Comparative Immunomodulatory E cacy of Rosemary and Fenugreek Against Escherichia Coli- Infection via Suppression of In ammation and Oxidative Stress in Broilers


 Broilers are frequently infected with Escherichia coli (E.Coli) bacteria, which often leads to the emergence of many diseases and high economic losses. Hence, the present study was performed to assess the comparative efficacy of dietary rosemary and fenugreek, under E.Coli-infection in broilers, via evaluation of growth performance, biochemical indices, immunological response and histo-morphological changes. Eighty Cobb broilers were allotted to four equal groups (n=20 chicks/group); control non-infected (CN), control infected (CI), rosemary infected (RI) and fenugreek infected (FI) groups. RI and FI groups showed a significant elevation in their body weight and body weight gain compared with CI group. Moreover, both groups revealed a significant decline in serum aspartate and alanine aminotransferase activities, as well as uric acid and creatinine levels. Significant decreases of total antioxidant capacity, catalase and superoxide dismutase activities were noted among CI chicks. Moreover, distinctly higher levels were evident in both RI and FI groups. Immunomodulatory markers assessment showed a significant increase in immunoglobulin G with a significant decline in interleukin-6 level in both RI and FI groups, with the lowest IL-6 value within FI group. Histopathological evaluations focused on the deleterious effect associated with E-Coli infection of broilers’ liver, kidney, intestine, spleen, bursa of fabricius, and thymus. A partial histological improvement was noticed among RI group, and nearly normal tissues were recorded in FI group. Overall, these findings suggest the ability of fenugreek to mitigate the adverse effects of E.Coli-infection on broiler performance and tissue pictures, through improvement of the chicken general health condition.


Introduction
In the global poultry industry, Escherichia coli (E-Coli) bacteria may lead to great economic losses each year due to high morbidity and mortality rates (Lau et al., 2010). E. coli infection is usually prevented or controlled with antibiotics. However, the emergence of the antibiotics and their continued use in poultry diets has raised many problems of varying severity including environmental pollution as well as the cross-resistance that may pose substantial risks for human health (Asai et al., 2011;Ghozlan et al., 2017). Notably, antibiotic growth inducers, in broiler feedings, have been banned by the European Commission. Therefore, many researchers have conducted their research studies to explore the potential use of effective alternatives to maintain livestock health. One possibility is the application of probiotics, prebiotics and herbaceous plants or their essential oils (Sarica et al., 2007), to replace antibiotics without negative impact on health or any loss of productivity (Demir et al., 2003;Maiorano et al., 2016 ). Herbal medicine has gained a wide interest in the feed industry, through their appetite and digestion-stimulating, . It is composed of 0.5% volatile oil, avonoids (diosmetin, genkwanin, diosmin, glucoside and luteolin), phenolic acids (rosmarinic, chlorogenic, neochlorogenic, labiatic and caffeic acids), carnosic acid, rosmaricine and isorosmaricine, triterpenic acids and others (Khan and Abourashed, 2010).

Materials And Methods
Experimental chicks, E-coli strain and natural products Eighty, one-day-old, apparently healthy commercial Cobb broiler were obtained from Alasma Masr Poultry Company, Egypt. E-coli strain O78 was obtained from Animal Health Research Institute, Ismailia, Egypt. Each chick inoculated with 0.5 ml of E. coli O78 bacterial inoculum, colony-forming unit (CFU), at 7 days old as 0.25 ml intranasal and 0.25 ml by eye drop route. Rosemary leaves and fenugreek seeds were purchased from local commercial market of herbs and medicinal plants (Al-kateb Company, Egypt), were grounded by the home blender, and then were added to the balanced ration by 5 gm plant powder/kg diet for each.
Birds grouping and treatment schedule Upon arrival, birds were weighed and kept under standard hygienic conditions in oor pens covered with unused wood shavings litter, with free access to the balanced commercial basal ration and fresh tap water ad-libitum until the end of the experiment. The temperature was adjusted according to the age, 32 o C at the rst week and decreased 2 o C per week. All chickens were vaccinated according to the vaccination schedule. Birds were then allotted into four equal groups (n = 20 chicks/group) as follow; control non-infected (CN) group, fed on balanced commercial ration free from any feed additives. Control infected (CI) group, fed on balanced commercial ration free from any feed additive but experimentally infected with E-Coli at one week of age (Majo et al., 1997). Rosemary infected (RI) group, fed on balanced commercial ration supplied with rosemary at the level of 0.5 % (5 gm rosemary leaves powder /kg ration) from one day to 6 weeks old (Ghazalah and Ali, 2008) and experimentally infected with E-Coli at one week of age. Fenugreek infected (FI) group, fed on balanced commercial ration supplied with fenugreek at the level of 0.5 % (5 gm fenugreek seed powder /kg ration) from one day to 6 weeks old (Elbushra, 2012) and experimentally infected with E-Coli at one week of age.

