Virulence genes of salmonella

Food Microbiology

Foodborne Pathogens: Hygiene and Safety View all 49 Articles

University of Teramo, Italy

Faculty of Veterinary Medicine, University of Belgrade, Serbia

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Original Research ARTICLE

1 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Malaysia
2 Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security (ITAFoS), Universiti Putra Malaysia, Serdang, Malaysia
3 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia
4 Department of Diagnostic and Allied Science, Faculty of Health and Life Science, Management and Science University, Shah Alam, Malaysia
5 Division of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
6 Department of Agricultural and Food Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, Malaysia
7 Novel Antibiotic Laboratory, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
8 Department of Science and Technology Studies, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

The aim of the present study was to investigate the prevalence of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium in retail beef from different retail markets of Selangor area, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 240 retail beef meat samples (chuck = 60; rib = 60; round = 60; sirloin = 60) were randomly collected. The multiplex polymerase chain reaction (mPCR) in combination with the most probable number (MPN) method was employed to detect Salmonella spp., S. Enteritidis and S. Typhimurium in the meat samples. The prevalence of Salmonella spp., S. Enteritidis and S. Typhimurium in 240 beef meat samples were 7.50, 1.25, and 0.83%, respectively. The microbial loads of total Salmonella was found in the range of 5 cfu/mL (data not shown). The optimized mPCR reaction mixture (25 μL) contained 2 μL of DNA template, 5 μL of 5 × PCR buffer, 2.5 μL of 25 mM MgCl₂, 0.5 μL of 10 mM deoxynucleotide triphosphate (dNTP), 0.5 μL of 1.2 μM primer mix and 14.2 μL of deionized water. The mixture was then treated with 0.3 μL (1.5 U) Taq DNA polymerase. PCR amplification was performed in triplicate with the following conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 45 s, annealing at 53°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 7 min. The positive controls used were S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076. Escherichia coli ATCC 25922 was used as a negative control.

The turbid MPN tubes were confirmed to be Salmonella by plating on selective CHROMagar Salmonella (CHROMagar Microbiology, Paris, France) and Xylose Lysine Deoxycholate (XLD) (Merck, Darmstadt, Germany) agar plates, and incubated at 37°C for 24 h. All the Salmonella isolates were then serotyped by slide agglutination using polyvalent “O” and “H” antisera (BD, Franklin Lakes, USA) at Veterinary Research Institute (VRI), Ipoh, Malaysia in accordance with the Kauffmann-White scheme.

The antimicrobial susceptibility was evaluated according to Clinical and Laboratory Standarts Institude (2012) by using disc diffusion method. Briefly, isolates were cultured aerobically in 10 mL Mueller-Hinton (MH) broth (Merck, Darmstadt, Germany) at 37°C for 24 h. Overnight cultures, grown on MH broth (OD adjusted to 0.5 MacFarland unit), were swabbed evenly with sterile non-toxic cotton swab on MH agar plates and left to dry for 2 to 4 min. Then, antimicrobial sensitivity discs were placed on the culture by using a disk dispenser and incubated at 37°C for 24 h. The tested antimicrobials were amoxicillin/clavulanic acid (AMC, 30 μg), amoxycillin (AML, 30 μg), ceftazidime (CAZ, 30 μg), cephazolin (KZ, 30 μg), ciprofloxacin (CIP, 5 μg), erythromycin (E, 15 μg), chloramphenicol (C, 30 μg), ampicillin (AMP, 10 μg), penicillin (P, 10 μg), streptomycin (S, 10 μg), tetracycline (TE, 30 μg), kanamycin (K, 30 μg), gentamicin (CN, 10 μg), vancomycin (VA, 30 μg), nalidixic acid (NA, 30 μg), and suphamethoxazole/trimethoprim (SXT, 25 μg) (Oxoid, Hamphire, United Kingdom). The multiple antibiotic resistance (MAR) index was calculated as “a/b,” where “a” the number of antibiotics for a particular isolate was resistant and “b” the total number of antibiotics tested (Krumperman, 1983).

