Comparative immunomodulatory efficacy of rosemary and fenugreek against Escherichia coli infection via suppression of inflammation and oxidative stress in broilers

Broiler chickens are frequently infected with Escherichia coli (E. coli) bacteria, which often leads to the emergence of many diseases and high economic losses. Hence, the current study was conducted to assess the relative efficacy of dietary rosemary and fenugreek, under E. coli infection in broilers and their ability to replace antimicrobials without any loss of productivity or negative influence on broiler health, via evaluation of growth performance, biochemical indices, immune 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. The RI and FI groups revealed a significant elevation in their body weight and body weight gain compared with the CI group. However, both groups showed a significant decline in serum aspartate and alanine aminotransferase activities, as well as uric acid and creatinine levels. A significant decrease in total antioxidant capacity, catalase and superoxide dismutase activities was noted among CI chicks. Moreover, distinctly higher activities were evident in both RI and FI groups. Assessment of immunomodulatory markers showed a significant increase in immunoglobulin G along 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. Partial histological improvement was noticed among RI group, and nearly normal tissues were recorded in FI group. Overall, the obtained findings suggest the ability of fenugreek to mitigate the adverse effects of E. coli infection on broiler performance and tissue profiles, by improving the general health status of the broiler chickens.


Introduction
In the global poultry industry, Escherichia coli (E. coli) lead to significant economic losses each year (Lau et al., 2010). E. coli infection is usually controlled with antibiotics. However, the emergence and continued use of the antibiotics in poultry feed have raised cross-resistance that poses substantial risks for human health ( Asai et al., 2011;Ghozlan et al., 2017). One possibility is the application of probiotics, prebiotics and herbaceous plants or their essential oils (Sarica et al., 2007), to replace antibiotics without negative health impact or any loss of productivity (Demir et al., 2003;Maiorano et al., 2016).
Rosemary (Rosmarinus officinalis), a widespread household plant, is used as a natural additive to animal feed with its antibacterial, antifungal and antioxidant activities (Genena et al., 2008;Mohamed et al., 2016a), as well as a flavoring agent for food, beverages and cosmetics preparations ( Ibarra et al., 2010). It possesses a number of therapeutic applications in medicine for the treatment or management of various pathological conditions such as inflammatory diseases, respiratory and GIT disorders (Al-Kassie and Abd- Al-Jaleel, 2011;El-Boshy et al., 2015). It is mainly composed of 0.5% volatile oil, flavonoids (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). Jafarı-sales and Pashazadeh (2020) indicated that 1.8 cineole and α-pinen are the highest essential oils in rosemary and have a remarkable anti E. coli activity, so it could be used as a suitable alternative to synthetic antibiotics. Furthermore, Ojeda et al. (2013) reported a relationship between the antibacterial activity of different rosemary essential oils against Gram-positive and Gram-negative bacteria, and they related their activities to the changes in membrane permeability and disruption of the E. coli cell membrane in vitro.
The emergence of drug-resistant E. coli indicates the possibility of chicken as a source of antibiotic resistance pool for humans and a major ecological risk to the environment. Therefore, in this paper, the authors report on the in vivo ameliorative effects of broiler ration supplementation with two medicinal plants, rosemary leaves (Rosmarinus officinalis) and fenugreek seeds (Trigonella foenum-graecum), challenging with experimental infection of E. coli for 6 weeks, on growth performance, some selective biochemical, immunological and antioxidant parameters, along with histopathological changes associated with hepatic, renal, intestinal, splenic, thymic and bursal tissues.

Experimental broiler chicks, E. coli strain and natural products
Eighty, 1-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. E. coli colonies were grown in nutrient broth for 24 h at 37 °C, and viable number was adjusted to 4 × 106 colony forming units (CFU). Each chick was inoculated with 0.5 ml of E. coli O78 bacterial inoculum, at 7 days old of age as the following: 0.25 ml intranasal and 0.25 ml via 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 g plant powder/kg diet for each.

Birds grouping and treatment schedule
Upon arrival, birds were weighed and kept under standard sanitary conditions in floor pens covered with unused wood shavings litter, with free access to the balanced commercial basal ration (Table 1) and fresh tap water ad libitum until the end of the experiment. The temperature was adjusted according to age, 32 °C at the first week and then decreased 2 °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 1 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 g rosemary leaves powder/kg ration) from 1 day to 6 weeks old (Ghazalah and Ali, 2008) and experimentally infected with E. coli at 1 week of age. Fenugreek infected (FI) group, fed on balanced commercial ration supplied with fenugreek at the level of 0.5% (5 g fenugreek seed powder/kg ration) from 1 day to 6 weeks old (Elbushra, 2012) and experimentally infected with E. coli at 1 week of age.

