ANTICOCCIDIAL ACTIVITY OF BLACK CUMIN (NIGELLA SATIVA) IN RABBITS

ANTICOCCIDIAL ACTIVITY OF BLACK CUMIN (NIGELLA SATIVA) IN RABBITS

SHAKER A. SEDDIEK^* and AMIRA M.M. METWALLY^**

^*Animal Health Research Institute-Banha Provincial Lab. (Avian Diseases)

^**Animal Health Research Institute-Banha Provincial Lab. (Biochemistry)

INTRODUCTION

Nigella sativa (N. sativa) (Family: Ranunculaceae), also known as black cumin, is a herbaceous annual plant used in traditional medicine for thousands of years because of its anticonvulsive (Rahman et al., 2008), antibacterial (Ali et al., 2012), antiinflammatory (Juhas et al., 2008) and antioxidant (Tripathi et al., 2012). It has beneficial effects in nervous system in rats (Hosseini et al., 2012), hyperlipidemia (Sabzghabaee et al., 2012), liver malignancy (Abdel-Hamid et al., 2012) and renal injury after acetaminophen treatment (Ahmed and El-Mottaleb, 2012).

Although numerous studies have been conducted to determine the effects of N. sativa seeds on numerous diseases and microbes, only a few studies have examined their effect on parasitic infections. Most of these studies indicated that treatment with N. sativa reduces the rate of parasitic egg lying and improves liver functions of infected animals with schistosomiasis (El-Shenawy et al., 2008). Additional studies indicated that N. sativa is effective against trematodes, cestodes and nematodes (Abu-El-Ezz, 2005). N. sativa oil has anthelmintic effect in rats infected with Trichinella spiralis (Abu-El-Ezz, 2005) and Aspiculuris tetraptera and Hymenolepis nana (Ayaz et al., 2007).

E. stiedae is one of the protozoal parasites with severe infectivity in rabbits and causes hepatic coccidiosis affects body weight and feeding rates that can result in death (Omata et al., 2001). N. sativa seeds can be used in oil emulsions to safely and effectively treat E. stiedae infection in rabbits with no side effects (Baghdadi and Al-Mathal, 2011). El-Sayed et al. (2005) reported that seeds of N. sativa contain thymolhydroquinone and volatile oil in pharmacologically effective amounts. They found that feeding of chicks with seeds (till 10 %) yielded no differences of blood plasma chemistry which are indicative of liver and kidney functions, higher body weight and could not prevent signs of coccidiosis and shedding of oocysts of E. tenella.

Black cumin seeds up to 2 % improved performance and immunity of birds (Nasir, 2009) and in form of oil (0.5%), seeds (1%) or meal (10%) improved physiological performance, body reaction and biochemical parameters of chicks (Hermes et al., 2011). Combination of N. sativa seeds oil and aqueous garlic extract had potent effect on AST, ALT, urea and creatinine in mice (El-Shenawy et al., 2008). N. sativa improved activities of liver related enzymes resulted in better feed conversion rate (FCR) (Nasir, 2009) and reduced the adverse effects of benzo[a]pyrene-induced immunotoxicity in broilers (Latif et al., 2011). Omega 3 and N. sativa seeds oil might prevent oxidative stress in rats (Attia et al., 2011).

Black cumin (2 or 3%) decrease cholesterol in egg yolk (Aydin  et al., 2008), had a beneficial effect on growth performance in chicks (Durrani et al., 2007) and improved broiler health if mixed with Artemisia leaves (Khalaji et al., 2011). Also, it improved liver through disappearance of bleeding between hepatic lobules with inflammatory cells in portal area and reduction in various stages of parasites in bile ducts of rabbits (Baghdadi and Al-Mathal, 2011).

This study was undertaken to investigate, anticoccidial effect of N. sativa seeds oil by studying parasitological, biochemical, economical parameters and examining liver tissue structure of rabbits infected with E. stiedae.

