Isolation and Molecular Identification of Salmonella pullorum from Broiler Chicken in Iraqi Fields

1. Introduction

Pullorum disease (PD) isconsidered one of the most dangerous bacterial infections and causes substantial financial losses in the poultryindustry, affects food security in Iraq and is caused by Salmonella enterica serovar Gallinarum biovars Pullorum ( 1 ). This bacteria is rod Gram-negative and prefer to be present in the liver, spleen, and reproductive tract ( 2 ). Common symptoms of PD include gasping, lack of appetite, lethargy, ruffled feathers, loud chirping, lameness, and diarrhea s in young chicks, and effects on the reproductive systeminclude a significant decline in egg output, fertility, and hatchability in adult birds ( 3 ). Salmonella pullorum is isolated using both selective and non-selective enrichment methods ( 4 ). Biochemical testing, on the other hand, carbohydrate fermentation tests, and PCR to determine the genomic DNA of bacterialserotyping were used to determine infection ( 5 ). Salmonellasp. colonies show distinct characteristics subject tobiochemical examinations, whichexplain the metabolic process of Salmonella pullorum so that they can be used as confirmatory tests for bacterial isolation ( 4 ). Salmonella pullorum is not like the otherSalmonellasp. in the fermentation of glucose, ornithine, and lycine and gives negative results with urea, lactose, citrate, indol, and sorbitol ( 2 ). Various forms of culture media are employed, such as triple sugar iodine, Salmonella Shigella agar, McConkey agar, Mortality indole ornithine, Simmons citrate, Methyl Red-Voges Proskauer, and Eosin Methylene Blue ( 2 , 4 ). PCR tests have been proven as diagnostic confirmatory Salmonella detection tools in broiler and layer chickens in terms of Workload reductions andreducing the time it takes to diagnose ( 6 ). In this study, samples (dropping, cloacal swabs, and visceral organs) taken from broiler chickens were examined for the isolation of Salmonella pullorum bacteria by culture, biochemical identified, and confirmed by PCR assay for 16S rRNA gene.

2. Materials and Methods

2.1. Sample Collecting

(100) samples were collected randomly from the broiler chicken fields of Baghdad (from 12 broiler chicken flocks of different ages for 8 districts province. Each sample includedncludingcloacal swabs (n=65), visceral organs (n=15), and dropping (n=20) collected in aseptic condition and placed in a container made of sterilized plastic in a refrigerated box until delivered to the laboratory.

2.2. Identification and Isolation of Salmonella pullorum

The samples were cultured in a non-selective Peptone water brothfor 24 hours at 37 °C and 10 ml Tetrathionate broth for 24 hours at 41.4 °C ( 2 ). The cells were then cultivated on a specific broth (Himedia's Selenite F broth) and incubated at 37 °C for 24 hours. A loopful of each broth was culturedstreaked across the surface of MacConkey agar plates (Oxoid), Xylose Lysine Desoxycholate agar (Himedia), Salmonella and Shigella agar (Himedia), Brilliant green agar (LAB), and chromogenic agar (Conda) for further incubation at 37 °C for 24 hours ( 2 ). Gram's stainingwas used to determine the staining features of the isolated bacteria and the purity of the culture (according to the manufacturer of the Gram's stain kit). Biochemical assays on Triple Sugar Iodide (TSI) agar, Motility Indole Ornithine agar, Simmons Citrate agar, MacConkey agar, Salmonella Shigella agar, and Eosin Methylene Blue agarwere used to indicate the species of isolates ( 7 ).

2.3. Molecular identification

Extraction of genomic DNA was done bystool DNA Kit was used to extract DNA from the samples (Bioneer, Korea). The kit was used following the directions of the company ( 8 ). The DNA was measured and certified with a NanoDrop before being stored at -20ºC until the subsequent tests were run. The 16S rRNA gene was employed as a critical objective of Salmonella pullorum and was detectedusing particular primers GenBank: FR686362.1 (Table 1). The PCR conditions were utilized according to table 2. The results of the PCR test were conducted on a 1% agarose gel with ethidium bromide dye and electrophoresed before being viewed with a UV.

2.4. Sequencing

The process of purifying PCR products from the PCR gel was carried out using the EZ EZ-10 Kit (Biobasic, Canada). Two samples of PCR products were sent to Macrogen Co. in Korea to sequence the 16S rRNA gene (AB DNA-based sequencing system). The Neighbor Distance Phylogenetic tree analysis was utilized to analyze the sequencing findings and create the phylogenetic tree utilizing NCBI-based websites and Mega v6 (Mega version 6) (Figure 1).