Growth performance parameters
Each chick was individually weighed at the beginning and end of the experiment (1 day and 6 th week old), respectively. Body weight gain (BWG) was calculated at 6 th week of the experiment by subtracting the body weight between two consecutive weights. Feed consumption (FC) was calculated by subtracting the amount of feed remaining at the end of the 6 th 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 for each group to obtain the feed conversion ratio (FCR) (Nobo et al., 2012).

Blood and tissue sampling
At the end of the experiment, five chicks were randomly obtained from each group for blood and tissue specimens' collection. Approximately 3 ml of blood samples was obtained by puncturing the heart of each bird, collected in a plain centrifuge tube and then used for the preparation of serum for assay of biochemical, immunological and antioxidant parameters (TAC and CAT). After blood sampling, chicks were gently sacrificed, 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 °C for the antioxidant assay.

Sera biochemical parameters
Blood sera were then used for assessment of hepatic and renal injury biomarkers. ALT and AST activities were 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 was performed 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).

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
Liver, kidney, intestine spleen, bursa and thymus samples, obtained from sacrificed birds of all groups, were freshly collected and then fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5-7-µm thickness and finally stained with hematoxylin and eosin (H&E) for histopathological examination. Routine histological procedures were carried out according to the method of Suvarna et al. (2012).

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

Results
The main clinical symptoms, observed among the E. coliinfected birds, were dullness, depression, raffled feathers, huddling, reduced fed and water consumption, which appeared within 24 h post infection. Respiratory signs were developed 2-3 days post infection, as sneezing, rhinitis and wet eyes. As summarized in Table 2, mortality was highest in CI group (25%) followed by that of RI (10%), and then the lowest percentage was recorded within FI (5%).

Growth performance parameters
Growth performance parameters was summarized within Table 3 (supplementary data). The CI group showed a significant decline in BW and BWG compared to CN. Meanwhile, RI and FI groups revealed a significant increase in BW and BWG compared to CI, with more significance in FI group. Moreover, non-significant change was reported in FI group compared to CN (Fig. 1A, B). Concerning FC and FCR, CI group showed a significant decrease when compared with CN. Additionally, a significant 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, D).

Cholesterol and glucose levels
CI group showed a significant increase and a significant decrease in cholesterol and glucose levels, respectively when compared to CN ( Fig. 2A,B). RI and FI groups revealed a significant decline in cholesterol level compared to CI ( Fig. 2A). RI group showed a significant increase, while FI group revealed a significant decrease in glucose level in comparison with CI (Fig. 2B). RI group showed a nonsignificant change in glucose level when compared to CN (Fig. 2B).

AST and ALT activities, UA and Cr concentrations
As shown in Fig. 3, the CI group showed a significant increase in AST and ALT activities along with a significant elevation in UA and Cr levels when compared to CN. On the other hand, both RI and FI groups showed a significant decline in their levels compared to CI, with the least recorded value in FI.

Immunological profile
Pointing to IL-6 and IgG levels, the CI group revealed a significant increase in their levels compared to CN (Fig. 4A, B). Meanwhile, both RI and FI groups showed a significant decrease in IL-6 level ( Fig. 4A) when compared to CI, along with a significant elevation in IgG level (Fig. 4B) when compared 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
The CI group showed a significant decrease in hepatic and renal SOD activities when compared to CN. In contrary, RI and FI groups showed a significant elevation of SOD levels in comparison with CI (Fig. 5). A non-significant change in hepatic and renal SOD levels was noted among CN, RI and FI (Fig. 5).