All Salmonella isolates collected in this study were screened for the presence of virulence genes using PCR. The primers, the size in base pairs of the respective amplification products and the references used for detection of six virulence genes are presented in Table 1. The virulence genes under study were invA, pefA, hilA, sopB, stn, and spvC. Positive (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) and negative control (E. coli ATCC 25922) were conducted in the detection procedure. To evaluate the reproducibility of the experiments, PCR amplification and electrophoresis experiments were carried out in triplicate.

Table 1. PCR primers used for amplification of virulence genes in Salmonella isolates.

All measurements were carried out in triplicate. Minitab (v. 14) statistical package (Minitab Inc., State College, PA) was used to determine if there was any significant difference between the prevalence of Salmonella in beef meat from wet market and hypermarket. For all analysis, P Keywords: beef meat, Salmonella, multiplex PCR, prevalence, antimicrobial resistance, virulence gene

Citation: Thung TY, Radu S, Mahyudin NA, Rukayadi Y, Zakaria Z, Mazlan N, Tan BH, Lee E, Yeoh SL, Chin YZ, Tan CW, Kuan CH, Basri DF and Wan Mohamed Radzi CWJ (2018) Prevalence, Virulence Genes and Antimicrobial Resistance Profiles of Salmonella Serovars from Retail Beef in Selangor, Malaysia. Front. Microbiol. 8:2697. doi: 10.3389/fmicb.2017.02697

Giovanna Suzzi, Università di Teramo, Italy

Milan Zivko Baltić, Faculty of Veterinary Medicine, University of Belgrade, Serbia
Giorgia Perpetuini, Università di Teramo, Italy

Department of Veterinary Public Health, College of Veterinary Science and Animal Husbandry, AAU, Anand - 388 001, Gujarat, India

Abstract

The aim was to detect virulence gene associated with the Salmonella serovars isolated from pork and Slaughterhouse environment.

Salmonella isolates (n=37) used in this study were isolated from 270 pork and slaughter house environmental samples collected from the Ahmedabad Municipal Corporation Slaughter House, Ahmedabad, Gujarat, India. Salmonella serovars were isolated and identified as per BAM USFDA method and serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh, India). Polymerase chain reaction technique was used for detection of five genes, namely invA, spvR, spvC, fimA and stn among different serovars of Salmonella.

Out of a total of 270 samples, 37 (13.70%) Salmonella were isolated with two serovars, namely Enteritidis and Typhimurium. All Salmonella serovars produced 284 bp invA gene, 84 bp fimA and 260 bp amplicon for enterotoxin (stn) gene whereas 30 isolates possessed 310 bp spvR gene, but no isolate possessed spvC gene.

Presence of invA, fimA and stn gene in all isolates shows that they are the specific targets for Salmonella identification and are capable of producing gastroenteric illness to humans, whereas 20 Typhimurium serovars and 10 Enteritidis serovars can able to produce systemic infection.

Introduction

Pork is one of the most widely eaten meats in the world, accounting for about 38% of meat production worldwide, although consumption varies widely from place to place [1]. Most of the pork consumer’s peoples are from tribal areas and pork is mainly consumed in the northeastern states of India. The present production of meat in India is estimated at 6.27 million tons in 2013 [2], which are 2.21% of the world’s meat production. The meat production has increased from 764,000 tons in 1970-71 to 6.27 million tons in 2010 in India, which is 2.21% of the world’s meat production. The contribution of meat from a pig is 5.31% [2]. According to the Food and Agriculture Organization of the United Nations, world’s pork production reached 114.2 million tons in 2012. Asia is the principal region, accounting for almost 60% of world pig meat production, World meat production is anticipated to expand modestly in 2013 to reach 308.3 million tons, an increase of 4.2 million tones or 1.4%, compared with 2012 [3].

Food safety hazards caused by food-borne pathogens such as Salmonella remain a major problem for the food industry. Salmonellosis is an important health problem and a major challenge worldwide having greater significance in developing countries [4]. Pork and pork products are recognized as an important source of human salmonellosis [5]. Salmonella is an important cause of food-borne (alimentary) health problems in humans [6]. The risk of Salmonella might differ between the production systems, caused by components of the husbandry systems affecting disease development and pathogen shedding or differences in the level of resistance to the pathogen [7]. The increased consumption of pork coupled with the high prevalence of enteropathogens in the swine industry suggests a rise in food-borne illness cases which can lead to human food-borne illness and loss of product shelf-life.

