Detection of Human and Fish Viruses in Marine Gastropods.

Marine gastropods represent a major food source for higher trophic levels and an important source of animal protein for humans. Like bivalve molluscs, gastropods can accumulate several types of contaminants; however, the bioaccumulation of microorganisms, particularly viruses, has been poorly investigated in these animals. This study focused on gastropods (Tritia mutabilis, Bolinus brandaris and Rapana venosa) collected during the fishing season from 2017 to 2021 in the north-western Adriatic Sea, and on clams (Ruditapes philippinarum) harvested in the same geographical area, in order to evaluate the presence of human and fish viruses in their tissues. A virological investigation was carried out on the digestive gland using molecular methods. The presence of hepatitis A virus was detected in one sample, whereas noroviruses were not present in the investigated specimens. Regarding fish viruses, it was possible to detect the presence of nervous necrosis virus (NNV) in 26.5% of the analyzed gastropods; however, the histological examination did not show any pathological changes in the nervous tissue in both NNV-positive and -negative batches. As a whole, the investigated gastropods showed the ability to bioaccumulate viruses; however, lower contamination by human viruses compared to bivalve molluscs was pointed out, posing a minor concern to human health.

Hazard Identification Related to the Presence of Vibrio spp., Biogenic Amines, and Indole-Producing Bacteria in a Non-Filter Feeding Marine Gastropod (Tritia mutabilis) Commercialized on the Italian Market.

Tritia mutabilis is a carrion-feeder edible marine gastropod with an open circulatory system. Therefore, biological, and chemical contaminants associated with the feed can reach all body tissues. The aim of the present study was to investigate the possible association of these characteristics with some food safety hazards. Vibrio spp. load, and the prevalence of pathogenic V. parahaemolyticus, V. vulnificus, and V. cholerae, were investigated. Moreover, biogenic amines (BAs) and indole-producing bacteria (IPB), markers of seafood decomposition, were quantified for the first time in an edible carrion-feeder. Overall, 49 batches were analyzed (38 from retail, and 11 from primary production). The Vibrio spp. load resulted of 5.64 ± 0.69 log10 CFU g-1 at retail, and 5.27 ± 0.74 at harvest but all batches resulted negative for pathogenic Vibrio. Histamine, putrescine, cadaverine, and tyramine were detected both at harvest and at the retail level. Their sum (BAs Index) showed a mean value of 50.45 and 65.83 mg Kg-1 in batches at harvest and at retail, respectively. IPB were detected at harvest and upon refrigeration for three days (T1-T3). The mean load resulted in 2.52 ± 0.85 log10 MPN g-1 at T0, 3.31 ± 1.23 at T3 in batches immediately refrigerated, and 3.22 ± 1.18 at T3 in batches previously immersed in clean seawater. Our results contribute to identifying food-borne hazards for T. mutabilis that may be related to the retention of biogenic amines and indole-producing bacteria due to carrion feeding.

Multifactorial Causes of Chronic Mortality in Juvenile Sturgeon (Huso huso).

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii, Shewanella putrefaciens, Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae.

Analysis of the sanitary survey 2015-2017 conducted in the gulf of La Spezia (Italy): Reclassification of the areas of production of live bivalve molluscs.

The sanitary survey is aimed at classifying and monitoring the production areas of live bivalve molluscs (LBM) and it is performed using standards that are provided by the Centre for Environment, Fisheries and Aquaculture Science's Guide to Good Practice. In this study, data from the sanitary survey carried out by the Asl5 Spezzino on the production areas of the gulf of La Spezia during the period 2015-2017 were analysed. The number and type of the analysis performed both on the total sampling and on the individual target species, as well as the number and type of found non-compliance (assessed on both mandatory parameters and on parameters fixed by the local monitoring plan) were considered. Data were also compared with those from the sanitary survey 2012-2014. Appropriate statistic tests were used to evaluate data from E. coli and Norovirus monitoring. Overall, 4306 analysis were performed, especially on the species M. galloprovincialis (89%) and they were mostly focused on to the search of biological agents. 160 NC were detected. Most of the NC concerns the Norovirus's positivity (93.75%) in M. galloprovincialis and C. gigas. A correlation between the levels of E. coli and rainfall/ seasonality (higher levels in the colder months) was proved, especially in the sampling points located in the inner part of the dam and in the Portovenere Bay. Class B was reconfirmed for M. galloprovincialis, the production areas of C. gigas were reclassified as A and those of V. verrucosa were definitively closed. The sanitary survey was therefore confirmed as a useful tool for reclassification and for monitoring LBM production areas.

Evidence of fish and human pathogens associated with doctor fish (Garra rufa, Heckel, 1843) used for cosmetic treatment.

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well-known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.

