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34.33± 0.667 a*. 157.7± 3.930 b*. Acanthocytes. 1.333 ± 0.333. 1.000 ± 0.577. 1.667± 0.3333. Echinocytes. 3.667± 0.667. 27.00± 2.000a*. 42.00± 3.000 b*.

Alexandria Journal of Veterinary Sciences www.alexjvs.com AJVS. Vol. 55 (2): 40-51. Oct. 2017 DOI: 10.5455/ajvs.276968

Biochemical and Histopathological Changes in Nile Tilapia, Oreochromis niloticus at Lake Edku Mohamed Mourad 1, Mahmoud Tanekhy2*, Ekbal. Wassif3, Hanem Abdel-Tawab3, and Afaf Mohamed1 1

Department of Fish Physiology, National Institute of Oceanography and Fisheries, Alexandria, Egypt. 2 Department of Fish Diseases, Faculty of Veterinary Medicine, Alexandria University 3 Department of Zoology, Faculty of Science, Assiut University, Egypt.

ABSTRACT Key words: Biochemical; Histopathology; Oreochromis niloticus; Lake Edku

Correspondence t: Mahmoud, Tanekhy [email protected] hoo.com

The main objective of this study is to assess some biochemical and histopathological changes in Nile Tilapia, Oreochromis niloticus caught from Lake Edku. A decrease in serum total protein, albumin, total lipids, bactericidal activity, ALT and AST was observed in El-Maadyah and Edku drain regions compared to recovery group, while the levels of serum glucose and lysozyme activity were increased. The frequencies of micronucleus (MN) and nuclear abnormalities (NAs) in fish erythrocytes showed an increase of micronuclei, lobed nuclei and fragmented apoptotic nuclei while notched nuclei were decreased. Finally, the frequencies of morphologically altered erythrocytes (MAEs) of fish showed an increase in swelled cells, vacuolated cytoplasm; microcytes; echinocytes; teardrop like; sickle cells and nuclear retraction. On the other hand, the main prominent hepatic lesions were parasitic granuloma, encapsulated parasitic cysts by connective tissue, fatty degeneration, congestion and necrosis. The histopathological analysis of muscle sections of both polluted sites showed edema, splitting of muscle fibers, hyalinized muscle fibers, fatty tissue, necrosis and infiltration of lymphocytes. From the above, we can conclude that the contaminated environment of fish could cause detrimental effects on fish tissues and immune system causing great economic losses for the natural resources.

concentrations induce biochemical, morphological and tissue histological alterations that may critically influence the quality of fish (Kaoud and El-Dahshan, 2010). Therefore, uses of biochemical changes are useful biomarkers because they are most sensitive indices of the stress state of an organism (Ramesh et al., 2009). Also monitoring of blood parameters has considerable diagnostic value acts as pathophysiological reflector of the general status of the body and important in diagnosing the functional and structural status of fish exposed to toxicants (Adhikari et al., 2004). In addition, Fish erythrocytes are more sensitive to water pollution compared to other biological endpoints (Sharma et al., 2007), as the peripheral erythrocytes are in direct contact to the toxicant during its transportation (Ruas et al., 2008). As well, it was found that the peripheral blood of fish

1.

INTRODUCTION Water pollution is a matter of great concern throughout the world due to existing of a wide range of pollutants that can threat the public water supplies and cause the damage to the aquatic life (Van et al., 2003). Whereas the aquatic ecosystems are the principal recipients of industrial, anthropogenic, agricultural fertilizers and pesticide pollutants that mainly released directly or indirectly (Rajeshkumar and Munuswamy, 2011), accordingly aquatic organisms are subjected to extensive stress impacts may affect on molecular and cellular components such as enzymes or impairing functions such as osmoregulation, metabolism, immune response and hormonal regulation (Barton and Iwama, 1991). In addition, it was reported that water pollutants prolonged exposure even in very low 40

