Polymerase chain reaction fails to detect Listeria species from wild and cultured Nile tilapia (Oreochromis niloticus) caught from a large freshwater lake in Zambia

¹Department of Environmental and Occupational Health, Levy Mwanawasa Medical University, Lusaka 33991, Zambia, ²Department of Disease Control, School of Veterinary Medicine, University of Zambia, Lusaka 32379, Zambia, ³Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 Ås, Norway, ⁴Department of Biomedical Sciences, School of Veterinary Medicine, University of Zambia, Lusaka 32379, Zambia, ⁵Department of Epidemiology and biostatics, Levy Mwanawasa Medical University, Lusaka 33991, Zambia

^&Corresponding author
Prudence Mpundu, Department of Environmental and Occupational Health, Levy Mwanawasa Medical University, Lusaka 33991, Zambia

Introduction    

Listeriosis is a severe foodborne disease caused by L. monocytogenes, a gram-positive bacterium belonging to the phylum firmicutes [1]. Fish and fish products contribute about 5.5% to 12.1% of confirmed listeriosis cases in the United States [2]. Previous studies [3-5] reported significant variations of 0.55% to 38% in the prevalence of Listeria species in raw fish samples collected from fish carcasses and fish markets in Nigeria, Iran, and India. In Africa, disease outbreaks linked to the consumption of specific foods are underreported, compared to water-related, because of inadequate water supply or treatment in many regions.

Most countries in the Southern part of Africa, including Zambia, do not differentiate between foodborne and waterborne diseases (outbreaks). L. monocytogenes is naturally present in the environment and tolerate a broad range of environmental stressors. In water and soil environments, L. monocytogenes integrates into multispecies biofilms. Its sturdiness allows L. monocytogenes to survive in refrigeration food production environments making it a significant food production concern. Raw fish contaminated with L. monocytogenes pose a public health concern mainly when inadequately heat treated or served as ready to eat food.

Listeria species identification involves culture methods based on selective enrichment followed by plating and characterization of single isolates. Rapid molecular tests, like Polymerase Chain Reaction (PCR), are available now. For instance, the housekeeping prs gene for the genus Listeria which encodes phosphoribosyl pyrophosphate synthase [6]. Although the prs gene does not differentiate different Listeria species, it nevertheless acts as an indicator of the genus Listeria in samples [6]. The most common tilapia species farmed in cages is Oreochrome niloticus. Due to its ease and cost-effectiveness, tilapia cage culture has boosted freshwater fish farming and economic interest in tropical countries. Tilapia is among the world's most frequently consumed fish types, and its farming improves food security, including income generation. Cultivation practices have raised concerns that pathogenic bacteria can contaminate the waters and confer a risk of foodborne disease. However, knowledge of the prevalence of Listeria species in tilapia caught from a large freshwater lake in Zambia is limited. Therefore, this study aimed to determine the presence of Listeria species circulating in freshly caught tilapia from a large freshwater lake in Zambia using PCR.

Methods     

Study design: a cross-sectional study design was conducted between the periods of February to April 2021.

Study settings: Siavonga district is located along lake Kariba on the North shore in the Southern Province of Zambia, 16° 32' South of the equator and 28° 44' East. Most fish produced comes from aquaculture cage farms. The Siavonga district was chosen for the study due to its large number of aquaculture cage farms and capture fisheries. All the sites we selected for sampling the owners had to consent and fish availability during the study period. At capture, fisheries sampling was at three of the six recorded major sites along lake Kariba in the Siavonga district. These we coded as sites 1, 2, and 3. Out of 15 aquaculture cage farms in the Siavonga Town Council, only three gave consent for sampling. The coding for the sites was 4, 5, and 6 (Figure 1).

