1-3]. The disease appears as cotton wool-like water moulds in the order Saprolegniales are those tufts on the body surface causing destruction of the skin water moulds in the genera Achlya and Saprolegnia that and/or fins due to cellular necrosis by hyphal can be pathogens of many fish species and their eggs penetration and that is generally restricted to the [8-10]. epidermis and dermis. Most lesions are caused by In Egypt, saprolegniosis constitute one of the Saprolegnia species that why the disease is called most important disease causing troubles in freshwater saprolegniosis, however, other Saprolegniaceae including cultured fish with several economical losses [11-13]. Achlya may be primarily cause a clinically identical Recently, mass mortalities associated with an outbreak saprolegniosis [4-6]. of saprolegniosis was recorded among Nile tilapia, Corresponding Author: Walid H. Hassan, Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62512, Egypt. Tel:

2 Oreochromis niloticus, fish farm in Beni-Suef All fish experiments were conducted in 10 L aquaria Governorate, Egypt, where as several Saprolegnia and at 25±2 C. Five tilapia individuals weighting 40±2 g Achlya isolates were found to be associated with the were placed in each aquarium 24 h prior to the disease incidence. experiments. The present study was conducted to evaluate the pathogenicity and the pathology of two selected isolates Experimental Design: Two sets of 4 aquaria (10 L) with of Saprolegnia and Achlya, isolated during the outbreak 5 fish in each were prepared. The first set was designated of saprolegniosis, against immature stages of Nile tilapia. for the experimental infection with A. proliferoides BSN-M005. Two aquaria were used for exposure to the MATERIALS AND METHODS two concentrations of zoospores of A. proliferoides BSN-M005, one was used for exposure to mycelial mats, Fungal Strains: For artificial infection, Achlya while the fourth one was used as negative control. proliferoides BSN-M005 and Saprolegnia diclina The second set was designated for the experimental BSN-M003 were used for testing their pathogenicity in infection with S. diclina BSN-M 003 and treated at the juvenile Nile tilapia. Both isolates were isolated from same manner as mentioned above. outbreak of saprolegniosis in cultured Nile tilapia. To induce infection, all fish groups including the The tested strains were grown in Petri dishes on glucose negative control were subjected to ami-momi (net-shake) yeast-extract (GY) agar and incubated at 20 C. The treatment [16] before being exposed to the zoospores or zoosporic stages of the tested fungi were obtained by mycelial mats of the tested fungi. Briefly, fish were shaken inoculating agar blocks 1 X 1 cm (3~4 blocks) with in the air in a fan-shaped scoop net for 2 min and then actively growing mycelia of each fungus into Petri dishes rapidly washed before being returned to their aquaria. containing 20 ml of GY broth. The cultured plates were Then, one liter of water was taken from each tested kept at 20 C for 24 hours. The growing mycelia were cut aquaria and replaced by 1 L of both low or high and washed repeatedly in sterilized tap water (TW) then, concentration of the tested fungal zoospore suspensions transferred into 20 ml of fresh sterilized TW and kept for 2 to obtain zoospore concentrations of 2 X 10 (low) and 18~24 hours at 20 C [14]. The zoospores were harvested, 5 2 X 10 (high) spore/ml. Control aquaria were treated as counted with a Neubauer chamber (ERMA, R, Tokyo) and experimental ones with exception. The exception is that 3 adjusted to two concentrations that were 2 X 10 and 2 X 1 L of aquaria water was replaced by 1 L of sterilized 6 10 spore/ml. Tufts of the growing mycelia in TW of TW. For exposure to fungal mycelia, 2 g wet weight of both tested fungi were also used for experimental mycilial mats of each tested fungi was warped in a piece infection. of gauze and hanged into their tested aquarium corners. The aquaria were checked each day after exposure for Fish: All experiments were performed at Aquatic 10 days, considering the day of exposure as 0-day and Laboratory Unit, Fish Department, Faculty of Veterinary dead and moribund fish were removed for examination. Medicine, Beni-Suef University, Beni-Suef, Egypt. All fish remaining at the end of the 10-day period were Sub-adult Nile tilapia with an average weight of 40±2 g removed for examination. and 14±2 cm length of both sexes were obtained from a private tilapia hatchery at Beni-Suef Governorate, Egypt. Pathological Examination: All fish were examined for The fish were maintained in 500 L fiberglass tank, the gross pathological changes and those exhibiting lesions water quality and temperature were monitored daily and were subjected to routine necropsy. Then, tissue kept within the acceptable range for tilapia. The fish were specimens from moribund and dead fish including all the fed commercial pellet feed (Joe-Trade Factory, El-Asher body with special intention to head, trunk muscles, gills men Ramadan, Cairo, Egypt) twice daily at a ratio of 2% of and fins were excised and fixed in 10% neutral buffered their body weight and kept under observation for 2 weeks formalin solution, embedded in paraffin and sectioned at prior to the experiment. Feeding was stopped three days 4~5 µm. The sections were stained with methanamine prior to starting the experiment following the various silver nitrate-grocott s variation and they counter-stained recommendations for the maintenance of fish bioassay with haematoxylin and eosin (Grocott-H and E) and/or described by Ellsaesser and Clem [15]. light green (Grocott-Light green). 189