MATERIALS and METHODS

Preparation of E. stiedae oocysts:

E. stiedae oocysts were collected from liver and gallbladder of infected rabbits and concentrated using flotation method (Heelan and Ingersoll, 2002), sporulated and stored in 2.5% pot. dichromate at 4°C until used.

Nigella sativa seed oil:

Nigella sativa seed oil used in this study was produced by PharcoPharmaceutical Company (Egypt, Alexandria) in the form of soft gelatinous capsules. Each capsule contains 450 mg of N. sativa seed oil. N. sativa seed oil was given to the rabbits by stomach tube after its extraction from the capsules.

Animals:

A total of 60 male New Zealand white rabbits, aged approximately 2 months and weighing of 954.92±3.79 gm (mean ± SE), were bred at Faculty of Agriculture, Moshtohor, Benha University. Rabbits were free from coccidian infection as determined by coccidian-free fecal samples over 7 consecutive days using the concentration flotation technique. The rabbits were weighed and distributed by ranking method (Gardiner and Wehr, 1950) into 5 equal groups of 12 rabbits each. Rabbits were separately housed in wire-floored batteries under sanitary conditions with controlled humidity, temperature, light periods (12-h light, 12-h dark cycles) and fed balanced diet ad libitum.

Experimental design:

First group (G1) was left as uninfected and untreated (negative control), second group (G2) was left as uninfected and treated with N. sativa seed oil. Rabbits of third (G3), fourth (G4) and fifth (G5) groups were inoculated with 1x10⁴ sporulated oocysts of E. stiedae per each through a gastric tube (Toulah, 2000). Third group (G3) was left untreated and considered as positive control. At day 14 post inoculation (PI), the rabbits of fourth group (G4) were treated with          N. sativa seed oil in a dose of 500 mg/kg BW (Farah et al., 2004) for successive 14 days (PI). Meanwhile, the rabbits of fifth group (G5) were given N. sativa seed oil for 7 days before infection and continued for 28 days PI with the same dose. Administration of N. sativa seed oil was applied orally every morning after an overnight fast and before feeding.

Fecal oocysts:

Fecal oocysts were collected, concentrated using the flotation technique and counted using a modified McMaster technique (Long and Rowell, 1975) on days 0, 7, 14, 21, 28 and 35 PI. The reduction % of oocysts was determined based on the number of oocysts per gram of feces in control and treated groups.

Bile oocysts:

Bile samples were then collected from the gall bladders of the sacrificed rabbits of all groups at 14 days post treatment (PT) and the oocysts of E. stiedae were counted per 1 ml bile (Peeters et al., 1981).

Blood parameters:

Blood samples were collected from ear veins of rabbits at 28 day PI. The serum was prepared and kept in deep freezer at -20 ºC until analysed. The biochemical parameters of the blood were determined in the serum by using commercial kits for total protein, albumin, cholesterol, AST and ALT. Serum globulin was calculated as difference between total protein and albumin.

Body weight:

Rabbits were weighed at the beginning of the experiment (0 day) and on days 7, 14, 21, 28 and 35 PI and their growth performances (body weights and body weight gains) were recorded (Davis et al., 1986).

Pathological study:

Liver lesions were scored according to Peeters and Geeroms (1986) and the livers were weighed after they were removed from five slaughtered rabbits on day 28 PI. The relative liver weight for each rabbit was calculated as the percentage of the total body weight (Gomez-Bautista et al., 1986). Fresh liver tissue specimens of the sacrificed rabbits on the 28^th day PI were fixed in 10% formalin, dehyderated with ethanol, cleared and embedded in paraffin wax. Section of 5-7 µm thickness were prepared and stained with hematoxylin and eosin (H&E) for the histopathological examination (Erdogmus and Eroksuz, 2006).

Statistical analysis:

The differences among experimental treatments were tested at P ≤ 0.05 by one-way ANOVA according to Duncan (1955) using the computer software program called.

RESULTS

Table 1: Mean number of oocysts/g feces (mean ± SE), overall mean and reduction percent in five groups of rabbits under experiment (n = 12).

