A total of sixty V. parahaemolyticus strains isolated from local cockles (Anadara granosa) were investigated by their antibiotic resistance patterns and plasmid profiles. The isolates showed multiple resistances towards most of the antibiotics tested. All strains of V. parahaemolyticus isolated harbored 1-3 plasmids, with sizes ranging from 2.7 to 54 kb. All V. parahaemolyticus strains showed high multiple antibiotics in frequencies of 0.58 – 0.94 indicating that the strains were derived from high-risk sources. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. These findings are essential because of the suggested involvement of seafood especially shellfish and environment in transmission of this pathogen to human. Thus, indicating that seafood may be a source of food- acquired antibiotic resistant bacteria to consumer.
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.
This study evaluates the sanitary and physico-chemical quality of Sg. Jarum Mas shellfish waters in order to establish its suitability as a model farm. Seawater and shellfish from nine stations (4 shellfish harvesting waters, 4 surrounding waters and 1 control site) were collected and analyzed monthly from September 2004 - September 2005. The results show that shellfish harvesting waters in Sg. Jarum Mas can be classified as ‘approved’ and ‘conditionally approved’. Hepatitis A virus was not detected in any of the shellfish examined. Dinophysis caudata and Pseuodonitzshia spp. were the most common harmful alga species observed. Harmful species that are known to produce toxins and cause shellfish poisoning such as Alexandrium spp., Gymnodinum spp., Pyrodinium sp. and Prorocentrum spp. were not detected. The physico-chemical characteristics of shellfish waters in Sg. Jarum Mas imply that they are suitable for aquaculture activity of moderately tolerant species such as shellfish.
The shrimp paste called belacan is a traditional umami taste condiment extensively used in Malaysia that is rich in glutamate and 5'-nucleotides. The aim of this study was to determine the concentration of glutamate and 5'-nucleotides of various types of foods prepared with belacan and to measure their sensory attributes. The concentration of free glutamic acid found in different brands of belacan was 180-530mg/100g and in local dishes 601-4207mg/100g. The total amount of 5'-nucleotides in belacan samples ranged from 0.85 to 42.25μg/g. A Quantitative Descriptive Analysis (QDA) using a list of 17 sensory attributes showed a good correlation between belacan concentration in the final food and a range of positive sensory attributes, except for bitter, sweet, sour taste and astringency. Belacan also contains bitter, sweet and sour compounds that change the positive attributes of belacan at higher concentrations. The highest aroma attributes were linked to nasi goreng belacan (belacan fried rice) while the highest flavour attributes were found in sambal belacan. There was a 32 folds significant increase of umami attributes with the addition of belacan to final foods. The optimum amount of belacan was 0.45% for asam pedas (tamarind flavoured dish with belacan), 18% for sambal belacan (chilli belacan), 1.5-2.5% for kangkong goreng belacan (stir fried water convolous with belacan), and 2% for nasi goreng belacan.
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.
Aimed of this study was to determine the presence of Vibrio cholerae in cockles (Anadara granosa)
from different coasts in Malaysia and to measure the biosafety of V. cholerae in raw cockles at wet market in Malaysia using the polymerase chain reaction (PCR) in combination with the most probable number (MPN) method. A total of 100 samples from 4 different wet markets in the West and East were examined for the presence of V. cholerae. The prevalence of V. cholerae between the two coasts was not significant different. In fact, the 74% of samples from West coast area was found positive while the 69% for samples collected in the East coast. West coast samples showed a prevalence of 60% for the wet market A=, 64% for B=, 88% for C= and 84% for the market D); East coast samples showed the same percentage with 72% for the wet markets E, F and H, followed by wet market G with 60%.With the MPN-PCR method, using 80 samples of raw cockles obtained from 4 wet markets, the occurrence of V. cholerae detected was of 95%. The frequency of V. cholerae in raw cockles obtained from wet market I and L was higher (100%) compared to other wet market (Wet market B=, 90%; Wet market C=, 95%).The density of V. cholerae detected in all samples ranged from 24000 MPN/g, but most of the samples (24 samples) were in category >24000 MPN/g concentration. V. cholerae was present in raw cockles in higher number. Hence, these results demonstrate the presence of pathogenic V.cholerae in cockles harvested and reveal the potential risk of illness associated with their consumption. This study will be the first biosafety assessment of V. choleare in raw cockles in Malaysia and it will provide significant insights about Malaysian scenario.
