Isolation, Identification and Characterization of Salmonella spp. from Chicken purchased at Wad Madani City, Gezira State, Sudan

Full Length Research Article

Isolation, Identification and Characterization of Salmonella spp. from Chicken purchased at Wad Madani City, Gezira State, Sudan

Abdel Moneim E. Sulieman1,2*, Farha E. Dafallah3, Eitimad H. Abdel-Rahman1, Nawaf I. Alshammari1, Sohair A. Shommo4, Salwa.E.Ibrahim5

Adv. life sci., vol. 8, no. 1, pp. 98-102, November 2020
*- Corresponding Author: Abdel Moneim E. Sulieman (Email: abuelhadi@hotmail.com)
Authors' Affiliations

 1. Department of Biology, Faculty of Science, University of Hail, Hai'l – Saudi Arabia
2. Department of Food Engineering and Technology, Faculty of Engineering and Technology, Gezira University, Wad-Madani – Sudan
3. Center of Biosciences and Biotechnology, Faculty of Engineering and Technology, Gezira University, Wad-Medani – Sudan
4. Department of Home Economic, Faculty of Education, University of Khartoum, Khartoum – Sudan
5. Department of Home Economic, College of Home Economic, King Khalid University (KKU), Abha – Saudi Arabia
 [Date Received: 11/09/2020; Date Revised: 11/11/2020; Date Published Online: 25/11/2020]

Abstractaa download_button
Introduction
Methods
Results
Discussion
References 

Abstract

Background: Salmonella is a potential human pathogen that causes salmonellosis, a food-borne disease. Addressing these major food safety and public health issues with effective monitoring of food-borne pathogens and dietary measures.

Methods: A cross-sectional study was conducted in the local markets of Alsug Alkabeer (AB), Alsug Ashabi (AK), and Alsug Alsageer (AS) sites in Wad Madani state, Sudan to detect and assess Salmonella infection in raw and cooked chicken samples.

Results: The results showed that the maximum number of different Salmonella species was recovered from raw and cooked specimens of chicken obtained from AB, AK and AS which were 6.5, 4.4, and 4cfu/g, respectively. In addition, Salmonella spp. in the locally reared chicken is significantly (p < 0.05) higher than that of farm poultry. On the other hand, the highest Salmonella count was recorded in AS, AK and AB egg samples which were 5.9, 3.5, and 2.3cfu/g, respectively. Interestingly, eggs from local sources of chicken had significantly (p < 0.05) higher numbers of Salmonella spp. compared to those from farm sources. Six species of Salmonella were described, namely: Salmonella typhi, S. cheers, S. arizonae, S. enteritidis, S. pullorum, and S. gallinarum.

Conclusion: High occurrence of Salmonella spp. in this study might be attributed to the low hygienic measures in the poultry retail markets during slaughtering and/or handling processes. Further studies are required and should be conducted within Gezira state to assess food safety.

Keywords: Food-borne disease; Pathogens; Biochemical test; Contamination; Food safety; Microbial growth; Food poisoning

Introduction6th button-01

Irresistible microbial sickness is a major cause of death in so many countries of the continent, particularly the developing countries. The aim of food safety has now been shifted by developed countries from investigative causes of food-borne diseases to proactive food, contamination prevention, and counteractive processes [1]. Salmonella has been recognized as a harmful food and waterborne pathogen that can infect humans and animals through extreme pain and mortality. Salmonella has three different entry pathways that lead to gastrointestinal disease by infected cooked foods (such as poultry, grains, eggs, and milk), environment litter and fertilizer, and consumption of contaminated raw fruits and vegetables [2-10]. The majority of human salmonellosis is common to both wild and domestic animals, thus, food of animal origin is a source of salmonellosis, typically between unprocessed poultry and prepared food products, i.e. through cross-contamination in food catering or at home. While all Salmonella is a non-host-adapted serotypes that cause most of the food-borne Salmonella emerge [11, 1, 12].  Handled raw poultry meat naturally host bacteria, most of which provoke poultry meat deterioration. Food-borne pathogens, such as Salmonella serotypes, Campylobacter jejuni, Listeria monocytogenes, C. perfringens and S. aureus can harbor food-borne pathogens [13, 10]. As disease-related meat, the food-borne disease outbreaks reported in poultry and poultry products to be ranked first and second in most countries around the world, respectively, and third in the United States [14]. Nevertheless, Salmonella infections of livestock and poultry products have been reported in Sudan [15, 16, 8].  Many studies have reported Salmonella outbreaks in relation to meat or eggs from poultry). A critical manifestation in poultry industry is the vertical transmission of infections from breeding hens to poultry meat as an epidemiology of Salmonella species infections [14, 17, 6].

Regardless of the coordinated efforts for the eradication of typhoid, malnutrition and related problems caused by Salmonella, it continues a major general medical issue around the world. Because most of Salmonella diseases are derived from the ingestion of unsafe food, a possible explanation for the prevalence of safe antimicrobial Salmonella is indicated by antimicrobial specialists in the feeding of affected creatures [4].