              Oocyst output (---x10⁴ oocysts/gram feces)

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil   

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.        

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa.          - LSD = Least significance difference.  - NS = Non significant.   - (*) = Significance at P ≤ 0.05.  - 0 day = Day of infection.

Table 2: Mean number of oocysts/ml bile (mean ± SE) in five groups of rabbits under experiment (n = 12).

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.          -G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.                 

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa. 

- LSD = Least significance difference.                                    - (*) = Significance at P ≤ 0.05.            

Table 3: Lesion scoring in five groups of rabbits under experiment (n=10). (Mean ±SE).

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil 

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.    

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.  

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa.       

- LSD = Least significance difference.                                    - (*) = Significance at P ≤ 0.05.           

Table 4: Some biochemical parameters in five groups of rabbits under experiment (n=10). (Mean ±SE).

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil   

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.  

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.  

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa. 

- LSD = Least significance difference.         - (*) = Significance at P ≤ 0.05.        - NS = Non significance at P ≤ 0.05. 

Table 5: Growth performance parameters (gm) (body weight and body weight gain) in five groups of rabbits under experiment (n=20). (Mean ±SE).

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil    

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.      

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.    

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa.    

- LSD = Least significance difference.                 - (*) = Significance at P ≤ 0.05.                 - 0 day = Day of infection.     

- NS = Non significance at P ≤ 0.05.    -Growth performance according to: Davis et al (1986)

Table 6: Relative liver weights in five groups of rabbits under experiment (n=10).

-L.W. = Liver Weight                                                              -L.W. /B.W. = Liver weight / Body weight 

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil    

-G3=Infected and not treated (+Ve control                             -G4= Infected and treated with N. sativa seed oil for 14 days PI.       

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI. 

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa.     

- LSD = Least significance difference.                                    - (*) = Significance at P ≤ 0.05.           

Table 7: Mean number of developmental stages (per microscopic field) in lumen of bile duct in five groups of rabbits under experiment (n=10). (Mean ±SE).

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.     

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.     

-Means with different alphabetical superscripts in the same row are significantly different (at P ≤ 0.05) and vice versa.       

- LSD = Least significance difference.                                    - (*) = Significance at P ≤ 0.05.           

Table 8: Mortality in five groups of rabbits under experiment.

-G1=Not infected and not treated (-Ve control)                      -G2= Not infected and treated with N. sativa seed oil

-G3=Infected and not treated (+Ve control)                            -G4= Infected and treated with N. sativa seed oil for 14 days PI.

-G5= Given N. sativa seed oil for 7 days before infection and continued for 28 days PI.       

Figure 1: Photomicrograph of rabbit liver tissue of none infected (normal) and treated with N. sativa seed oil group (G2): showing a normal central vein (A) (H&E, 400 X), showing a normal portal area containing portal vein, hepatic artery and bile duct (B) (H&E, 400 X), showing a hyperplastic proliferation of the epithelial lining of bile duct (C) (H&E, 200 X) and (D) (H&E, 400 X).

Figure 2: Photomicrograph of rabbit liver tissue of positive control group (infected and untreated) (G3) on day 28 PI: showing a dilated, congested central vein with necrosis of its endothelial lining (A) (H&E, 200 X), showing necrosis of hepatocytes and hemorrhages in portal area with inflammatory reaction (B) (H&E, 400 X), showing papilliform projections of epithelium into the lumen of bile duct containing very large number of developed oocysts (C) (H&E, 200 X), and showing different developmental stages of E. stiedae in the lumen of bile duct (D) (H&E, 400 X).

Figure 3: On day 28 PI, photomicrograph of rabbit liver tissue of infected group and treated with N. sativa seed oil for 14 days PI (G4): showing incomplete healing of its endothelial lining and healing of hepatic tissue (A) (H&E, 400 X), presence of little hemorrhages in the portal area with mild vacuolar degeneration of hepatocytes (B) (H&E, 400 X), multiple papillary fronds in the bile ducts with the presence of moderate number of the parasite stages from the lumen of bile duct (C) (H&E, 200 X), and (D) (H&E, 400 X).