Sample size and sampling: sampling was done through the collection of fish to be sampled based on the two main fishing types found in the Siavonga district: aquaculture cage farms and capture fisheries. To get equal variation, we collected one hundred and fifty tilapia swabs from capture fisheries (n=75) and aquaculture cage farms (n=75). Swab samples from each fish came from the exterior surface and gills, independently sampled at the landing stage. We used random sampling at both the capture fisheries and the aquaculture cage farms to pick the individual tilapia for swabbing from the involved sites.

Sample collection and processing: we used a standard procedure for collecting swab samples from the fish, briefly, a sterile metal template to pre-mark a 5cm² x 5cm² area for swabbing. After swabbing the fish with a moist cotton swab, the swab was inserted back into a screw-cap tube containing Amies transport media. We recorded metadata such as farmed or wild-caught, collection site, and collection date. Sample storage was at -4°C before transportation, usually within 72hrs after sampling; we took it to the Microbiology laboratory at the School of the Veterinary Medicine University of Zambia. Samples were assigned laboratory codes upon receipt. Immediately after arrival, the swabs from the tubes were transferred into 9ml of pre-enriched broth (Oxoid) and incubated at 37°C for 48hrs. During the cultivation and identification of Listeria species, the L. monocytogenes strain ATCC19118 we used as a positive control.

Isolation and identification of Listeria species: the protocol for the isolation of Listeria species is previously described by Mpundu P et al. [7]. Briefly described as follows; firstly, a fish sample was inoculated into 9ml of pre-enriched broth (Oxoid) and incubated at 37°C for 24hrs. Later 1ml of pre-enriched broth (Oxoid) was transferred into 9ml of Fraser broth (Oxoid) (enriched with Listeria selective supplement) and vortexed for 1min, followed by incubation at 37°C for 48hrs. A loop full of the Listeria enriched culture was inoculated onto the surface of Listeria selective agar (Oxoid), incubated at 37°C for 48hrs, and observed for colonies with morphology typical for Listeria species, i.e., green-blue color, with a possible greenish sheen. Presumptive Listeria species colonies were sub-cultured on Nutrient Agar (Oxoid) incubated at 37°C for 24hrs to obtain pure colonies. The quality of the agar plates was tested by cultivating the L. monocytogenes strain ATCC19118. We performed standard biochemical tests on the purified presumptive Listeria species: Gram staining, catalase, sulphur indole motility, oxidase, urease, Voges Proskauer, and methyl-red tests.

Deoxyribonucleic acid (DNA) extraction and polymerase chain reaction identification of Listeria: polymerase chain reaction assays were performed on presumptive Listeria isolates to verify their identity. Chromosomal deoxyribonucleic (DNA) was isolated from the isolates grown on nutrient agar using the ZYMO Research quick DNA miniprep kit, following the manufacturer's instructions. For PCR detection of Listeria species, a partial 370bp fragment of the prs gene was targeted by using the primer pair prs -F (5'-GCT GAA GAG ATT GCG AAA GAA G-3') prs -R (5'-CAA AGA AAC CTT GGA TTT GCG G-3') [6]. PCR was under the following conditions initial denaturation at 94°C for 1 min, 30 cycles at 94°C for 45 s, 53°C for 45 s, 72°C for 2 min, and final extension step at 72°C for only 5 min. Finally, the PCR product was separated on a 1.5% agarose gel stained with ethidium bromide to visualize a potential 370 bp band. We used the L. monocytogenes strain ATCC19118 for quality control.

Data analysis: all the obtained data from the tilapia fish carcasses were entered in the excel datasheet and imported to Stata version 15 (Stata cop, College Station, Texas, U.S.A). The primary outcome was the presence of the Listeria species contamination from tilapia fish swabs. We calculated the prevalence of presumptive Listeria species contamination as the proportion of the total tilapia fish swabs collected that were contaminated phenotypically.

Ethics: approval of ethics was acquired from Excellence in Research Ethics Committee reference no. (Ref.no.2020-Jan-006). We sought permission from Siavonga Town Council and maintained the confidentiality of all sampling sites involved throughout this study.