3 RESULTS External Clinical Signs and Mortality Pattern: Artificial infection of Nile tilapia was done using A. proliferoides BSN-M005 and S. diclina BSN-M003 and the results varied considerably with the two fungi used. Certainly, none of the fish challenged to low zoospore concentrations and those challenged with mycelial mats of both tested fungi died or showed clinical signs Fig. 1: Moribund immature Nile tilapia, O. niloticus, characteristics to saprolegniosis. Furthermore, during shows cotton-like mycelial growth 4 days after post-mortem examination, none of these fish showed challenging with S. diclina BSN-M 003 Arrows. external or internal gross lesions. They seemed to be as Scale bar 5cm apparently healthy as the fish in the negative controls. On gross observation, fish groups challenged with high zoospore concentrations of tested S. diclina strains as well as A. proliferoides became listless, showed erratic swimming, rose to the water surface or rested on the bottom of their aquarium. Fish challenged with S. diclina started to die on the day 2 after challenge showed no characteristic external signs, while on the days 3-7 both dead and moribund fishes had mycelial growth on various Fig. 2: Immature Nile tilapia, O. niloticus, died 4 d after parts of the body surface especially on the head, dorsal being challenged with S. diclina BSN-M 003. Note and caudal fins (Figs. 1and2). Finally, all fish in that group the saprolegniosis lesion on tail fin and eye were died within 8 days. On contrary, mortalities among Arrows. Scale bar in cm fish challenged with A. proliferoides were slightly delayed and started at the day 6, extended till the end of the experiment (10 days) and associated with cotton like, whitish color lesions scattered on the body surface (Figs. 3and4). As a matter of fact, the cumulative mortality rates of the tested fish groups that were exposed to high zoospore concentrations of A. proliferoides BSN-M005 and S. diclina BSN-M003 were 60 % and 100 %, Fig. 3: Moribund immature Nile tilapia, O. niloticus, respectively (Table 1). All control negative groups that showing cotton-like mycelial growth scattered on were exposed only to ami-momi treatment as well as those the head and body surface 6 days after that exposed to mycelial mats showed survival rates of challenging with A. proliferoides BSN-M % and that was evident till the end of the experiments. Arrows. Scale bar 5cm In contrast, fish group that exposed to high zoospores of S. diclina BSN-M003 experienced cumulative mortality rates of 20, 40, 20 and 20% on the days 2, 4, 6 and 8 after challenge, respectively. On the other hand, fish group exposed to high zoospores of A. proliferoides BSN-M005 experienced cumulative mortality rates of 20, 20 and 20% on the day 6, 8 and 10 after challenge respectively. Fig. 4: Immature Nile tilapia, O. niloticus, died 8 days after Histopathological Changes: Generally, the being challenged with A. proliferoides BSN-M005. histopathological changes associated with experimental Note the saprolegniosis lesion on head, dorsal and infection with S. diclina BSN-M003 and A. proliferoides tail fin Arrows. Scale bar in cm 190

4 5 1 Table 1: Pathogenicity of A. proliferoides BSN-M005 and Saprolegnia diclina BSN-M 003 to immature Nile tilapia exposed to 2 X 10 spore ml Mortality % Days After infection A. proliferoides BSN-M005 S. diclina BSN-M Total Fig. 5: Slight destruction of the epidermal layer of immature Nile tilapia, O. niloticus, exposed only to ami-momi treatment (arrows). Scale bar = 50µm Fig. 8: Head of Immature Nile tilapia, O. niloticus, 6 days after challenge with A. proliferoides BSN-M005. Note superficial penetration of the fungal elements to the epidermal layer. Grocott-Light green. Scale bar = 50µm Fig. 6: Extensive edema of the hypodermis radiating away from the invading mycelia of S. diclina BSN-M 003 resulting in marked myofibrillar degeneration Fig. 9: Invading fungal elements of A. proliferoides BSN- Arrows. Scale bar 50µm Grocott-Light green M005 penetrating only epidermal and dermal layers Arrows. Note the hyperplasic proliferations and edema around the invading mycelia. Scale bar 30µm. Grocott-HandE) BSN-M005 were loss of the epidermis, edema of the hypodermis and different degrees of degenerative changes in the underling musculature. In contrast, ami-momi treatment frequently caused damage and loss to the epidermal layer of the treated fishes (Fig. 5) and, therefore, facilitates the establishment of the infection. Fig. 7: Hyaline degeneration of myofibrillar cells and loss The histological examination of the saprolegniosis of nuclei, focal myofibrillar necrosis and edema lesion of the tested fish after exposure to high (*) together with massive mycelia penetration of concentration of S. diclina BSN-M003 zoospores showed S. diclina BSN-M 003 Arrows. Scale bar 30µm. the fungus radiated away from the focus of the infection Grocott-HandE and subsequently penetrated the basement membrane, 191

