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Article

Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR

by
Bryan Tanyag
1,2,*,
Jerick Jann Quiambao
1,2,
Aira Angeline Ko
1,2,
Amarjet Singh
1,2,
Flordeliza Cambia
1 and
Ulysses Montojo
1
1
Fisheries Postharvest Research and Development Division-Seafood Safety and Quality Section, National Fisheries Research and Development Institute, Quezon City 1103, Philippines
2
Integrated Research Laboratory, National Fisheries Research and Development Institute, Quezon City 1103, Philippines
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2021, 1(3), 510-519; https://doi.org/10.3390/applmicrobiol1030033
Submission received: 22 September 2021 / Revised: 22 October 2021 / Accepted: 27 October 2021 / Published: 29 October 2021
Browse Figures
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Abstract

:
Manila Bay contributes significantly to the Philippines’ economy through industry, trade, tourism, and agriculture. However, microbiological contamination in the bay is putting it at risk in the present. Pathogen contamination of the water poses a serious threat to food safety, implicating public health. To address these concerns, the present study examined the prevalence of Salmonella in six different aquaculture commodities, Perna viridis (mussel), Crassostrea iridalei (oyster), Scylla serrata (crab), Penaeus spp. (shrimp), Oreochromis niloticus (tilapia), and Chanos chanos (milkfish), as well as in environmental water from growing areas. A tandem approach of culture-based and real-time PCR methods for the isolation and identification of Salmonella was carried out. To accurately identify the isolates, a real-time PCR Taqman assay based on the invA gene was used. Among the fishery resources examined, no positive samples were observed from C. chanos and O. niloticus. In addition, Salmonella was found in twenty (20) samples, representing 16.26% of all aquaculture commodities collected. Furthermore, Salmonella was found in 13.58% of the 81 environmental water samples examined. P. viridis had the highest prevalence of 55.55% out of 18 samples examined. Samples contaminated with Salmonella failed to meet the regulatory limits set by BFAR FAO 210 series 2001 and EC No. 2073/2005. In addition, it was observed that the sample matrix had a significant impact on the presence of Salmonella (p < 0.05). However, the spatial and temporal distribution of Salmonella did not vary greatly (p > 0.05). This study underscores the importance of imposing strict policies by regulatory bodies to prevent diseases, thus avoiding severe health implications.

1. Introduction

Aquaculture is a major source of seafood comprising 110.2 million tons with an estimated value of USD 243.5 billion in 2016 [1]. In 2012, aquaculture met about 50% of the global demand for fish and fishery products, with about 90% of aquaculture products coming from the Asian region. In the Philippines, aquaculture produced 3.1 million tons of fish, crustaceans, mollusks, and other aquatic animals valued at USD 3.92 billion [2]. Aquaculture was a major contributor to the country’s total fish production in 2019, accounting for 41% of the total value. In addition, provinces along the Manila Bay area such as Bataan, Bulacan, Pampanga, and Cavite had aquaculture production valued at approximately USD 28,000, 58,000, 502,000, and 6000 million, respectively [3].
Manila Bay is characterized as a semi-enclosed body of water that represents a significant part of the country’s economy through industry, commerce, tourism, and agriculture. Fish ponds and pens/cages are widespread in the surrounding provinces along the bay. According to the BFAR and BAS (2003), the total area of fish ponds and pens/cages along Manila Bay was 37,050.68 and 549.00 ha, respectively.
However, these highly regarded benefits of Manila Bay to the country are currently under threat. Runoff from nearby households is being dumped straight into the bay, causing adverse effects to the surrounding waters, specifically microbiological contamination. Microbiological studies conducted in the area have shown high concentrations of coliforms and Escherichia coli [4,5]. Contamination of the bay with pathogens poses a significant threat to aquaculture in the area. One of these human pathogens includes Salmonella, which are Gram-negative, facultatively anaerobic bacteria belonging to the family Enterobacteriaceae [6]. Salmonellosis or infection with Salmonella causes enteritis, abdominal pain, nausea, gastrointestinal problems, and even life-threatening diseases such as typhoid and paratyphoid fever [7,8]. Most outbreaks of salmonellosis in different geographic areas such as Europe, the United States, and other Asian countries were attributed to fish or seafood [9]. The introduction of this pathogen to fish and fishery resources is associated with the contamination of aquatic environments or during handling in processing plants.
Since Salmonella is pathogenic and the primary cause of foodborne disease outbreaks in the country [10,11], it is critical to examine aquaculture commodities from the bay for potential contamination. Moreover, assessing these microbiological risks to food safety is essential to implement control measures to protect public health. Thus, a preliminary study on assessing the prevalence of Salmonella in fish and fishery resources from aquaculture farms in Manila Bay was conducted. It sought to determine the prevalence of Salmonella in fishery resources from aquaculture farms in Manila Bay using culture-based methods and the Taqman assay real-time PCR method using the invA gene specific for Salmonella. Observed results were compared to the regulatory limits set by the Bureau of Fisheries and Aquatic Resources (BFAR), Fisheries Administrative Order (FAO) 210 series of 2001, and European Commission (EC) No. 2073/2005. In addition, the seasonal (wet and dry) and spatial (Cavite, Bulacan, Pampanga, Bataan) distribution of Salmonella in aquaculture farms in Manila Bay were also determined. Data obtained will serve as baseline information on the prevalence of Salmonella in fish and fishery resources collected from Manila Bay aquaculture ponds, which is essential for monitoring and recommending policy to mitigate public health risks.

