Saprolegniosis is one of the most important oomycetes infections that can cause huge economic losses in cultured ecosystems (Van West 2006; Phillips et al. 2008). Our results clearly demonstrated the pathogenicity of S. ferax to Nile tilapia associated with higher mortalities. The present results were in agreement with (Stueland et al. 2005) who found that two of seven Saprolegnia spp. strains, tested for their pathogenicity to Atlantic salmon Salmo salar, caused 89% and 31% cumulative mortality in challenged salmonids. These strains were significantly more pathogenic than the other strains tested. Moreover, (Hussein and Hatai 2002) reported that the cumulative mortalities of the different salmonids fish groups exposed to 2 × 105 spore/L concentrations of S. salmonis NJM 9851 were 90% for brown trout, 93.3% for sockeye salmon and 100% for rainbow trout, masu salmon, and Japanese char, however; all salmonid species exposed to 2 × 105 spore/L concentrations of S. parasitica NJM 9868 had a cumulative mortalities of 100%. Saprolegnia lesions were in form of cottony like masses on different sites on fish body mainly in the dorsal region and on the dorsal and adipose fins (Yanong 2003) which in same event with our results lesion appeared mostly in all sites which are exposed to scarification during descaling process. The death mainly occurred due to the osmotic failure caused by the damaged epidermis (Bruno and Poppe 1996).
Our results showed that Potassium permanganate as a chemical treatment lowering the mortality to 15% in the 2nd week and 0 in the 3rd week; compared to 42.5 and 20% of mortality by 2nd and 3rd week in the control positive group, respectively. The most effective treatment for controlling saprolegniosis is the malachite green; however, since it has several drawbacks (Schreier et al. 1996) it’s not permitted for use in fish-farming in most countries. Thus, the need to search for alternative compounds as effective as malachite green is imperative.
Potassium permanganate is used for ectoparasites, bacteria, fungal disinfections on the skin and gill of fishes, it act as an oxidant through decreasing BOD of water by oxidize the organic matter and the amount of oxygen will be increase by using potassium permanganate (Noga 2010). (Marking et al. 1994) examined two In the same event, (Rasowo et al. 2007) investigated the effect of formaldehyde, sodium chloride, potassium permanganate and hydrogen peroxide treatment for saprolegniosis in catfish (C. gariepinus) eggs and found that the best hatchability performance was when eggs treated with 2 ppm potassium permanganate for 30 min (96.7%). (Darwish et al. 2009) evaluate the efficacy of KMnO4 at a dose of 2.0 mg/L above the potassium permanganate demand for 2 h duration against Columnaris infection in channel catfish using different route of administration and found that using KMnO4 simultaneously with challenge give 99% survival while using KMnO4 postchallenge enhanced survival (85%) than positive control (78%); which conclude overall efficacy of KMnO4 as a treatment against skin infection. (Marking et al. 1994) reported that potassium permanganate at two concentrations 50 and 100 mg/l decreased fungal infection of rainbow trout eggs but rate of hatching didn’t increase. Our results were supported by (Thomas-Jinu and Goodwin 2004) who found that KMnO4 reduced columnaris mortality from 100% to 69%.
