Samples were collected from various poultry farms in Baghdad, yielding 50 specimens from two-week-old chickens that showed signs of diarrhea. These samples were carefully transported in a cold container to the laboratory within two hours. Each fecal sample was placed in a sterile test tube that contained 1 g of the sample, 10 ml of normal saline, and 0.1 ml of the sample suspension. This mixture was then inoculated onto MacConkey agar (HiMedia, India) and Salmonella Shigella agar (HiMedia, India). The inoculated media were incubated at 37°C for 24 to 48 hours—the Vitek 2 compact system was used for diagnosing the bacteria and antibiotic sensitivity test.

DNA extraction

Transfer 1 to 2 ml of cultured cells into a 2 ml tube. Centrifuge the cells at 13,000 rpm for 1 minute to pellet them. DNA extraction was performed using the G-spin kit (Intron Biotechnology, cat. no. 17045). Electrophoresis was conducted to analyze the DNA fragments post-extraction and to assess the results of the PCR interaction in the presence of standard DNA, allowing for the identification of the product size of the PCR interaction on the Agarose gel.

Purity and concentration of DNA

The concentration and purity of DNA are assessed through absorbance measurements of micro-volume samples utilizing the Nano Drop spectrophotometer (Nabi/Korea). Purity is evaluated using the 260/280 ratio, with optimal purity falling between 1.80 and 2.00.

Primers

The primers were lyophilized and dissolved in ultrapure ddH2O to create a stock solution with a final 100 pmol/μl concentration. This stock solution was stored at -20°C to prepare a working concentration of 10 pmol/μl. The working primer solution was made by combining 10 μl of the stock with 90 μl of ddH2O, resulting in a final volume of 100 μl. The primers were sourced from IDT (Integrated DNA Technologies, USA). Target size for amplification was 1250 bp using the primer sets of 16S rRNA 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TACGGTTACCTTGTTACGACTT-3' ( Mohammed & Al-Samarraae, 2021).

PCR Amplification Analysis

PCR amplification was performed in a 25 μl volume in the presence of iNtRON's Maxime PCR PreMix Kit that included Taq polymerase, PCR buffer, gel loading buffer, and dNTPs. The mixture consisted of 1.5 μl template DNA, one μl each primer at a concentration of 10 pmol, and 16.5 μl distilled water. Amplification was performed using a Mastercycler (Eppendorf, Germany). The PCR process began with the initial denaturation at 94°C for five minutes. Subsequently, it proceeded with a denaturation at 94°C for 45 seconds, an annealing at 56°C, an extension at 72°C for one minute, and a final extension at 72°C for seven minutes. The products of PCR amplification of 16S rRNA forward and reverse primers were separated on agarose gel electrophoresis (1.5% agarose, 5 volts/cm ²) in 1x TBE buffer for 1 hour and 30 minutes. The molecular value standard was a DNA marker (100 bp) of Bioneer (Korea), and a gel documentation system to observe.

The PCR products were analyzed through 2% agarose gel electrophoresis and visualized under ultraviolet light (302 nm), after staining with either ethidium bromide or Red Stain. Macrogen Korea carried out gene sequencing. A homology search was performed utilizing the Basic Local Alignment Search Tool (BLAST), accessible through the National Center for Biotechnology Information (NCBI) at ( http://www.ncbi.nlm.nih.gov) and via the BioEdit program.

The C. violaceum Killed Whole Cell Antigen (CKA) was constituted as described in reference ( Haq et al., 2022). Bacteria were cultivated on nutrient agar at 37°C for 48 hours, then exposed to Gram staining and microscopic examination for their morphological integrity. The bacteria were washed with PBS (pH 7.2) three times after 3000 rpm centrifugation for 20 minutes and treated with a 0.3% formalin PBS (pH 7.2) solution. The combination was incubated at 37°C for one to two hours and then stored overnight at 4°C. Following three more washes using PBS (pH 7.2), the bacterial suspension was centrifuged for the second time at 3000 rpm for 20 minutes to prepare the CKA. The colony-forming units of the CKA were enumerated by the McFarland tube method, and protein concentration was quantified based on reference ( Kielkopf et al., 2020).

Aloe Vera extract was obtained from a local supplement manufacturing company (Alemad Factory) and prepared at a concentration of 100 mg/ml.

Twenty-four Swiss mice weighing 14-15 grams were randomly divided into eight groups. The first group was immunized with a subcutaneous injection of 1.5 × 109 CFU/ml of the CKA antigen. The second group was immunized with the same amount of CKA antigen combined with 100 mg/ml of aloe vera subcutaneously. As a negative control, the third group was given subcutaneously 1 milliliter of PBS (pH 7.2). On day 14 post-immunization, the antigen's initial and second booster doses were administered. After immunization, serum samples were collected on day 21 to measure IgG and IL-6 levels using the Elabscience enzyme-linked immunosorbent assay (ELISA) kit tailored for mice (Elabscience, China), following the manufacturer's protocol. Optical density was measured at a wavelength of 450 nm (Socimed Sarl, France), and the concentrations of IgG and IL-6 were determined through interpolation from a standard curve, with results expressed in pg/mL. A pilot study was conducted to determine infectious dosages for C. violaceum using the McFarland tube method, preparing three dilutions (1.5 × 10 ⁸, 3.0 × 10 ⁸, and 6.0 × 10 ⁸ CFU/ml) with a total of 24 mice divided into three groups of eight. Group 1 was infected orally with 1.5 × 10 ⁸ CFU (0.1 ml), Group 2 received 3.0 × 10 ⁸ CFU (0.1 ml orally), and Group 3 was also infected with 3.0 × 10 ⁸ CFU (0.1 ml orally). Clinical symptoms and mortality rates were observed over three days post-infection, confirming that the infectious dose was 1.5 × 10 ⁸ CFU/ml. Mice infected with C. violaceum at 21 days post-immunization (1.5 × 10 ⁸ CFU/ml, orally) were monitored for clinical signs every 6 to 8 hours over a week. Ten days after infection, the serum was taken and the mice euthanized for histopathological examination of the vital organs; liver, kidney, spleen, and intestine were collected and fixed in a 10% formalin solution immediately after extraction ( Snyder et al., 2022).

