Clinical Signs and Postmortem (PM) Lesions of E. coli Infection: Experimental infection with E. coli revealed suggestive clinical signs and PM lesions after 48 hr. post-infection in the form of depression with whitish diarrhea.
PM lesions revealed liver enlarged and congested and distended gallbladder and cecum. Such changes were less prominent in the agrimos-infected group (see Table 1).
Growth Performance: The feed supplemented with agrimos® in the third and fourth groups significantly (P ≤ 0.05) increased the body weight (BW) and total gain along the whole experiment and improved feed conversion ratio (FCR) compared to the other groups (see Table 2). On the other hand, the above-mentioned parameters were decreased significantly (P ≤ 0.05) in the control-infected group as compared to the other groups.
Serum Parameters Related to Liver Function: In Table (3) there were significant (P ≤ 0.05) increases in the activities of alanine transaminase (ALT) and aspartate aminotransferase (AST) enzymes in the group of broiler chickens infected with E. coli compared to the control non-infected group. However, serum total protein and albumin were significantly (P ≤ 0.05) decreased. Agrimos® supplementation did not affect serum ALT, AST, and albumin but the serum total protein and globulin affected significantly (P ≤ 0.05) when compared with the control non-infected group.
Serum Antioxidant Enzymes Activity: Serum MDA level was significantly (P ≤ 0.05) increased in broiler chickens infected with E. coli (see Table 4). However, serum catalase (CAT) and superoxide dismutase (SOD) activities were significantly (P ≤ 0.05) reduced compared to the other groups. Agrimos® supplementation significantly (P ≤ 0.05) decreased serum lipid peroxidation (MDA) with significant (P ≤ 0.05) increased serum CAT and SOD when compared to the control-infected group.
Immune Response against Newcastle Disease (ND): Immune response to vaccination of ND, which evaluated by Hemagglutination Inhibition (HI) titer revealed a difference in log2 of GM. There was a significant difference in HI titer in the second week after infection between each of the E. Coli infected group and the control negative group, and the agrimos infected group. In addition, the agrimos group significantly differs about the other three groups.
After vaccination in the second and third week, this pattern was observed where no difference was recorded between each of the E. coli infected group, control non-infected, and agrimos® infected group. Besides, the agrimos group significantly differs about the other three groups (see Table 5).
Colonization of E. coli: The colonization of E. coli in the control infected group in different organs showed several rates of 67%, 44%, 22%, 44%, and 53% in the respiratory tract, liver, gallbladder, spleen, and fecal swab, respectively (see Table 6). However, in the agrimos infected group, the colonization of E. coli in different organs showed reduced rates of 22%, 33%, 22%, 11%, and 33% in the respiratory tract, liver, gallbladder, spleen, and fecal swab, respectively.
The Weight of Immune Organs: At zero, 15, and 35 days of age, the weight of the immune organ versus body weight was evaluated (see Table 7 and Figure 1). On days 15 and 35, the results of thymus weight showed a significant increase (P ≤ 0.05) in the agrimos non-infected and infected groups in comparison with the other groups. However, there was a significant decrease (P ≤ 0.05) in the control infected group compared to the control non-infected group.
The results of spleen on day 15 showed that there was a significant increase (P ≤ 0.05) in the agrimos infected group compared to the other groups. Furthermore, there was a significant increase (P ≤ 0.05) in the agrimos® non-infected group compared to the control non-infected and infected groups. However, on day 35, the agrimos non-infected group witnessed a significant increase in the spleen’s weight (P ≤ 0.05) compared to the other groups. As well, the control infected group had a significant increase (P ≤ 0.05) compared to the control non-infected group on day 15 but a significant decrease (P ≤ 0.05) on day 35.
The bursa’s weight was significantly increased (P ≤ 0.05) in the agrimos non-infected group compared to the other groups but it was significantly decreased (P ≤ 0.05) in the control infected group compared to the other groups on days 15 and 35.