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is a convenient and reliable tissue used as an endpoint in the assessment of the genotoxic effects of pollutants, (Udroiu, 2006). Genotoxic alterations estimating the presence of micronucleus and other nuclear abnormalities as well as morphological alterations of erythrocytes serving as an index of cytotoxicity frequently used as diagnostic tools to evaluate the health status of the fish exposed to a complex mixture of contaminants in the aquatic habitats (Corredor-Santamaría et al., 2016). The histopathological biomarkers can also be used as indicators for pollutant effects on aquatic organisms and are a reflection of the general health of the aquatic organisms and considered closely related to the other stress biomarkers (El-Bakary et al., 2011). This study concerned with the evaluation of the general health status of Nile tilapia, O. niloticus exposed to a complex mixture of pollutants using biochemical, Immunological genotoxic, cytotoxic and histopathological biomarkers.

After 48 hours blood samples were collected from the caudal vein of El-Maadyah region group and Edku drain group. While collected after a full of month from the recovery group using disposable syringes then centrifuged at 5000 rpm for 10 min to separate serum samples used for the determination of glucose level (Kaplan, 1984), total protein level (Henry, 1964), albumin level (Doumas et al., 1971), globulin (Coles, 1974), total lipid level (Kagawa et al.,1982), liver function enzyme activity (ALT&AST) according to Reitman and Frankel, (1957), and kidney function (creatinine level) according to Bartels et al., (1972). Immunological parameters (lysozyme and bactericidal activities) were determined according to Engstad et al., (1992); Rainger and Rowley, (1993), respectively. All of the biochemical and Immunological parameters were measured using specific reagent kits purchased from biotech and biomerix Company. 2.4. Micronucleus (MN) test, binuclei (BN), nuclear abnormalities (NAs) and morphologically altered erythrocytes (MAEs)

2. MATERIALS AND METHODS 2.1. Fish samples

Blood smears were obtained by blood samples were taken from caudal vein using disposable syringes and smeared on clean microscopic slides, fixed in absolute methanol after air drying at room temperature. Slides were stained with hematoxylin and eosin then dehydrated in 95% and 100% absolute ethyl alcohol then cleared in xylene and permanently mounted by DPX (Pascoe and Gatehouse, 1986). Three slides per fish from each fish 000 cells were scored under 000× magnification (Osman et al., 2011) to determine the frequencies of MN, BN and NAs as described b Çavaş et al., (2005). In addition to different patterns of morphological alterations of erythrocytes (MAEs) analysis. Frequencies were expressed per 1000 cells. For the scoring of micronuclei, the following criteria were adopted according to Çavaş et al., (2005), the MN must be separate, circular or ovoid bodies, non-refractive and having the same staining pattern as the main nucleus and the MN diameter should be less than one-third of the main nucleus.

A total number of 200 apparently healthy Oreochromis niloticus with average bod weight of g fish and cm in length collected at two sites of Lake Edku, first site was El-Maadyah region and the second site was Edku drain. 2.2. Experimental design Fishes were divided into three groups the first group represented the experimental group of ElMaadyah region, the second one represented Edku drain and the third group represented the recovery group (El-Maadyah region), were left in aquaria and acclimatized for 48 hours. Continuous aeration using electric air pumping compressors was maintained in each aquarium throughout the period of this study. After acclimatization period the aquaria of the recovery group supplied with chlorine free tap water according to Innes, (1966) and was left for a month. Fish wastes were cleaned by siphoned with three quarters of the aquarium’s water which was replaced by aerated water from the water storage tank. Fishes were fed on a commercial diet containing 21% crude protein. The diet was daily provided at a fixed feeding ratio of 3% of body weight of fish as described by Eurell et al., (1978).

