A trial for treatment of ichthyophonosis in cultured oreochromis niloticus using fucus and neem plants nadia a. Abd el-ghany1 and hoda m. L. Abd alla2




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8th International Symposium on Tilapia in Aquaculture 2008


A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN CULTURED OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
NADIA A. ABD EL-GHANY1 AND HODA M. L. ABD ALLA2


  1. Dept. of Fish Diseases, Animal Health Research Institute, Dokki, Giza, Egypt.

  2. Animal Health Research Institute, Zagazig. Lab. Egypt.

Abstract

This is first trial that carried out in Egypt on the treatment of Ichthyophonosis in Oreochromis niloticus. Clinically naturally infected O niloticus with Ichthyophonus spp. was observed as spherical multinucleate spores white and creamy in colour were very variable in size, found in liver, spleen and kidney and presence of spores freely with mucus in gills, the germinating stages, hyphae and endospores were also found.

Different cultures were used in growth of Ichthyophonus hoferi spores as Eagles minimum essential medium (MEM), thioglycollate medium and Sabourauds dextrose broth. In addition, solid media as Sabourauds dextrose agar, all media were supplemented by different concentration of fetal bovine serum. Healthy O.niloticus was infected experimentally by spores of Ichthyophonus spp MEM-10 pH 3.5 culture as 1ml/ fish. Mortality, clinical signs and postmortem changes in experimentally infected fish were recorded. The results of biochemical analysis and hematology showed increased plasma levels of cortisol, eosinophils and monocytes while decreased total protein, albumin, total globulin and lymphocytes in group infected with Ichthyophonosis. In the present study, we investigated the effect of crude extract of Azadirachta indica (neem) leaves at dose 5ppm /kg ration and Fucus vesiculosus extract at dose 2g/kg ration in controlling of such disease in fish. Approximately, most of these parameters increased in infected fish treated with neem and fucus .The present data concluded that neem is more effective than fucus and qualifies as a safe and efficient in the prevention of Ichthyophonosis in fish.

INTRODUCTION

Ichthyophonus disease is recognized to be of economic significance in both fish cultured and wild fishes (McVicar, 1999). Its causative agent is Ichthyophonus hoferi, that has a very broad host spectrum and is known to infect marine and cultured fish, causing granulomatous systemic disease in vascularized organs such as heart, spleen, liver and kidneys (Spanggaard et al., 1994).The variability in clinical signs is undoubtedly related to the site of the fungal invasion, including abnormal swimming patterns, some fish develop a dark colour. Also, it is the cause of many unidentified deaths in fishes (Jones and Dawe, 2002).

Diagnosis of Ichthyophonosis depends on external and internal alterations, isolation of infective fungus which needs special condition and nutrients and histopathology for the infected organs. More recently, the application of medicinal herbs in disease management is gaining momentum because herbal treatment is cost –effective and has minimal side effect.

Azadirachta indica (neem) is one of the most widely tropical plants and its leaves contain several chemical and biological active ingredients (Erratum, 2001), about ten herbs are most commonly used in China to treat fish diseases such as gill rot, white mouth disease, Aeromonas hydrophilia in Carp and fungal pathogen as Aphanomyces invadans (Siddiqui et al., 1992 and Harikrishnan et al., 2005).

Fucus vesiculosus is marine algae sulfated polysaccharide with a wide variety of biological activities, that revealed anti-inflammatory and antiviral (Hayashi et al., 1996) antibacterial (Sakai, 1999) antifungal (Miles et al., 2001) anticoagulant and immunomodulating (Kuznetsova et al., 2003) antitumor effects (Skibola et al., 2005) and antioxidant (Veena et al., 2007).

The present study was carried out to evaluate the effect of Azadirachta indica (neem) and Fucus vesiculosus in the ration of Nile tilapia (O.niloticus) in treating Ichthyophonus disease.