2. Materials and Methods

2.1. Sampling Area

The collection of samples was conducted in pre-identified aquaculture ponds along Manila Bay (Figure 1). Sampling sites were established in the provinces of Orani (120.53 E, 14.81 N) in Bataan; Obando (120.89 E, 14.68 N) and Paombong (120.78 E, 14.85 N) in Bulacan; Bacoor (120.92 E, 14.49 N) and Kawit (120.90 E, 14.45 N) in Cavite; and Sasmuan (120.62 E, 14.92 N) in Pampanga.

2.2. Sample Collection

The collection of samples was performed monthly during the dry season (April to June 2019) and wet season (July to September 2020). A total of 123 aquaculture commodity samples and 81 environmental water samples were collected aseptically from aquaculture ponds, pens, and shellfish growing areas that included finfishes, bivalves, and crustaceans. Species identified were the following: 24 Perna viridis (mussel), 15 Crassostrea iridalei (oyster), 23 Penaeus spp. (shrimp), 27 Scylla serrata (crab), 17 Chanos chanos (milkfish), and 17 Oreochromis niloticus (tilapia). The collected samples were placed in individual sterile stomacher bags and then placed again inside a clean, leak-proof, properly labeled re-sealable bag. Water samples of approximately 250 mL were collected in a composite from three sampling points in the pond. Then, samples were transferred into appropriately labeled sterile 250 mL borosilicate bottles containing 0.25 mL, 3 percent sodium thiosulfate [12]. Aseptic techniques were observed throughout the collection. All samples collected were placed in an ice chest and temperature kept at 0–4 °C during transport to preserve the integrity of the samples prior to laboratory analysis.

2.3. Detection of Salmonella

2.3.1. Sample Preparation and Isolation of Salmonella

Samples were prepared in accordance with Chapter 1 of the US FDA Bacteriological Manual [13]. To extract the meat, samples were dissected and shucked with sterile tools. Furthermore, Salmonella detection was carried out using the methods described in US FDA Bacteriological Manual Chapter 5. Twenty-five (25) grams of meat were transferred aseptically into a sterile stomacher bag, and 225 mL of lactose broth (LB) was added to each sample and homogenized for 2 min. Each sample was then transferred to a sterile 250 mL borosilicate glass bottle and incubated for 60 ± 5 min at room temperature. The pH of the samples was determined and adjusted if necessary to 6.8 ± 0.2. An aliquot of 0.1 mL enriched LB sample was transferred to Rappaport-Vasiliadis (RV) broth and another 1 mL to tetrathionate (TT) broth for selective enrichment. RV broth was incubated for 24 ± 2 h at 42 ± 0.2 °C, and TT broth, for 24 ± 2 h at 43 ± 0.2 °C. Selective enrichment in RV and TT was performed to eliminate competing bacteria from the sample.

2.3.2. Blanks and Controls

Salmonella Typhimurium ATCC 13,311 and Salmonella Typhimurium BIOTECH 1826 were used as positive controls, whereas for negative control, Staphylococcus aureus BIOTECH 1582 was used with slight variations from the procedures described in US FDA Bacteriological Manual Chapter 5. Specific ATCC cultures mentioned in the US-FDA BAM were not used in this study. Lastly, uninoculated medium was used as sterility control or as blank for conventional analyses, while molecular grade water was used for the real-time PCR analysis.