Growth performance parameters
Each chick was individually weighed at beginning and end of the experiment (one day, 6th week old), respectively. Body weight gain (BWG) was calculated at 6th weeks of the experiment by subtracting the body weight between two successive weights. Feed consumption (FC) was calculated /6 weeks by subtracting the amount of feed remaining at the end of the 6th week from the amount of feed given at the beginning of the experiment. The amount of feed consumed was then divided by the weight gain of each group to obtain the Feed conversion ratio (FCR) (Nobo et al., 2012).

Blood and tissue sampling
At the end of the experiment, ve chicks were randomly obtained from each group for blood and tissues collection. About 3 ml of blood samples were obtained by the heart puncture of each bird and collected in a plain centrifuge tube and used for the preparation of serum for assay of biochemical, immunological and antioxidant parameters (TAC and CAT). After blood sampling, chicks were gently sacri ced and small specimens from the liver, kidney, intestine, spleen, thymus and bursa were obtained for histopathological examination. Furthermore, parts of liver and kidney were stored at -20 o c for the antioxidant assay (SOD).

Sera biochemical parameters
Blood sera were used for assessment of hepatic and renal injury biomarkers. ALT and AST activities was determined colorimetrically according to the method of Crowley ( 1967). UA was determined by uricase -POD enzymatic colorimetric method according to Fossati et al. ( 1980). Cr occurred by photometric colorimetric test for kinetic measurement, methods without deproteinization, using readymade kits as described by the method of Owen et al., ( 1954). Serum glucose was determined according to Kinoshita et al. ( 1979). Cholesterol was determined by the enzymatic colorimetric method; CHOD-POD according to the method described by Allain et al. (1974).
Cytokines estimation IL-6 was assessed and IgG concentrations were calculated according to the method of Koivunen and Krogsrud (2006).

Evaluation of antioxidant indices
SOD concentration was measured according to Koivunen and Krogsrud (2006). CAT was assessed according to Wheeler et al., (1990), meanwhile TAC was calculated according to Hariane and Moya (2015).

Histopathological examination
Specimens of liver, kidney, intestine spleen, bursa, and thymus of sacri ced birds from all groups were freshly collected and then xed in 10% neutral buffered formalin, embedded in para n, sectioned at 5-7 µm thickness and nally stained with Hematoxylin and Eosin for histopathological examination.
Routine histological processing was carried out according to the method of Suvarna et al., (2012).

Statistical analysis
Data collected from biochemical, immunological and antioxidant assays of treated groups were statically analyzed in compare to control group for the mean and standard error using statistical software program (SPSS for windows, version 16, USA). Difference between means of different experimental groups were carried out using one-way ANOVA with Duncan multiple comparison tests. Dissimilar superscript letters in the same column show a signi cance (P > 0.05) (Landau and Everitt, 2004).

Result
The main clinical signs observed among the E-coli infected birds were dullness, depression, ra ed feathers, huddling, reduced fed and water consumption, which appeared within 24 hrs. post infection. Respiratory signs were developed 2-3 days post infection; as sneezing, rhinitis and wet eyes. As summarized in Table 1, mortality was highest in CI group (25%) followed by those of RI (10%) and then the lowest percentage were recorded within FI (5%). Moreover, non-signi cant change was reported in FI group compared with CN ( Fig. 1A, B). Concerning FC and FCR, CI group showed a signi cant decrease when compared with CN. Additionally, a signi cant decline of FC and FCR was noted among RI and FI groups in comparison with CI, with the less FC and FCR in RI group (Fig. 1C, B).

