Three species of commonly eaten shellfish found in Malaysian coastal waters were examined for the presence of common bacterial enteropathogens. Vibrio parahaemolyticus, non-agglutinating vibrios, and various serotypes of enteropathogenic E. coli were isolated from a large proportion of them. Salmonella were isolated in two instances. High colony counts with evidence of faecal contamination indicated the strong possibility of pulltion being the cause for the presence of these enteropathogens. Methods of cooking and eating these shellfish enhance their likelihood of acting as vehicles of diarrhoeal disease.
Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.
Detection of diatom frustules in bone marrow (diatom test) is used for diagnosing ante-mortem drowning where the usual signs of drowning are not present in dead bodies recovered from water. However, controversies over the reliability of diatom test results are continuing. There have been indications on the possibilities of diatoms entering into systemic circulation from atmospheric air, food and drink. While diatoms have been demonstrated in the gut content of edible marine forms such as shrimps and clams, the present study, for the first time, provides empirical evidence on the prevalence as well as abundance of diatom frustules in the samples of cooked non-vegetarian foodstuffs that impend human consumption in Kelantan, Malaysia. It is found that 50 g each of cleaned and cooked prawns and of clams impending human consumption contain about 8360 and 29,054 diatom frustules, respectively. A person accustomed to prawn and clam food would be ingesting an estimated 2 million diatoms in a single year. Considering the suggestion that detection of five diatom frustules in 10 g of bone marrow would suffice for concluding drowning as mode of death, and the fact that there is yet no proof that diatom frustules do not enter into the human systemic circulation through the digestive tract, the estimated number of diatom frustules routinely ingested acquires significance since entry of a few of such ingested frustules into the systemic circulation can lead to false positive test results. The findings of this research raise two important issues: first, population based routine food related diatom ingestion requires to be estimated, and, second, studies have to be initiated to categorically prove or disprove the possibility of entry of diatom frustules into the systemic circulation via the digestive tract.
Allergy to different classes of mollusks, including squid, which are members of the class Cephalopods has been reported. Tropomyosin, a major muscle protein, is the only well-recognized allergen in squid. The aim of this study was to characterize IgE-binding proteins of local Loligo edulis (white squid) consumed in Malaysia. Protein profiles and IgE-binding proteins were detected by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) and immunoblotting using sera from 23 patients with positive skin prick test to raw squid extract. SDS-PAGE of the raw extract exhibited 21 protein bands (10-170 kDa) but those ranging from 19 to 29 kDa and 41 to 94 kDa were not found in the cooked extract. Immunoblotting of raw extract demonstrated 16 IgE-binding bands, ranging from 13 to 170 kDa. A heat-resistant 36 kDa protein, corresponding to squid tropomyosin, was identified as the major allergen of both extracts. In addition, a 50 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. Our findings indicate that the allergen extract used for diagnosis of squid allergy should contain both the 36 kDa and 50 kDa proteins.
The accumulation of paralytic shellfish toxins (PSTs) in contaminated shellfish is a serious health risk making early detection important to improve shellfish safety and biotoxin management. Capillary electrophoresis (CE) has been proven as a high resolution separation technique compatible with miniaturization, making it an attractive choice in the development of portable instrumentation for early, on-site detection of PSTs. In this work, capillary zone electrophoresis (CZE) with capacitively coupled contactless conductivity detector (C(4)D) and UV detection were examined with counter-flow transient isotachophoresis (tITP) to improve the sensitivity and deal with the high conductivity sample matrix. The high sodium concentration in the sample was used as the leading ion while l-alanine was used as the terminating electrolyte (TE) and background electrolyte (BGE) in which the toxins were separated. Careful optimization of the injected sample volume and duration of the counter-flow resulted in limit of detections (LODs) ranging from 74.2 to 1020 ng/mL for tITP-CZE-C(4)D and 141 to 461 ng/mL for tITP-CZE-UV, an 8-97 fold reduction compared to conventional CZE. The LODs were adequate for the analysis of PSTs in shellfish samples close to the regulatory limit. Intra-day and inter-day repeatability values (percentage relative standard deviation, n=3) of tITP-CZE-C(4)D and tITP-CZE-UV methods for both migration time and peak height were in the range of 0.82-11% and 0.76-10%, respectively. The developed method was applied to the analysis of a contaminated mussel sample and validated against an Association of Official Analytical Chemists (AOAC)-approved method for PSTs analysis by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) after pre-column oxidation of the sample. The method presented has potential for incorporation in to field-deployable devices for the early detection of PSTs on-site.