Sera antioxidant indices
The CI group revealed a non-significant decline in TAC and CAT levels compared with CN group (Fig. 6A, B). RI and FI groups showed a significant increase of TAC and CAT levels compared to CI one (Fig. 6A, B). On the other hand, non-significant 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 selected organs of experimental broilers. The histological structures of all previously mentioned organs in response to different treatments were illustrated in Figs. 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 hepatocytes, Kupffer cell hyperplasia, mononuclear leukocytic infiltration 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, fibrous connective proliferation was observed around the portal area admixed with mononuclear leukocytic infiltration (Fig. 7B). RI birds' liver revealed very mild degenerative changes in hepatocytes with mononuclear cellular infiltration. The central vein was 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 infiltration; 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 infiltration 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 was 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 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).

head arrows). H&E stain
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 infiltration (mainly heterophils, 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 infiltration 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 significant 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 fine 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 Fig. 12 Representative photomicrograph of broiler bursa of Fabricius. A; CN group, B; CI group, C; RI group and D; FI group. Here, lymphoid follicle (thick arrow); cortex (CO); medulla (M); marked lymphocytic depletion (LD); interfollicular connective tissue (I). H&E stain 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 filled the lamina propria of each fold. Each bursal follicle was composed of 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 karyopyknosis. 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. Herbal plants are used in animal nutrition as appetite, digestion stimulants, physiological functions stimulants, prevention and treatment of certain pathological conditions and antioxidants ( Mohamed et al., 2016a;2016b;Ismaiel et al., 2017;Abdellatief et al., 2017;Emam et al., 2018;Farouk et al., 2020;Gad et al., 2021). The current study focused on the comparative efficacy of rosemary and fenugreek as feed additives, growth promoters, immunostimulants and tissue protective agents against E. coli infection in broilers.
The decreasing effect of E. coli infection on B.W., B.W.G, FC and FCR, noted in the present study, may be attributed to colonizing of E. coli in the intestinal wall and secreting toxins and so affects intestinal integrity which reflected on feed intake and so on weight gain (Gomis et al., 1997;El-Baky et al., 2014). This assumption is proved by intestinal histopathological examination where E. coli badly affect intestinal tissues with atrophy, sloughing and necrosis of intestinal villi and glands along with leukocytic infiltration, which came in accordance with Moursi et al. (2008).
Moreover, the present findings indicated a decreasing of BW among RI birds compared with CN group, which came in agreement with Hernández et al. (2004), Abd El-Latif et al. (2013) and Soltani et al. (2016). This likely was due to reduction in feed intake that results from the strong flavor of rosemary which needs 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 fiber particularly, cellulose which may hampered nutrient utilization by chickens (Barelli, 2013;Soltani et al., 2016). Opposite 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 ( Yesilbag et al., 2011). On the other hand, fenugreek cleared an elevating effect on BW and BWG, which comes in agreement with Park and Kim (2015). Meanwhile, it is not in harmony with the results of Saki et al. (2014) and Patel et al. (2014). Our finding may be attributed to the fenugreek controlling effect on potential pathogens in gut microflora, thus moving 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 were clearly confirmed 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 the current study, E. coli infection resulted in an increase in AST, ALT, UA, Cr and cholesterol, with a decline in glucose level. These findings are in accordance with Zaki et al. (2012) and Abdel Ziz et al. (2016) who recorded that E. coli infection in chickens resulted in significant increase in liver enzymes (AST and ALT) activities. Our results also were in complete harmony with those reported by Joan and Pannel (1981), who stated that the E. coli infection produced alteration in cellular permeability due to changes in cell membrane which allows the escape of these enzymes into serum in abnormal high level. Our findings are magnified by histopathological examination of hepatic and renal tissues which are expressed as hepatocytic vacuolar degeneration and marked necrobiotic changes of hepatic tissue, along with renal tubular degenerative changes. This histopathological figures came in agreement with Moursi et al. (2008).
On contrary, rosemary succeeded to decrease AST, ALT, UA and Cr levels, which was proved by histopathological examination that revealed mild degenerative changes in hepatocytes. Similar data were obtained by Albasha and Azab (2014) and Mohamed et al. (2016a) who recorded the protective effect of rosemary supplementation against cadmium, gentamicin and lead acetate induced hepatorenal toxicity, respectively. Azab et al. (2016) related the hepatoprotective effect of rosemary to its principal antioxidant constituents (rosmarinic acid, diterpenoids such as carnosic acid, carnosol, carotenoid and alpha-tocopherol) which inhibit free radicals' generations. Also, Mohamed et al. (2016a) related the renal protecting effect of rosemary to synergistic interactions between its individual components with its antioxidant properties. Moreover, rosemary showed hypercholesteremic properties in RI group, which were also recorded by Ghazalah and Ali (2008) and Polat et al. (2011),who related it to leaves defatted portion rich in fibrous content that prevents intestinal cholesterol absorption. Additionally, fenugreek mediated a decrease of AST, ALT, UA, Cr and glucose levels than CI, which is magnified by histopathological examination that revealed near normal hepatocellular and renal architecture. These results were also recorded by Mamoun et al. (2014) and Park and Kim (2015). Mentreddy (2007) related the hypoglycemic effect of fenugreek to the steroidal saponins, alkaloids and 4-hydroxyisoleucine soluble dietary fiber fraction, exerting delaying effect on sucrose digestion and inhibition of carbohydrate hydrolyzing enzyme, as well as stimulating insulin secretion from the β pancreatic cells.
Our findings of increased IgG and IL-6 levels in CI group besides came in accordance with Eleiwa et al. (2011). The microbial pathogens stimulate the immune responses which produce cytokine IL-6, favor B-cell maturation and produce neutralizing antibodies IgG that neutralize bacterial toxins (D' Elios et al., 2011). Since, the efficacy 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-specific stress factors.
Generally, herbs rich in flavonoids, vitamin C and carotenoids improve the immune system and present immunostimulant effect through enhancement of phagocytic activity, modulation of cytokine secretion, histamine release, immunoglobulin secretion, plasma myeloperoxidase and lysozyme activity increase (Mirzaei-Aghsaghali, 2012). Rosemary was able to increase IgG and decrease IL-6 in RI birds; these results agreed with Da Rosa et al. (2013) who related its anti-inflammatory activity to the effect on decreasing the proinflammatory cytokines with increasing the anti-inflammatory cytokine in mice that suffered from pleurisy. Additionally, fenugreek succeeded to increase IgG and decrease IL-6 in FI group; this immunostimulant effect 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, use of antioxidants herbs in broiler feed is important not only for their health, but also for the oxidative stability of their meat products (Fellgenber and Speisky, 2006).
Rosemary can elevate the SOD, CAT and TAC in RI birds. Soltani et al. (2016) related the antioxidant properties of rosemary to the high polyphenols containing hydroxyl groups that probably stop free radical formation. Furthermore, Polat et al. (2011) observed the greatest activity of SOD through broiler supplementation of rosemary in comparison to vitamin E. Moreover, fenugreek abled to suppress oxidative stress indicated by the increase in SOD, CAT, and TAC level in FI birds. Maharana and Dadhich (2016) related these findings to the oxidative stress suppression, reduction in cell apoptosis and fibrosis to trigonelline present in fenugreek. Additionally, Mohammadzadeh et al. (2015) recorded an elevation of catalase enzyme activities after treating rats with acetaminophen-liver toxicity by fenugreek.