Cholesterol and glucose levels
Concerning cholesterol and glucose levels, CI group showed a signi cant increase and a signi cant decrease in cholesterol and glucose levels, respectively when compared CN ( Fig. 2A, B). RI and FI groups revealed a signi cant decline in cholesterol level compared with CI ( Fig. 2A). RI group showed a signi cant increase, while FI group revealed a signi cant decrease in glucose level in comparison with CI (Fig. 2B). RI group showed a non-signi cant change in glucose level when compared with CN (Fig. 2B).
AST and ALT activities, UA and Cr concentrations As shown in gure 3, CI group showed a signi cant increase in AST and ALT activities along with a signi cant elevation in UA and Cr levels when compared with CN. On the other hand, both RI and FI groups showed a signi cant decline in their levels compared with CI, with the least recorded value in FI.

Immunological pro le
Pointing to IL-6 and IgG levels, CI group revealed a signi cant increase in their levels compared with CN ( Fig. 4A, B). Meanwhile, both RI and FI groups showed a signi cant decrease in IL-6 ( Fig. 4A) when comaped with CI, along with a signi cant elevation in IgG level (Fig. 4B) when comapared with CN and CI groups, with the lowest value of IL-6 and the highest IgG level obtained from FI group.

Hepatic and renal SOD activities
Regarding hepatic and renal SOD activities, CI group showed a signi cant decline in their levels when compared with CN. In contrary, RI and FI groups showed a signi cant elevation of SOD levels in comparison with CI (Fig. 5). A non-signi cant change in hepatic and renal SOD levels was noted among CN, RI and FI (Fig. 5).

Sera antioxidant indices
With regard to antioxidant indices, CI group revealed a non-signi cant decline in TAC and CAT levels compared with CN group (Fig. 6A, B). RI and FI groups showed a signi cant increase of TAC and CAT levels compared with CI one (Fig. 6A, B). On the other hand, non-signi cant changes in TAC and CAT activities were recorded among RI and FI groups (Fig. 6A, B).