5 extended into the dermis and, in most cases, continued to concentrations of zoospores of both tested fungi showed grow into the hypodermis and musculature. Extensive different levels mortalities. However fish group exposed edema of hypodermis was present, radiating away from to S. diclina BSN-M003 showed high cumulative the mycelial mass into non-affected areas. The edema mortalities (100 %) than those exposed to A. proliferoides extended into the muscle mass, resulting in marked BSN-M005 (60 %) indicating that S. diclina BSN-M003 myofibrillar degenerative changes. The initial change was infection can cause severe destruction of the superficial hyaline degeneration of myofibrillar cells, followed by loss tissue and the direct cause of death is probably related to of nuclei together with focal myofibrillar necrosis the massive osmoregulatory problems [6, 18]. (Figs. 6 and 7). Frequently, changes in the underling The histopathological demonstrations of the musculature ranged from complete sarcoplasmic loss to saprolegniosis lesions of tested fish that exposed to high mild degeneration and fungal elements were observed zoospores of S. diclina BSN-M003 showed the radiating penetrating the entire musculature. One the other hand, fungal elements first destroy the epidermis followed by for the tested fish that exposed to zoospores of penetration of basement membrane and subsequent A. proliferoides BSN-M005, only mats of hyphae attached invasion into the dermis, hypodermis and subcutaneous the surface of epidermis were observed (Figs. 8 and 9) musculature (Figs. 6 and 7). The epidermal cells and together with necrosis and degenerative changes close to collagen fibers were necrosed and degenerated close to hyphae. In other sites, fungal elements were observed the hyphae. These pathological findings were consistent penetrate into dermal layer but never reach the underling with those previously described [2, 16, 18]. musculature. As far as we know, these pathological findings probably referred to enzymatic activities (chymotrypsinincluding DISCUSSION like, serine proteases) of such opportunistic fungi Saprolegnia as well as Achlya were also likely In earlier studies conducted by Hussein and Hatai to play a role in their pathogenicity, their virulence and [16], saprolegniosis was successfully induced in 5 species invasiveness against their hosts [19-22]. On the other of salmonids by using ami-momi treatment and hand, the hitopathological picture related to fish that confirmed that the method was extremely useful as the exposed to high zoospores of A. proliferoides BSN-M005, method of artificial infection. In this study, the revealed superficial invasion of the fungal elements only ami-momi treatment was applied to induce artificial to the epidermis and to a little extend to hypodermal layer infection with A. proliferoides BSN-M005 and S. diclina (Figs. 8 and 9). The possible explanation is may be related BSN-M003 in sub-adult Nile tilapia. Interestingly, data to the lake of enzymatic activities of A. proliferoides generated with the presented study showed that none of BSN-M005. This is may be supported by what is reported the fish groups challenged with low zoospores or mycelial by Kales [23] who found that S. ferax and Saprolegnia mats of either tested fungi died, nor did those in the spp. had a proteolytic activity more than A. ambisexualis. control groups, although they were exposed to It is clear from the present study that S. diclina ami-momi treatment at the same level of the tested BSN-M003 is highly pathogenic than A. proliferoides groups. Similar results were obtained by Hatai and BSN-M005 to Nile tilapia. Even though the virulence of Hoshiai [2] who used the same technique for examining the two species was different, further studies on the factor the pathogenicity of S. parasitica and S. diclina to (s) involved in virulence of fish-pathogenic strains of immature Coho salmon and they also recorded that no Saprolegniales are required. mortalities occurred among fish groups exposed only to ami-momi treatment. On contrary, the results obtained ACKNOWLEDGEMENTS by other authors in relation to the use of ami-momi treatment to induce saprolegniosis are contradictory. The authors would like to thank Fish department, Thus, Fregeneda et al. [17] elucidated that the ami-momi faculty of Veterinary Medicine, Beni Suef University, for treatment predisposes the experimental infection of supporting this research work. immature rainbow trout with Saprolegnia; however, it could kill small fish. REFERENCES The results in (Table 1) show that A. proliferoides BSN-M005 and S. diclina BSN-M003 could be pathogenic 1. Neish, G.A. and G.C. Hughes, Fungal Disease to Nile tilapia. All fish groups exposed to high of Fishes. Neptune City, T. F. H. Publication. 192

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