Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Finding Genetic Differences in Salmonella enterica Isolated from Poultry

Zaman Kareem Hanan1*, Ali A. Abdulkareem2, Noor Hassan Ghargan1, Adnan Abd Azeez3

1College of Medicine, University of Thi-Qar, Nassiryhia, Iraq; 2Department of Animal Production, Faculty of Agriculture and Marshes, University of Thi-Qar, Iraq; 3College of Dentistry, University of Thi-Qar, Nassiryhia, Iraq.

Received | July 24, 2025; Accepted | October 08, 2025; Published | October 15, 2025

*Correspondence | Zaman Kareem Hanan, College of Medicine, University of Thi-Qar, Nassiryhia, Iraq; Email: [email protected]

Citation | Hanan ZK, Abdulkareem AA, Ghargan NH, Azeez AA (2025). Finding genetic differences in Salmonella enterica isolated from poultry. J. Anim. Health Prod. 13(s1): 600-606.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.s1.600.606

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Since chicken meat is one of the most basic food sources for humans, livestock is among the most crucial economic sectors in underdeveloped countries (Abdulkareem, 2020; Al-Jebory et al., 2024; Salman et al., 2024a, b). Poultry are susceptible to a wide range of diseases, including viral and bacterial ailments such as salmonellosis (Naji and Hanna, 1999; Al-Saeedi et al., 2024; Ajafar et al., 2024). If dangerous microorganisms are present, the carcass’s traits and attributes may suffer (Jaffar and Abdulkareem, 2022). Enteric infections are a major source of illness and death worldwide. Gram-negative enteric microbes are thought to be mindful for approximately 3 million fatalities around the world each year from loose bowels and gastrointestinal illness (Donnenberg, 2000). Salmonellae is a gram- bacterium that causes serious illnesses in humans and domestic animals (Makela and Hormaeche, 1997; Eisenstein, 1999). There are around 2668 distinct Salmonella enterica serotypes. A major cause of food-borne disease outbreaks and a worldwide public health issue, the condition can harm both people and animals (Akbarmehr, 2011).

Salmonella enterica is the pathogen that causes salmonellosis. Salmonellosis, which occurs when feed or water sources are taken after being contaminated by the urine or feces of animals that may act as Salmonella reservoirs, causes significant morbidity and mortality worldwide (Raffatellu et al., 2008; Akbermehr, 2012). Depending on the host’s susceptibility, Salmonella enterica infections presented a range of clinical signs (Crump et al., 2015; Majowicz et al., 2010). These include bacteremia, E.F, gastroenteritis, and ASC (Burns et al., 1997; Andrews-Polymenis et al., 2010). Although human salmonellosis ordinarily up as a self-limiting nourishment harming, it can moreover sometimes result in a major systemic ailment or E. fever. Contaminated food is the essential implies of transmission for non-typhoidal sicknesses, developed Salmonella, a zoonotic ailment that influences a large number of animals reservoir. The foremost common of these poultry animal (Akbarmehr, 2011).

Non-typhoidal Salmonella is responsible for an estimated 1 million infections, 20,000 hospitalizations, and 378 deaths per year. Three million children are killed by it every year in both wealthy and developing countries (Cardinale et al., 2005; Scallan et al., 2011), Salmonella-specific PCR using invA primers was found by Lampel et al. (2000) to be sensitive, quick, and specific for detecting Salmonella in a variety of clinical samples. Furthermore, Kunduk et al. (2006) reported that PCR is a quick, accurate, and precise way to diagnose typhoid (Feyer and Karmi, 2013). The invasion of host epithelial cells is dependent on Salmonella’s invA gene, which contains sequences unique to the genus Salmonella. It has also been confirmed to be a basic PCR target with significant diagnostic applications. The molecular approaches are the most straightforward and economical. This gene is consequently a promising target for the detection of Salmonella from different forms of biological material, as was previously mentioned, the aimed of study to pinpoint the invA gene responsible for cell invasion offer atomic clarification of it. This was accomplished by utilizing the Sanger arrangements procedure to decide the developmental tree of the Salmonella segregates beneath consider and analyze the sequencing of the intensified quality portion.

Materials and Methods

DNA extraction and PCR

Follow the directions of the manufacturer (Geneaid Biotech, Taiwan), DNA extraction was performed The PCR specifically targeting InvA gene was performed using the master mix (Table 1) and the thermoprofile outlined in Table 2. Afterwards, PCR products were run on a 1.5% agarose gel, they were stained for 45 minutes at 95 volts in 1X TBE buffer.

