1. Introduction
1.1 Instructions
Salmonella is a Gram-negative, facultatively anaerobic bacterium that is a member of the family Enterobacteriaceae that is found in the environment (Steve Yan et al., 2004). Within environment, Salmonella species can be found in a broad variety of locations; nevertheless, the intestinal tracts of animals are the primary habitat for these bacteria (Ellermeier & Slauch, 2006). The ingestion of infected items, such as eggs, milk, and chicken meat, can lead to the transmission of the Salmonella bacteria among individuals (Gillespie et al., 2003).
It is estimated that twenty percent (20%) of all poultry products around the world are infected with Salmonella. Salmonella able remain for an extended period of time in the habitats of animals and humans, as well as in facilities, due to the production of biofilms (Vestby et al., 2009). S. enteritidis and S. typhimurium serovars have been discovered in the majority of the salmonellosis outbreaks that have been attributed to the eating of poultry products (Vose et al., 2011).
Polymerase Chain Reaction (PCR) is a powerful technique used to investigate outbreaks of food-borne illnesses and identify specific pathogens (Riyaz-Ul-Hassan et al., 2004; Tayeb et al., 2020). PCR offers rapid outcomes and a notable level of specificity. Combining routine PCR testing with standard culture methods should enhance the accuracy of determining the existence of this disease in grill carcasses (Carrasco et al., 2012). The primary cause of Salmonella contamination in carcasses is the presence of Salmonella in the digestive system, skin, and feathers of chickens. This contamination can occur along to the processing line.
This paper was carried out on the frozen poultry meat at the central laboratory of the Ibrahim Khalil border, Zakho, Duhok region, to assess the level of contamination of poultry carcasses with Salmonella spp., specifically S. typhimurium and S. enteritidis. The contamination was evaluated using a combination of culture and triplex PCR methods, which involved the detection of prot6E and fliC genes.
2. materials and methods
2.1 Mode of sampling
The sampling was performed at the Central laboratory of Ibrahim Khalil border Duhok region, For the purpose of this investigation, a total of one hundred and ten (110) frozen poultry samples were collected using cluster sampling techniques. This process was carried out between summer and autumn of 2023.
2.2 Bacterial isolation and identification
The protocol was conducted according to the ISO 6579-2020 manuals and a previously published protocol (Sharif & Tayeb, 2021). Briefly, 25 g of frozen meat sample were added to 225 mL Buffer peptone water (BPW) (CONDALAB, SPAIN) and then were incubated at 37℃ Celsius degrees for 18 h. Then, one mL of each re-enriched sample was put into 9.9 mL of Rappaport-Vassiliadis soya peptone broth (RVS broth), and the mixture was then incubated at 41.5 ℃ for 24 hr, and 1 mL was transport to a10 mL of Muller-Kauffmann-Tetrathionate Novobiocin (MKTTn, Merck, Germany), then incubated at 37 ℃ for 24 h. Following the incubation period, a loopful of each culture was streaked onto Xylose Lysine Deoxycholate agar (XLD agar) (Condalab-Spain) Salmonella Chromogenic Medium which was then preserved at 37 ℃ for 24 h. The urease and triple sugar iron (TSI), lysine decarboxylase, were utilised in order to validate the presence of presumed Salmonella colonies through biochemical analysis (Jamshidi et al., 2010).
2.3 Extraction of DNA
After the results of the biochemical tests indicated that the bacterial colonies were really Salmonella species, they were grown on nutrient agar for an overnight incubation. For DNA extraction from the suspected colonies, a previous published protocol was followed (Ameen et al., 2016). Briefly, a small amount of Salmonella from agar plates was mixed with 100 μL of sterile distil water in a 1.5 mL Eppendorf tube and vortexed to create a bacterial suspension. Following this, the bacterial suspension underwent boiling at 95-100ºC for 10 a min and subsequent centrifugation at 10,000x g for 10 min. The resulting supernatant served as the DNA template for qPCR.
