Possibilities of using mussels (Mytilus galloprovincialis ) to predict rotavirus contamination in Albania
Published Online: Sep 20, 2023
Page range: 339 - 345
Received: Mar 30, 2023
Accepted: Sep 14, 2023
DOI: https://doi.org/10.2478/jvetres-2023-0053
Keywords
© 2023 Arta Lugaj et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Rotavirus is a highly contagious virus that can spread easily through contaminated food and water. It is a major cause of severe diarrhoea in children worldwide, as rotaviruses are a major cause of acute gastroenteritis and can lead to dehydration, malnutrition, and even death. The virus is able to persist in the environment because of its stability and resistance to disinfection, making it a persistent threat to public health (15). Rotavirus is a double-stranded RNA virus that is non-enveloped, consists of 11 double-stranded RNA molecules, and belongs to the
The outer capsid contains two proteins, VP7 and VP4, which are used to classify rotavirus strains into different serotypes (8). These proteins are also targeted by neutralising antibodies produced by the immune system following a natural infection. Group A rotaviruses are responsible for most cases of severe disease and deaths, particularly in low-income countries with poor sanitation and limited access to healthcare (5). Vaccination is an effective strategy to protect against rotavirus and other enteric viruses, especially in areas where the disease is endemic. Rotavirus vaccines have been shown to be highly effective in preventing severe rotavirus disease and have been approved by the WHO for global use. RotaTeq, a pentavalent vaccine, and Rotarix, a monovalent vaccine, are currently the two vaccines that are widely used.
The widespread use of these vaccines has led to a significant reduction in the burden of rotavirus disease globally (2, 29). Nevertheless, previous studies have demonstrated the presence of rotavirus in mussels from various locations. Since shellfish are widely consumed all over the world, this raises concerns about the potential for human exposure to this and other pathogens (20). Shellfish, particularly mussels, are a common source of foodborne illnesses caused by viral pathogens, including rotavirus. Mussels are particularly resistant to chemical and bacteriological pollution and can accumulate xenobiotics in their tissues in proportion to the environmental concentration. They can also trap and accumulate bacteria and viruses present in seawater and act as passive carriers of human pathogens. Mussels use their ciliated gill epithelia and mucous membranes to filter and sieve food particles from the water (10, 24, 28). This ability to filter water and concentrate pathogens makes mussels a useful bioindicator for detecting microbial contamination in seawater (24, 27). While mussels have been primarily used as indicators of chemical pollution, they can also be used to monitor viral contamination. Regular testing of water and shellfish can help to prevent outbreaks of foodborne illness and ensure the safety of seafood for human consumption. Typically, the concentration of viruses in seawater is very low, which makes their detection challenging and requires the concentration of large volumes of water (4, 18, 28). Overall, the prevention and control of viral contamination of food and water requires a multi-faceted approach, including proper waste water treatment, improved hygiene practices, and vaccination programmes. By implementing these measures, we can reduce the incidence of viral infections and protect public health.
The study aimed to investigate the presence of rotavirus in
Butrinti Lake, one of the study areas, is 16.3 km2 in size, has been intensively used for aquaculture of mussels (
Fig. 1.
Total numbers of collected
Coordinates of the investigated sampling sites
| Off the Cape of Stillo | Butrinti Lake | |
|---|---|---|
| Sampling 2015 | 39°41’19.7”N | 39°45’09.3”N |
| 19°59’28.1”E | 20°01’35.3”E | |
| Sampling 2019 | 39°41’16.6”N | 39°45’22.6”N |
| 19°59’05.8”E | 20°01’53.6”E | |
| Sampling 2021 | 39°41’26.5”N | 39°45’18.1”N |
| 19°59’49.4”E | 20°02’22.6”E |
Reverse transcription polymerase chain reaction (RT-PCR) was used to detect enteric viruses in the
The digestive glands of these selected individuals were removed, frozen in liquid nitrogen, and stored at −80°C until they were tested by molecular biology techniques at the Laboratory of Environmental Physiology at the Department of Biology of the University of Padua and at the Laboratory of Wildlife Diseases at the Faculty of Veterinary Medicine, of the Agricultural University of Tirana.
The methodology used to concentrate viruses and purify viral RNA from digestive gland tissue was adopted from a previously published paper (23). The tissues were homogenised in MilliQ water supplemented with Proteinase K (Sigma-Aldrich, St. Louis, MO, USA), and the resulting homogenate was incubated at 37°C for 1 h. After Proteinase K was inactivated at 65°C for 15 min and centrifuged at 3,000 rpm for 5 min, the supernatant was used for viral RNA purification and was purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and 8 M LiCl to remove glucidic contaminants, according to the manufacturer’s protocol (14). The purified RNA was then quantified using a spectrophotometer and assessed for integrity by capillary electrophoresis. An ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used for quantification, and an Agilent 2100 Bioanalyzer was used for capillary electrophoresis with an RNA 6000 Nano kit (Agilent Technologies, Palo Alto, CA, USA). The first strand of cDNA was reverse transcribed at 42°C for 1 h from 1 μg of total RNA in a 20 μL reaction mixture containing 1 μL of ImProm-II Reverse Transcriptase (Promega, Madison, WI, USA) and 0.5 μg of Random Primers (Promega). For generic detection of Group A rotaviruses, an RT-PCR method based on amplification of a VP7 fragment was used. The primers selected were VP7-fw (5’-TAAATGAAT GGTTATGTAACCCAAT-3’; position 527–551 of human wild-type strain) and VP7-re (5’- AATCCG CTACTTTTCTCTTGG-3’; position 829–808 of human wild-type strain). The PCR programme consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, followed by a final elongation step at 72°C for 10 min. To verify the successful amplification of viral genetic material, the PCR amplicons were purified using the NucleoSpin gel extraction and PCR clean-up 2-in-1 kit (Macherey-Nagel, Düren, Germany), ligated into the pGEM-T Easy Vector (Promega), and cloned in XL1-Blue
The experimental data were analysed using SPSS version 20.0 (IBM, Armonk, NY, USA). StatGraphics Plus software (StatGraphics Technologies, The Plains, VA, USA) calculated the descriptive statistics as mean, standard deviation, and minimum and maximum values for the studied parameters at each sampling point.