This study was undertaken to assess the levels of trace metals (As, Cd, Cu, Pb, and Zn) in two common species of cockles (Anadara granosa and Anadara inaequivalvis) from two coastal areas in Thailand (Pattani Bay) and Malaysia (the Setiu Wetlands). A total of 350 cockles were collected in February and September 2014. Trace metals were determined by Inductively Coupled Plasma Mass Spectrometry. We observed that cockles in both areas had a higher accumulation of metals in September. Notably, the biota-sediment accumulation (BSAF) of Cd was highest in both areas. A strong positive correlation of Cd with the length of the cockles at Pattani Bay (r(2) = 0.597) and the Setiu Wetlands (r(2) = 0.675) was noted. It was suggested that As could be a limiting element (BSAF < 1) of cockles obtained from Pattani Bay. In comparison with the permissible limits set by the Thailand Ministry of Public Health and the Malaysia Food Regulations, mean values of As, Cd, Cu, Pb, and Zn were within acceptable limits, but the maximum values of Cd and Pb exceeded the limits for both areas. Regular monitoring of trace metals in cockles from both areas is suggested for more definitive contamination determination.
Numerous prevalence studies and outbreaks of Vibrio parahaemolyticus infection have been extensively reported in shellfish and crustaceans. Information on the quantitative detection of V. parahaemolyticus in finfish species is limited. In this study, short mackerels (Rastrelliger brachysoma) obtained from different retail marketplaces were monitored with the presence of total and pathogenic strains of V. parahaemolyticus. Out of 130 short mackerel samples, 116 (89.2%) were detected with the presence of total V. parahaemolyticus and microbial loads of total V. parahaemolyticus ranging from <3 to >10(5) MPN/g. Prevalence of total V. parahaemolyticus was found highest in wet markets (95.2%) followed by minimarkets (89.1%) and hypermarkets (83.3%). Pathogenic V. parahaemolyticus strains (tdh+ and/or trh+) were detected in 16.2% (21 of 130) of short mackerel samples. The density of tdh+ V. parahaemolyticus strains were examined ranging from 3.6 to >10(5) MPN/g and microbial loads of V. parahaemolyticus strains positive for both tdh and trh were found ranging from 300 to 740 MPN/g. On the other hand, antibiotic susceptibility profiles of V. parahaemolyticus strains isolated from short mackerels were determined through disc diffusion method in this study. Assessment of antimicrobial susceptibility profile of V. parahaemolyticus revealed majority of the isolates were highly susceptible to ampicillin sulbactam, meropenem, ceftazidime, and imipenem, but resistant to penicillin G and ampicillin. Two isolates (2.99%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.41 which shown resistance to 7 antibiotics. Results of the present study demonstrated that the occurrence of pathogenic V. parahaemolyticus strains in short mackerels and multidrug resistance of V. parahaemolyticus isolates could be a potential public health concerns to the consumer. Furthermore, prevalence data attained from the current study can be further used to develop a microbial risk assessment model to estimate health risks associated with the consumption of short mackerels contaminated with pathogenic V. parahaemolyticus.