 

Table 1: Master mix for the preparation of PCR solution

Reagents	Volume
Primer forward	1.5 µl
Primer reverse	1.5 µl
DNA template ( <250ng)	1 µl
Free water	16 µl
Total	20 µl

 

DNA sequencing of PCR amplicons

The PCR amplicon sequences were acquired from the terminal in accordance with the instructions provided by the sequencing provider (Macrogen Inc. Geumchen, Seoul, South Korea). By further analyzing only clear chromatographs obtained from ABI sequence data, the annotation and variations were confirmed to be free of PCR or sequencing artifacts. By comparing the observed nucleic corrosive arrangements of the bacterial tests with the reference arrangements that were retrieved from the bacterial database, the virtual areas and other points of interest of the obtained PCR fragments were determined.

Interpretation of sequencing data

BioEdit Assemble Edit Suite v. 7.1 (D.M. WI, USA) was used to modify the PCR products’ sequencing findings, modified, and evaluated alongside the sequences within the corresponding reference database. The nucleic acids sequences detected were counted within both the PCR amplicons and the corresponding regions within the reference genome. Each revealed variant within S. enterica genes was described by means of SnapGene Viewer 4.0.4 (https://www.snapgene.com).

Phylogenetic tree

This analyse constructed a particular phylogenetic tree according to (Zhang et al., 2000) and comprehensive tree was constructed as outlined by the Hanan et al. (2021); Gharkan et al. (2023).

Results

The invA gene variant was found in 15 samples with amplicons that were roughly 286 bp long. All of the

 

Table 2: The condition of PCR for invA gene amplification.

Monoplex gene	oC/ Time					Cycle number
	Initial denaturation	Cycling condition			Final extension
		Denaturation	Annealing	Extension
inv A	95/5min	94/40 s	58/60s	72/90s	72/10 min	30

 

Table 3: The 286 bp PCR amplicons used to access a subset of the InvA quality within the genomic DNA groups of S. enterica (GenBank accession number NC_003197).

Amplicon	Reference locus sequences (5′ - 3′)	Length
InvA gene	*TCATCGCACCGTCAAAGGAACCGTAAAGCTGGCTTTCCCTTTC-CAGTACGCTTCGCCGTTCGCGCGCGGCATCCGCATCAATAATAC-CGGCCTTCAAATCGGCATCAATACTCATCTGTTTACCGGGCATA-CCATCCAGAGAAAATCGGGCCGCGACTTCCGCGACACGTTCTG-AACCTTTGGTAATAACGATAAACTGGACCACGGTGACAATAGA-GAAGACAACAAAACCCACCGCCAGGCTATCGCCAATAACGA-ATTGCCCGAACGTGGCGATAATTTCAC**	286 bp

 

* Refers to the forward primer sequences (placed in a forward direction).

 

 

amplified amplicons were checked for clean, distinct, and sharp bands before sending for sequencing. By utilizing NCBI impact, the sequencing responses illustrated the intensified products confirmed manually. The NCBI BLASTn motor uncovered a tall degree of arrangement closeness between the sequenced tests and S. enterica arrangements with respect to the 286 bp PCR amplicons of the as of now focused on InvA arrangements. The NCBI BLASTn motor appeared that the InvA quality sequences coding locale was mostly secured by roughly 99% homology with the expecting target. The highlights of these groupings were underscored inside the increased groupings (Table 3) by differentiating the gotten DNA groupings (GenBank acc. NC_003197) with the watched DNA arrangements of the tests beneath examination.

The 286 bp PCR amplicons used to access a subset of the InvA quality within the genomic DNA groups of S. enterica (GenBank accession number NC_003197) as are displayed in Table 3 along with their length and location. A total of 19 nucleic acid differences were found in the alignment findings of the 286 bp samples when compared to the matching S. enterica referencing sequences (Figure 1). These sequences were created by matching the most related sequences that were deposited in the NCBI database (GenBank accession NC_003197) with the samples that we examined.

According to the analyzed InvA sequences in the bacterial samples under examination, a phylogenetic tree was generated. The three inspected tests (LC762569, LC762570, and LC762571) were included in this phylogenetic tree beside the other stored DNA groupings. These tests were adjusted with their profoundly related groupings in Tamura-Nei mode. There were sixteen adjusted nucleic corrosive groupings within the tree because it was as of now shaped. The inspected InvA groupings were assembled into various neighboring phylogenetic branches based on the hereditary arrangements of S. enterica, showing a incredible bargain of inconstancy in this life form with regard to the inspected InvA groupings (Figure 2). S. enterica subsp. enterica serovar Ouakam, S. enterica subsp. enterica serovar Newport, S. enterica subsp. enterica serovar Enteritidis, and S. enterica subsp. enterica serovar Typhimurium were also included, along with other relevant serovars. All of these subspecies, however, belonged to the enterica serovar.

 

Table 4: The plan of the observed contrasts inside the 286 bp of the InvA amplicons in comparison with the NCBI reference groupings (GenBank acc. no. NC_003197).