The Statistical Packages of Social Sciences (SPSS) program was used to detect the effect of different groups and periods on study parameters. LSD-Least significant difference (two-way) was used to compare the means significantly in this study ( George & Mallery, 2019).

The analysis of fifty fecal samples from diarrhea cases revealed that seven isolates, representing 14%, tested positive, as indicated in Table 1.

All strains of C. violaceum manifested as black colonies on SS agar due to their ability to produce H2S after 24 hours ( Figure 1). On MacConkey agar, the isolates appeared as pink colonies due to lactose fermentation ( Figure 1) and non-producing pigmentation on nutrient agar ( Figure 1). Additionally, all isolates tested positive for oxidase and catalase.

The Vitek 2 Compact system was employed to identify and assess the antimicrobial susceptibility of C. violaceum, yielding a 98% probability of identification, as illustrated in Table 2.

When standard DNA is present, the PCR interaction's outcome allows for the product size differentiation resulting from the PCR process on the Agarose gel ( Figure 2).

The concentration of C. violaceum DNA was measured at 42.5 ng/ml, with a purity value of 1.937. One isolated strain was examined to identify the presence of 16S rRNA genes. As a result, the strain showed a positive amplification of 1250 bp on an agarose gel, while no amplification was detected in the negative control ( Figure 3).

The NCBI website ( http://www.ncbi.nlm.nih.gov) was used to analyze the sequences. There was 98% homology between the C. violaceum reference strains and the isolated strains in GenBank (Accession No. MW282912.1) Figure 4 Table 3.

The results regarding IL-6 levels across the various groups (G1 and G2) indicated no significant differences (P<0.05) at 21 weeks post-immunization, with measurements of 34.98 ±0.97 pg/ml for G1 and 35.37 ±0.54 pg/ml for G2 when compared to the control negative group, which had a level of 30.93 ±0.62 pg/ml. Furthermore, Group 3 (control negative) demonstrated a significantly elevated IL-6 concentration (P<0.05) ten days following infection with C. violaceum, reaching 945.06 ±9.87 pg/ml. Additionally, both G1 and G2 showed significant increases in IL-6 levels (P<0.05) ten days post-infection, measuring 287.54 ±13.75 pg/ml and 302.11 ±14.68 pg/ml, respectively, as detailed in ( Table 3) and ( Figure 5).

The findings related to IgG levels among the different groups (G1 and G2) revealed significant variations (P<0.05) at 21 weeks after immunization. Specifically, G1 presented an average of 34.98 ±0.98 pg/ml, while G2 showed a higher average of 55.43 ±0.49 pg/ml, in contrast to the negative control group, which recorded a level of 11.93 ±0.70 pg/ml. Additionally, Group 2 exhibited a notably increased IL-6 concentration (P<0.05) ten days post-infection with C. violaceum, reaching 105.61 ±2.15 pg/ml. In comparison, G1 attained a level of 84.19 ±1.60 pg/ml, whereas G3 reached only 32.39 ±1.16 pg/ml, as outlined in ( Table 4) and ( Figure 5).

Histopathological examination after 10-day post-challenge showed that all groups under study were affected with different histopathological changes: The G1 CKA: the liver section revealed normal parenchyma with well-conserved hepatic cords and sinusoids, normal liver histology, no pathological lesions identified ( Figure 6). The spleen section showed the presence of well-formed granulomas—collections of epithelioid histiocytes (elongated, pale cytoplasm) often surrounded by lymphocytes and increased vasculature with some fibrous septae ( Figure 7). The kidney section showed histologically normal renal cortex, and no apparent pathology was present in this section ( Figure 8). The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip, extremely mild mucosal injury ( Figure 9). The G2 CKA- Aloe vera: the liver section showed mild portal inflammation with lymphocytic infiltrates in and around portal tracts, no significant lobular inflammation or hepatocyte necrosis ( Figure 10). The spleen section revealed Lymphoid hyperplasia or possible lymphoma and dense lymphocytic infiltrate ( Figure 11). The kidney section showed no obvious lesions such as necrosis, inflammatory cell infiltration, fibrosis, or glomerular sclerosis ( Figure 12). The intestinal section showed moderate subepithelial space formation and epithelial lifting to the sides of villi ( Figure 13). The G3 Control negative (infected = C+): the liver section showed dense infiltrate of lymphocytes in the portal area extending into the periportal area, and interface hepatitis is seen (piecemeal necrosis), scattered lobular inflammation, and mild hepatocyte ballooning ( Figure 14). The spleen section showed preserved follicular hyperplasia, tubules appeared dilated with cytoplasmic vacuolization, and moderate lymphocytic infiltration in interstitial areas ( Figure 15). The kidney section revealed Diffuse lymphocytic infiltration, focal necrosis, and congestion in zones, glomerular structure observed to be mildly disturbed, severe damage to parenchymal tissue evident ( Figure 16). The intestinal section revealed massive epithelial lifting and few villi with denuded tips, demonstrating advancing mucosal injury ( Figure 17) Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. .