2.5. Histopathological examination Samples of liver and dorsal white muscle of (3 surviving fish of each group) were taken and immediately fixed in 10% neutral buffered formalin. The fixed tissues were processed routinely for paraffin embedding technique. Embedded tissues were

2.3. Blood sampling, determination of biochemical and immunological parameters 41

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sectioned at 4-5 µ in thickness and then stained by following stains: Harris's haematoxylin and eosin stain (H&E) (Bancroft and Steven, 1982) and Milligan’s trichrome stain for the connective tissue fibers (Milligan, 1946) and examined microscopically. 2.6. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by multiple comparison Tuke ’s test using a software program Graph PadPrism 5.01 Software). 3. RESULTS and DISCUSSION 3.1. Biochemical parameters: 3.1.1. Glucose level: variation in blood glucose level is the most important index of secondary stress response in fish (Kumar et al., 2016). The present study revealed a significant increase in serum glucose level p ˂ 0 0 in Edku drain region in comparison to the recovery group (Table, 1). While it was insignificant p ˃ 0 0 between El-Maadyah and the recovery group. Similar findings were recorded in fishes exposed to heavy metals (Mekkawy et al., 2010). The alterations in the glucose level might be related to liver damage, renal injury, lack of nutrition and synthesis of glucose from extra hepatic tissue proteins and amino acids (Öner et al., 2008). In addition, chemical contaminants modulate the

carbohydrate metabolism, resulting in hyperglycemia by stimulating the glycogenolysis in fish (Levesque et al., 2002; Osman et al., 2010). 3.1.2. The total protein level: a significant decrease p ˂ 0 0 in total protein level h poproteinemia in El-Maadyah region compared to recovery group. Zaghloul et al., (2006) found a significant decrease in serum total proteins when they studied the effect of copper toxicity on three fish species: O. niloticus, C. gariepinus, and T. zillii. This decrease might be due to the destruction of protein-synthesizing subcellular structures and the decrease or inhibition of the liver protein synthesis (Fontana et al., 1998). The loss of protein from damaged kidneys might be resulted in hypoproteinemia (Gad, 2005). Also, the higher energy demand of the body to counter stress may induce an increase in protein catabolism, a process in which both blood and structural protein are converted to energy during toxicant thereby reducing the serum protein (Kori-Siakpere et al., 2011) and/or due to other several pathological processes involving renal damage, elimination in urine, changes in hepatic blood flow and/or plasma dissolution (Gluth and Hanke, 1985).

Table (1): Changes in serum levels of Nile Tilapia, O. niloticus caught from Lake Edku parameter

Recovery group N = 10

El- Maadyah N = 10

Edku drain N = 10

Glucose (Mg/dl)

73.8 ± 0.772

76.2 ± 0.727

76.6 ± 0.733 b*

Total protein(Gm/dl)

6.19 ± 0.139

5.73 ± 0.090 a*

6.18 ± 0.066

Albumin(Gm/dl)

3.61 ± 0.041

3.37 ± 0.056 a*

3.37 ± 0.050 b*

Globulin (Gm/dl)

2.580 ± 0.144

2.360 ± 0.086

2.810 ± 0.098

Total lipids(Gm/l)

6.1 ±0.623

4.45±0.095 a*

4.47 ± 0.047 b*

ALT (Iu/l)

61.90 ± 0.948

55.60 ± 0.499 a*

55.50 ± 0.619 b*

a*

70 ± 0.919 61.20 ± 0.757 (Iu/l)± SEM. * Significant Data representAST means at P < 0.05. n= number of samples.

63 ± 0.615 b*

Creatinine(Mg/dl)

1.160 ± 0.031

1.380 ± 0.051 a*

0.93 ± 0.037 b*

Lysozyme activity (Unit/ml)

0.110 ± 0.003

0.120 ± 0.006

0.114 ± 0.004

Bactericidal activity (S. I.)

