Journal of Medicine in the Tropics

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 18  |  Issue : 2  |  Page : 98--102

Isolation and polymerase chain reaction detection of virulence invA gene in Salmonella spp. from poultry farms in Jos, Nigeria


Joseph Aje Anejo-Okopi1, Samson Ejiji Isa2, Onyemocho Audu3, Idowu O Fagbamila4, Jacob Chire Iornenge1, Ifeanyi Stella Smith5,  
1 Department of Microbiology, University of Jos, Jos, Nigeria
2 Department of Medicine, Infectious Diseases Unit, Jos University Teaching Hospital, Jos, Nigeria
3 Department of Epidemiology and Community Health, College of Health Sciences, Benue State University, Makurdi, Benue, Nigeria
4 Bacterial Research Division, National Veterinary Research Institute, Vom, Plateau State, Yaba, Lagos, Nigeria
5 Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria

Correspondence Address:
Joseph Aje Anejo-Okopi
Department of Microbiology, University of Jos, Jos
Nigeria

Abstract

Background: Salmonella serovars are one of the most common food-borne pathogens, and poultry consumption is responsible for the majority of routes of infection worldwide. There is a paucity of documented data regarding the prevalence of virulence determinant genes in Salmonella serovars in Nigeria. The aim of the study was to isolate Salmonella spp. in selected poultry farms in Jos Metropolis, Plateau State, Nigeria. Methodology: A total of eighty samples were conveniently collected from 18 commercial poultry. The samples were from poultry droppings, egg shells, workers«SQ» hands, and feeds. The samples were examined for the presence of Salmonella by standard microbiological techniques. The isolates were phenotypically confirmed using biochemical characterization and virulence gene determined by polymerase chain reaction (PCR). Results: The overall isolation percentage of Salmonella species was 28.75% (23/80). DNA extraction was carried out on the isolated 23 Salmonella isolates and 11 successfully quantified. Of the 11 isolates, ten (91.0%) successfully amplified using the invA gene-specific primers by PCR method. The result indicates the presence of Salmonella in poultry farms, and this posed a major concern for public health. Conclusion: The result showed that the use of PCR amplification of virulence genes in suspected Salmonella spp. from poultry farms proved to be efficient and could serve as an alternative rapid tool for the detection of Salmonella spp. Further large studies with the use of more virulence genes are needed to understand the Salmonella epidemiology in poultry farms that serves as a major protein source of the nation.



How to cite this article:
Anejo-Okopi JA, Isa SE, Audu O, Fagbamila IO, Iornenge JC, Smith IS. Isolation and polymerase chain reaction detection of virulence invA gene in Salmonella spp. from poultry farms in Jos, Nigeria.J Med Trop 2016;18:98-102


How to cite this URL:
Anejo-Okopi JA, Isa SE, Audu O, Fagbamila IO, Iornenge JC, Smith IS. Isolation and polymerase chain reaction detection of virulence invA gene in Salmonella spp. from poultry farms in Jos, Nigeria. J Med Trop [serial online] 2016 [cited 2021 Feb 26 ];18:98-102
Available from: https://www.jmedtropics.org/text.asp?2016/18/2/98/192237


Full Text

 Introduction



Salmonellosis is a significant public health issue in sub-Saharan Africa including Nigeria. In the developed and developing countries, nontyphoidal Salmonella spp. from poultry farms are responsible for the majority of domestically acquired food-borne illness. [1],[2],[3] The digestive tract, skin, and feather of poultry are considered to be the major sources of food-borne bacteria; contamination of meat products is, therefore, possible during slaughter, feather plucking, and handling. [4] Reports from Jos, Nigeria, showed high prevalence rates (32.5%-51.1%) of Salmonella spp. from commercial poultry farms floor, meat, and eggs using conventional isolation methods. [5],[6] The incidence of Salmonella spp. in poultry dropping, feeds, eggs, and chicken meat continues to be on the increase with high morbidity, and in some cases death. [7] The most commonly identified causative agents of food-borne salmonellosis are the nontyphoidal Salmonella Typhimurium and Salmonella Enteritidis. Both serotypes have the ability to colonize the reproductive organs of hens which cause major food-borne illness, and S. enteritidis has been the most implicated serovars worldwide. [8] The illnesses are mild and self-limiting in immunocompetent humans, but can be severe with fatal outcome in immunocompromised individuals such as those with HIV/AIDS. [9] An earlier study in Jos using conventional culture reported a high prevalence of Salmonella serovars from poultry farms. [10] Although useful for producing isolates for molecular diagnosis, conventional cultures are time consuming and labor‐intensive and fail the purpose of producing a rapid and reliable result necessary for public health protection.

The polymerase chain reaction (PCR) method is rapid and reliable for the detection and identification of Salmonella food-borne pathogen, and the Salmonella virulence gene (invA) has been found suitable target for PCR amplification. [11],[12] In this study, we aimed to isolate and detect invA gene from poultry farm samples in Jos Metropolis, Nigeria.