2.4 Triplex PCR method
The PCR process was conducted in a final volume of 25 μL, which included 2.5 μL of 10x PCR buffer comprising 500 mM of KCL and 200 mM of Tris–HCL, 1.25 μL of deoxynucleotide triphosphate (10 mM), 1.5 μl of MgCl2 (2 mM), 0.5 μL of Taq DNA polymerase (Fermentas), 2 μL of extracted DNA, and 0.5 μL of each primer. A Rotor-Gene Q7 (Qiagen kit) was used to perform the amplification reaction and cycling conditions. The procedure consisted of an initial incubation at 95 °C for five minutes, then following by thirty-five cycles of denaturation at 94 °C for sixty seconds, annealing at 56 °C for thirty seconds, extension at 72 °C for thirty seconds, and a final extension period for ten minutes at 72 °C (Jamshidi et al., 2009).
Primers of S141 and S139, which were target for the invA gene, were utilised for the testing of the genus Salmonella. Primers of prot6e-6 and prot6e-5, which were specific for the prot6E gene, were utilised for the identification of S. enteritidis. Additionally, Tym and Fli15primers, which were targetted for the fliC gene, were utilised for the identification of S. typhimurium (Table 1) (Afshari,et al.2018)
These two strains of S. typhimurium (ATCC: 14028) and S. enteritidis (ATCC-13076) served as positive controls obtained from New standard company, while sterile distilled water served as the negative control.
Table 1: displays the oligonucleotide sequence utilised as primers in the triplex-PCR (Qiagen triplex Kit)
|
Primers |
Sequences (5′–3′) |
Target gene |
|
S139-F |
GTG AAA TTATCG CCA CGT TCG GGC AA |
InvA |
|
S141-R |
TCA TCG CAC CGT CAA AGG AAC C |
|
|
Fli15-F |
CGG TGT TGC CCA GGT TGGTAAT |
fliC |
|
Tym-R |
ACT CTT GCT GGC GGT GCG ACTT |
|
|
Prot6e-5-F |
ATA TGG TCG TTG CTGCTT CC |
Prot6e |
|
Prot6e-6-R |
CATTGT CCA CCG TCA CTTTG |
F: forward primer; R: reverse primer. (Afshari,et al.2018)
3. results
3.1 Morphological characteristics of Salmonella
Based on the morphology of Salmonella colonies observed on both selective and non-selected media, a total of 14 (12%) meat samples were tested as positive for the presence of Salmonella. The distinctive characteristics of the colonies, such as colour, size, and shape, allowed for accurate differentiation and confirmation. The morphological characterization of the black and mauve colonies as shown in Figure 1.
Figure 1: Typical Salmonella Spp on chromogenic agar, showing mauve color (A), and on XLD agar red, black colonies (B).
3.2 Biochemical confirmation
The suspected samples were confirmed as positive by traditional culturing methods were confirmed by biochemical tests. The samples which were initially suspected of harbouring specific microorganisms were subsequently verified as positive through conventional culturing techniques, a confirmation that was further substantiated by biochemical assays
 |
Lysine ,urase test ,TSI as shown in Figure 2.
Figure 2: Biochemical test performance. Left side tube: urea is negative; in the middle: H2S gas production; right side tube: Lysin decarboxylase shows positive result.
3.3 Confirmation by PCR
We used real time PCR (Triplex kit) in this finding. Further confirmation was done by using PCR techniques (fragment of the invA gene). Using a triplex PCR assay, five of the fourteen isolates were determined to be related to S. enteritidis (fragment of the prot6e gene) (Figure 3), and four of the isolates were related to S. typhimurium (fragment of the fliC gene) (Figure 4). It was determined that the remaining isolates did not belong to the species S. enteritidis or S. typhimurium.
Figure 3: Shows the genus of Salmonella plus S. enteritidis and S. typhimurium detected by qPCR. The yellow channel is for S. typhimurium.