Histopathological evaluations
In all four treated-groups; hepatic, renal, intestinal, splenic, thymic and bursal specimens were processed further for histopathological analysis. Architectural changes were successively recorded in all select ed organs of experimental broilers. The histological structures of all previously mentioned organs in response to different treatments were illustrated in gures 7, 8, 9, 10, 11 and 12, respectively.
The microscopical examination of hepatic tissue sections, obtained from CN group, demonstrated normal hepatic architecture along with normally arranged hepatocytes separated by hepatic sinusoids and radiated from the central vein. The hepatocytes appeared crowded polygonal cells with centrally located spherically basophilic nuclei and acidophilic cytoplasm (Fig. 7A). In contrast, CI group showed thick hepatic capsule with marked degenerative changes among hepato cytes, Kupffer cell hyperplasia, mononuclear leukocytic in ltration around the central vein, evidence of marked congestion and dilatation of central vein and sinusoids as well as marked diffuse necrobiotic changes of hepatic tissue. Such degenerative changes were evidenced by vacuolar degeneration in some pyknotic hepatocytes. Moreover, brous connective proliferation was observed around the portal area admixed with mononuclear leukocytic in ltration (Fig. 7B). RI birds' liver revealed very mild degenerative changes in hepatocytes with mononuclear cellular in ltration. The central vein slightly dilated and congested compared to control group (Fig. 7C). Meanwhile, FI birds' liver showed near normal hepatocellular organization and architecture, with very mild degenerative changes of some hepatocytes and less mononuclear cell in ltration, others showed regeneration in the rest of the cells. Additionally, the central vein appears normal (Fig. 7D).
The selected renal sections obtained from CN group revealed normal renal histological structures of the glomeruli and surrounding tubules (Fig. 8A). However, infected birds showed marked degenerative changes of tubular cells and areas of mild interstitial in ltration of mononuclear leukocytic cells were noticed among the renal cortex of this treated group. Additionally, congestion of the renal blood vessels and inter-tubular capillaries were also observed along with extravasated RBCs among this group (Fig.  8B). The degenerative changes of tubular cells are indicated by vacuolar and hydropic degeneration. Additionally, individual epithelial cells were shrunken with pyknotic nuclei. Concerning RI birds, the kidneys showed mild congestion of the renal blood vessels and inter-tubular capillaries. Additionally, the lining epithelium of the convoluted tubules was mostly appeared degenerated (Fig. 8C). The degenerated changes of renal structures were seen to be disappeared in FI group which exhibited near normal renal features (Fig. 8D).
Control untreated birds revealed normal intestinal architecture with uniform intestinal villi lined by columnar epithelium with goblet cells in between, as well as intestinal glands located between the bases of villi in intestinal mucosal layer (Fig. 9A). Even the intestine of infected birds showed vacuolation, atrophy, sloughing and necrosis of intestinal villi along with leukocytic in ltration (mainly hetrophils, macrophage and lymphocyte) associated with edema and necrosis of the muscularis mucosa (Fig. 9B).
The intestinal tissue architecture of RI birds revealed some degenerative changes of the intestinal architecture but less than that picture recorded in an infected group alone (Fig. 9C). Meanwhile, FI birds showed normal villus architecture with mild cellular in ltration in intestinal mucosa and sub-mucosa when compared with the control (Fig. 9D).
Histological examination of splenic sections obtained from CN broilers showed no difference in splenic architecture enclosing normal white and red pulps (Fig. 10A). Additionally, the splenic red pulp formed mainly from cords of reticular and blood cells associated with immunocompetent cells; macrophages and lymphocytes. The white pulp is the splenic lymphatic tissue, composed mainly of lymphoid follicles with periarterial sheath (Fig. 10A). Meanwhile, the infected group showed noticeable pathological changes among splenic parenchyma when compared to control group. These changes include lymphocytic depletion and degeneration (Fig. 10B). Additionally, massively congested areas within the splenic red pulp were noted. Marked increasing of the area red pulp on the expense of the white one was also recorded among this infected group. RI group showed a signi cant difference from that of infected birds without any treatment including relative improvement of white pulp containing small-sized lymphoid follicles with mild to moderate congestion of splenic blood vessels along red pulp (Fig. 10C). Splenic parenchyma restored its architecture to almost the normal picture and appeared to be regenerated after fenugreek treatment with mild congestion of splenic blood vessels (Fig. 10D).
The present light microscopic study of thymic sections from control untreated birds revealed thin connective tissue capsule surrounded the gland, numerous ne septa of connective tissue originated from the capsule were divided the organ into incompletely separated lobules. Each lobule organized into a peripheral cortex and a central medulla with numerous thymocytes, few macrophages, and diffuse Hassall's corpuscles found (Fig. 11A). On the other hand, the thymus of CI group showed marked lymphocytic depletion when compared with the thymus of the control non-infected group along with blood vessels congestion and extravasated haemo biotic cells (Fig. 11B). Lymphocytic necrotic areas were also noted near the area of thymic cortex concomitant with an irregular arrangement of thymic cells within cortex and medulla. Hence, the boundaries between the cortex and medulla were mingled together. Both RI and FI revealed thymic architectural improvements but the best pictures were observed in the infected group treated with fenugreek (Fig. 11C, D).
It is clearly noticed that the bursal sections obtained from CN group showed normal longitudinal mucosal folds projected into the lumen covered by follicular epithelium, numerous follicles lled the lamina propria of each fold. Each bursal follicle was composed a peripheral cortex and a central medulla. The cortex composed mainly of many closely packed small lymphocytes meanwhile medulla contained fewer cells of various sizes (Fig. 12A). Meanwhile that of the infected bird's revealed mild to moderate lymphoid depletion with severe diffuse edema of the interfollicular connective tissue in the lamina propria (Fig.  12B). In the medulla of the follicles, some lymphocytes showed karyopy knosis. Regarding RI group, there was still tendency of interfollicular edema and mild lymphoid depletion among the examined sections (Fig. 12C), however, FI group showed an improvement of the degenerative changes when compared with infected group with less edematous area among the interfollicular connective tissue (Fig. 12D).