Adjuvant treatment as immunostimulant improved the mortality rates from 27.5% to 10% from the first to the second week compared to the control positive which encountered 30% and 42.5% of mortality in week 1and 2. Furthermore, no mortalities were recorded in the 3rd week in FCA group. In addition, FCA treatment showed to some extent a better rate for fish survival compared to KMnO4 treatment in the second week after challenge. To our knowledge, there are no studies for using FCA alone as immunostimulant to enhance fish immunity infected with saprolegniosis or investigate its effect regarding the antioxidant response in fish. However, Many co-workers have shown that the adjuvant has a great effect on innate immune response; For example, injection of FCA induces nonspecific protection against several bacterial pathogens and ciliate pathogen of fish (Olivier et al. 1985; Kajita et al. 1992). (Olivier et al. 1985) showed increased resistance of coho salmon (Oncorhynchus kisutch) to Aeromonas salmonicida after injection of the same preparation of FCA that we used in our studies (i.e., with killed Mycobacterium butyricum). Thus, this augmentation of the immunity reflected on the host response to evade the infection. In the same context, (Harikrishnan et al. 2009) found that a prior administration of a triherbal mixure of medicinal plant compounds, azadirachtin (Az), camphor (Ca) and curcumin (Cu), affected mortality rate positively upon changing with virulent starin of Aphanomyce invadans. On the contrary, (Kunttu et al. 2009) evaluate the efficacy of two immunostimulants, yeast b-glucan and b-hydroxy-b-methylbutyrate (HMB) to treat the Fingerling rainbow trout experimentally infected with Flavobacterium columnaris, and found that given orally both stimulants enhanced the levels of immune function parameters, but did not improve survival in challenge at any concentration of the stimulants used. Intra peritoneal injection of β-glucan increased parameter values several fold, but no beneficial effect of injected glucan on survival was observed. (Zahran et al. 2012) have found that adjuvant parenteral administration in channel catfish could elect a nonspecific defence by expressing antimicrobial polypeptides histone like protein-1 (HLP-1) at a distant site (skin) HLP-1 known with its potent bactericidal, parasiticidal and funigicidal activity (Robinette et al. 1998).
Saprolegnia infection resulted from many stress factors that fish are exposed, these factors lowering its resistance and enhance fish susceptibility to infection, Saprolegnia act through secreting proteolytic like enzymes that alter integument integrity and facilitate penetration (Peduzzi and Bizzozero 1977).
Antioxidant activity in our study showed different patterns between both treatments to some extent; but as general both treatment had general same effects regarding the antioxidant response. NO is an important regulating signaling molecule, it produced endogenously from L-arginine and molecular oxygen by the enzyme nitric oxide synthase (Bogdan 2001). NO mediate its biological activity by binding with different targets such as, heme groups, cysteine residues, and iron and zinc clusters, thus a certain regulation of NO production is required to mediate its biological effect. When NO level is too high that may indicate presence of toxicity, stressful conditions, infectious agent that all participate in occurrence of the disease (Farrell et al. 1992; Ignarro 2000; Vladutiu 1995). In the present result, NO level significantly increased in the infected group, while with both treatments they significantly decreased to within the normal value. A similar response was observed in rainbow trout inoculated with a virulent strain of Renibacterium salmoninarum compared with avirulent strains (Campos-perez et al. 2000), In the same trend (Acosta et al. 2004) found enhanced response in serum concentrations of stable nitric oxide (NO) metabolites in small weight gilthead sea bream (Sparus aurata) (30-75 g body weight) lasting from 6 h to six days post-infection with a peak at 24 h; when inoculated of with a sublethal dose of different Photobacterium damsela e subsp. piscicida (Pdp) strains (DI-21 and 94/99), however; no such response was detected in larger fish (150-600 g).
GSH is an important antioxidant and the amount of GSH present could reflect the antioxidant potential of an organelle (Liu et al. 2008). GSH activities were significantly higher during the challenge in the infected group. This could be attributed to the induction of Reactive oxygen species (ROS), and so more antioxidant enzymes are released to compensate the damage produced by infection (Di Giulio et al. 1989; Storey 1996). (Wilhelm Filho et al. 2002) found that gill tissues are one of the main contributors of ROS generation in fish. This explanation is supported also by other studies by (Thomas and Wofford 1984; Gallagher et al. 1992; Thomas and Juedes 1992) where they demonstrated higher level of GSH following exposing fish to stressor. (Oliveira et al. 2008) showed a higher adaptive competence expressed as antioxidant defenses activation, namely GSH and glutathione peroxidase (GPX) in Liver against different concentration of Phenanthrene. However, SOD level was significantly reduced upon challenge and that could be due to disruption of erythrocyte membranes by ROS caused hemorrhage and loss of antioxidant enzymes in human immunodeficiency virus (HIV-infected humans); and the typical symptom in grass carp hemorrhage virus (GCHV) infected grass carp is erythrocyte hemolysis and bleeding in the muscle may relate to the significantly decreased SOD and catalase (CAT) activities (Zhang et al. 2003) when the grass carp were infected with GCHV. Interestingly, a significant decrease in SOD was also observed in white spot syndrome virus (WSSV) infected Penaeus monodon (Chang et al. 2003). Our results in accordance with (Kim et al. 2009) who examined the effect of β-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression in grass carp challenged with grass carp hemorrhage virus (GCHV) and found that the SOD and CAT activities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with β-glucan than in infected. Similar results were obtained in Wister rats when injected mangiferin I/P at different dose to overcome cyclophosphamide toxicity; showing that Cyclophosphamide significantly lowered the superoxide dismutase and catalase (CAT) activities in lymphocytes, polymorphonuclear cells (PMN) and macrophages, while the administration of mangiferin significantly protected the activities of SOD and CAT, demonstrating the antioxidant mechanism to exist in its immunoprotective role.