MATERIAL AND METHODS

Naturally infected Fish

A total number of 100 Oreochromis niloticus were collected from a private fish farm Giza Governorate previously suffered from Ichthyophonosis with body weight range of 50-60 g. for isolation of fungi, and transferred to Fish Diseases Department at the Animal Health Research Institute. Body weight and length, clinical signs and postmortem changes were recorded (Innes 1966).



Fungal examination

Fresh smears of liver, heart, spleen, kidneys and gills were examined by light microscope immediately or few hours after death for the presence of spores or any other stages of Ichthyophonus. The inoculum used consisted of fungal material removed aseptically from suspected organs to culture medium (Faisal et al., 1985).

According to Rand and Cone (1990), the inoculum was cultured aseptically in different broths, Eagles minimum essential medium (MEM, Sigma M5775), thioglycollate medium (TGC, Oxoide) and Sabourauds dextrose broth medium (SDB, Difco) using sterile tubes with 5ml medium/tubes. The fungus was also inoculated into solid media of Sabourauds dextrose agar (SDA, Difco). All media were supplemented by different concentration of fetal bovine serum (FBS, Sigma F4010), the percentages of FBS and the resulting pH of each medium are listed in Table 1. All media were supplemented by penicillin at 100UI/ml and with streptomycin at 100ug/ml.The culture in tubes and plates were incubated at 14 0C for 15days.

Identification of the fungal growth was performed by microscopically examination of wet mount preparation and stained by lactophenol cotton blue (LPCB) as described by (Spanggaard et al., 1995).The isolates were subcultured in MEM-10 pH 3.5 for experimental infection.

Table 1. List of the media used for in vitro culture


Medium

FBS%

pH

MEM-10

10

3.5

MEM-10

10

7

TGC-10

10

6.6

SDB-10

10

3.5

SDA-1

1

5.5

Trial for treatment in experimental infection

Rations

Standard commercial ration formulated to contain 32%crude protein (8.0% menhaden fish meal, 45.0% soybean meal, 25.0 cornmeal, 14.0% wheat middling) and 5.6% crude lipid. The ration was supplemented with 5ppm of Azadirachta indica (neem) extract/kg ration and 2 g Fucus vesiculosus extract /kg ration. Dietary ingredients were thoroughly mixed in a Hobart mixer and extruded through a 2.5-mm diameter diet in a Hobart meat grinder. The pellets were air-dried at room temperature, broken into small pieces, sieved to obtain appropriate size and stored in a freezer at -8°C until used.



Experimental fish

A total number of 150 apparently healthy Nile tilapia fingerlings were transported from a private commercial fish farm to fish diseases department at the Animal Health Research Institute and acclimated to the laboratory conditions in glass aquaria supplied with an air and dechlorinated tap water at 21 ±1°C for a month before the start of the experiment. At the end of acclimation period, 30 fish were randomly collected and subjected to mycological examination as previously mentioned to confirm that these fish were free from infection.



Experimental Design

One hundred and twenty O. niloticus were divided into two groups. The first group (20 fish) was designated as a negative control. The second one (100 fish) was infected by using stomach tube containing I. hoferi MEM-10 pH 3.5 culture as 1ml/fish for one time according to Dorier and Degrang, (1961). The mortality and clinical abnormalities were recorded during one month of infection. At the end of 30 days of infection, some of the infected fish (15) were scarified for mycological examination to verify the presence of infection and the survived fish (60) was subdivided into three equal sub-groups. Group A was represented as infected positive control. Group B was fed on ration containing 5ppm of Azadirachta indica (neem) extract/kg ration (Harikrishnan et al, 2005). Group C was fed on ration containing 2 g Fucus vesiculosus extract /kg ration (Farias et al., 2004). The treatment was continued for three months. At the end of experiment, all survivors were examined for infection as previously mentioned.