44.9 ± 0.752

38.5 ± 0.764 a*

35.6 ± 0.581 b*

Data represent means ± SEM. * Significant at (P < 0.05) N= number of samples. a: represent significant difference between recovery vs. El-Maadyah b: represent significant difference between recovery vs. Edku drain

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Table (2): The frequencies of Micronucleus (MN) and nuclear abnormalities (NAs) in erythrocytes of O. niloticus Genotoxicity

Recovery group N=3

El-Maadyah N=3

Edku drain N=3

Micronuclei (MN)

1.333 ± 0.333

5.333 ± 0.333 a*

11.00 ± 1.000 b*

Binuclei (BN)

1.333 ± 0.333

2.333 ± 0.333

0.667 ± 0.333

Kidney-shaped nuclei Blebbed nuclei Lobed nuclei Notched nuclei Hook-shaped nuclei Fragmented apoptotic nuclei

1.333 ± 0.667

1.333 ± 0.333

2.000 ± 0.0

2.333 ± 0.333 1.667 ± 0.333 2.333 ± 0.333 0.0 ± 0.0

2.333 ± 0.333 3.667 ± 0.882 1.667 ± 0.333 0.667 ± 0.333

1.333 ± 0.3333 4.667 ± 0.333b* 0.333 ± 0.333b* 0.0 ± 0.0

0.0 ±0.0

1.000 ± 0.0a*

1.333 ± 0.333b*

Vacuolated nuclei

4.333 ± 0.667

3.667 ± 0.882

24.33 ± 5.667b*

Data represent mean /1000 cells ± SEM. * Significant at (P < 0.05) N= number of samples. a: represent significant difference between recovery vs. El-Maadyah b: represent significant difference between recovery vs. Edku drain

Table (3): The frequencies of morphologically altered erythrocytes (MAEs) of O. niloticus cytotoxcicty

Recovery group N=3

El- Maadyah N=3

Edku drain N=3

Swelled cells

3.000 ± 0.0

15.00± 2.082a*

8.000± 0.5774

Vacuolated cytoplasm

52.67 ± 2.667

77.33± 7.333 a*

166.0± 2.000b*

Microcytes

21.67± 1.667

34.33± 0.667 a*

157.7± 3.930 b*

Acanthocytes

1.333 ± 0.333

1.000 ± 0.577

1.667± 0.3333

Echinocytes

3.667± 0.667

27.00± 2.000a*

42.00± 3.000 b*

Tear-drop like

3.000± 0.0

8.333± 1.333 a*

5.667± 0.667

Sickle cell

1.667 ± 0.333

11.67± 1.667 a*

19.67± 0.333b*

Nuclear retraction

3.667± 0.667

55.33± 5.333 a*

5.000 ± 0.0

Fused cells

2.000± 0.0

3.000 ± 0.0 a*

1.333± 0.333

Data represent mean /1000 cells ± SEM. * Significant at (P < 0.05) N= number of samples. a: represent significant difference between recovery vs. El-Maadyah b: represent significant difference between recovery vs. Edku drain

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In addition, due to increased lipolysis (Ghosh and Chatterjee, 1989) and detoxification mechanism during stress (Neff, 1985). On the other hand, there was insignificant decrease (p > 0.05) in protein level in Edku drain group compared to the recovery group. 3.1.3. Albumin level: there was general significant decrease p ˂ 0 0 in albumin in both polluted sites when compared to recovery group. This may resulted from loss through urine or feces, increased catabolism or impaired synthesis, (Nguyen, 1999). This is in agreement with (El-Fayoumi and Abd Allah, (2003). Hypoalbuninemia reflects the active inflammation and serious hepatic and renal damage. The latter damage cannot prevent albumin from the blood into urine and being lost or to the liver necrosis as a result of toxicant which led to leakage from liver into the blood and/or inhibition of liver enzymes (Mahmoud et al., 2012). 3.1.4. Globulin Globulin measurement is of considerable diagnostic value as it relates to general nutritional status, liver function, and the vascular system integrity. There was insignificant decrease p ˃ 0 0 in globulin level in El-Maadyah region compared to recovery group. While there was insignificant increase p ˃ 0 0 in Edku drain compared to recovery group. The parasites infested fish can have a detrimental effect on the immunological response and resulted in a decline in the values of the total protein, Albumin and globulin as a result of nutrient material consumption by the parasites (Eissa et al., 2012). 3.1.5. Lipids: are important source of energy, the present study revealed a significant decrease in serum total lipid p ˂ 0 0 in both sites when compared to recovery group (Table, 1). This is in accordance with Abu-El-Ella (1996), who found a decrease in muscle and serum total lipids in grass carp; Ctenopharyngodon idella exposed to cadmium and imposed this decrease to the great energy demand to confront this stress. This might also be due to the decline in insulin levels because insulin has a greater effect on lipogenic and protogenic pathways (ElNaggar et al., 1998).