Preliminary investigation on the microbiological quality of edible marine gastropods of the Adriatic Sea, Italy.

According to the European Legislation, marine gastropods placed unprocessed on the market must comply with the same requirements established for live bivalve molluscs but, being considered not filterfeeding and unable to concentrate fecal contaminants, they may be harvested outside the classified areas. Despite this statement, little scientific information is available on the microbiological quality of these animals. The aim of the present study was to investigate 28 batches of edible snails of the Adriatic Sea, namely Nassarius mutabilis and Bolinus brandaris, with respect to i) smell and viability, by a method here reported; ii) the bacterial component of the whole body referred to E. coli, Vibrio spp., V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus. A total of 21 batches of N. mutabilis and 7 batches of B. brandaris were analyzed. Batches of both species retrieved from the primary production were all largely composed of viable animals, had saltwater/neutral smell, and showed mean value of Vibrio spp. of 5,34 and 5,79 log10 UFC g-1 in N. mutabilis and B. brandaris respectively. 47% of the batches of N. mutabilis retrieved from the market, were largely composed of dead animals, had acrid/nasty smell, and showed mean value of Vibrio spp. of 6,53 log10 UFC g-1. E. coli, V. vulnificus and V. cholerae were never detected, but all samples were positive for V. alginolyticus. One sample of B. brandaris was positive for V. parahaemolyticus genotyped by PCR at the specie level (ToxR+) and positive for the thermostable direct hemolysin gene (tdh+).

Molecular characterization and drug susceptibility of non-O1/O139 V. cholerae strains of seafood, environmental and clinical origin, Italy.

Toxigenic and antimicrobial susceptibility patterns and genetic relatedness of 42 non-O1/O139 V. cholerae strains, the majority of them isolated from seafood and marine water of the Adriatic sea, Italy, and 9 clinical strains, two of which with seawater of the Adriatic as the source of infection, were studied. All strains had hlyA El Tor gene but lacked ctxA gene. Four and two isolates, respectively, also had stn/sto and tcpA Class genes. More than 90% of strains showed susceptibility to cefotaxime, ciprofloxacin, cloramphenicol, tetracycline, trimethoprim + sulfamethoxazole and intermediate or full resistance to tetracycline and erythromycin. Six strains of seafood and clinical source were multi-drug resistant. PFGE analysis allowed to type all the strains with 50 banding patterns. Twenty-one strains, 11 and 8 from seafood and seawater, respectively, and 2 of clinical origin, were grouped into 9 different clusters. We report the presence of toxigenic and multidrug resistant non-O1/O139 V. cholerae strains in Adriatic, some of which genetically related, and support that they represent a potential reservoir of toxin and antibiotic resistance genes.

Preliminary study on the antimicrobial susceptibility pattern related to the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area.

V. vulnificus is a Gram-negative bacterium, commonly found in estuarine and coastal habitats, that can infect humans through seafood consumption or wound exposure. This study represents the first attempt to correlate the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area, with their antimicrobial susceptibility patterns. On the whole, 40 V. vulnificus strains, isolated from shellfish (n=20), different coastal water bodies (n=19), and the blood of a Carretta carretta turtle (n=1), were utilized. All strains were positive for the species-specific genes vvhA and hsp, with high variability for other markers: 55% (22 out of 40) resulted of the environmental (E) genotype (vcgE, 16S rRNA type A, CPS2 or CPS0), 10% (4 out of 40) of the clinical (C) genotype (vcgC, 16S rRNA type B, CPS1), and 35% (14 out of 40) of the mixed (M) genotype, possessing both E and C markers. The antimicrobial susceptibility was assayed by the diffusion method on agar, according to the Clinical Laboratory Standards Institute (CLSI), utilizing the following commercial disks (Oxoid): ampicillin (AMP), ampicillin- sulbactam (SAM), piperacillin (PRL), cefazolin (KZ), cefotaxime(CTX), ceftazidime (CAZ), imipenem (IPM), meropenem (MEM), amikacin (AK), gentamicin(CN), tetracycline(TE), ciprofloxacin (CIP), levofloxacin (LEV), trimethoprim-sulfamethoxazole (SXT), and chloramphenicol (C). 75% of the strains, (n=30) including all C strains, was sensitive to all the tested antibiotics, whereas E strains showed intermediate sensitivity to AK (2 strains), CIP and CAZ (1 strain), TE (1 strain) and resistance to KZ (1 strain), and 4 M strains showed I to AK.

Occurrence of Vibrio Parahaemolyticus, Vibrio Cholerae and Vibrio Vulnificus in the Clam Ruditapes Philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy.

Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagarTM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin b-colistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh); V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC), V. parahaemolyticus (toxRP) and V. vulnificus (vvhA). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05).