Blood samples

Five blood samples were obtained from each group by cutting tail in hepranized tube then were separated and kept at 20±1 °C until used for biochemical analysis. Estimation of cortisol was carried out after Pickering, (1981), Triiodothyronine (T3) and Tetraiodothyronine (T4) according to (Abroham 1981) using Radioimmunoassay kits.

Serum total protein albumin and total globulins were performed using 7150 Automatic blood chemistry analyzer (Ciba- Corning Diagnostic Crop). Total leucocytic count (TLC) was estimated according to (Stoskoph, 1993) and differential leucocytic count was carried out after (Hibiya, 1982).

Histopathological Examination

Tissue specimens from suspected examined organs were fixed in 10%buffered formalin, embedded in paraffin, sectioned and stained with Haematoxylin and Eosine (H&E) as well as periodic Acid Schiff methods (PAS) according to (Roberts 1978).



Statistical analysis

The obtained data were analyzed statistically by analysis of variance according to (Snedecor and Cochran 1982).



RESULTS

Clinical picture

The general clinical signs in infected fish were represented as sluggish movements, darkening of the colour, ulceration of skin, depressions in the bones of the head above and under the eyes (Fig. 1). Post-mortem examination of the infected fish revealed presence of white, well defined, macroscopic nodules of the size of a pin head in kidneys, spleen and liver (Fig. 2).



Mycological examination

Squash preparation of gills and internal organs of such fish revealed the presence of various life stages of Ichthyophonus hoferi in liver, kidneys and spleen. The recognized stages were distension of the spore wall was observed with the formation of a hyphae or new budding yeast like spores (Fig, 3&4). Development of uni or binucleated endospores and multinucleate spore (resting spore) which vary greatly in diameter and germinating flask shaped cyst or uninucleated endospores scattered between tissues of the organs (Fig 5).

The first isolation of Ichthyophonus from infected organs was achieved in all the media assayed, though optimal growths of the organism were observed in MEM-10 and SD broth-10 (Fig 6 and 7). Results of cultivation of infected materials on SDA-1 revealed hyphae growth both on the surface and into the substance from 4-6 days (Fig 8), the hyphae growth increased to full fill plate within 10-14 days post inoculation formation of hyphae and spores was observed and large elongated chlamydospores from the apex or middle of the hyphae appeared (Fig 9 and 10).

Germination of spores was observed in TGC-10 and SD-10 after 2-4 days of incubation. Long septate hyphae were formed and the cytoplasm was released from the spore to the newly formed hyphae (Fig 11). The growth of hyphae was higher on SD broth-10 PH 3.5 are a septate, characterized by oblong apices and extensive branching after one day of inoculation, hyphae developed bulbous tips, which rounded up to form large, thick-walled spores (Fig 12). Endogenous division of spores occurred, and spherical uni or bi-nucleate bodies were formed after cleavage.

The endospores were released by short and thick hyphae, after their release they grew, the nucleus divided, and multinucleate spores were formed. Ichthyophonus growths were optimal in MEM-10 and SD-10 with pH 7 and 3.5 respectively. The fungus was sub cultured in this medium for more than 1 year with monthly or bimonthly passages.

The examination of 100 cultured O. niloticus was classified as: 20 infected when the organism was isolated by invitro culture, 15 clinically infected or diseased when visible white lesions were observed on at least one organ and confirmed to be Ichthyophonus by culture, 45 sub-clinically infected when visible lesions were not apparent but Ichthyophonus was detected microscopically and 20 negative when Ichthyophonus couldn't be identified by any of the above techniques.



Experimental infection

O. niloticus were susceptible to Ichthyophonus hoferi where the cumulative mortality rate reached 25% in one month (Table, 2). Most of moribund O. niloticus showed emaciation, skin ulceration with abdominal distension in some cases. Post-mortem examination of moribund fish revealed presence of one to a few focal, creamy white patches in liver, spleen and kidney. Microscopic examination of infected organs revealed the presence of characteristic forms of Ichthyophonus hoferi similar of naturally infected fish. Also, fungal reisolation from infected organs on MEM-10 and SD-10 with pH 7 and 3.5 respectively were positive for Ichthyophonus hoferi.