Table 2: Show the prevalence of salmonella in chicken meat
|
Number of sample |
Total Salmonella spp detected |
Positive S. enteritidis |
Positive S. typhimurium |
Salmonella spp |
|
110 |
13 |
(5 out of 13) |
(4 out of 13) |
(4 out of 13) |
4. duscussion
Determining the prevalence of microorganisms in food is the initial stage in implementing techniques to prevent food poisoning. The study yielded a 12 % isolation rate of the Salmonella genus using both the culture technique and triplex PCR. Jamshidi et al. utilized the same procedure to isolate Salmonella from 11.66% of the samples. The contamination rate in other research demonstrated a prevalence of 33% (124 out of 376 samples) in meat chicken and the beef meat samples from market shops in Tehran (Dallal et al., 2010). Similarly, in England, 25% (60 out of 241 samples) of complete raw chicken samples were found to be contaminated (Jørgensen et al., 2002). In the USA, the contamination rate was observed to be 3% in poultry carcasses (Zhao et al., 2001). In Duhok region , Ahmed and his team in 2023 found around 68%of Salmonella spp in poultry carcasses.
The majority of Salmonella enterica infections in Iran occur in poultry, with serovar S. enteritidis being detected in 51.4% (35/68) of the samples (Zahraei-Salehi et al., 2005). Sixty percent of salmonellosis cases in Europeans and the majority of cases globally are caused by Salmonella enterica, serovar Enteritidis (Thorns, 2000). The majority of cases of Salmonellosis in the US are caused by S. typhimurium (Afshari et al., 2018).
The disparity between the findings of this study and those of previous research may be attributed to variables such as adherence to rigorous manufacturing standards and the conducting of Hazard Analysis and Critical Control Points (HACCP) during the slaughter process. Additionally, the specific stage of the process chosen for sampling, the time of year when the slaughter occurred, and the methodology employed for sampling and culturing could also contribute to the observed differences (Yin et al., 2016).
A previous study revealed that the lowest rate of Salmonella spp. contamination (6%) in slaughtered birds occurred after stunning, whereas the greatest contamination rate (52%) was observed prior chilling (Yin et al., 2016).
The main serovars used to manage risk factors of salmonellosis are S. enteritidis and S. typhimurium. These serovars can be transmitted from parent hens to their offspring at the hatchery (Heyndrickx et al., 2002). The findings indicated that S. enteritidis was present in 38% (5/13) of the samples, while S. typhimurium was detected in 30% (4/13) of the samples. The other isolates (35%) likely belonged to serovars that were not assessed in this study. According to a previous report, the prevalence of S. enteritidis contamination in poultry carcasses was found to be 1.8% (Ulloa et al., 2010).
Previous research revealed that 8.2% of the 1154 chicken meat samples and 12.8% of the chicken piece samples were contaminated (Alexandre et al., 2000). The most common serotype found was S. enteritidis. In another study performed in Mashhad-Iran, documented that the prevalence of S. typhimurium contamination in poultry carcasses was 8.2% and 6.10% (Jamshidi et al., 2009). The serological type S. enteritidis was shown to be the most prevalent in a study done by Mikołajczyk and Radkowski (Zhao et al., 2001). De Freitas and colleagues (de Freitas et al., 2010) detected the presence of S. enteritidis in 1.37% of poultry samples in Brazil using multiplex polymerase chain reaction (triplex PCR).
Currently, molecular approaches are highly accurate and sensitive in detecting and identifying Salmonella spp, particularly when dealing with a large number of samples (Malorny et al., 2007). Additionally, it has been noted that employing two procedures can substantially enhance the identification of isolates, as PCR is unable to differentiate between deceased and viable cells. Therefore, it is recommended to utilize PCR in conjunction with microbiological assays. In this investigation, both the classical methodology (enrichment broth, selective media, and biochemical tests) and the molecular technology were employed for detection.
Another study found that Salmonella was detected in 32 samples (16%) using standard culture methods and in 38 samples (19%) using the PCR approach. When the data from both methods were merged, the identification of the pathogen climbed to 45 out of the 198 samples (23%) (Whyte et al., 2002). On the other hand, a further finding was done on the isolation of Salmonella species in poultry products, revealing that 4.4% were contaminated based on traditional culturing methods and 1.8% when assessed using qPCR (Ameen et al., 2016).
conclusion
Ultimately, the findings of this study suggest that imported frozen chicken possesses the potential to transmit Salmonella spp. infections to individuals. Consequently, it is imperative for the poultry sector to prioritize the implementation of stringent control measures aimed at minimizing the transmission of infections during production operations.
Acknowledgements
The author would like to express their gratitude for the support provided by the Central Laboratory of Ibrahim Khalil Border in facilitating and contributing to this work.
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