Figure 1. The sequence analysis of the PCR products of S. pullorum targeted the 16S rRNA gene in NCBI

3. Results

In the current investigation, 75 of the 100 samples tested positive for Salmonella pullorum. At the beginning of the isolation stage, the culture medium of bacteria on peptone water was cloudy and had an unpleasant smell. Due to Salmonella's non-lactose fermentation capabilities, all isolates generated On MacConkey agar media, colorless, smooth, pale, and clear raised colonies; on the XLD and BGA plates, there were tiny black and pink colonies. The recovered bacteria had a tiny rod shape, were Gram-negative, and were solitary or paired in Gram's staining. Indole, ureas, and oxidase were all negative in all Salmonella; however, catalase, lysine iron agar, and simmone's citrate were all positive. Alkaline slant (red), acid butt (yellow), and H2S and gas production all indicated positive results on triple sugar iron agar (TSI) (Figure 2).

Figure 2.A: Salmonella -Shigella agar. B: Simmons citrate agar. C: Triple sugar iron agar. D: Motility test

Figure 3 shows that S. pullorum was found in samples using the PCR technique. The size of these products was 1500bp of the 16S rRNA gene. Sequencing of parts The existence of this bacteria in the positive samples were confirmed by sequencing the PCR-positive samples. The confirmed isolates (OM988162.1) exhibited 99.02 percent similarity to the NCBI isolate (MF445124.1) and 98 percent (MH352164.1), respectively, an isolate from Colombia when the phylogeny was examined. Furthermore, the current research isolates. Figure 4 depicts these findings in the form of a phylogenetic tree.

Figure 3.S. Pullorum PCR results targeting the 16S rRNA gene on an agarose gel. Ladder (100bp) M, the positive samples 1 and 2

Figure 4. Phylogenetic tree study of S. pullorum PCR results targeting the 16S rRNA gene

4. Discussion

Salmonella pullorum is one of the important bacterial pathogens that leads to high mortality in young birds, and in adult birds, it is associated with reduced egg laying, weight loss, and economic losses. The prevalence of S. pullorum infection in poultry samples was 75 percent, which is nearly identical to the number reported by AL-Iedani, Khudor ( 10 ) in Basrah region, where the prevalence of Salmonella was 80 percent and 92 percent, respectively. A survey of the most common digestive disorders in laying hens and parent stock was conducted in Mosul, and Salmonellosis accounted for 70% of the situations ( 11 ). This discrepancy could be attributed to variances in environmental and management factors. Its regional spread because the survey did not focus solely on Salmonella infection but also covered the most common digestive illnesses. The culturing and biochemical results are consistent with the findings of Ayesha, Mahmood ( 12 ) and Rumi, Rahman ( 13 ). On SS agar media, red and white colonies with black centers were seen in all Salmonella suspicious isolates. The same result was shown by Ashraf, Ahmed ( 14 ) and Abd El-Ghany, El-Shafii ( 15 ) - the Salmonella spp. Colonies Detailed diagnosis, on the other hand, may necessitate using sophisticated instruments such as PCR methods. The PCR approach was employed in this case to determine the presence of this bacterium in the chickens' feces. These findings are consistent with Xiong, Song ( 16 ) that devised a PCR approach to detect the presence of these bacteria in addition to the traditional laboratory methods. Furthermore, Saud and AL-Zuhariy ( 17 ), employed a quick and low-cost PCR method with the O and H antigen alleles as targets and reported success rather than relying on serological assays.

Furthermore, Majchrzak, Krzyzanowska ( 18 ), described a PCR approach that may be used to distinguish between Salmonella serovars. Sequencing and phylogeny were used to detect successful differentiation rates amongst Salmonella isolates ( 19 , 20 ); this is consistent with the recent findings, which demonstrated that one sample had 99 percent similarity to worldwide isolates. Current molecular and phylogenetic research analysis showed that S. pullorum is present in broiler chickens in the analyzed area, as well as evolutional traits and linkages to several global isolates. As a result, preventative programs to prevent salmonella infections must be considered in the case of broiler chickens.

Authors' Contribution

Study concept and design: R. G. H.

Acquisition of data: M. T. B.

Analysis and interpretation of data: M. T. B.

Drafting of the manuscript: M. T. B.

Critical revision of the manuscript for important intellectual content: R. G. H.

Statistical analysis: R. G. H.

Administrative, technical, and material support: R. G. H.

Ethics

We hereby declare all ethical standards have been accepted by the ethics committee of the University of Baghdad, Baghdad, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

We extend our gratitude to the College of Veterinary Medicine, the University of Baghdad, and Al-Nahda Veterinary Laboratories for the facilities they provided throughout the research period, through which we reached positive results in the isolation and characterization of bacteria.

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