Treatment effects

Treatment trials exerted zero mortality in Azadirachta indica (neem) extract while it was 25% in the treated group by Fucus vesiculosus extract

No fungus was reisolated from organs of O. niloticus after treatment with Azadirachta indica and isolated from organs after treatment with Fucus vesiculosus (Table, 3).

Haematological and biochemical analysis

The present data exhibited that O. niloticus infected with Ichthyophonus hoferi revealed a significant elevation in the levels of plasma cortisol, while plasma levels of T3 and T4 did not change than the control ones throughout all periods of the trial (Table, 4). At the same time the treated groups exhibited a significant increase in plasma levels of cortisol, T3 and T4 with both Azadirachta indica (neem) and Fucus vesiculosus throughout all the treatment periods except at the first month of the treatment with neem, T3 did not change significantly than the control group.

Table , (5) showed a significant reduction in the concentration of total protein, albumin and total globulin in fish infected with Ichthyophonus hoferi throughout all the experimental periods, except albumin at first month of infection did not appear any changes in comparison with control.

In addition, the to treated groups showed a significant increase in the concentration of total protein, total globulin in group treated with neem extract and albumin in group treated with fucus extract while albumin of neem extract treated group and globulin of fucus extract treated group did not change significantly at all periods treatment in comparison with the control groups.

Data in (Table, 6) illustrates the total and differential leucocytic counts, there was leucocytosis in Ichthyophonus hoferi infected fish at first month of infection while leucopenia was detected at second and third months of infection. Also this group appeared lymphocytopenia, neutropenia, monocytosis and eosinophila at all periods of the trial.

However treatment with Azadirachta indica (neem) and fucus vesiculosus exhibited elevation of the total leucocytic count and lymphocytes with decrease monocytes and eosinophils throughout all treated periods except at first and third month of neem extract treated group and first month of fucus extract treated group, eosinophils did not differs than the control .

The histopathological sections from infected organs with Ichthyophonus hoferi revealed that Kidney there were diffuse extraverted erythrocytes all over the renal tissue in between the tubule, the epithelium cells lining the renal tubules showed swelling with vacuolar cytoplasm Fig (13).

Liver there were severe dilatation of central veins and sinusoid. The hepatocytes showed different stages of necrobiotic changes with complete rupture of the cells associated with appearance of some fungus spores strongly positive PAS and inflammatory cells infiltration Fig (14-15).

Gills there were oedema with mononuclear leucocytic inflammatory cells as well as dilated blood vessels with presence of spores were noticed as strongly positive PAS Fig (16-17).

Discussion

Ichthyophonosis, having a wide host and is defined as one of the economic significant affections in fish culture and wild fisheries. In this study, majority of infected fish missed external lesions except for darkening of the colour, ulceration of skin, depressions in the bones of the head above and under the eyes as those nearly noticed by Kent et al., (2001). Sindermann and Scattergood (1954) described behavioral changes in salmonids, curvature of spine in herring and salmonids with darkening of the skin were all seen in few cases indicating that the fungus invade the hosts nervous system. In herring and trout roughening of skin were noted by Reichenback-klinke (1973). Internal examination of examined O.niloticus demonstrated light gray raised nodules in liver, spleen and kidneys, the organs richly supplied with blood, which indicate the systemic nature of Ichthyophonus. Squash preparation from these nodules demonstrated that the parasite seen were refractile thick double walled spores. There were developmental stages of Ichthyophonus hoferi observed as latent resting stage, yeast like stage and hyphae stage. Difference in the developmental pattern of Ichthyophonus hoferi were observed according to the conditions surrounding the fungus in tissue of fish host. According to McVicar (1982), such differences probably reflect the nutrients available and the conditions surrounding the developing fungus.