The virulence of Salmonella is linked to a combination of chromosomal and plasmid factors. Different genes such as inv, spv, fimA and stn have been identified as major virulence genes responsible for salmonellosis. Salmonella pathogenicity islands (SPIs) are large gene cassettes within the Salmonella chromosome that encode determinants responsible for establishing specific interactions with the host, and are required for bacterial virulence in a given animal like other pathogenicity islands. More than 20 SPIs have been described [8]. The chromosomally located invasion gene invA codes for a protein in the inner membrane of bacteria that is necessary for invasion of epithelial cells [9]. Whereas, an operon (spvRABCD), containing five genes, is present on plasmids commonly associated with some serotypes. One main function of the spv operon is to potentiate the systemic spread of the pathogen [10]. The spvC is virulence-related gene on the plasmid required for survival within host cell [11]. Some studies have provided evidence that the virulence plasmid plays a significant role in human disease [12]. Salmonella induced diarrhea is a complex phenomen on involving several pathogenic mechanisms, including production of enterotoxin. This enterotoxin production is mediated by the stn thus it plays a significant role in causing gastroenteritis by producing enterotoxin [13].

The purpose of this study was to evaluate the potential virulence of Salmonella isolates from eggs and poultry house environment by detecting the presence of the invA, spvR, spvC, fimA and stn virulence genes using the polymerase chain reaction (PCR).

Materials and Methods

Approximately, a total of 270 samples of pork and slaughterhouse environment will be collected from the Ahmedabad Municipal Corporation Slaughterhouse, Ahmedabad, Gujarat under aseptic precautions. The samples were collected in sterilized polyethylene bags and transported to the departmental P.G. Research Laboratory in an icebox for further processing and microbiological analysis. All the samples collected are shown in Table-1 .

Number of samples collected from different sources for isolation of Salmonella spp.

Type of sample	Number of samples
Muscles	30
Tonsils	30
Rectal swabs	30
Intestine	30
Lymph node	30
Water	30
Liver	30
Knife swab	30
Butchers hand swab	30
Total	270

Our study used Salmonella isolates (n=37) recovered from pork and Slaughterhouse environmental samples collected from the Ahmedabad Municipal Corporation Slaughterhouse, Ahmedabad, (Gujarat), India. 13 Salmonella enteritidis and 24 Salmonella typhimurium Salmonella serovars were isolated and identified as per BAM USFDA method [14] and serotyped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh, India). The DNA of isolates of Salmonella was prepared by boiling method. Approximately, loop full of culture was taken in microcentrifuge in 100 µl of sterilized DNAse and RNAse-free milliQ water (Millipore, USA). Then, vortexed and samples were heated at 95°C for 10 min, cell debris was removed by centrifugation and 3 μl of the supernatant was used as a DNA template in PCR reaction mixture. PCR was performed with four sets of primer pairs specific for the invasion gene invA, spvR gene, spvC gene, fimA gene and stn gene as shown in Table-2 .

Primer pairs used for virulence characterization of Salmonella isolates.

Primer pair target	Primer sequence (5’→3’)	Annealing temp (°C)	Length (bp)	Reference
invA	F: GTG AAA TTA TCG CCA CGT TCG GGC AA R: TCA TCG CAC CGT CAA AGG AAC C	63	284	[15]
spvR	F: CAG GTT CCT TCA GTA TCG CA R: TTT GGC CGG AAA TGG TCA GT	57	310	[16]
spvC	F: ACT CCT TGC ACA ACC AAA TGC GGA R: TGT CTT CTG CAT TTC GCC ACC ATC A	63	571	[17]
fimA	F: CCT TTC TCC ATC GTC CTG AA R: TGG TGT TAT CTG CCT GAC CA	56	85	[18]
stn	F: CTT TGG TCG TAA AAT AAG GCG R: TGC CCA AAG CAG AGA GAT TC	55	260	[20]