liver under the influence of pesticides. Their low levels could either be as a result of enzymatic inhibition, the reduction in the permeability of hepatic cells membrane by the toxicant forcing the enzymes to accumulate in the cells, or liver damage without any regeneration. (Yousafzai and Shakoori, 2011). Also, the decline observed in the AST level may be due to the fasting caused by the parasites infested fish ( Özdemir et al., 2016) as show in Table (1). 3.1.7. Kidney functions: Creatinine is a biomarker for kidney function. This study showed significant increase (p ˂ 0 0 in the creatinine level in ElMaadyah region compared to recovery group (Table, 1). These findings in agreement with Zaki et al., (2009), who found an increase in the creatinine level in O. niloticus due to cadmium exposure. The raised serum creatinine level may be attributed to kidney dysfunction (Alkaladi et al., 2015). Hadi et al., (2009) reported that the elevation of creatinine level might be induced by elevation of muscle tissue catabolism, glomerular insufficiency, or the impairment of carbohydrates metabolism. While there was a significant decrease p ˂ 0 0 between Edku drain and recovery group. This finding is in agreement with (Kotb et al., 2013) they found a significant decrease in creatinine level in fish, O. niloticus exposed to diclofop-methyl compared to control group. (Ogamba et al., 2011) reported that the decline in the level of creatinine suggests that it was completely used up by the muscle as a result of the stress induced by the toxicant. 3.2. Immunological parameters: 3.2.1. Lysozyme activity: There was insignificant increase (p > 0.05) in its level in both polluted sites compared to recovery group. 3.2.2. The bactericidal activity: There was a significant decrease p ˂ 0 0 of bactericidal activit in both contaminated groups as controlled with the recovery group due to the direct and indirect effects of contaminants on immune system of fish and lowered leukocytes. This result is similar to Tanekhy et al., (2016) who found that increased bactericidal activity has occurred in tilapia after addition of probiotics to contaminated water.

3.1.6. Liver functions: ALT and AST are very useful biomarkers in liver toxicological studies (Osman et al., 2010). The present study revealed a significant decrease (p ˂ 0 0 in ALT and AST enz mes in both polluted sites compared to recovery. Li et al., (2004) found decreased levels of ALT and AST in the fish

3.3. Blood alteration detection 3.3.1. Genotoxic effects: Micronucleus test (MN) with other erythrocyte nuclear abnormalities (ENAs) 44