 Methodology



The study was carried out in Jos Metropolis comprising three local government areas (Jos North, Jos South, and Jos East). The main occupation is agriculture with significant population involve in poultry farming. Two (Jos North and South) local governments out of three were visited for sampling. Before the enrollment, voluntary and informed consent was obtained from poultry farm owners and poultry farm handlers (individuals involved in poultry breeding, farming, and the loading and transport of poultry). Ethical approval was also obtained from Plateau State Specialist Hospital and approval from the Department of Veterinary Services, Ministry of Agriculture, Plateau State, Nigeria. Nine farms in each local government were conveniently selected from identified clusters after interview with the farm owners who consented to the study. The sample collection which was from October 2014 to January 2015, involved 18 commercial broiler/layer poultry farms, and all the farms practiced the deep litter management system. Each farm was visited twice each and a total of eighty samples including both poultry (droppings and egg shells swabs), humans (hand swabs), and feeds were collected without a previous history of vaccination against Salmonella. Samples were transported to the laboratory and were investigated for the presence of Salmonella spp. in the Bacteriology Laboratory, National Veterinary Research Institute, Vom, Plateau State, Nigeria.

Sample Collection

A total of eighty samples of poultry droppings, egg shells, workers' hands, and feeds were aseptically collected in clean polyethylene bags from 18 farm sites from different locations in the two local governments of Jos Metropolis from October 2014 to January 2015. The collected samples were transported to the laboratory for bacteriological analysis.

Microbiological Analysis

We used the standard microbiological laboratory techniques for isolation and identification of Salmonella serovars according to the International Organization for Standardization (ISO) 6579. [13] The aseptically swabs in sterile buffered peptone water were incubated at 37°C for 18 h. One mL from this preenrichment homogenate was inoculated into 10 mL of tetrathionate broth and incubated. The transfer of 0.1 mL of the same homogenate was made to Rappaport-Vassiliadis broth, incubated at 42°C for 24 h. A loopful of each broth was streaked on Salmonella-Shigella (S-S) agar, xylose lysine deoxycholate agar, and MacConkey's agar plates, incubated at 37°C for 24 h. Colonies suspected of Salmonella serovars were purified, morphologically and biochemically identified according to the guidelines of the ISO 6579. [13] Samples positive for Salmonella spp. through phenotypic and biochemical characterization were aseptically transferred to nutrient agar slant and incubated at 37°C for 24 h. Prepared bacterial smears from pure culture were fixed, Gram-stained, and examined microscopically, and stocks were kept at refrigerator condition for DNA extraction procedures.

DNA Extraction of Isolated Salmonella

0The bacteria isolates confirmed by biochemical tests were subcultured from the stored slants on Luria-Bertani agar for 24 h at 37°C, from which broth culture was prepared. From the prepared broth, DNA extraction by boiling method was carried out. 1.5 ml of the sample broth was centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and the pellets were washed twice with sterile water, and 200 μl of sterile water was added to the pellets, the pellets were vortexed to homogenize and boiled in a dry bath at 100°C for 10 min. This was followed by vortexing and centrifugation at 12,000 rpm for 5 min. The supernatant containing the DNA was transferred to another tube and stored at −20°C. The quantification and purity of the extracted DNA were estimated using a NanoDrop spectrophotometer. Of 23 positive isolates, 11 successfully quantified after extraction procedure. The quantified DNA extract supernatant was prepared and used for PCR with Salmonella specific reverse and forward invA primers of 521 bp.

Primers Set and Polymerase Chain Reaction Amplification

Salmonella-specific primers, forward and reverse have, respectively, the following nucleotide sequence based on the invA gene of Salmonella 5' GTG AAA TTA TCG CCA CGT TCG GGC AA-3'and 5' TCA TCG CAC CGT CAA AGG AAC C-3'. Reaction with these primers was carried out in a 25 μl amplification mixture consisting of 2.5 μl × 10 PCR buffer (500 mM KCl and 200 mM TrisHCl), 1.25 μl dNTPs (10 mM), 1.6 μl MgCl, 0.5 μl of each primer, 0.5 μl of Taq DNA polymerase (Fermentas), and 1.5 μl of extraction for each isolate were used in the reaction. Amplification was conducted in Master-gradient Thermo Cycler (Eppendorf). The cycle conditions were as follows: an initial incubation at 94°C for 60 s, followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 64°C for 30 s, and elongation at 72°C for 30 s, and finally 7 min of extension at 72°C. The amplified DNA products from Salmonella specific-PCR were further analyzed by electrophoresis on 1.5% agarose w/v gels stained with ethidium bromide and visualized using ultraviolet illumination. A 100 bp DNA ladder was used as a biomarker for PCR products as shown in [Figure 1].{Figure 1}