PCR amplifications were performed in a final volume of 25 µl containing DNA template (3 µl), ×2 PCR Mastermix (MBI Fermentas) (12.5 µl), 10 pmol/µl of each primer (MWG-Biotech AG, Germany) (1 µl) and 5.5 µl nuclease-free water. Amplification for invA gene was carried out as described by Kumar et al. [15] with minor modifications. The reaction conditions involved initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s. A final extension of 5 min at 72°C was employed. The amplification for spvR gene was carried out similarly by employing standardized annealing temperature. The fimA gene fragment was amplified at annealing temperature of 56°C and extension for 30 s. The spvC gene fragment was amplified at annealing temperature of 63°C and extension for 1 min. The amplification for stn gene was carried out employing same conditions as invA except annealing at 55°C. Amplification products were separated by electrophoresed on 2% agarose gel stained with 5 µg/ml of ethidium bromide with a 100 bp DNA ladder as molecular weight marker.

Results and Discussion

All 37 Salmonella isolates (13 of which belonged to serovar Enteritidis and 24 belonged to Typhimurium) contained the invasion gene invA, other studies having reported similar results [17,21-24], which was expected since the invA is an invasion gene conserved among Salmonella serotypes.

Similar to invA gene all isolates produced 260 bp DNA fragment specific for stn gene which was in agreement with other authors 25. Thus, all the Salmonella isolates were found highly invasive and enterotoxigenic.

The fimA gene was detected in all 37 isolate produced 85 bp DNA fragment. Which is similar to that of Naravaneni and Jamil [18,19] and this demonstrated that fimA gene has a high degree of sequence conservation among Salmonella serovars. This is very useful in the diagnosis of Salmonella organisms at the genus level.

The spvR gene was detected in 30 isolates belonged to Typhimurium and Enteritidis, which is similar to that of Araque [23] and this shows that the strains have the plasmid borne virulence characters that have ability to cause the systemic infection while spvC was not detected in any isolates, which is in contrast to that of Soto et al. [27] who found presence of spvC in all the isolates ( Table-3 ). Electrophoreses results of invA, spvR, fimA and stn gene are shown in Figures- Figures-1 1 - -4, 4 , respectively.

Virulence genes present in different serovars of Salmonella.

1 Discipline of Genetics, School of Life Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa

2 Virology and Microbiology Research Group, College of Pharmacy, City University College of Ajman, Al Tallah 2, Ajman, P.O. Box 18484, UAE

Associated Data

Abstract

Livestock are an important source of protein and food for humans, however opportunistic pathogens such as Salmonella spp. turn livestock into vehicles of foodborne diseases. This study investigated the prevalence of virulence genes in Salmonella spp. isolated from livestock production systems in two provinces of South Africa. During the period from May to August, 2018, a total of 361 faecal (189), oral (100), environmental (soil (36) and water (27)) and feed (9) samples were randomly collected from different animals (cattle, sheep, goats, pigs, ducks and chickens) that were housed in small-scale livestock production systems from Eastern Cape and KwaZulu-Natal Provinces in South Africa. Salmonella spp. were isolated and identified using microbiological and DNA molecular methods. Salmonella spp. were present in 29.0% of the samples of which 30.2% belonged to the Salmonella enterica species as confirmed by the positive amplification of the species specific iroB gene. Virulence genes that were screened from livestock-associated Salmonella were invA, iroB, spiC, pipD and int1. Statistically significant associations (p Keywords: Salmonella, zoonosis, pathogenicity, virulence, food-borne, livestock, Xylose-Lysine-Deoxycholate, pathogen, PCR, integron, infection, humans, one-health

1. Introduction

Salmonellae are facultative intracellular Gram-negative bacteria that cause high morbidity and mortality in a wide range of hosts including humans, birds, mammals, and insects [1]. Salmonellae are one of the most problematic, foodborne, and zoonotic pathogens that cause health threats and challenges to general human well-being [2]. Salmonella spp. reside in the gastrointestinal tract of warm-blooded animals. The bacteria cause salmonellosis in humans, a disease that is presented mostly by mild diarrhea, also well-known as food poisoning [3,4]. Salmonellosis may be fatal, depending on the dose of infection and the immune status of the infected individual [5]. In the United States, Salmonella spp. are currently on the top of the list of pathogens that cause foodborne infections [6]. In South Africa, Salmonella spp. are regarded as one of the leading causes of foodborne outbreaks [7]. Foodborne outbreaks were reported in South Africa due to consumption of animal and poultry contaminated products [8,9,10,11]. Salmonella infection causes economic losses in the agriculture sector and it negatively impacts food animals which are reared for the generation of income [12].