Therefore, the endospores formed in fish could be responsible for septicemia and subsequent infection of organs. The formation of new spores by budding like (yeast like) germination could be a means of proliferation with the granuloma and not an occasional developmental form as reported by Dorier and Degrang (1961). This dimorphism has been demonstrated in Mucor rouxii (Bartnicki-Garcia 1973), Histoplasma and Candida (Alexopoulos and Mims 1985).

Ichthyophonus growth and development was achieved in all the media assayed and was generally similar to that reported by other authors for Ichthyophonus hoferi or other forms of Ichthyophonus (Chien et al., 1979; McVicar 1982; and Okamoto et al,. 1985). Different development patterns were observed, according to the medium used, in spite of this polymorphic development, characteristics of fungi and related organisms (Bruns et al,. 1991).

The development in media with acid pH (SD, TGC and MEM-10) associated with germination of multinucleate spores, branching of the formed a septate hyphae and formation of terminal spores in the hyphal tips, was similar described by Okamoto et al. (1985), Ana and Pilar (1999) for Ichthyophonus hoferi grown in MEM-10 at the same pH value. In MEM-10 at the pH 7 we observed internal cleavage and release of endospores through short hyphae that was rather similar to pattern one described by the above mentioned authors in MEM-10 at the pH 7.The growth of fungus was achieved on Sabouraudes dextrose agar (SDA-1) grow into the substrate within 3-7 days. The growth become heavy and filled the plate within 2 weeks post inoculation. However Sindermann and Scattergood (1954) described germination of the inoculum from herring after 7-10 days on SDA-1within 3-20°C with optimum temperature 10°C. Resting spore germination may produce slender non-septated microhyphae and tubular club shaped macrohyphae. This may produced large spherical thick walled spores or cleaved to numerous hyphal (endospores), this agree with the finding of Chine et al., (1979) and Ana and Pilar (1999) .In our cultures we never observed motile forms such as those described by McVicar (1982) and Spanggaard et al., (1995), though some ovoid forms, apparently similar to those described by these authors, were sometimes present.

Experimentally infected O. niloticus showed nearly similar clinical signs, postmortem and histopathological alterations to those observed in naturally infected ones. The mortality percentages was 25% in the infected groups with Ichthyophonus cultured on MEM-10 adjusted at pH 3.5 for 30 days. On the other hand, the control group showed neither clinical signs and postmortem lesion nor mortality. Such findings were met by Ana and Pilar (1999), Jones and Dawe (2002) and Kocan and Hershberger (2006) who induced infections in different fish species by exposure to a pure culture of Ichthyophonus hoferi.

Treatment trials exerted zero mortality in Azadirachta indica (neem) extract and No fungus was reisolated from organs of O. niloticus after treatment.

These results were in agreement with those of Harikrishnan et al.,( 2005) who reported the inhibitory action of Azadirachta indica (neem) leaves and seeds extract on a wide spectrum of microorganisms including fungi as Aphanomyces invadans and also agreed with those of Tandan et al .,(1998) who reported the wound healing effect of neem because of increased cutaneous capillary permeability at the site of the wound. In group treated by Fucus vesiculosus extract mortality rate was 18.75% and fungus isolated from organs after treatment with Fucus vesiculosus. These results were in disagreement with Kubitza (2000) who explained that Fucus vesiculosus enhanced non specific immunity and diseases resistance.

Farias et al., (2004) recorded that Fucus increase diseases resistance in O. niloticus, Cyprinus carpio and shrimp Penaeus monodon respectively.

It has been presumed that disease constitutes an environmental stressor in fish Wedemeyer&Mcleady(1981).The obtained results revealed a significant increase in the levels of plasma cortisol in Ichthyophoniasis infected fish, while there were no changes in the levels of plasma T3 and T4 throughout all periods of the trial.

The increase in the cortisol levels in this study may be related to that cortisol production is an important factor underlying the course of infectious diseases in fish (Pickering, 1981).Also; Dunn (1989) indicated that infections activate the same type of neural response as physical stressors.