2.3.3. DNA Extraction

Extraction was carried out in accordance with the protocol outlined by the manufacturer of the Purelink gDNA extraction kit (Invitrogen, Carlsbad, CA, USA). DNA was extracted from 1 mL of 24-h RV enrichment. The purity and concentration of the extracted DNA were also determined prior to the real-time PCR assay using a Multiskan Sky® Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MD, USA). Concentrations of DNA in the samples were calculated based on Lambert-Beer’s equation at a wavelength of 260 nm. The absorption maximum of proteins at 280 nm was used to assess the purity of DNA samples.

2.3.4. Identification and Confirmation of Salmonella spp.

Culture-Based Method

Samples that were positive with selective enrichment were streaked onto Hektoen enteric agar (HEA) and incubated for 24 ± 2 h at 35 °C. Gram staining was performed on Salmonella spp. colonies using Grams Stain-Kit (HiMedia®, Mumbai, India). In addition, typical colonies were streaked on triple sugar iron agar (TSIA) and lysine iron agar (LIA). Purified colonies showing typical Salmonella growth (alkaline slant and acid butt, with or without the production of H2S) were subjected to biochemical tests using API 20E strips (BioMérieux, Marcy-l’Étoile, France).

Real-Time PCR Taqman Assay

The primers and probe set used were specific for invA gene of Salmonella spp. Sequences (Table 1) were derived from US FDA Bacteriological Manual Chapter 5 and were tested for specificity through a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, Accessed: 10 January 2019). These were synthesized by Integrated DNA Technologies (IDT). The 5’ end of the probe was labeled with a FAM reporter dye and the 3’ end with a BHQ quencher. The assay had a total reaction volume of 20 µL consisting of 10 µL Universal Mastermix II with UNG® (Applied Biosystems, Foster City, CA, USA), 0.6 µL of forward primer (300 nm conc.), 0.6 µL of reverse primer (300 nm conc.), 0.5 µL probe (250 nm conc.), 6.3 µL of tris-EDTA, and 2 µL of template DNA. A 2-min incubation initiated the amplification at 50 °C for optimal UNG enzyme activity, followed by a 10 min, 95 °C enzyme activation step for AmpliTaq Gold®. This was followed by 40 cycles of denaturation step for 15 s at 95 °C followed by subsequent annealing and extension step for 60 s at 60 °C. All reactions were run on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

2.4. Statistical Analysis

Resulting data were statistically analyzed using the Statistical Package for the Social Sciences (SPSS) version 21. The Pearson Chi-square test was used to determine any significant relationships between the positive PCR results and sampling locations as well as seasonal variations. The significance level was set at 95% confidence with margin of error of 0.05.

3. Results and Discussions

3.1. Detection of Salmonella by Culture-Based and Real-Time PCR Methods

The culture-based method was performed to confirm the presence of Salmonella in fish and fishery resources as well as in environmental waters collected from growing areas. The golden standard for detecting Salmonella is typically carried out in laboratories by conducting culture-based methods outlined in the US Food and Drug Administration—Bacteriological Analytical Manual (US FDA—BAM) guideline. Simultaneously, a real-time PCR assay was utilized to detect the presence of Salmonella in fish and fishery resources. Sensitivity of the method was determined by generating 10 trials of standard curves. Using pure DNA extracted from reference control organisms (Salmonella Typhimurium ATCC 13311, Salmonella Typhimurium BIOTECH 1826), the assay was able to identify the target invA gene in both strains. The method detected Salmonella at concentrations of 3 × 106 pg/µL, but detection had a lower limit of up to 3 pg/µL, only. The limit of detection was determined by preparing a serial dilution of the reference control strains and analyzing each dilution point in a 7-fold repeat. Specificity of the primers and probe used was also confirmed by testing against different non-target bacterial strains. A total of 30 samples were tested, 15 of which were spiked with low concentrations of Salmonella and the remaining with non-Salmonella strains. The results demonstrated that the invA gene specific for Salmonella could discriminate non-target organisms from the target Salmonella strains. Templates of non-target organisms, blank control, and negative control showed no amplification when tested using the assay.
Due to its high sensitivity and specificity, the invA gene has been used extensively in several studies with Salmonella as an organism of interest. This gene, which is found on pathogenicity island I, is essential because it codes for proteins in bacterial cell membranes required to invade intestinal epithelial cells [14,15]. It is detected in virulent pathogenic strains and established as an international standard for Salmonella identification [16]. The study was limited to only detecting Salmonella at the genus level. Different serotypes of Salmonella were not classified from the isolated bacteria. As a result, further investigation will be needed to determine the available Salmonella serovars in fish, fishery resources, and environmental water samples from growing areas. In addition, serotypes should be classified to categorize the isolates as typhoidal and non-typhoidal Salmonella strains specifically.
As observed, isolated Salmonella spp. from positive samples appeared as typical blue to blue-green colonies with black centers in HEA (Figure 2A). Moreover, isolated colonies gave an excellent Salmonella spp. classification result of 99.9% using API 20E strips (Figure 2B). Lastly, real-time PCR results showed that isolated colonies from fish and fishery resources and environmental water samples from growing areas were confirmed as Salmonella (Figure 3). Results showed that the positive control, Salmonella Typhimurium ATCC 13311, had a CT value of 18.12 ± 0.24 since 3.0 × 106 pg/uL Salmonella concentrations were prepared, contrary to the CT value of the sample having 29.64 ± 0.22, implying that lower Salmonella concentrations were detected. Lastly, both Staphylococcus aureus BIOTECH 1582 and the molecular grade water did not produce any amplification curve, suggesting a negative result.