Pyrodinium bahamense (var. compressum) has been the only dinoflagellate species that has caused major public health and economic problems in the Southeast Asian region for more than 2 decades now. It produces saxitoxin, a suite of toxins that cause Paralytic Shellfish Poisoning (PSP). A serious toxicological problem affecting many countries of the world, mild cases of this poisoning can occur within 30 minutes while in extreme cases, death through respiratory paralysis may occur within 2-24 hrs of ingestion of intoxicated shellfish. Blooms of the organism have been reported in Malaysia, Brunei Darussalam, the Philippines and Indonesia. The ASEAN-Canada Red Tide Network has recorded 31 blooms of the organism in 26 areas since 1976 when it first occurred in Sabah, Malaysia. As of 1999, the most hard hit country has been the Philippines which has the greatest number of areas affected (18) and highest number of Paralytic Shellfish Poisoning (PSP) cases (about 1995). Malaysia has reported a total of 609 PSP cases and 44 deaths while Brunei has recorded 14 PSP cases and no fatalities. Indonesia, on the other hand has a record of 427 PSP cases and 17 deaths. Studies on ecological/environmental impacts of these blooms have not been done in the region. Estimates of economic impacts have shown that the loss could be up to USD 300,000 day-1. Most of the data and information useful for understanding Pyrodinium bloom dynamics have come from harmful/toxic algal monitoring and research that have developed to different degrees in the various countries in the region affected by the organism's bloom. Regional collaborative research and monitoring efforts can help harmonize local data sets and ensure their quality and availability for comparative analysis and modeling. Temporal patterns of the blooms at local and regional scales and possible signals and trends in the occurrence/recurrence and spread of Pyrodinium blooms could be investigated. Existing descriptive and simple predictive models of Pyrodinium blooms can be improved and refined to help in the management of the wild harvest and aquaculture of shellfish in a region where the people are dependent on these resources for their daily food sustainance and livelihood.
Hepatitis A is a liver infection caused by the hepatitis A virus (HAV). Outbreaks of hepatitis A have been linked to the consumption of both raw and cooked shellfish. These outbreaks could induce a public confidence problem over shellfish safety and may result in important economic losses for the seafood industry. The work presented in this study investigated the presence of HAV in shellfish from Peninsular Malaysia. A total of 365 of cultured and wild shellfish from 36 sampling locations located throughout Peninsular Malaysia were examined using a commercial nucleic acid extraction and reverse transcription -polymerase chain reaction (RT-PCR) kit. HAV was not detected in almost all of the shellfish samples xamined. Only one cockle sample from Changkat, Seberang Perai was positive for HAV. The results suggest the absence of HAV or very low amount of HAV viral particles in most of the shellfish examined.
Polymerase chain reaction (PCR) technique was used to assay for the detection of specific genes in the genomes of the Aeromonas spp. isolated from environmental and shellfish sources, particularly aero and hlyA genes, responsible for aerolysin and hemolysin toxins production in this genus. The results showed that: (i) the 1500 bp amplicon of the hlyA gene was detected in 20/38 of the Aeromonas hydrophila, 13/38 of the A. caviae and 6/9 of the A. veronii biovar sobria isolates; (ii) the 690 bp amplicon of the aero gene was detected in 20/38 of A. hydrophila, 17/38 of A. caviae and 6/9 of A. veronii biovar sobria isolates; (iii) the nucleotide blast results of aerolysin gene sequences of the representative strains of A. hydrophila, A. caviae and A. veronii biovar sobria revealed a high homology of 94%, 95% and 95% with published sequences, respectively and ; (iv) the protein blast showed 97%, 94% and 96% homology when compared to the published sequences, respectively. The finding of A. hydrophila virulence genes in other members of the genus Aeromonas, may give a new perspective to the significance of these results. The method described here may be a useful detection tool to assist in further investigation of aero and hlyA genes in the genus Aeromonas, especially for food microbiologist.
Biochemical analysis was carried out for pH profiles, freshness in terms of K-values, amino acids profiles, total volatile bases (TVB), total volatile acids (TVA) and biogenic amines for fresh and preserved Macrobrachium rosenbergii. Results showed that pH profiles of Macrobrachium rosenbergii explain the inability of this parameter to be used to evaluate the quality of Macrobrachium rosenbergii. Thus changes in pH profiles of Macrobrachium rosenbergii should be combined with indicators such as total volatile acids and total volatile bases. Total volatile acids of the shrimps increased steadily in small amounts throughout the storage period. A rapid increase in TVB at 100C was detected due to the increase in total aerobic bacteria at elevated temperatures. The microbial activities caused the decrease in the amino acids arginine, lysine and histidine which correlated well with the increase in the corresponding biogenic amines such as putrescine, cadaverine and histamine respectively. Preservatives used in this study controlled the production of these biogenic amines without significantly altering the pH of preserved shrimp.