Although Salmonella is a major cause of foodborne diseases in South Africa, there are limited data on the disease since it usually causes self-limiting gastroenteritis and cases are rarely reported [8,12]. It has been reported that six out of seven Salmonella enterica serovar Enteritidis outbreaks that occurred in South Africa from 2013 to 2015 were of food origin [13]. It was reported that 141 (43%) out of the 327 foodborne outbreaks reported in South Africa between 2013 to 2017 were reported in warmer months from KwaZulu-Natal [7]. An outbreak of food origin caused by S. enterica serotype Virchow was reported at a school in South Africa [14].

Surveillance of Salmonella is frequently conducted by different organizations worldwide in order to study its prevalence and epidemiology [8,15]. In South Africa surveillance is mainly the responsibility of governmental departments such as the Department of Health as well as the Department of Agriculture, Forestry and Fisheries. In order to bolster the surveillance, government departments have collaborations with local universities and other research institutions. Salmonella species play a role in metabolism when it is in a non-virulent state [16]. Factors such as stressful conditions, environmental changes and mutations can trigger virulence in a bacterium thus turning a previously non-virulent strain into a pathogenic strain [17].

The Salmonella genus includes more than 2500 serological variants (serovars) and is broadly categorized into S. bongori and S. enterica species [18]. According to the U.S. Centers for Disease Control (CDC) S. enterica is further subdivided into six subspecies that are designated by taxonomic names such as S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. indica. S. enterica species is highly diverse consisting of more than 2600 serovars which are further divided into typhoidal Salmonella and non-typhoidal Salmonella (NTS), depending on the disease it causes [19,20]. Typhoidal Salmonella spp. are restricted to human hosts while non-typhoidal Salmonella spp. can infect a wide range of hosts [21]. Faecal shedding of NTS results in environmental contamination and transmission to humans, leading to disease outbreak [16]. NTS has a broad host range and is often associated with foodborne outbreaks in humans [21]. S. enterica serovar Typhimurium and S. enterica serovar Enteritidis are the most frequently reported pathogens in Salmonella outbreaks and the prominent cause of gastroenteritis in humans [22,23].

Salmonella pathogenicity is mediated by numerous genes such as invA, spiC and pipD, which code for effectors that induce successful host infection. Pathogenicity of Salmonella is expressed in three ways such as host cell invasion, intracellular survival and colonization [24]. Numerous virulence genes are essential for Salmonella pathogenesis and these genes are located on various elements of the genome including the chromosome, plasmids, integrated bacteriophage DNA, Salmonella pathogenicity islands (SPIs), and Salmonella genomic islands (SGIs) [19,25]. SPIs are large gene cassettes and only SPI-1 and SPI-2 (not all SPIs) encode a membrane-associated type III secretion system (T3SS) [26] which secretes a pool of 44 effector proteins [27], that alter the functioning of eukaryotic cells in order to facilitate bacterial pathogenicity inside the cell [28,29,30]. Previous studies reported that SPIs are acquired by horizontal transmission and vertically pass to new clones [31]. More than 20 SPIs have been characterized, with greater focus on SPI-1 and SPI-2 that function via encoded T3SS since they harbor host invasion and intracellular survival genes [29,32]. Inside the host cell, SPI-2 expresses genes that are important in intracellular survival, proliferation, and persistence in internal organs such as the spleen and liver [30,33]. Salmonella spp. use virulence genes and factors located in SPI-1 for cell invasion and to initiate its pathogenicity [29]. The invasion A (invA) is one of the most studied virulence factors that is also used as a biomarker for Salmonella spp. detection as it contains sequences that are unique to the genus Salmonella. [34] Invasion A is a factor in the outer membrane of Salmonella spp. that is responsible for entering the host epithelial cells in the intestines thus initiating infection [34]. The inv locus in S. enterica serovar Typhimurium was characterized and it was reported that invA is essential in the display of virulence in the intestine [35].