 Results



Macroscopically, Salmonella suspected isolates showed smooth red-colored colonies with black center on xylose lysine deoxycholate agar, it also appeared as deep blue (black like) colonies on Hektoen enteric agar, pale on MacConkey's agar, colorless smooth, transparent, and raised colonies and on S-S agar. On H 2 S, Salmonella produce colorless colonies with black centers. Microscopically, on staining, Salmonella appeared as Gram-negative, nonspore forming, and short rod shape. Biochemically, all isolates were nonlactose fermenting and negative oxidase, indole, and Voges-Proskauer and urea hydrolysis tests. The observed prevalence of Salmonella spp. from conventional method was 28.75% (23/80); droppings 11/80 (13.75%), egg shell swabs 5/80 (6.25%), and human hand swabs 7/80 (8.75%). The result of agarose gel electrophoresis showed PCR amplification products of DNA of invA gene (521 bp) of positive Salmonella spp. and ten (91.0%) of the 11 isolates were positive to invA specific gene and this indicated a clear abundance of the virulence gene in analyzed isolates [Figure 1].

 Discussion



Salmonella infection is a major bacterial disease in poultry and humans, causing significant economic loss and illnesses. Out of eighty samples, we isolated 23/80 (28.75%) of Salmonella spp. by conventional culture method and 12.5% confirmed by PCR. Salmonella invA gene specific primers PCR is a known and tested rapid, sensitive, and specific means for identifying Salmonella at the genus level in a variety of samples. The invA gene encodes a protein in the inner membrane of bacteria for invasion of epithelial cells of the host. [14] The use of invA specific gene in this study was to support the ability of the primer sets to confirm the isolated Salmonella spp. The PCR result confirmed Salmonella isolates identified by conventional culture method in the amplification of DNA fragments of 521 bp for the invA gene in ten (91.0%) Salmonella isolates, irrespective of serovar and sample type. This corroborates with several other reported studies that had also confirmed the successful detection of 100% of Salmonella isolates from poultry farm and products through the use of invA gene specific primers. [15],[16] The study also corroborates with some earlier work done using PCR methods, in which nontyphoidal Salmonella were isolated from animal, food, and human fecal samples in developing countries. [12],[17] The observed prevalence of Salmonella invA gene from poultry and poultry products was in variance with some other studies, which reported higher prevalence. [18],[19] This variation may be due to differences in study methodology; gene-specific involvement, sample types, sample size and hygiene practices in farms, and geographic locations. Our observed Salmonella virulence specific gene invA contamination from poultry farms was as expected higher than an earlier reported finding using conventional method, [5] and this could be as the result of the sensitivity of PCR employed. Salmonella spp. are known to infect poultry farms and products by a wide variety of species; however, one serovar may be predominant in a location for years before being replaced by another. The most common and clinically significant serovars harboring the virulence invA gene causing salmonellosis globally are S. Typhimurium and S. Enteritidis.[20]

The observed outcome of the PCR result from the 11 quantified isolates (12.5%) was high; 10/11 (91.0%) compared to the phenotypic method by culture (28.75%) used. This result suggests that the use of invA gene by PCR is fast, more sensitive and specific than the conventional methods, and it will be a good alternative method for the detection of Salmonella spp. in food and clinical samples. [21] Reported findings from within and other parts of the developing countries have reported wide range of prevalence of Salmonella in poultry products (3%-66%), [22],[23] which may indicate the need for improve hygiene and sanitary standards in poultry farms that could ultimately translate to quality poultry products. The conventional culture method is known to be the standard for bacterial detection in poultry products and other foods but generally takes longer time and could be less sensitive compared to molecular-based techniques. Therefore, the use of invA gene specific PCR method is a useful procedure for rapid identification of Salmonella from poultry and poultry products, whether in routine testing or research endeavors.

This is probably the first study employing PCR technique for the detection of Salmonella in poultry farms in Jos; however, there are important limitations. The poultry farms were conveniently selected and could have introduced bias. The sample size is small in addition to the lack of adequate training of poultry handlers which may culminate in poor farm management, and Nigeria is a large country and poultry farms vary in sizes and operational strategies which may contribute to Salmonella transmission and infection. Therefore, the generalizability of our study is limited; however, the finding has significant implications for public health.

 Conclusion



A significant proportion of our poultry farms and products are contaminated with Salmonella spp. with virulence specific gene (invA) which has economic and public health implications. The use of molecular methods to detect virulence-specific gene in Salmonella spp. can save labor hour from conventional methods and enhance rapid detection of Salmonella. It is, therefore, important to consider a mass food hygiene programs to prevent economic and health hazards. Furthermore, larger studies on the detection of Salmonella using invA gene and other virulence genes are suggested to establish a generalizable data. The findings also indicate that poultry handlers are to ensure best farm management practices to deliver safe poultry products and never to underestimate the risk of Salmonella contamination from poultry farms.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.

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