The main clinical signs observed among the E-coli infected birds were dullness, depression, raffled 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%).
Table (1): The mortality percentage (N= 20) in chicken administrated Rosemary and Fenugreek for 6weeks and experimentally infected with E-Coli.
Groups
|
Number of dead chicks
|
Mortality %
|
CN
|
0
|
0
|
CI
|
5
|
25
|
RI
|
2
|
10
|
FI
|
1
|
5
|
Growth Performance parameters
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 with CI, with more significance in FI group. Moreover, non- significant change was reported in FI group compared with 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, B).
Cholesterol and glucose levels
Concerning cholesterol and glucose levels, CI group showed a significant increase and a significant decrease in cholesterol and glucose levels, respectively when compared CN (Fig. 2A, B). RI and FI groups revealed a significant decline in cholesterol level compared with 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 non-significant change in glucose level when compared with CN (Fig. 2B).
AST and ALT activities, UA and Cr concentrations
As shown in figure 3, CI group showed a significant increase in AST and ALT activities along with a significant elevation in UA and Cr levels when compared with CN. On the other hand, both RI and FI groups showed a significant decline in their levels compared with CI, with the least recorded value in FI.
Immunological profile
Pointing to IL-6 and IgG levels, CI group revealed a significant increase in their levels compared with CN (Fig. 4A, B). Meanwhile, both RI and FI groups showed a significant decrease in IL-6 (Fig. 4A) when comaped with CI, along with a significant 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 significant decline in their levels when compared with 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
With regard to antioxidant indices, 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 with 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 figures 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 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 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 infiltration (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 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 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 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).