3.2. Prevalence of Salmonella in Fish and Fishery Resources and Environmental Waters

The presence of Salmonella in seafood has sparked the interest of many researchers because of the risk of foodborne outbreaks. Salmonella is a common source of foodborne disease in humans worldwide, and it is typically spread through contaminated food or water [17]. In this study, 20 of the 123 samples tested positive for Salmonella, representing 16.26% of all aquaculture commodities collected (Table 2). Furthermore, Salmonella was found in 13.58% of the 81 environmental water samples examined. Data analysis revealed that the prevalence of Salmonella varied among different sample types (p > 0.05). Kumar et al. (2009) suggest that the prevalence of Salmonella in individual seafood types was found to be quite variable, although they were collected from different locations with similar environments. As observed, Salmonella was most prevalent in Perna viridis, with 55.55% contamination. This was followed by Crassostrea iridalei, another bivalve, with a 19.05% prevalence. This is similar to the findings of previous studies, where bivalves were the most contaminated with Salmonella from all sample types [18,19,20,21]. Bivalves utilize their filter-feeding mechanisms to filter particles, such as bacteria, from the environment. This leads to the high occurrence of Salmonella in bivalves. The reported high prevalence of Salmonella in bivalves in this study could be associated with the small number of samples analyzed. Compared with the previous studies, Salmonella prevalence in bivalves only ranged from 2–4% [21,22]. Moreover, other samples with descending prevalence were Scylla serrata, environmental water samples from growing areas, and Penaeus spp. with 14.81, 13.58, and 8.70% prevalence, respectively. On the other hand, Salmonella was not detected in any of the examined Oreochromis niloticus and Chanos chanos samples. Contrary to the recent findings, Salmonella was isolated in various fish samples [23,24]. However, it was clearly stated in the study of Sheng and Wang (2021) that the prevalence of Salmonella varies among different countries due to various factors such as the fish species, geographic locations, sampling stages, sampling parts, sources, and fish product types. Moreover, only fish flesh samples were used in this study. This likely explains the absence of Salmonella in fish samples because a previous study revealed that Salmonella was mostly isolated from the fish viscera [25]. Data revealed that the sample matrix, such as fish, crustaceans, bivalves, or environmental waters, had a significant impact on the presence of Salmonella (p < 0.05).
The Philippines’ BFAR issued FAO No. 210 series of 2001 [26] establishing and requiring the absence of Salmonella in 25 g of fish products. A total of 20 bivalves and crustaceans failed to meet the requirements of the standard. Moreover, 14 bivalve samples failed the regulatory limit (EC No. 2073/2005) set by the European Union (2005), stating that Salmonella should be absent in 25 g of live bivalve mollusks that are placed on the market during their shelf-life [27]. With this information, policies should be strictly implemented to ensure public health safety in consuming seafood. Gastrointestinal symptoms acquired by consuming seafood are often underestimated and misdiagnosed. In addition, these health issues are frequently not addressed by Filipinos because most symptoms are self-limiting. These facts underscore the importance of imposing strict regulatory policies by regulatory bodies to prevent diseases, thus avoiding severe health implications.