Policy suggestion
Although herbal medicines have always been a form of therapy for livestock among poor farmers, in recent years, it gained extensive attention in the feed industry. Rosemary is a spice and medicinal herb, has been used as a tonic, stimulant and carminative as well as in treating dyspepsia, stomach pains, and nowadays as an antioxidant and anti-inflammatory agent, thus a therapeutic potential in treating many diseases condition. Historically, fenugreek was used as a medicinal herb having a nutritional value. Recent research studies have shown its effectiveness in promoting lean body mass, reducing blood glucose levels, lowering cholesterol and treating gastrointestinal disorders. In modern food technology, it is used as a food stabilizer, adhesive and emulsifying agent.

Managerial implication
The clinical study might have an implication for infectious research and might recommend against the use of a particular nutritive antibiotic. Therefore, implication signifies the impact of the current research, and recommendations might be concrete actions that the research proposes.

Conclusion
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, so many alternatives such as probiotics, prebiotics, exogenous enzymes, antioxidants and herbaceous plants have been investigated to replace antimicrobials. Medicinal plant is added to feed as extracts obtained from dried plants or parts of the plants; its effect depends largely on the dosage used by active components.
Considering the obtained findings, it can be concluded that rosemary or fenugreek supplementation is beneficial in improvement of both biochemical and histological alterations induced by E. coli infection in broilers. However, the present study suggests the protective, anti-inflammatory, antioxidant and immunomodulatory effects of rosemary or fenugreek on E. coli-induced toxicity; the most protective efficacy was recorded in infected chicks treated with fenugreek. Moreover, the use 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 findings, further investigations are needed to explore each rosemary phenolic substances and fenugreek seeds chemical components' mechanism of action against E. coli toxicity in broilers.