The PCR results and descriptive statistics obtained from analysing
Fig. 2.
Percentages of rotavirus-infected
Fig. 3.
Percentages of rotavirus-infected
Fig. 4.
Percentages of rotavirus-infected
There were no specimens collected from Butrinti Lake in the autumn because of the high water temperatures, which may have impacted the presence of rotavirus.
In total, the percentage of infected individuals in the sea off the Cape of Stillo in 2015 was 44%, in Butrinti Lake in 2019 it was 36%, and in 2021 it was 23%.
SPSS version 20.0 (IBM, Armonk, NY, USA) was used to calculate the descriptive statistics values at each sampling point at Cape of Stillo and Butrinti Lake, as shown in Table 2. According to these summary statistics, we can emphasize that a higher variance in Cape of Stillo suggests greater dispersion of data points, whereas in Butrinti Lake, the degree of variability in 2021 is higher than in 2019.
Descriptive statistics from Butrinti Lake and Cape of Stillo in 2015, 2019 and 2021
| Study area | N | Range | Mean | Std. deviation | Variance |
|---|---|---|---|---|---|
| Cape of Stillo (2015) | 9 | 80 | 44.111 | 31.0743 | 965.611 |
| Butrinti lake (2019) | 9 | 25 | 35.733 | 10.00587 | 100.118 |
| Butrinti lake (2021) | 9 | 66.7 | 23.144 | 24.9266 | 621.335 |
We used a successful molecular method, namely reverse transcription polymerase chain reaction (RT-PCR), for the detection of enteric viruses in shellfish from
According to the results, rotavirus was present in wild Mediterranean mussels from three sampling sites off the Cape of Stillo, three sampling sites from Butrinti Lake in 2019, and two sampling sites 1 and 2 from Butrinti Lake in 2021, but not in sampling site 3 from Butrinti Lake in 2021. This information suggests that there is a presence of rotavirus in certain areas and years within the aquaculture system in Albania. Further investigation and monitoring will be necesary in the future in order to gain a better understanding of mussel cultivation in the aquaculture system in Albania. This can help determine the extent of rotavirus contamination, its potential impact on mussel populations, and the measures that may need to be taken to manage the presence of rotavirus in the future.
However, it is important to note that there were no specimens collected in autumn from Butrinti Lake because of mussel mortality caused by increased water temperature. It is also worth noting that shellfish tend to accumulate microorganisms during periods of low water temperatures, which can result in a higher incidence of viral gastroenteritis through shellfish consumption during these periods (26). This may explain the seasonality of shellfish-borne viral diseases. Additionally, rotavirus is heat-stable and more resistant to chlorine disinfection than bacteria, which is important to consider for food safety measures. Based on the results of the Cape of Stillo sampling sites, it appears that the percentage of rotavirus-infected mussels was higher in June and September than in August, which was likely because of the lower water temperatures during these periods. Sampling sites 1 and 3 showed higher percentages of infected mussels than site 2, which may indicate site-specific factors contributing to the presence of rotavirus. The results obtained from the Cape of Stillo samples had a wide distribution, which may be linked to the presence of areas used for marine aquaculture in the Greek part (at least 2 km from the Cape of Stillo) of the Greek–Albanian maritime boundary region. Coastal and estuarine areas with marginal domestic pollution can also create ideal conditions for viral transmission through shellfish like Mediterranean mussels. The overall 44% presence of rotavirus in the mussels collected from the sea off the Cape of Stillo is a significant finding and highlights the importance of monitoring and assessing the potential risks associated with consuming these shellfish.
It is important to note that rotavirus is a significant cause of gastroenteritis, particularly in children under the age of 5. An outbreak of acute gastroenteritis was reported by the Public Health Institute in Tirana in 2014. Rotavirus was detected in 21% of samples, more frequently in children under 2 years of age, who accounted for 80.8% of all positive cases (19). More recently, a study conducted in Albania, the Salento peninsula in southern Italy, and different hospitals in Rome found that 31.3% of stool samples collected in Tirana were positive for rotavirus, while the positivity rate was 78.3% in the Salento peninsula and 40.3% in Rome (1). These findings suggest that rotavirus remains an important cause of gastroenteritis in the region and highlight the need for continued monitoring and control measures to reduce the incidence of infections caused by this virus.
It is important to note that the detection of rotaviruses in shellfish should be considered an indicator of human faecal pollution and the possible presence of other human viruses. Therefore, it is recommended to conduct further investigations in Albanian coastal areas to determine the extent of the contamination and the potential risk to public health. Additionally, the current regulations should be revised to include the detection of other pathogens, including viruses, in order to ensure the safety of shellfish and protect public health. It is important to prioritise public health concerns in the management of aquaculture and the regulation of shellfish harvesting and consumption.
This study found that mussels (
These findings are consistent with those of a previous study conducted by different authors that evaluated the use of mussels in biomonitoring marine environments. We proved the presence of rotavirus in