One of the most important genes is iroB, a Fur-regulated gene located in a large DNA region which is used in the detection of S. enterica subspecies enterica [36,37]. Previous studies which detected typhoid and non-typhoid Salmonella by PCR used invA and iroB together with flagellar genes [38,39]. Furthermore, iroB was used to detect Salmonella from blood in another study [40]. The IroB gene is a member of the iroA (iroBCDEN) gene cluster which is responsible for the synthesis and transport of enterobactin, a siderophore produced by Salmonella spp. and is essential for iron uptake inside the host [41]. Besides enabling bacterial iron uptake, expression of the iroA cluster also facilitates the host immune escape by interrupting macrophage homeostasis [42]. The specific role of iroB is to encode glucosyltransferase which glucosylates enterobactin [41]. Enterobactin glucosylation contributes to the virulence of the bacteria by preventing the host antimicrobial protein (lipocalin-2) from sequestering the siderophore [43,44].

spiC is another gene in the SPI-2 that is essential for intracellular survival and host defense escape [45]. Macrophages are important innate immune barriers which defend the host against infections and their function is activated by gamma interferon and facilitated by factors such as cytokines and eicosanoids [46]. Upon activation, macrophages kill pathogens that are capable of surviving inside them. In order to escape the host’s defense, spiC is involved in the signal transduction pathway which expresses the suppressor of cytokine signaling, leading to gamma interferon signaling inhibition [45]. It was reported that spiC is also involved in the translocation of effectors into the cytosol of macrophages [47].

The SPI-5 harbors six genes in which mutations in four of these genes were reported to radically lower enteropathogenicity [48,49]. pipD is one of the genes in the SPI-5 that is involved in inflammatory enteritis by coding a cysteine protease homolog that is essential in the long term systemic infection [48,49,50].

Gastroenteritis is the most common disease caused by non-typhoidal Salmonella. This disease usually resolves without treatment but it can be systemic in severe cases and require antimicrobial treatment. There is, however, an enormous challenge with using antibiotics as Salmonella is one of the ‘superbugs’ which are resistant to several classes of antibiotics [51]. The antimicrobial resistance phenotype is attributed to the possession of class 1 integron by some of the Salmonella serovars.

The class 1 integron is a mobilizable cluster of antimicrobial resistance genes found in Salmonella genomic island [52,53,54,55]. Class 1 integrons are made up of integrase gene, a primary recombination site and a promoter region [56]. The role of int1 is to recombine gene cassettes (associated with antibiotic resistance), which are only transcribed in an integron since they lack a promoter [57,58]. Class 1 integron carries gene cassettes for resistance to antibiotics such as those which were used as first line treatment for salmonellosis. The presence of class 1 integrons carrying gene cassettes in virulent Salmonella spp. increases the threat to humans as it limits the treatment options available [59,60]. Infections by non-tyhpoidal Salmonella spp. affect both developing and developed countries. Studies and incidences revealed that food animals are the carriers of NTS and are potential zoonotic sources of infection to humans [61,62,63]. Against this background, this study focused on the detection and determination of the prevalence of virulent Salmonella spp. in livestock production systems in the KwaZulu-Natal and Eastern Cape Provinces in South Africa.

2. Materials and Methods

The study was approved by the Animal Research Ethics Committee of the University of Kwa-Zulu Natal (Reference numbers AREC/051/017M, AREC 071/017 and AREC 014/018). The field sampling protocols, samples collected from animals, and the research were conducted in full compliance with Section 20 of the Animal Diseases Act of 1984 (Act No 35 of 1984) and were approved by the South African Department of Agriculture, Forestry and Fisheries DAFF (Section 20 approval reference number 12/11/1/5 granted to Prof. Dr. ME El Zowalaty).

During the autumn and winter months of the year 2018, a total of three hundred and sixty-one (361) oral, faecal, soil, water and feed samples were randomly collected from different animal hosts such as cattle, sheep, goats, pigs, ducks and chickens. The animals were housed in small-scale commercial farms in Flagstaff (O.R Tambo, Eastern Cape), Verulam (eThekwini, KwaZulu-Natal) and the South Coast (Amandawe and Mtwalume, UGU, KwaZulu-Natal) as depicted in Figure 1 .

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