The thymus’ and bursa’s weight in the control non-infected group were significantly (P ≤ 0.05) decreased but the spleen’s weight was significantly (P ≤ 0.05) increased. The control-infected group showed a significant (P ≤ 0.05) increase in the weights of thymus, spleen, and bursa on day 15, and then showed a significant (P ≤ 0.05) decrease on day 35. The agrimos® non-infected and infected groups showed a significant (P ≤ 0.05) increase in the weights of spleen and bursa on days 15 and 35 but showed a significant (P ≤ 0.05) increase in the weight of thymus on day 15 only which returned to its normal size on day 35.
The Pathological Findings of Broiler Chickens: The histopathological examination of the spleen, bursa, thymus, liver, and duodenum of the chicks of the control non-infected group (on days 15 and 35 after infection) showed no obvious histopathological alterations (see Figure 2a and 2b). Concerning the control infected group (on day 15), the bursa showed variable degrees of epithelial hyperplasia, degeneration, and ulceration in some cases. These changes were associated with subcortical fibrous tissues proliferation in some cases (see Figure 2c, 2d and 2e). The thymus of this group showed a narrowing in the cortical width and an increase in the medulla in most cases. Some other cases had the appearance of clear areas or holes that contained small dark nuclei (a defining characteristic of the apoptosis in the lymphoid organ) (see Figure 2f). No microscopical changes were detected on the spleen of this group. Regarding the control infected group (on day 35), similar changes were seen on day 15 but more holes were detected in the thymus. Also, the focal area of round cells aggregation was seen in the liver of this group (see Figure 3a). The duodenum of this group showed hyperplasia on the epithelial cells lining the intestinal villi accompanied with vacuolation (see Figure 3b)
The histopathological examination of the bursa of Fabricius in most cases in the agrimos non-infected group on day 15 (4 out of 5) showed normal atypical fold (plica) (see Figure 3c). Some of these cases showed a highly dilated germinal center and narrow cortex, associated with a mild degree of epithelial lobulation. One case showed follicular lymphocytic depletion. Similar histopathological changes were seen on the bursa of Fabricius in the agrimos non-infected group on day 35. One case showed pores within the cortex of many plicae (see Figure 3d), and this was associated with interfollicular edema. Regarding the thymus gland in both times (days 15 and 35), no obvious histopathological alterations were seen on the thymic lobe except in few cases (2 out of 10), which showed thrombus formation within the thymic vasculature (see Figure 3e). Concerning the caecal tonsil of the agrimos® non-infected group on days 15 and 35, just epithelial sloughing was seen in some cases (see Fig. 3f), and no obvious histopathological alterations were viewed on the lymphoid nodules (lymphoid aggregation) (see Figure 4a). Two cases (out of 10) showed a necrotic cyst (see Fig. 4b). No obvious microscopical changes were seen on the spleen of this group (see Figure 4c). The microscopical examination of the liver of this group showed mild focal areas of round cell aggregation (see Figure 4d) in two cases (out of 10).
The histopathological examination of the bursa of Fabricius in most cases in the agrimos infected group on days 15 and 35 after infection (2 out of 4) showed normal morphological appearances but 2 cases showed interfollicular edema (see Figure 4e) and epithelial hyperplasia and folding associated with mucous (goblet) cell activation. Regarding the thymus gland in both times (days 15 and 35), no obvious histopathological alterations were seen on the thymic lobe except in few cases (1 out of 4) which showed focal areas of necrosis and hyalinization (see Figure 4f). No obvious microscopical changes were seen on the spleen of this group (see Table 8 and 9).
Gene Expression Related to E. coli Infection and Agrimos Supplementation: The results of Q-PCR revealed that the expression levels of nuclear factor-kappa (NF) and tumor necrosis factor-alpha (TNF) genes of liver tissues were significantly (P ≥ 0.05) increased in the group infected by E. coli (Figure 5) compared to the negative control group.
In the agrimos® non-infected group, the gene expression of NF and TNF in liver tissues did not show significant differences in comparison with the control non-infected group. The levels of such genes were similar to those of the control non-infected group.
It was found that the NF and TNF expression levels were significantly (P ≥ 0.05) reduced due to agrimos supplementation when compared to those of the infected group without supplementation. This shows the protective effect of agrimos supplementation on the expression of the above-mentioned genes of the infected broiler chicks.