First Multi-Year Retrospective Study on Vibrio Parhaemolyticus and Vibrio Vulnificus Prevalence in Ruditapes Philippinarum Harvested in Sacca Di Goro, Italy.

The present work describes a retrospective study aiming to verify a possible correlation between the environmental conditions (temperature, salinity and dissolved oxygen), the abundance of Vibrio spp., and the prevalence of V. parahaemolyticus and V. vulnificus in the Manila clam R. philippinarum harvested in Sacca di Goro, Emilia-Romagna Region, Northern Italy. On the whole, 104 samples, collected in the period 2007-2015 and submitted to microbiological analyses (isolation and genotyping), have been reconsidered for Vibrio spp. load, V. parahaemolyticus prevalence (total, gene marker toxRP; potentially pathogenic, gene markers tdh and/or trh) and V. vulnificus prevalence (total, gene markers vvhA and hsp) together with environmental data obtained from the monitoring activity of the Emilia-Romagna Regional Agency for the Prevention, the Environment and the Energy. Environmental data have been processed to calculate the median of each, assessing the seasonal range of seawater temperature (warmer months: April-October, T°C >16.45°C; cooler months November-March, T°C <16.45°C), salinity (27 psu), and dissolved oxygen (< or >8.2 mg/L). Total V. vulnificus, total and potentially pathogenic V. parahaemolyticus were present respectively in the 11.5, 29.8 and 6.7% of the samples. The Vibrio spp. load (mean value of 4.69±0.65 log10 colony forming unit g-1) and the prevalence of potentially pathogenic V. parahaemolyticus, were not significantly correlated to the environmental conditions (P>0.05), whereas the prevalence of both total V. vulnificus and total V. parahaemolyticus was significantly higher in the warmer period (P<0.05), without correlation with salinity and dissolved oxygen values (P>0.05).

Fecal shedding of thermophilic Campylobacter in a dairy herd producing raw milk for direct human consumption.

Factors affecting the fecal shedding of thermophilic Campylobacter in Italian dairy farms were investigated in a 12-month longitudinal study performed on a dairy farm authorized to sell raw milk in Italy. Fifty animals were randomly selected from 140 adult and young animals, and fecal samples were collected six times at 2-month intervals. At each sampling time, three trough water samples and two trough feed samples also were collected for both adult and young animals. Samples were analyzed with real-time PCR assay and culture examination. Overall, 33 samples (9.7%) were positive for thermophilic Campylobacter by real-time PCR: 26 (9.2%) of 280 fecal samples, 6 (16.6%) of 36 water samples, and 1 (4.2%) of 24 feed samples. Campylobacter jejuni was isolated from 6 of 280 samples; no other Campylobacter species was isolated. A higher (but not significantly) number of positive fecal samples were found in younger animals (11.33 versus 6.92% of adult animals), and a significantly higher number of positive water samples were collected from the water troughs of young animals. A distinct temporal trend was observed during the study period for both cows and calves, with two prevalence peaks between November and December and between May and July. Several factors such as calving, housing practices, herd size, management practices forcing together a higher number of animals, and variations in feed or water sources (previously reported as a cause of temporal variation in different farming conditions) were excluded as the cause of the two seasonal peaks in this study. The factors affecting the seasonality of Campylobacter shedding in the dairy herds remain unclear and warrant further investigation. The results of the present study indicate that special attention should be paid to farm hygiene management on farms authorized to produce and sell raw milk, with increased surveillance by the authorities at certain times of the year.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach.

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.

Detection of Vibrio splendidus and related species in Chamelea gallina sampled in the Adriatic along the Abruzzi coastline.

Vibrio species are an important and widespread component of marine microbial communities. Some Vibrio strains are potentially pathogenic to marine vertebrates and invertebrates. The aim of this study was to identify vibrios, in particular Vibrio splendidus and related species, isolated from clams (Chamelea gallina) collected along the coasts of the Abruzzi region from May to October 2007. The isolates obtained were phenotyped and classified as belonging to the genus Vibrio. The strains underwent biochemical testing in accordance with Alsina's scheme for V. splendidus identification. Molecular analysis of the 16S-23S intergenic space region and recA gene was used to identify V. splendidus and related species. All the samples examined were found to contain halophylic Vibrio species, with V. alginolyticus, V. splendidus-related species and V. mediterranei most commonly found. A polymerase chain reaction of the 16S-23S intergenic space region and sequencing of the recA gene from isolates confirmed that phenotyping of Vibrio species is not sufficient to distinguish between different species. Differentiation of the highly related species among V. splendidus-related clusters remains an important issue. In this regard, our data suggests sequencing the recA genes was far more discriminatory than sequencing 16S rDNA for this purpose.