Thus, it’s evident that the KMnO4 and adjuvant a promising tools in a protecting against immunological tissue injury may be through the regulation of antioxidant enzyme activities, thus potentiating the cellular antioxidant capacity. Moreover, it emphasize on the role of adjuvant as an immunoprotective role mediated through the inhibition of reactive intermediate-induced oxidative stress in lymphocytes, neutrophils and macrophages. Adjuvant affect macrophage activity and phagocytosis (Olivier et al. 1986a) and thus the respiratory burst activity due to an increase in the antioxidant oxidation level in phagocytes which is an important indicator of innate immune response (Miyazaki 1998).
Biochemical analysis, revealed changes in level of Albumin, AST but no changes have been found in total protein, globulin or ALT. AST level was markedly increased in the infected group compared to KMnO4 and FCA groups, while ALT didn’t change. Others studies were in support to our results, they found that increased activity of AST, CK, and LDH are related to venipuncture, which is done through the musculature of the caudal peduncle. While, Sorbitol dehydrogenase and ALT appear to be present in low concentration in skeletal muscle and may be better indicators of hepatocellular damage and this indicate that there were no substantial damage to internal organ as the liver (Tripathi et al. 2003). Also, Biochemical changes in fish included significant hyperglycemia, hyponatremia, and hypochloridemia were evident in koi (Cyprinus carpio)experimentally infected with Flavobacterium columnare infection (Tripathi et al. 2005).
Albumin level exhibited a moderate decrease in the infected group compred to the control one, while it showed significant reduction by week 2 and 3 in both KMnO4 group, but in FCA the albumin level matched within the control one. This may happen as a result of epidermal damage due to Saprolegnia also, through skin ulcers present on skin surface, these can represent a portal of loss of plasma protein or that excess of water diffused to the fish body through these sites resulting in slight heamodilution (Tripathi et al. 2005). Our results were in accordance with (Ruane et al. 2002) who observed reduction in plasma protein levels in common carp after confinement and in red sea bream Pagrus major following acute handling stress (Biswas et al. 2006). In salmonids, a reduction in serum protein concentration has been observed when the fish were infected or stressed (Melingen et al. 1995; Møyner et al. 1993). These differences are possibly species-specific effects of environmental conditions on serum/plasma protein and globulin concentrations in fish.
Plasma ion concentration in the present study showed variations in their levels; Na+ and K+ was increased in the infected group and decreased to some extent in the other groups. This is also can be explained in the same context which account for damaged epidermis and loss of the integument integrity and so increase in the permeability with loss of this ions or being diffused from water into fish body. Changes in biochemical parameters in hybrid tambacu fish naturally parasitized by Dolops carvalhoi (Crustacea, Branchiura), a fish louse were in form of increases in MCHC, plasma glucose levels, serum protein, sodium and chloride levels, number of monocytes and PAS-positive granular leukocytes (PAS-GL), when compared with values in control fish (Tavares-Dias et al. 2007). Freshwater fish body is hypertonic to the surrounding environment and so upon infection with Saprolegnia and subsequent skin damage this could results in influx of the water into fish body, potentially resulting in osmoregulatory failure and disruption in the electrolytes homeostasis (Tripathi et al. 2005).