Microbiological quality analysis of freshwater prawns from three sampling sites in Peninsular Malaysia viz: Site 1- Kg. Jumbang, Negri Sembilan; Site 2- Kg. Cangkat Tin, Perak and Site 3- Kg. Cenderiang, Perak for total mesophilic and psychrophilic aerobic counts, proteolytic bacterial counts, histamine producing bacteria, cadaverine producing bacteria and putrescine producing bacteria in the prawns and pond water for the three sites showed that the microbiological quality of freshwater prawns is related to the microflora of pond water in which they were grown. The initial bacterial counts indicated the values were in the range of log 4+ CFU/g for all samples. Total mesophilic and psychrophilic counts of the head regions were higher than that of the body regions for all prawn samples and types of growth media tested. All samples showed an increase in counts with time and temperature of storage up to log 7+ CFU/g for mesophilic counts after 12 hours at ambient, 6 days at 10 ± 2°C and 12 days at iced storage. The samples from Site 2 had relatively higher counts compared to the other two sites which correlated well with the levels determined in the pond water. Similar trends were observed for psychrophilic counts but at lower values for the different types of media studied.
Effects of preservatives on quality changes and shelf life of shrimp during iced storage indicated that boric acid, lactic acid and sodium metabisulphite managed to inhibit psychrophilic bacteria and biogenic amines formation in prawns while maintaining the mesophilic counts at lower levels during iced storage.
Vibrio cholerae still represents a significant threat to human health worldwide despite the advances in hygiene, consumer knowledge, food treatment and food processing. In Malaysia, statistics in year 2009 have shown that among the food and water borne diseases, food poisoning has the highest incidence rate of 36.17 per 100,000 populations and with a mortality rate of 0.01 per 100,000 populations. In this study, 22 seafood samples comprising of fish, squid, crustacean and mollusks purchased from wet market and supermarket were analyzed. The Most Probable Number (MPN) and real time PCR was used to enumerate the Vibrio cholerae in seafood sample. The results showed that MPN-real time PCR of the samples from wet market had a maximum of >1100 MPN/g compare to 93 MPN/g enumerated from the MPN plate. The MPN-real time PCR in the samples from supermarket indicated 290 MPN/g as compared to 240 MPN/g enumerated from the MPN plate. The standard curves showed that there was a good linear correlation between the Ct values. The minimum level of detection of Vibrio cholerae standard DNA at targeted gene was 3 x 10-5 ng/μl.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
The status ofhve l1eavy metals: cadmium, lead copper, zinc and mercury were determined in seafood and its products imported_ti·om Thailand via Bukit Kayu Hiram, Kedah, Flame Atomic Absorption Spectrophotometer was used to determine the level of these heary metals except for mercury, where the cold vapor technique was used, Randomized sampling was done according to a predetermined sampling plan based on the previous years consignments. Data collected were compared with the maximum permitted level of -metal contaminants in fish and fish products- ofthe Fourteenth Schedule (Regulation 38) of Food Act I983 and Food Regulations I 985 to ascertain compliance. lt was noted form this study, that the level of metals detected in seafood and its products had a very wide range, The levels detected for cadmium was at a range of 0. 00] - 3.9/2, 0.07 — 0.29, 0,04 - 4,4 mg/kg in fish, shellfish and cuttlefish respectivelv. In general, cadmium level in some samples was notably higher particularly in shellfish. All samples had lead level less than the permitted value except for fish where the highest value detected was 3.28 mg/kg. The level of copper and zinc was higher than the permitted value in octopus, prawn and crab, Mercury level in all samples analvzed was found to be below the detection level. As for fish samples, zinc level was found to be higher whereas copper was within the limit. However, it was also noted that the level of all the heavy metals in jiozen jish was within the permitted limit.
