Prevalence of Salmonella spp. in Oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Prevalence, seasonal occurrence and antimicrobial resistance of Salmonella in poultry retail products in Greece

Aims: To detect the prevalence, the seasonal occurrence and distribution of Salmonella serotypes in poultry products and to determine the resistance profile of Salmonella isolates. Method and Results: A total of 96 skin-on chicken carcasses and 30 liver samples were analysed between May 2007 and May 2009 from twenty-two different commercial farm brands found in retail market countrywide. Salmonella was isolated from 38 (39·5%) of 96 chicken carcasses and from 10 (33·3%) of 30 liver samples. Higher isolation rate (60·4%) was observed in carcasses detected during summer (May to October), and lower isolation rate (18·7%) was observed in carcasses detected during winter (November to April); in liver samples, the positive rates were 53·4 and 13·2%, respectively. Twelve serotypes were detected with the serotypes Hadar, Enteritidis and Blockley being the most prevalent at 29·2, 22·9 and 12·5%, respectively. Nine of 11 Salm. Enteritidis isolates occurred during summer. Of 48 isolates, 38 (79%) were resistant to one or more of the antimicrobial agents used. The highest resistance rates were found to the following antimicrobials: streptomycin (64·5%), tetracycline (56·2%), nalidixic acid (39·5%), ampicillin and rifampicin (33·3%). Conclusions: The relatively high Salmonella spp. contamination rates of raw chicken meat and liver have been detected. Salm. Enteritidis isolates peaked in summer, increasing the risk to human health. Antibiotic resistance of Salmonella still remains a threat as resistance plasmids may be extensively shared between animal and humans. Significance and Impact of the Study: The study enabled us to improve the data on the seasonal occurrence of Salmonella and to determine the antimicrobial pattern profile and trends in Salmonella strains isolated from poultry retail products in Greece.     

Biochemical biomarkers in gills of mangrove oyster Crassostrea rhizophorae from three Brazilian estuaries

Responses of biochemical biomarkers were evaluated in gills of immature adult mangrove oyster Crassostrea rhizophorae collected in three estuarine regions along the Brazilian coast. In each region, ten oysters were collected in one reference site (R) located far from pollution sources, and in two polluted sites (P-I and P-II sites) located in another water body with similar characteristics. P-I site is located close to recognized pollution sources while P-II site is in the same water body, but far from pollution sources. At the Paranaguá Bay (Southern Brazil), polluted sites receive domestic, harbor and phosphate fertilizer plant discharges. High lipid peroxides (LPO) content was observed in winter oysters from the P-I site. In summer, higher catalase activity was observed in these oysters. In the Piraquê region (Southeastern Brazil), polluted sites receive domestic and agricultural effluents. Lower total oxyradical scavenging capacity (TOSC) towards peroxyradicals was observed in summer oysters from both P-I and P-II sites. In the Itamaracá region (Northeastern Brazil), polluted sites receive paper mill and caustic soda and chlorine factories effluents. Increased glutathione S-transferase (GST) activity was observed in oysters from the P-I site in both summer and winter. At Paranaguá Bay (higher latitude), no seasonal differences were observed in oysters from the R site, suggesting that temperature was not an important factor influencing biomarkers levels. Lower GST activity was observed in oysters from the R site of the Itamaracá Bay (lower latitude) in winter and summer. Taken together, data obtained point to responses of biomarkers in oysters from polluted sites of the three estuarine regions analyzed, indicating the need for future monitoring of the biological effects of contaminants in these environments. They also point to the relevance to consider both season and latitude as factors influencing biomarker responses in environmental contamination monitoring programs.     

Prevalence of Perkinsus marinus (dermo), Haplosporidium nelsoni (MSX), and QPX in bivalves of Delaware's inland bays and quantitative, high-throughput diagnosis of dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.     

Chronic infections of Haplosporidium nelsoni (MSX) in the oyster Crassostrea virginica

Oysters inhabiting areas enzootic for the parasite Haplosporidium nelsoni (MSX) are exposed to infective particles each summer, and it is often difficult to distinguish newly acquired lesions from older infections. To study long-term parasitism without the complication of new infections, MSX-infected oysters were moved from Delaware Bay to a disease-free area on the New Jersey coast. Because infections seen after the transfer were acquired in Delaware Bay during a known infective period, it was possible to determine how long oysters remained infected and how they were affected by chronic parasitism. Chronic infections displayed the same seasonal cycle (high levels in winter and late spring, low levels in summer and early spring) that occurs in enzootic areas with annual reinfection. Within individual oysters, chronic MSX became localized, relapsed into general infections, and then became localized again in a sequence that was probably controlled by temperature. Some experimental oysters survived with MSX for at least 4 years. Hemocytosis persisted in these oysters, and their poor condition suggested that chronically infected individuals would have lowered resistance to additional stress.     

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.     

Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.     

Epidemiology and potential land-sea transfer of enteric bacteria from terrestrial to marine species in the Monterey Bay Region of California

Marine mammals are at risk for infection by fecal-associated zoonotic pathogens when they swim and feed in polluted nearshore marine waters. Because of their tendency to consume 25-30% of their body weight per day in coastal filter-feeding invertebrates, southern sea otters (Enhydra lutris nereis) can act as sentinels of marine ecosystem health in California. Feces from domestic and wildlife species were tested to determine prevalence, potential virulence, and diversity of selected opportunistic enteric bacterial pathogens in the Monterey Bay region. We hypothesized that if sea otters are sentinels of coastal health, and fecal pollution flows from land to sea, then sea otters and terrestrial animals might share the same enteric bacterial species and strains. Twenty-eight percent of fecal samples tested during 2007-2010 were positive for one or more potential pathogens. Campylobacter spp. were isolated most frequently, with an overall prevalence of 11%, followed by Vibrio cholerae (9%), Salmonella spp. (6%), V. parahaemolyticus (5%), and V. alginolyticus (3%). Sea otters were found positive for all target bacteria, exhibiting similar prevalences for Campylobacter and Salmonella spp. but greater prevalences for Vibrio spp. when compared to terrestrial animals. Fifteen Salmonella serotypes were detected, 11 of which were isolated from opossums. This is the first report of sea otter infection by S. enterica Heidelberg, a serotype also associated with human clinical disease. Similar strains of S. enterica Typhimurium were identified in otters, opossums, and gulls, suggesting the possibility of land-sea transfer of enteric bacterial pathogens from terrestrial sources to sea otters.     

First isolation of Nocardia crassostreae from Pacific oyster Crassostrea gigas in Europe

In summer 2006 an extensive mortality of Pacific oysters Crassostrea gigas occurred in Lake Grevelingen, the Netherlands. A sample of Pacific oysters was investigated for the presence of shellfish pathogens as potential causes of the mortality. Yellow-green lesions were observed in several oysters upon clinical inspection. Histopathology showed that 6 out of 36 oysters had a suspected bacterial infection, including 4 Nocardia-like infections. Two bacterial species, Vibrio aestuarianus and Nocardia crassostreae, were isolated from haemolymph samples and identified using PCR and sequencing of the 16S rRNA gene. This is the first isolation of N. crassostreae from shellfish in European waters. The near full-length 16S rRNA sequence of this Dutch Nocardia sp. isolate was identical to other known N. crassostreae isolates from the west coast of North America. The primary cause of oyster mortality was thought to be the physiological stress from environmental conditions, including prolonged high water temperatures and low oxygen levels. The multiple bacterial species isolated from the diseased Pacific oysters may have been a secondary cause.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.