3.3. Prevalence of Salmonella between Sampling Locations

Table 3 shows the prevalence of Salmonella among the different sampling locations. Results suggest that Salmonella was most prevalent in Bulacan, wherein 22.92% (n = 11) out of 48 samples collected were positive. In addition, Bataan had a prevalence of 20.83% (n = 10) out of 48 samples. It was followed by Cavite, having a prevalence of 10.00% (n = 5) out of 50 samples. Finally, Pampanga had the least prevalence observed, as only 8.62% of the samples were contaminated with Salmonella. Data analyses revealed that the prevalence of Salmonella did not vary significantly (p > 0.05). Thus, no significant differences were observed in all provinces.
Approximately 13.92% of the Philippines’ population inhabits the Manila Bay area [28]. Areas such as Manila, Las Piñas City, Parañaque City, Pasay City, Navotas, Bataan, Bulacan, Pampanga, and Cavite are among the cities and municipalities surrounding the coastal areas of Manila Bay. A study conducted by Levantesi et al. (2012) revealed that high concentrations of Salmonella could be attributed to densely populated areas. Thus, differences in Salmonella concentrations were observed from different sampling points in their study. Because Salmonella is transmitted via the fecal-oral route, bacteria can enter the aquatic environment directly with the feces of infected humans or animals or indirectly, e.g., via sewage discharge or agricultural land run-off [29]. Anthropogenic activities directly contribute to Salmonella contamination of fish and fishery resources. Because the above-mentioned cities and municipalities surrounding Manila Bay are densely populated, it is expected that the prevalence of Salmonella does not vary significantly between sampling areas (p > 0.05).

3.4. Seasonal Distribution of Salmonella in Fish and Fishery Resources and Environmental Waters

Figure 4 shows the prevalence of Salmonella during the wet and dry seasons across different sample types. As observed, Perna viridis had the highest prevalence of Salmonella during the wet season. Out of nine Perna viridis samples collected, 77.78% were contaminated with Salmonella. This was followed by Crassostrea iridalei, Scylla serrata, environmental water samples, and Penaeus spp. having 33.33, 13.33, 11.11, and 7.69% prevalence, respectively. On the contrary, the prevalence of Salmonella was different during the dry season. Perna viridis had the highest prevalence of 33.33%, followed by both Scylla serrata and environmental water samples with a similar 16.67% prevalence. Lastly, Crassostrea iridalei and Penaeus spp. had 13.33 and 10.00% prevalence, respectively. It was noted that both Oreochromis niloticus and Chanos chanos were negative for Salmonella during the dry and wet seasons.
Based on the results of this study, no significant difference was observed between dry and wet seasons (p > 0.05). This may be attributed to the limited sampling collections conducted throughout the study period since sample collection depends on the harvest time of pond owners. Contrary to the findings of various researchers, Salmonella is most prevalent during the dry season [30,31,32,33]. The increased prevalence of Salmonella during the dry season is affected by various environmental factors such as host shedding, enhanced persistence of Salmonella in warm temperatures, and an increase in storm events [34]. However, studies conducted by Simental and Martinez-Urtaza (2008) and Luo et al. (2015) correlate the presence of rain to the occurrence of Salmonella in coastal areas [35,36]. They have concluded that rain is a distinguishing factor that transports Salmonella spp. in the marine environment, leading to high bacterial loading through run-off. Thus, establishing the seasonal pattern of Salmonella is complex, as its occurrence may be influenced by various factors [37]. Further studies should be carried out to establish the temporal distribution of Salmonella in Manila Bay.

4. Conclusions

Manila Bay is contaminated with Salmonella, as seen from the collected samples of fish and fishery resources and environmental waters in growing areas. The observed prevalence of Salmonella demonstrated that contamination varies among each sample type. In addition, the filter-feeding mechanism of bivalves contributed to their high Salmonella prevalence. The researchers propose that more research be conducted to establish the association of contamination in aquaculture commodities with their growing areas. Moreover, sources of contamination should also be examined in the future. Risk assessment studies should also be conducted to assess the risks associated with the consumption of seafood. The findings in this study can be used by competent authorities to strengthen regulatory frameworks that address the health and safety of consumers and at the same time promote fair trade practices.