High consumer demand for shellfish has led to the need for large-scale, reliable shellfish supply through aquaculture or shellfish farming. However, bacterial infections which can spread rapidly among shellfish poses a major threat to this industry. Shellfish farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. The extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in shellfish. This study aimed to investigate the incidence of pathogenic Vibrio parahaemolyticus and determine the antibiotic resistance profile as well as to perform plasmid curing in order to determine the antibiotic resistance mediation. Based on colony morphology, all 450 samples tested were positive for Vibrio sp; however, tox-R assay showed that only 44.4% (200/450) of these were V. parahaemolyticus. Out of these 200 samples, 6.5% (13/200) were trh-positive while none were tdh-positive. Antibiotic resistance was determined for all V. parahaemolyticus identified against 14 commonly used antibiotics and the multiple antibiotic resistance index (MAR) was calculated. The isolates demonstrated high resistance to several antibiotics tested- including second and third-line antibiotics- with 88% resistant to ampicillin, 81% to amikacin,70.5% to kanamycin, 73% to cefotaxime, and 51.5% to ceftazidime. The MAR index ranged from 0.00 to 0.79 with the majority of samples having an index of 0.36 (resistant to five antibiotics). Among the 13 trh-positive strains, almost 70% (9/13) demonstrated resistance to 4 or more antibiotics. Plasmid profiling for all V. parahaemolyticus isolates revealed that 86.5% (173/200) contained plasmids - ranging from 1 to 7 plasmids with DNA band sizes ranging from 1.2 kb to greater than 10 kb. 6/13 of the pathogenic V. pathogenic strains contained plasmid. After plasmid curing, the plasmid containing pathogenic strains isolated in our study have chromosomally mediated ampicillin resistance while the remaining resistance phenotypes are plasmid mediated. Overall, our results indicate that while the incidence of pathogenic V. parahaemolyticus in shellfish in Selangor still appears to be at relatively reassuring levels, antibiotic resistance is a real concern and warrants ongoing surveillance.
Some diatoms of the genera Pseudo-nitzschia and Nitzschia produce the neurotoxin domoic acid (DA), a compound that caused amnesic shellfish poisoning (ASP) in humans just over 30 years ago (December 1987) in eastern Canada. This review covers new information since two previous reviews in 2012. Nitzschia bizertensis was subsequently discovered to be toxigenic in Tunisian waters. The known distribution of N. navis-varingica has expanded from Vietnam to Malaysia, Indonesia, the Philippines and Australia. Furthermore, 15 new species (and one new variety) of Pseudo-nitzschia have been discovered, bringing the total to 52. Seven new species were found to produce DA, bringing the total of toxigenic species to 26. We list all Pseudo-nitzschia species, their ability to produce DA, and show their global distribution. A consequence of the extended distribution and increased number of toxigenic species worldwide is that DA is now found more pervasively in the food web, contaminating new marine organisms (especially marine mammals), affecting their physiology and disrupting ecosystems. Recent findings highlight how zooplankton grazers can induce DA production in Pseudo-nitzschia and how bacteria interact with Pseudo-nitzschia. Since 2012, new discoveries have been reported on physiological controls of Pseudo-nitzschia growth and DA production, its sexual reproduction, and infection by an oomycete parasitoid. Many advances are the result of applying molecular approaches to discovering new species, and to understanding the population genetic structure of Pseudo-nitzschia and mechanisms used to cope with iron limitation. The availability of genomes from three Pseudo-nitzschia species, coupled with a comparative transcriptomic approach, has allowed advances in our understanding of the sexual reproduction of Pseudo-nitzschia, its signaling pathways, its interactions with bacteria, and genes involved in iron and vitamin B12 and B7 metabolism. Although there have been no new confirmed cases of ASP since 1987 because of monitoring efforts, new blooms have occurred. A massive toxic Pseudo-nitzschia bloom affected the entire west coast of North America during 2015-2016, and was linked to a 'warm blob' of ocean water. Other smaller toxic blooms occurred in the Gulf of Mexico and east coast of North America. Knowledge gaps remain, including how and why DA and its isomers are produced, the world distribution of potentially toxigenic Nitzschia species, the prevalence of DA isomers, and molecular markers to discriminate between toxigenic and non-toxigenic species and to discover sexually reproducing populations in the field.
Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.