In contrast to, Rand and Cone (1991) found that cortisol did not change in fish infected with Ichthyophonus hoferi, whereas the same authors supported the present study with thyroid hormone where they recorded that T4 did not differ in Ichthyophonasis infected fish than control. In addition, the present data exhibited increased levels of cortisol, T3 and T4 in both treated groups throughout all experimental periods but T3 did not exhibit any change at the first month in group treated with neem. Concerning, elevation of these hormones after treatment with neem and fucus may be these herbals induce the production of cytokines in a variety of cells (Talwar et al., 1997 and Hirayasu et al., 2005) and these cytokines modulate the neuroendocrine functions of the hypothalamus as well as acting on the pituitary, thyroid, pancreas, adrenal glands and gonads to modulate hormone release (Kennedy & Jones, 1991). It is worthy to mention that corticosteroids has biphasic effect on immune system, they produce an initial inhibition of macrophage and T cells development following a stressor stimulus but prolonged release of corticosteroids(by lower concentration) may stimulate the immune response (Brown, 1994).

Also thyroid hormones (T3 & T4) stimulate the release of thymic hormones and maturation of T lymphocytes (Berczi, 1986). The present data demonstrated decreased concentration of total protein, albumin and total globulin in fish infected with Ichthyophonus hoferi. '

In accordance with this result Rand and Cone (1990) found depressed protein in rainbow trout infected with Ichthyophonus hoferi and this may be attributed to pathology associated with high numbers of resting spores in kidney, spleen, heart and liver, as supported by histopathological results in this study.

Interestingly, these parameters increased significantly with both treated groups except albumin in group treated with neem and total globulin in group treated with fucus throughout all the treatment periods. This may be referred to herbal medicines help to heal the lesion (Harikrishnan et al. 2003) and neem may induce the state of non-specific increased resistance in fish.

In this data, infected group showed significantly increase in the numbers of circulating leucocytes after first month with monocytosis at all experimental periods, while there were leucopenia after second and third month with lymphocytopenia and neutropenia throughout all periods of the trial, Similarly Harikrishnan etal.,(2005) found that increased the total leucocytic count in the common carp infected with fungal pathogen, Aphanomyces Invadans suggesting that the production of white blood cells proportionate to any stress of infection, where pathogen may induce endotoxin and tissue injury which is a potent stimulant to initiate the proliferation, differentiation, and activation of B lymphocyte and macrophage (Xiang etal.,2008). Leucopenia after long period of infection in the present data is consistent with Rand and Cone (1990) and Qureshi et al., (2001) who recorded leucopenia in fish having fungal infection, suggesting that leucopenia is a generalized host response and supports that there is cell mediated response to wall of the resting spore ( Mc Vicar, 1982).

Interestingly, the fish treated with neem or fucus exhibited a significant increase in the total leucocytic count and lymphocytes., these results were supported by Upadhyayet , etal.,(1992)who reported that treatment with neem (Azadirachta indica)resulted in the maximum number of leucocytes and macrophages exhibiting enhanced phagocytic activity .Furthermore Kuznetsova et al.,(2003) who found that fucoidan from brown algae Fucus evanesces stimulated phagocytic and bactericide activity at leucocytes of mice peritoneal exudates.

The histopathological changes induced by Ichthyophonus hoferi in fish were nearly agreed with those mentioned by Shaheen and Easa (1996) in wild and cultured tilapia.

Based on aforementioned results, the following conclusions could be recommended as the effective role of neem (Azadirachta indica) in the treatment of Ichthyophonosis in O. niloticus fish is more better than fucus since neem stimulated both humoral and cell mediate immunity and succeeded for the first time to eradicate all the Ichthyophonus spores in fish after three months of treatments, while fucus stimulated cell mediate immunity only and succeeded to eradicate about 75% of Ichthyophonosis spores.



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