Author Contributions

Conceptualization, U.M.; Writing—Original Draft, J.J.Q. and A.A.K.; Writing—Review and Editing, B.T., F.C. and U.M.; Investigation, B.T., J.J.Q., A.A.K. and A.S.; Methodology, B.T., J.J.Q., A.A.K. and A.S.; Formal Analysis, B.T. and J.J.Q.; Visualization, B.T., J.J.Q.; Project Administration, U.M.; Funding Acquisition, U.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Acknowledgments

This study was funded by the BFAR as a component of the project titled “Assessment of heavy metals, pathogenic bacteria, and other pollution indicators in Manila Bay aquaculture farms”, under the Manila Bay Rehabilitation and Restoration in compliance with the Supreme Court Mandamus (G.R. 171947-48). The authors would also like to thank the Local Government Units of Orani (Bataan), Obando (Bulacan), Paombong (Bulacan), Bacoor (Cavite), Kawit (Cavite), and Sasmuan (Pampanga) for their assistance in the collection of samples of aquaculture commodities and environmental water. Likewise, we would like to extend our gratitude to the Seafood Safety and Quality Section staffs of NFRDI-FPHRDD for assisting us in the collection and analyses of samples.

Conflicts of Interest

The authors declare no conflict of interest.

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Applmicrobiol 01 00033 g001 550
Figure 1. Map of sampling sites around Manila Bay.
Figure 1. Map of sampling sites around Manila Bay.
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Figure 2. (A) Salmonella spp. colonies in HEA that appear as blue-green colonies with black centers; and (B) Salmonella spp. biochemical reactions on API 20E strips.
Figure 2. (A) Salmonella spp. colonies in HEA that appear as blue-green colonies with black centers; and (B) Salmonella spp. biochemical reactions on API 20E strips.
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Figure 3. Data output from real-time PCR analysis.
Figure 3. Data output from real-time PCR analysis.
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Figure 4. Seasonal prevalence of Salmonella.
Figure 4. Seasonal prevalence of Salmonella.
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Table
Table 1. Sequences of Primers and Probe for Salmonella.
Table 1. Sequences of Primers and Probe for Salmonella.
invA Gene BasesSequence
PrimersForward205′-AAC GTG TTT CCG TGC GTA AT-3′
Reverse205′-TCC ATC AAA TTA GCG GAG GC-3′
Probe 205′/FAM/TGG AAG CGC TCG CAT TGT GG/BHQ/-3′
Table
Table 2. Prevalence of Salmonella per sample type.
Table 2. Prevalence of Salmonella per sample type.
Sample TypeNo. of Samples ExaminedPositive (%)
Bivalves
Perna viridis *1810 (55.55)
Crassostrea iridalei214 (19.05)
Crustaceans
Scylla serrata274 (14.81)
Penaeus spp.232 (8.70)
Finfishes
Oreochromis niloticus160 (0)
Chanos chanos180 (0)
Environmental Water8111 (13.58)
Total20431 (15.20)
* significant.
Table
Table 3. Prevalence of Salmonella per sample location.
Table 3. Prevalence of Salmonella per sample location.
Sample TypeNo. of Samples Examined Positive (%)
Bulacan4811 (22.92)
Bataan4810 (20.83)
Cavite505 (10.00)
Pampanga585 (8.62)
Total20431 (15.20)
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Tanyag, B.; Quiambao, J.J.; Ko, A.A.; Singh, A.; Cambia, F.; Montojo, U. Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR. Appl. Microbiol. 2021, 1, 510-519. https://doi.org/10.3390/applmicrobiol1030033

AMA Style

Tanyag B, Quiambao JJ, Ko AA, Singh A, Cambia F, Montojo U. Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR. Applied Microbiology. 2021; 1(3):510-519. https://doi.org/10.3390/applmicrobiol1030033

Chicago/Turabian Style

Tanyag, Bryan, Jerick Jann Quiambao, Aira Angeline Ko, Amarjet Singh, Flordeliza Cambia, and Ulysses Montojo. 2021. "Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR" Applied Microbiology 1, no. 3: 510-519. https://doi.org/10.3390/applmicrobiol1030033

APA Style

Tanyag, B., Quiambao, J. J., Ko, A. A., Singh, A., Cambia, F., & Montojo, U. (2021). Prevalence of invA Gene of Salmonella spp. in Fish and Fishery Resources from Manila Bay Aquaculture Farms Using Real-Time PCR. Applied Microbiology, 1(3), 510-519. https://doi.org/10.3390/applmicrobiol1030033

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