In Sudan, the broiler chicken population has been estimated to be 22.5 million chicks [18] and Khartoum State generates 90% of Sudan’s production [19, 20]. However, the traditional sector (small farms) produced about 60% of total broiler production and the rest was provided by the new sector (companies). However, many people in Sudan rear household chicken and eat their produce (meats and eggs) locally or sell it on the local market. Therefore, the purpose of this study was to isolate, detect and assess Salmonella infection in raw and cooked chicken samples from Wad Madani City, Sudan.

Methods6th button-01

Study Area

This study was carried out at Wad Madani, capital of Sudan's Gezira State. The town is situated on the west side of the Blue Nile between 14 ° 24'N- 14.4 ° N longitude and 33 ° 31'E- 33.517 ° E latitude, 136 km southeast to Khartoum.

Samples Collection

Raw and cooked chicken products samples were collected from Alsug Alkabeer (AB), Alsug Ashabi (AK) and Alsug Alsageer (AS) local market at Wad Madani city during the period 2016-2017. The original sources of the chicken and egg samples were either local farms or local houses from where they had been brought to the market. All samples were stored in sterile ice bags and forwarded to Department of Botany and Agricultural Biotechnology, Faculty of Agriculture, Khartoum University, Sudan.

Culture preparations for Salmonella Isolations

For preparation of serial dilution, 9 ml of sterile distilled water was poured aseptically into five test tubes each and 1 ml of the initial sample (chicken and egg) was added to the first tube giving 1:10 dilution. Again, 1 ml was transferred from the first tube, added to the second tube, and thoroughly mixed. Procedure continued until the fifth test tube. Each sample was diluted from 10-1 to 10 - 5. Further analyses were carried out on the samples. 25 grams of the sample were sterilized, aseptically weighed, and thoroughly mixed with 225 ml of sterile nutrient broth then was grown at 37 °C for 24 hours. Additionally, 10 ml of solution was added to 100 ml of sterile selenite cysteine broth aseptically drawn. The broth was placed in an incubator at 37°C for 24 hours; a decimal dilution series was prepared in 0.1% peptone solution in the surface covered with 0.1 ml amount of dilution onto pre-poured pre-dried plate of bismuth sulphite agar (BSA).  In order to promote Salmonella growth, the agar plates were incubated at 37°C for 72 hours. Thereafter the discrete black metallic sheen colonies were the viable colonies of Salmonella that were counted by colony counter and the results were expressed as colony forming unit per gram (cfu/g).

Salmonella Isolation and Identification

Salmonella pure colonies were streaked onto sterile agar plates with nutrients and were incubated for 24 hours at 37°C. The pure colonies of Salmonella isolates were sub-cultivated in nutrient agar slopes and incubated for 24 hours at 37°C, and then the plates were kept in the refrigerator at 4°C until it was used for biochemical testing. Purified isolates have been identified according to Cowan and Steel [21].

Salmonella Biochemical Identification Tests           

For the detection and characterization of Salmonella isolates, biochemical measures were used as laid down by Harrigan [22]; Juneja et al. [23]. These tests included Gram stain test, catalase test, nitrate reduction test, Vogs- Proskauer (VP) test (acetone production), citrate use test, urease test, indole test, motility test, sugar fermentation, casein hydrolysis, starch hydrolysis and methyl red test.

Statistical Analysis

Biochemical research results were entered into Microsoft Excel, edited, coded and analyzed using Statistical Software (SPSS version 19.5 for Windows). The collected data was recorded as arithmetic means ± standard mean error (SEM). Then after, Student’s t-test and the chi-square test were used to make comparisons and correlations between explanatory variables, respectively. Therefore, the mean and percentage of Salmonella were used for quantitative testing, and the Student’s t-test and the chi-square to test variables significance at P < 0.05. 

Results6th button-01

Enumeration of Salmonella

Tables (1-3) indicate Salmonella counts from cooked samples of chicken, eggs and chicken shawarma obtained from three separate locations (Alsouq Alkabeer, Alsouq Alshabi and Alsoug Alsageer).

It has been observed that the highest Salmonella spp. count was recorded after 120 days of storage in cooked chicken samples collected from Alsoug Alshabi (6.5 cfu / g), followed by Alsoug Alkabeer samples (4.4 cfu / g) and finally Alsoug Alsageer samples (4 cfu / g mean). There was, however, a significant difference between the different locations (F= 40.95; Fcrit=4.46; P-value=0.0063) with a big difference between the storage times (F= 12.46; Fcrit=3.84; P-vaue=0.001628).

The highest Salmonella spp. count was recorded in samples from shawarma (Table 2) collected from Alsoug Alshabi (mean 6.4 cfu / g) in 120 days storage, followed by Alsoug Alkabeer (mean 3.9 cfu / g) and lastly Alsoug Alsageer (mean 2.3 cfu / g). Table (2) also revealed that the times stored for Shawarma samples were substantially influenced by the Salmonella count (F= 12.85; F-crit= 3.84; P-value= 0.00), in addition to the significant differences between locations (F= 169.35; F-crit= 4.46; P-value= 0.00). Contamination of cooked chicken and eggs with Salmonella can be caused by the use of contaminated raw materials and/or unhygienic measures during storage, as well as long-term maintenance at room temperature of poultry feed [24].