Figure 4: On day 28 PI, photomicrograph of rabbit liver tissue of infected group and given N. sativa seed oil for 7 days before infection and continued for 28 days PI (G5): showing healing of the central vein endothelium. The hepatocytes laminate regained their original radial shape (A) (H&E, 400 X), disappearance of hemorrhages from the portal area as well as disappearance of vacuolar degeneration of hepatocytes (B) (H&E, 400 X), showing almost disappearance of the parasite stages from the lumen of the bile duct (C) (H&E, 200 X), and (D) (H&E, 400 X).

DISCUSSION

This study indicates effectiveness of N. sativa for control of coccidiosis in rabbits by a dose of 500 mg/kg BW. At day 28 PI, infected rabbits treated with N. sativa seedoil recovered their appetite, started to gain weight, became active again and recovered from diarrhea with feces returning to its beady form. Similar results were reported by Al-Gamdi (2003) when used 250-500 mg/ kg BW; Baghdadi and Al-Mathal (2011) when used 400 mg/ kg BW in rabbits and El-Dakhakhny et al. (2000) when used 800 mg/kg BW orally for 4 weeks in albino rats. However, dose of20, 40 and 100 mg/kg BW were effective in treating schistosomiasis through decreasing of number of shedding eggs and reducing of liver damage in mice (Tantawi and Mostafa, 2003).

At 28 day PI, Table 1 showed a marked decrease in fecal oocysts count in group G5 (0.67±0.19) when compared with those of G3 (95.50±2.72) and G4 (14.67±0.36). Meanwhile, at 35 day PI, there is disappearance of oocysts in the feces of group G5 (0.00±0.00) when compared with those of G3 (63.50±3.49) and G4 (7.25±0.41). Overall mean of oocysts output in G5 was 3.33±0.37 with reduction of 91.88% versus to 41.01±4.26 (reduction of 0 %) in G3 and 10.24±1.11 (reduction of 75.03%) in G4. This may be attributed to presence of powerful antioxidants and alkaloid nigellicine in N. sativa seed oil that could limit the growth and development of    E. stiedae stages leading to reduction of oocysts formation and their appearance in the feces. Theeffective material in N. sativa seed oil in G5 may have beenmore concentrated than in G4, thereforehaving a more rapid and robust effect. In G5, the      E. stiedea parasite exposed to the N. sativa seedoil for long time (28 successive days) versus short time (14 days) in G4. Similar results were reported by Abu-El-Ezz (2005) on Trichinella spiralis in rats, Ayaz et al. (2007) on Aspiculuris tetraptera and Hymenolepis nana in naturally infected mice, El-Shenawy et al. (2008) on S. mansoni in mice and Baghdadi and Al-Mathal (2011) on E. stiedae in rabbits.

At 28 day PI, Table 2 showed significant reduction of oocyst count in the bile of group G5 (7.58±0.47) with reduction of 92.11% when compared with those of G3 (96.08±3.36) (reduction of 0 %) and G4 (22.08±0.80) (reduction of 77.02 %). This may be due to the effect of thymoquinone substance present in the N. sativa seed oil that could inhibit the growth and development of E. stiedae invasive stages (sporozoaites and merozoites) resulted in reduction of oocysts formation in the bile. These results are supported by findings of Mahmoud et al. (2002), Rahman et al. (2008) and Baghdadi and Al-Mathal (2011) on E. stiedae in rabbits.

At 28 day PI, Table 3 showed significant decrease in the liver lesion scoring in G5 (1.80±0.20) when compared with that of G3 (3.80±0.13) and G4 (2.30±0.15). This indicates that the N. sativa seed oil was highly effective at reducing the liver lesions in G5 when compared with groups G3 & G4. Furthermore, rabbits infected with E. stiedae showed remarkable improvement in liver tissue histopathology on day 28 PI {in rabbits given           N. sativa seed oil for 7 days before infection and continued for 28 days PI (G5)}. In addition, there was a huge reduction in various stages of parasite in liver tissue, resulting in reduced lesions in the liver (Table, 3); consequently the fecal and bilary oocyst output were markedly reduced. Similar results were reported by Baghdadi and Al-Mathal (2011) on E. stiedae in rabbits.