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and morphologically altered erythrocytes (MAEs) assays are used by several authors for detecting cytogenotoxic potential of various pollutants in field as well as laboratory conditions (Fenech et al., 2011; Anbumani and Mo-hankumar, 2012). The present study showed an increase of micronuclei, lobed nuclei and fragmented apoptotic nuclei while notched nuclei were decreased as shown in Table (2) and Fig. (1). Micronuclei are cytoplasmic chromatin masses appear as small nuclei outside the main nucleus, which can be formed from either the chromosome fragments or whole chromosomes that lag at cell division due to the lack of centromere, damage, or a defect in cytokinesis during the anaphase stage of cell division and fail to become incorporated into daughter cell nuclei (Heddle et al., 1991; Harabawy and Mosleh, 2014) The blebbed-nucleated cells and binuclei are considered as genotoxic analogs of micronuclei show a similar origin (Serrano-Garcia and MonteroMontoya, 2001). Crott and Fenech, (2001) proposed that the blebbed nuclei may be a precursor of micronuclei. Accordingly, the other forms of abnormal nuclei recorded in the present study such as lobed nuclei and kidney-shaped nuclei may represent different precursors of micronuclei or binuclei phenomena (Harabawy and Mosleh, 2014). In addition fragmented-apoptotic cells can be considered as one of the principal mechanisms in elimination of micronucleated damaged cells Mičić et al., 2002). Although the mechanisms responsible for NAs have not been fully explained, the formation of different NAs may represent a way by which the cell reduces any amplified genetic material from the nucleus (Shimizu et al., 1998). 3.3.2. Cytotoxic effects (the frequencies of morphologically altered erythrocytes (MAEs)

depending on the nature and kind of the toxic agents, time of exposure, fish species, different areas, season, etc. (Okonkwo et al., 2011; Osman et al., 2011); these variations probably related to the chemical kinetics of the toxins and to the hemopoietic cycle speed (Kumar, 2012). The obtained findings showed variations in the frequencies of MN, NAs and MAEs of both polluted groups compared to recovery group are in agreement with those of other studies revealed that, the frequencies were raised in fishes inhabiting the polluted sites or exposed to toxicants in the lab (Harabawy and Mosleh, 2014). 3.4. Histopathological findings: Liver: the histopathological analysis of recovery fish liver stained with (H&E) exhibited normal histological appearance of liver with Polygonal shaped Hepatocytes having spherical nucleus located among blood sinusoids forming cord-like structure. Also hepatopancreas contacted with hepatocytes. Moreover, liver sections stained with Milligan trichrome stain showed no connective tissue between hepatocytes (Fig.2A-B). While several hepatic lesions were identified in the fish liver of both polluted sites as shown in (Fig., 3 and 5). From the most prominent lesions identified in the fish liver include parasitic granuloma (local productive inflammation) showing parasite in the center, hyperplastic cells, macrophage infiltration and surrounded by fibrous tissue probably is the result of a host defense reaction. This is in agreement with (Olivero et al., 2013). The presence of the parasitic granuloma was an inflammatory cell response causing the walling off of the pathogen within the infected tissues (Elkesh et al., 2013), as well as presence of encapsulated parasitic cysts by connective tissue as shown in Fig.,(3). This is in accordance with El-Naggar et al.,(2009). In addition to fatty degeneration, congestion and necrosis. Muscle: sections of recovery group stained with (H&E) showed normal muscle bundles with muscle fibers with nuclei at the periphery of the muscle fibers. In addition, the examination of muscle sections of recovery group stained with Milligan trichrome stain showed small amount of interstitial connective tissue between muscle bundles (Fig., 2C-D). On the other hand, the histopathological analysis of muscle sections of the both polluted sites showed several pathologic alterations as shown in Fig.,(4; 6) include edema, splitting muscle fibers, hyalinized muscle fibers, fatty tissue, necrosis and infiltration of lymphocytes.

The present study showed an increase in swelled cells, vacuolated cytoplasm; microcytes; echinocytes; tear-drop like; sickle cells and nuclear retraction as shown in Table, (3) and Fig.(1). The formation of echinocytes may be due to changes in protein confirmation in the erythrocyte membrane (Anbumani and Mo- hankumar, 2012). Ateeq et al., (2002); Mekkawy et al., (2011) demonstrated that the erythrocytes exhibited cytoplasmic vacuoles as a result of unequal distribution of hemoglobin. Da Silva Souza and Fontanetti, (2006) proposed that the formation of microcyte as a way to eliminate the MN from the cell. The frequencies rates of MN, NAs in addition to (MAEs) may exhibit significant variations, 45