Regarding Table 3, the highest Salmonella spp. count reported in the collected egg samples was from Alsoug Alshabi (mean 5.9 cfu / g) in 120 days storage, followed by Alsoug Alkabeer (mean 3.5 cfu / g) and lastly Alsoug Alsageer (mean 2.3 cfu / g). Overall, eggs can become infected with the penetration of Salmonella during or after oviposition [25].

Table (3) also revealed that the various storage times did not significantly affect the Salmonella counts (F= 0.80; F-crit= 3.84; P-value = 0.32668), and there were no significant differences between the various locations (F= 1.29; F-crit= 4.46; 0.32668).

Salmonella spp. isolated from various samples in this study were comparable to the results reported by Zhao et al. [11]; Beach et al. [1]; and Salsbury [26].  Salmonella was isolated in 19–54% of cattle carcasses, 1.9% of beef samples at retail and 4.2% of retail chicken samples [11, 1]. As well as large amounts of human salmonellosis being specifically related to human interaction with wild and domestic animals. Anyway, livestock feeding stuffs are vectors for salmonellosis [27-30].

Salmonella Quality and quantities

A total of 242 Salmonella spp. isolates from the samples collected from different locations, and   sources (local and farm) are shown in Fig. (1).

As indicated in Fig. (1) and (2), the prevalence of Salmonella was the highest among cooked eggs (23.96%) followed by cooked chicken (20.66%), local chicken (11.57%), local egg (10.33%), farm eggs (5.37%) and farm chicken (4.95%). From the 242 Salmonella isolates, 39 isolates (16.1%) were identified as S. enteritis, 70 (28.9%) were S. pullorum, 42 (17.3%) S. gallinarum, 48 (19.7%) were S. typhi, 15 (6%) were S. Arizona and 28 (12%) S. paratyphi. Compare to the present study, S. pullorum (28.9 per cent) was the most frequently species of Salmonella among the samples (Fig. 2). As well as, S. Arizona has been isolated from 5 out of 7 examined sources, with the remaining Salmonella species isolated from all sample sources. However, S. Pullorum was the most common species among cooked chicken, cooked eggs, farm chicken, farm eggs and local eggs.

 

 

Figures & Tables


 

 

 

 

 

 

 

 

 

 

Discussion6th button-01

As for the species Salmonella isolated from local and farm poultry, five species of Salmonella (S. typhi, S. enteritids, S. paratyphi, S. pullorum and S. gallinarum) have been distributed and found in farm poultry, while four species (S. typhi, S. enteritids, S. pullorum and S. gallinarum) have been found in local chicken. However, more than 95 per cent of Salmonella cases have been recorded as foodborne diseases. From the results obtained, in Alsug Alkabeer and Alsug Alshabi, the six species of Salmonella isolates (S. typhi, S. enteritids, S. arizona, S. paratyphi, S. pullorum and S. gallinarum) were collected from cooked chicken while all species, except S. enteritids have been noted in Alsug Alsageer. As for the samples of cooked eggs, the various species of Salmonella were isolated.

Chicken contamination with Salmonella (Fig. 2) was still far higher than that recorded by El Hussein et al. [31], Yagoub [16]  and Elsafi et al. [8] , which were 9.2%, 6.2%, 3.4%, respectively. In addition, our findings were significantly higher than reports from other countries, such as Nepal 14.5% [32], Canada 14% [7], and South Africa 19.2% [33], and Turkey 12% from [34].

Many developing countries have shown a comparatively higher prevalence of Salmonella in humans, food, and animals such as 73.3% in Egypt [31] , 68.2% in Ethiopia, 51.2% in Argentina, 25.9% in Korea, and 72% in Thailand [5]. It is vital to perceive that the prevalence and distribution of Salmonella serovars varies from location to location [35] and isolation rates vary depending on the location in which the research was conducted; the sampling program and the limit detections for the methodologies [36].     

The high occurrence of Salmonella spp. in our study could be observed due to the low hygienic measures noticed in the poultry retail markets of Wad Madani (Sudan) during slaughtering, de-feathering, gutting, cadaver cutting, scalding, and handling. Such methods can lead to the cross contaminations among the safe and clean ones. Furthermore, the absence of veterinary supervision may lead to the slaughtering of diseased birds. Therefore, to irradiate contamination with Salmonella and other foodborne pathogens, it is strongly recommended to enhance hygienic practices during chicken rearing, processing and handlings. Furthermore, it also advised to investigate the health status of food handlers on premises that may have had spreaders of foodborne illnesses or asymptomatic organisms.

Author Contributions

All authors designed the experiments. A.M.E, and F. E. D performed the experiments. E. H. A and A.M.E analyzed the data. N.I. A, A.M.E and S. A .S. wrote the manuscript. All authors read and approved the manuscript.

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgment

The authors express their sincere thanks to the staff and technicians of the Center of Biosciences and Biotechnology, Faculty of Engineering and Technology, Gezira University, Wad-Madani, Sudan, and Department of Biology, Faculty of Science, University of Hail for their unlimited assistance and support.

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