Sample	Variant	Position in the PCR fragment
S1, S2,S3, S4, S7, S8,S10,S11, S12,S13, S14, S15	A34T	34
S5, S6	A35 del	35
S1, S3, S10 S4,S13 S1,S2 S3, S4, S5,S6,S7, S8,S9,S10,S11, S12,S13, S14, S15	C36G C36A T 42del	36 36 42
S1, S2,S3,S4, S8,S9,S10, S13, S14 S5, S7, S11, S112 S1,S2 S3, S4, S5, S7, S8,S9,S10,S11, S12,S13, S14, S15 S1,S2 S3, S4, S5, S7, S8,S9,S10,S11, S12,S13, S14, S15 S11,S12 S5,S11,S12 S4,S10,S14 S5,S8,S12 S7,S11,S12,S15 S8,S13 S1,S2,S3,S4,S9,S10 S6,S7,S8,S11,S12,S13,S14,S15 S1,S2,S3,S4,S5,S6,S7,S8,S9,S10,S11,S12,S13,S14,S15	A69del A69G A70ins G71ins G91ins A119ins A124G G223A C253T C278G C278T T279G T279C G280C	69 69 70 71 91 119 124 223 253 278 278 279 279 280

 

 

Submission in NCBI

InvA gene sequences were submitted in NCBI’s Gene Bank and are available under accession numbers: LC762569, LC762570, and LC762571.

Discussion

Investigating S. enterica’s genetic variants was the aim of the most recent study. To do this, 15 bacterial samples (designated S1 to S15) were collected from grill chicken, ROSE 308 faeces, and were analysed for the InvA sequences. Salmonella’s invasion of intestinal epithelial cells depends on the invA gene, which codes for a protein in the bacterial inner cell wall (Al-Kaaby et al., 2014; Sharma and Das, 2016; Shanmugasamy et al., 2011). Salmonella’s invA gene is also utilised in the molecular diagnostics of the bacterium because it comprises a distinct sequence of this genus (Karim et al., 2020).

One of the numerous chromosomal virulence genes that were utilised to detect Salmonella in poultry faecal samples was the invA gene. The PCR assay of Salmonella serovar also targeted the invA gene (Jamshidi et al., 2009), and the invA gene was grouped in the islands of Salmonella species’ pathogenicity (Daigle, 2008). The virulent invA gene, which encoded a protein present in the inner layer of the bacterial membrane and had sequences that were specific to all salmonella serovars, is what allowed the bacteria to infiltrate the host’s epithelial cells (Daigle, 2008).

Salmonella-specific sequences can be found in the invA sequence of enteric bacteria, It has been found to be a good PCR target with possible diagnostic applications (Mohamed, 2013). Different distributions of the analysed samples were caused by the neighbouring places in the phylogenetic tree being attributed to the rate of variations within those samples. According to the InvA gene chromatograph file data, there are deletion mutations in several locations along this sequence, such as A35, T42, A69, A71, and 119del. Additionally, nucleotide insertion was found in a few different locations within the InvA gene, such as: A nucleotide in position 70 and G in the majority of samples; G nucleotide in position 91 of the S11 and S12 samples; and A nucleotide in position 119. The local isolate S. enterica (LC762570.1) exhibited a closed relationship and 100% similarity with reference isolates S. enterica subsp. enterica seovar Typhimurium (LC318972.1, LC318422.1) and S. enterica subsp. Enterica seovar Ouakam, as indicated by the phylogenetic tree of the InvA gene. The observed phylogenetic distances (tree scale 0.001) between the integrated organisms and local isolates within this clade are noteworthy since they clearly show that the incorporated bacterial genomes have progressive homology. To determine the relationship between Iraqi isolates and the higher query cover, which exceeds 90% of national isolates, the maximum likelihood and minimum evolution methods were used to create the phylogenetic tree for each gene.

Taken together, the finding of this study highlight the emerging genetic variations in the S. enterica and may offer foundational information for improved diagnostics and vaccine prepration to contain the infection in future.

ACKNOWLEDGEMENT

I extend my sincere gratitude to everyone who contributed to the success of this scientific research, with special thanks to the Deanship of the College of Agriculture and Marshes at the University of Dhi Qar.

NOVELTY STATEMENT

This study investigates the novel molecular characteristics of Salmonella isolated from poultry in Dhi Qar province. The research aims to determine its resistance to antibiotics and other emerging propertie.

AUTHOR’S CONTRIBUTION

ZKH: Designed the study and conducted DNA sequence analysis using bioinformatics tools.

AAA: Performed laboratory experiments including DNA extraction and PCR amplification.

NHG: Contributed to statistical analysis and figure preparation.

AAA: Supervised the research, provided academic guidance, and reviewed the manuscript.

Generative AI and AI-assisted technology statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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