This study was conducted to improve the formulation of chili shrimp paste (CSP) based on sensory evaluation in terms of acidity, source of acid and coarseness of chili paste and to improve the production process of CSP. The effectiveness of dimethyl dicarbonate (DMDC) as a microbial reduction agent was also evaluated. To produce CSP with different coarseness, a milling machine was used. Two types of preference test were conducted, i.e. ranking and hedonic. The preferred pH level was 4.0, the best acid source was kalamansi juice, and the most preferred coarseness for chili paste was when milling plates with a gap of 120 µm was used. DMDC has no effect on microbial reduction due to the presence of fat globules in CSP which hindered the inactivation action. Milling can substitute pounding as it is much faster and can produce a uniform CSP with higher volume.
Vibrio parahaemolyticus is prevalent in tropical marine environment in all seasons and can cause seafood-borne gastroenteritis. A total of 251 suspected isolates were tested including 60 from frozen shrimp, 50 from cultured live shrimp, 67 from sediments of culture ponds and 74 from water were subjected to polymerase chain reaction (PCR) targeting the toxR gene for confirmation as V. parahaemolyticus. Of the 128 toxR positive isolates, 15% of the isolates from culture environment (from live shrimp, sediments and water) and 7% of frozen shrimp samples were positive for the tdh and trh genes. Since urease production could be a marker of trh but not tdh in V. parahaemolyticus, a total of 189 of the 251 suspected V. parahaemolyticus isolates were tested for urease production and 41% of the isolates were found to be positive for urease production. However not all urease positive strains of V. parahaemolyticus were positive for either tdh or trh genes. Detection of virulent strains in shrimp culture environment in Malaysia suggests a probable risk for health of people consuming raw shrimp.
The concentrations of cadmium, copper, zinc and lead, in the total soft tissues of green-lipped mussel Perna viridis of a wide range of sizes (2-11 cm), were determined from a population at Pasir Panjang. The metal contents (μg per individual) and concentrations (μg per g) of cadmium, lead, copper and zinc were studied in P. viridis to find the relationships with body sizes. Smaller and younger mussels showed higher concentrations (μg per g) of Cd, Pb and Zn than the larger and older ones. The results of the present study showed that the plotting of the metal content, against dry body flesh weight on a double logarithmic basis, gave good positive straight lines; this observation is in agreement with Boyden’s formula (1977). This indicated that P. viridis showed a different physiological strategy for each metal being studied, which is related to age.
Hepatitis A virus infection occurs globally and is causing a public health concern, primarily in developing countries due to its persistent circulation in the environment. The improved sanitary condition and increase in awareness of personal hygiene have led to the marked reduction of HAV prevalence in industrialized countries during childhood and to a shift of the infection towards adulthood. HAV is an environmentally stable, positive single stranded RNA virus that is primarily transmitted by the fecal-oral route, person to person contact or ingestion of contaminated food and drink. One of the main causes leading to HAV infection is epidemiologically linked to the consumption of raw or undercooked shellfish particularly oysters and clams. Due to their filter-feeding style, these shellfishes readily concentrate viruses from the surrounding water containing municipal sewage, and as a consequence pose a health threat to consumers. Therefore, development of detection techniques possessing the requisite sensitivity and specificity for the practical routine monitoring purposes is of great importance necessary for the protection of shellfish-consuming public. Nucleic acid based method such as reverse transcription PCR has emerged as the popular method of choice in view of its rapidity, accuracy and
sensitivity in contrary of the time-consuming conventional cell culture and hybridization techniques. However, detection of hepatitis A virus is firstly hampered by the non-cytophatic effect of wild type HAV strain, secondly, the low concentration of viral genome present in the environmental sample which requires effective isolation and concentration of virions and lastly the labor-extensive purification and thorough removal of the abundance of the PCR inhibitors which will unfavorably reduce the efficiency of PCR detection.
Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to atleast three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticusfrom cockles in Padang, Indonesia.
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.
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.
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.
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.
DNA damaging effect of the salted and fermented food products (salted fishes, dried shrimps and shrimp pastes) collected from three different locations in Malacca namely Pantai Puteri, Batang Tiga and Kelemak on the DNA of the Chang liver cells were evaluated via Alkaline Comet Assay. Treatment at 62.5 mg/ml following 24 hours of incubation was used based on the preliminary cytotoxicity data. Percentage of damage to the DNA was calculated using software for scoring based on the tail moment and tail intensity (severity of the DNA damage). Hydrogen peroxide was used as positive control at 0.1 mM following 30 minutes of incubation in 4 C. The results showed that the methanol extracts of shrimp pastes and salted fish from Pantai Puteri, exhibited a higher DNA damage (shrimp pastes - TM - 8.33 ± 2.19; TI - 31.67 ± 5.84, salted fishes - TM - 2.25 ± 0.86; TI - 9.25 ± 1.55) and were expressed as (shrimp pastes) 56.66 ± 8.74% of DNA damage and methanol salted fish extracts from the same location showed 13.00 ± 2.84% of the DNA damage on Chang liver cells compared to the other extracts. Values for methanol extract of shrimp pastes from Pantai Puteri were comparable to the positive control - Hydrogen peroxide (TM- 9.50 ± 1.50; TI - 30.50 ± 2.50). On the other hand, aqueous salted fishes extract from Pantai Puteri (TM - 1.33 ± 0.42; TI - 8.67 ± 2.42) and shrimp pastes extracts from Kelemak (methanol extract - TM -1.75 ± 0.15; TI -7.50 ± 0.50, aqueous extract - TM - 1.00 ± 0.00; TI - 5.00 ± 0.00) showed slightly high value for tail moment and tail intensity as compared to negative control (TM - 0.29 ± 0.05; TI - 2.50 ± 0.29). Values for methanol extracts of shrimp pastes from Pantai Puteri were comparable to the positive control (TM- 9.50 ± 1.50; TI - 30.50 ± 2.50). In conclusion, our results demonstrate genotoxic damage induced by few salted and fermented food extracts in Chang liver cell.
This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.
Seafood samples obtained in seafood markets and supermarkets at 11 sites selected from four states in Malaysia were examined for the presence of nine potentially pathogenic species from the genus Vibrio between July 1998 and June 1999. We examined 768 sample sets that included shrimp, squid, crab, cockles, and mussels. We extensively examined shrimp samples from Selangor State to determine seasonal variation of Vibrio populations. Eight potentially pathogenic Vibrio species were detected, with overall incidence in the samples at 4.6% for V. cholerae, 4.7% for V. parahaemolyticus, 6.0% for V. vulnificus, 11% for V. alginolyticus, 9.9% for V. metschnikovii, 1.3% for V. mimicus, 13% for V. damsela, 7.6% for V. fluvialis, and 52% for a combined population of all of the above. As many as eight Vibrio species were detected in shrimp and only four in squid and peel mussels. The overall percent incidence of any of the eight vibrios was highest (82%) in cockles (Anadara granosa) among the seafoods examined and was highest (100%) in Kuching, Sarawak State, and lowest (25%) in Penang, Pulau Penang State, among the sampling sites. Of 97 strains of V. cholerae isolated, one strain belonged to the O1 serotype and 14 to the O139 serotype. The results indicate that the various seafood markets in Malaysia are contaminated with potentially pathogenic Vibrio species regardless of the season and suggest that there is a need for adequate consumer protection measures.
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.
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.