Discussion
The prohibition of nutritive antibiotic use in Europe, as well as the increased awareness of the consumers, triggered a need for natural and safe feed additives to achieve better poultry production. , this likely was due to reduction in feed intake that resulting from the strong avor of rosemary which need an adaptation period for accommodating oral and nasal sensing, preparing the gastrointestinal tract for food reception, and modulating digestive secretions and gut motility. Additionally, it may be also due to the fact that rosemary leaves contain high crude ber particularly, cellulose which may hampered nutrient utilization by chickens (Barelli, 2013;Soltani et al., 2016). Oppositely to the results of Mathlouthi et al. (2012) who recorded good growth performance effects of rosemary, that may be due to the difference in the used rosemary form, source, and concentration . On the other hand, fenugreek cleared an elevating effect on BW and BWG, which come in agreement with Park and Kim (2015). Meanwhile, it not harmony with that results of Saki et al. (2014) and Patel et al. (2014). Our nding may be attributed to fenugreek controlling effect on potential pathogens in gut micro ora, thus move the animals from immune defense stress to increase absorption of essential nutrients, improving the digestive capacity of the small intestine and thereby helping animals to grow better, as mentioned by Hashemi and Davoodi (2011). Such results clearly con rmed by histopathological evaluation that revealed less degenerative changes in RI intestine and normal villus architecture in fenugreek infected intestinal tissue. This good histological picture came in accordance with Gurkan et al. ( 2015).
In . Since, the e cacy of immune system in chickens mainly depends on the bursa of fabricius and thymus for lymphocytic differentiation and initiating humeral and cellular-immune responses. So the marked bursal and thymic lymphocytic depletion, induced by E.coli experimental infection, was previously reported by Madian et al. (2008) who reported an immunosuppressive effect of E-coli. Meanwhile, Nakamura et al. (1986) related this depletion to a combination of direct effects of E-coli toxic components and non-speci c stress factors.
Generally, herbs rich of avonoids, vitamin C, and carotenoids bene ts the immune system, and presenting immunostimulant effect through; enhanced phagocytic activity, modulation of cytokine secretion, histamine release, immunoglobulin secretion, plasma myeloperoxidase and lysozyme activity increase (Mirzaei-Aghsaghali, 2012). Rosemary abled to increase IgG, decrease IL-6 in RI birds, these results are agreed with Da Rosa et al., (2013) who related its anti-in ammatory activity to effect on decreasing the proin ammatory cytokines with increasing of the anti-in ammatory cytokine in mice suffered from pleurisy. Additionally, fenugreek succeeded to increase IgG, and decrease IL-6 in FI group, this immunostimulant effects is related to high total phenolic content following both fenugreek gastric and duodenal digestion (Jayawardena et al. 2015).
E-coli endotoxin resulted in elevating the systemic cytokines (TNF and IL-6), which enhance production of superoxide anion, release of lysozyme, H 2 O 2 and chemotaxis, as an adaptive mechanism to decrease reactive oxygen formation, besides increasing its uptake, resulting in the production of potent oxidant bactericidal agents (Dutta and Bishayi, 2009). Meanwhile, when the stress is too high, antioxidant activity is decreased and apoptosis is activated (Surai, 2015), which cleared the decrease in the SOD, CAT and TAC levels in our E-Coli infected group. Generally, antioxidant supplementation resulted in increased interleukin levels, elevated total lymphocytes, increased killer cell activity and antibody response to antigen stimulation. Moreover, using of antioxidants herbs in broiler feed is not important only for their health, but also for the oxidative stability of their meat products (Fellgenber and Speisky, 2006).

Conclusion
Considering the obtained ndings, it can be concluded that rosemary or fenugreek supplementation is bene cial in reduce and improve the biochemical and histological alterations induced by E. coli-infection in broilers. However, the present study suggests the protective, anti-in ammatory, antioxidant, and immunomodulatory effects of rosemary or fenugreek on E. coli-induced toxicity; the most protective e cacy was recorded in infected chicks treated with fenugreek. Moreover, the using of fenugreek as feed additive may be a good strategy against oxidative stress induced by E. coli, knowing that it is prohibited to administer in case of hypoglycemia. To strengthen these ndings, further investigations are needed to explore the mechanism action of rosemary and/or fenugreek against E. coli toxicity in broilers.