Results     

Of the 150 tilapia samples collected from captured fisheries and aquaculture cage farms, 2.0% (3/150) were phenotypically indicative of Listeria species contamination. None of the presumptive Listeria isolates were prs positive. However, the positive control samples gave the expected 370 bp on the agarose gel, which secured reliability. The highest prevalence of presumptive Listeria isolates occurrence was found in samples from capture fisheries 2.7% (2/75) (95% CI: -1.048 - 6.453), aquaculture cage farms 1.3% (1/75) (95% CI: 1. 284 - 3.916) (Table 1).

Discussion     

The present study determined the prevalence of Listeria species contamination of tilapia from lake Kariba in the Siavonga district in Zambia. In the present study, none of the surface swab samples collected from fresh tilapia carcasses belonged to the genus Listeria on PCR. To the best of our proof, the present study is the first that investigated Listeria species contamination in tilapia from a large freshwater lake in Zambia. A similar study in India showed that Listeria species surface swabs had 72.4% of tropical fish and fishing environments [8]. The significant difference deduces from the differences in the sampled fish; Jeyasekaran and others collected sample swabs from fish markets and fish processing units. Onjong and others conducted a study on the microbiological safety of fresh tilapia in value chains, and a 2% prevalence of L. monocytogenes sampled at landing was recorded [9]. The differences in isolation with this present study deduction are from the sampled tilapia fish characteristics and techniques used in the isolation method. This present study utilized the prs gene to confirm the Listeria genus, a housekeeping gene found in all Listeria species. At the same time, Onjong and co-workers used VITEK 2 system version 0.8.01 biomerieux, Inc. Hazelwood, MO. These two methods may yield different results due to differences in sensitivity [10]. Further, this study only collected swabs at the landing stage without multiple processes.

Human activities occurring near sampled water bodies may thus influence the level of Listeria contamination in the fish. In the Siavonga district, cattle and crop farming are rare, and most people solely depend on fishing for a living. The low level of Listeria might partly be due to the lack of extensive farming activities in this area. The none-detection of Listeria contamination on PCR observed in the present study could also be explained because sampling was at the primary production stage. Furthermore, sampling was from a single lake, where environmental factors are assumed to be similar, previous studies reporting a higher prevalence of Listeria species mainly sampled fish from different water bodies.

Climatic conditions, mainly rainfall, contribute to bacterial contamination of surface water. The collection of samples was during the rainy season, when the probability of isolating Listeria species is usually higher than during the dry season, as reported in other studies. However, Hansen and others (2006) failed to find significant seasonal variations of L. monocytogenes levels in water from freshwater fish farms [11]. The principle limitation of this study is the low sample size. However, some studies have used less than ten (10) samples of fish from freshwater and have been able to isolate Listeria species. Additionally, the other limiting factor may link to the approach used. Including entire fish or the enteric contents would have increased the likelihood of isolating Listeria. On the other hand, large freshwater bodies have a practical dilution effect. Another insidious factor that we might have inadvertently ignored may have significantly affected the final results.

Conclusion     

Results from the study intimate the likelihood of low levels of Listeria species in wild-captured and cultured tilapia from lake Kariba in the Siavonga district of Zambia. However, conducting further studies across different seasons is recommended. Additionally, there is need to repeat the same study with an increased sample size and inclusion of enteric organs and whole fish analysis to increase isolation rates.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Prudence Mpundu conceptualized the study and wrote the manuscript. Marina Elisabeth Aspholm and Musso Munyeme: validation of the molecular work protocol, and manuscript proofreading. John Bwalya Muma, Nawa Mukumbuta, and Walter Muleya contributed to manuscript editing and proofreading. All the authors have read and agreed to the final manuscript.

Table and figure     

Table 1: presumptive Listeria species results in tilapia fish from captured fisheries and aquaculture cage farms from Kariba, a large freshwater lake, Zambia (n=150)

Figure 1: map of the Siavonga district showing the study areas

References