In this study, Table 4 showed that liver enzymes {ALT (13.94±0.12), AST (27.82±0.17)}, total cholesterol (129.89±0.23) and creatinine (0.886±0005) were significantly decreased in G5 when compared with G4 {ALT (16.59±0.21) and AST (33.95±0.13)}, total cholesterol (140.04±0.24), creatinine (0.962±0.006) and with G2 {ALT (12.26±0.05), AST (23.81±0.24)}, total cholesterol (126.59±0.20), creatinine (0.860±0.007). However, total protein (5.16±0.04), albumin (2.70±0.01) and globulin (2.46±0.04) in G5 were increased and directed toward normal level when compared with G4 (4.92±0.03, 2.61±0.02& 2.31±0.05, respectively) and G2 (5.40±0.06, 2.76±0.03 & 2.64±0.08, respectively). This may be attributed to presence of thymoquinone group thatprotected liver cells from damage consequently liver functions as AST and ALT were improved. Similar results were reported by Mahmoud et al. (2002) and El-Shenawy et al. (2008) in mice. However, N. sativa seeds (till 10 %) yielded no differences of blood plasma chemistry in chicks (El-Sayed et al., 2005). Black cumin at level of 2 or 3% would decrease cholesterol in egg yolk (Aydin  et al., 2008). Addition of N. sativa in form of oil (0.5%), seeds (1%) or meal (10%) to broiler diets under stress increased total protein, albumin and globulin; reduced mortality, cholesterol, AST; and ALT and improved RBCs, WBCs and Hb (Hermes et al., 2011), prevented oxidative stress and improved biochemical parameters (Attia et al., 2011), decreased cholesterol (Zaoui et al., 2002 and Isik et al., 2011) and counterbalanced renal injury in rats (Ahmed and El-Mottaleb, 2012). Also daily administration of            N. sativa seed oil (800 mg/kg orally for 4 weeks) improved AST, ALT and bilirubin but caused a significant decrease in cholesterol in albino rats (El-Dakhakhny et al., 2000).

In Table 5, the uninfected rabbits that received          N. sativa treatment for 35 days (G2) showed significant body weight and cumulative weight gains starting from day 14 till end of experiment (2196.67±6.45, and 1176.33±9.34, respectively) when compared with (G1) (2078.33±6.61 and 1124.33±8.13, respectively). This may be due to the N. sativa seeds contain appetizing materials that may increase food intake. Similar results were reported by El-Abhar et al. (2003). In groups (G5) and (G2), rabbits that received the N. sativa seedoil gained weight more rapidly. Specifically, rabbits in G5 (1965.00±7.48) increased in weight by 22.77 % as compared to G3 (1491.71±10.58), whereas G2 rabbits (2196.67±6.45) increased in weight by 33.91 % on day 35 PI. The difference in weight gain was very significant between the two treated groups (G5 and G4) (1004.33±8.08 versus 922.67±12.35, respectively). This may be attributed to the higher percentage of fatty materials in N. sativa seedoil. This agrees with that of El-Abhar et al. (2003) and Khalaji et al. (2011). Broilers receiving 40 g kg (-1) of black seed in feed had a significant effect on body weight gain, feed intake and FCR (Durrani et al., 2007). N. sativa seeds (up to 2 %) improve performance, health and immunity of birds with less mortality (Nasir, 2009). In this study, disease symptoms were improved and body weights increased within 2 weeks of treatment, indicating recuperation from E. stiedae infection. Similar results were reported by Ghanem et al. (2008).