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Fig. 1. Erythrocytes of O. niloticus, stained with H & E; showing (A) normal erythrocytes, (B-C) Micronuclei (MN) with different size and positions in the erythrocytes, (D-E) different shapes of binucleated erythrocytes, (F) blebbed nucleus, (G) kidney shaped nucleus, (H–I) different shapes of lobed nuclei, (J) notched nucleus, (K)vacuolated nucleus, (L) hook-shaped nucleus, (M) fragmented-apoptotic nucleus, (N) swelled cell, (O-P) different shapes of vacuolated cytoplasm, (Q) microcyte, (R) acanthocyte, (S-T) different shapes of echinocytes, (U) teardrop-like erythrocyte, (V) sickle cells, (W) nuclear retraction ,(X-Y) different shapes of fused erythrocytes.

Fig. (2): (A, B) Photomicrograph of liver section of the recovery group A: showing normal polygonal shaped hepatocytes (H), portal vein (PV) and hepatopancreas (HP) contacted with hepatocytes (H) (H&E X 400). B: showing normal polygonal shaped hepatocytes (H) and no connective tissue between hepatocytes (Milligan trichrome stain X 400). (C, D): Photomicrograph of recovery muscle C: showing normal muscle bundles with muscle fibers with nuclei (N) at the periphery of the muscle fibers. (H&E X 400). D: showing small amount of interstitial connective tissue between muscle bundles (CT) (Milligan trichrome stain X 400).

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Fig. (3): Photomicrograph of liver section of El-Maadyah group A: showing necrosis (N), acute massive necrosis, shrinkage and dissociation of cell (short arrow) and parasitic granuloma (local productive inflammation) (Gr); B: showing marked dilation in blood vessel (Di), necrosis (N) and pycnotic cells (Pn); C: showing infiltration of eosinophilic granular cells (EGCs), thrombosis (Th) and necrosis (N); D: showing parasitic granuloma (local productive inflammation) (Gr), aggregation of melanomacrophage (Mm), necrosis (N) and eosinophilic granular cells (EGCs); E: showing aggregation of melanomacrophage (Mm), degenerated pancreatocytes (DPCs) and necrosis (N) (H&E X 400). F: parasitic cysts surrounded by fibrous connective tissue (C) and pericellular connective tissue (CT) (Milligan trichrome stain X 400).

Fig (4). Photomicrograph of muscle section of El-Maadyah group A: showing wavy appearance of muscle fibers (arrow); B: showing splitting muscle fibers (SMF) and Edema (E); C: showing hyalinized muscle fibers (HMF) (H&E X 400). D: showing fatty tissue (F) and interstitial connective tissue between bundles (CT) (Milligan trichrome stain X 400).

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Fig. (5): Photomicrograph of liver section of Edku drain group A: showing hemolysis (Hs) and necrosis (N); B: showing fatty degeneration (F); kupffer cells (K) and karyolysis (Kl); C: showing necrotic hepatocytes (N); congested blood vessel (Cn) and karyolysis (Kl); D: showing congested portal vein, congested blood vessels (Cn), loss of contact between pancreatocytes and hepatocytes (blue arrow) and necrosis of hepatocytes (N); E:showing dilated blood vessel (Di), necrosis of hepatocytes (N) and kupffer cells (K) (H&E X 400). F: showing connective tissue (CT) (Milligan trichrome stain X 400).

Fig (6). Photomicrograph of muscle section of Edku drain group A: showing edema in the connective tissue between bundles of nerve fibers (arrow), splitting muscle fibers (SMF) and edema (E); B: showing splitting muscle fibers (SMF), necrosis (N) and infiltration of lymphocytes (IF); C: showing splitting muscle fibers (SMF) and edema between muscle bundles (E) (H&E X 400). (D): showing edema between muscle bundles and nerve bundles (E) and connective tissue (CT) (Milligan trichrome stain X 400).

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