In Table 6, the mean liver weights and the relative liver weights of the infected and untreated rabbits (G3) (113.43±1.01 and 0.076, respectively) were significantly higher than the uninfected and untreated rabbits (G1) (30.47±0.430 and 0.015, respectively), and uninfected and treated rabbits (G2) (30.53±0.29 and 0.014, respectively) throughout the duration of the experiment. The liver weights were not significantly different between the infected groups (G1 and G2) (30.47±0.30 and 30.53±0.29, respectively) on day 28 PI. However, following N. sativa treatment, liver weights were drastically decreased in G5 (76.05±0.60) as compared with G4 (90.53±0.71) and G3 (113.34±1.01) on day 28 PI. Similar results were reported by Baghdadi and Al-Mathal (2011).

In Table 7, the mean number of developmental stages of E. stiedae per microscopic field was significantly decreased in G5 when compared to G4 and G3 (2.00±0.37 versus to 9.1±0.55 and 74.5±5.01, respectively). This attributed to the anticoccidial effect of N. sativa seed oil on E. stiedae parasite after use for long time (28 days PI). Similar results were mentioned by Baghdadi and Al-Mathal (2011).

In Table 8, the mortality of infected rabbits with       E. stiedae was 8.33 % in G5 (1/12), 16.67% in G4 (2/12), 41.67% in G3 (5/12) PT. However, there were no mortalities in Groups 1 & 2. Similar results were reported by Hermes et al. (2011) in poultry and Baghdadi and Al-Mathal (2011) in rabbits.

Normal tissue structure of hepatocytes, portal area, and bile ducts was present in liver samples from uninfected and treated rabbits with N. sativa seed oil (G2), regardless of treatment, on day 28 PI (Fig. 1, A-D). This ensures that there may be no toxic effect of N. sativa seed oil on liver cells even for a long time treatment. Similar results were reported by Baghdadi and Al-Mathal (2011) in rabbits and Attia et al. (2011) in rats.Classical pathological changes of hepatic coccidiosis in liver of infected rabbits (G3) were present on day 28 PI (Fig. 2, from A to D). Rabbit liver of G3 showed a dilated congested central vein with severe necrosis of endothelial lining and haemorrhagic areas (Fig. 2, A), necrosis of hepatocytes and haemorhages in portal area with inflammatory reaction (Fig. 2, B), papilliform projections of epithelium into the lumen of bile duct containing very large number of developed oocysts (Fig. 2, C), and different developmental stages of      E. stiedae in the lumen of bile duct (Fig. 2, D). Similar results were reported by Baghdadi and Al-Mathal (2011).

On day 28 PI, liver of treated rabbits with N. sativa seed oil for 14 successive days (G4) showed incomplete healing of congested central vein and endothelial lining encircling central vein (Fig. 3, A), presence of little hemorrhages from portal area with mild vacuolar degeneration of hepatocytes (Fig. 3, B), multiple papillary fronds in the bile ducts were recorded with the presence of moderate number of the parasite stages from bile duct lumen (Fig.3, C&D). Similar results were reported by Abdel-Hamid et al. (2012) in rabbits who reported that the methanolic extract of N. sativa has a chemopreventive effect against progression into liver malignancy. Meanwhile, the liver of the rabbits given N. sativa seed oil for 7 days before infection and continued for 28 days PI (G5) showed complete healing of the congested central vein (Fig.4, A), disappearance of hemorrhages from portal area (Fig.4, B), and almost disappearance of the parasite stages from the lumen of the bile duct although presence of hyperplasia in the columnar epithelial cells of bile ducts which may be recovered by time (Fig.4, C&D). Similar results were reported by Baghdadi and Al-Mathal (2011) on E. stiedae in rabbits, El Shenawy et al. (2008) on      S. mansoni in mice.

The present results indicate that N. sativa seeds oil can be used to safely and effectively against             E. stiedae in rabbits with no side effects although presence of hyperplasia in the columnar epithelial cells of the bile ducts that may be recovered by time. In addition to, improvement of growth performance as well as liver and kidney functions. Future studies would be focused on determining the active materials within N. sativa treatments, optimal doses and mode of action.

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