Biohazards Assessment in Large-Scale Zonal Centrifugation

A study was conducted to determine the biohazards associated with use of the large-scale zonal centrifuge for purification of moderate risk oncogenic viruses. To safely and conveniently assess the hazard, coliphage T₃ was substituted for the virus in a typical processing procedure performed in a National Cancer Institute contract laboratory. Risk of personnel exposure was found to be minimal during optimal operation but definite potential for virus release from a number of centrifuge components during mechanical malfunction was shown by assay of surface, liquid, and air samples collected during the processing. High concentration of phage was detected in the turbine air exhaust and the seal coolant system when faulty seals were employed. The simulant virus was also found on both centrifuge chamber interior and rotor surfaces.     

Impact of Transport Crate Reuse and of Catching and Processing on Campylobacter and Salmonella Contamination of Broiler Chickens

The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).     

Predation by gulls on crabs in rocky intertidal and shallow subtidal zones of the Gulf of Maine

Most organisms in intertidal areas are marine in origin; many have distributions that extend into the subtidal zone. Terrestrial predators such as mammals and birds may exploit these animals during low tide and can have considerable effects on intertidal food webs. Several studies have shown that avian predators are capable of reducing densities of sessile and slow-moving intertidal invertebrates but very few studies have considered avian predation on mobile invertebrate predators such as crabs. In this study, we investigated predation by Great Black-backed Gulls (Larus marinus Linnaeus) on three species of crabs (Cancer borealis Stimpson, Cancer irroratus Say, and Carcinus maenas Linnaeus). The study was at Appledore Island, ME (a gull breeding island) and 8 other sites throughout the Gulf of Maine, including breeding islands and mainland sites. On Appledore Island, intertidal and subtidal zones provided over one-third of prey remains found at gull nests, and crabs were a substantial proportion (∼ 30% to 40%) of the total remains. Similarly, collections of prey remains from intertidal areas indicated that crabs were by far the most common marine prey. C. borealis was eaten far more often and C. irroratus and C. maenas less often than expected at each site. Comparing numbers of carapaces to densities of crabs in low intertidal and shallow subtidal zones at each site, we estimated that gulls remove between 15% and 64% of C. borealis during diurnal low tides. The proportion of C. borealis eaten by gulls was independent of proximity to a gull colony. Approximately 97% of the outer coast of Maine is within 20 km of a breeding island. Thus, a lot of gull predation on crabs may occur throughout the Gulf of Maine during summer months. Crabs are important predators of other invertebrates; if predation by gulls reduces the number of crabs in intertidal and shallow subtidal areas, gulls may have important indirect effects on intertidal food webs.     

Recovery of Bacteriophage from Contaminated Chilled and Frozen Samples of Edible West Coast Crabs

Edible West Coast crabs (Cancer magister and C. antennarius) were contaminated with bacteriophage and then held in a chilled or frozen state. Results indicated a significant survival of virus regardless of storage conditions.     

Very high expander processing of maize on animal performance,digestibility and product quality of finishing pigs and broilers

The present study investigated the effect of hydrothermic maize processing and supplementation of amino acids (AA) in two experiments. In total, 60 barrows and 384 broilers were fed four diets including either unprocessed (T1), or hydrothermically processed maize, that is short- (T2), or long-term conditioned (LC) (T3), and subsequently expanded maize of the same batch. Assuming a higher metabolizable energy (ME) content after processing, the fourth diet (T4) contains maize processed as treatment T3, but AA were supplemented to maintain the ideal protein value. Performance, digestibility and product quality in both species were assessed. Results show that in pigs receiving T4 the average daily feed intake was lower compared with the other treatments, whereas no difference was observed in broilers. The T3 improved the feed conversion rate compared with T1 (P<0.10) for both species. In contrast, average daily gain (ADG) (1277 g/day for T2 and 1267 g/day for T3 v. 971 g/day for T1) was only altered in pigs. The hydrothermic maize processing increased the apparent total tract digestibility (ATTD) of dry matter, starch and ether extract after acid hydrolysis. This may be a consequence of higher ATTD of gross energy in the finishing phase for both animal species, suggesting a higher ME content in diets with processed maize. The higher ME content of diets with processed maize is supported also by measurements of product quality. Supplementation of AA in T4 enhanced the loin depth in pigs as well as the amount of breast meat in broilers. Further effects of processing maize on meat quality were the reduced yellowness and antioxidative capacity (P<0.10) for broilers, likely due to the heat damage of xanthophylls and tocopherols. Processing also increased springiness and chewiness (P<0.10) of the broilers breast meat, whereas the loin meat of pigs showed a decreased lightness and yellowness (P<0.10) in meat when hydrothermic processed maize was used (for T2, T3 and T4). LC processed maize (T3) showed the lowest springiness in pork, however the supplementation of AA in T4 did not show differences between the treatments. Shown results demonstrated positive effects of hydrothermic processing of maize on animal performance and digestibility in both species. However, effects on carcass characteristics and product quality differed. The negative effects on product quality could be partly compensated with the AA supplementation, whereas a change in meat colour and reduced antioxidative capacity was observed in all groups fed hydrothermic maize processing.     

A systemic microbial disease in the Dungeness crab,Cancer magister,caused by a Chlamydia-like organism

A Chlamydia-like agent was diagnosed in Dungeness crabs, Cancer magister, collected from Willapa Bay and northern Puget Sound, Washington. Over a 5-year period, infected crabs were found during the months of December through March. Infection by the organism is systemic, resulting in greatly hypertrophied cells with appressed nuclei. Unlike Chlamydia or other Chlamydia-like organisms, the present microbe has a great affinity for connective tissue or connective tissue cells rather than epithelial cells. Transmission electron microscopy revealed the presence of several developmental stages that are similar to chlamydial stages, in addition to what appear to be slightly altered or aberrant stages.     

Hermit crabs as bioindicators of recent tributyltin (TBT) contamination

Evaluation of the assimilation pathway and depuration time of a given pollutant by aquatic species is important to understand the dynamics of this substance in the biota, and to search for potential ecological indicators. In the present study, the uptake pathway and depuration time and rate of the pollutant tributyltin (TBT) were investigated in the omnivorous hermit crab (Clibanarius vittatus). The assimilation and uptake pathway were investigated using hermit crabs collected in an area free of TBT. The crabs were held in the laboratory for 45 days, under one of four treatments: procedural control (PC) - water and food without TBT; T1 - water with and food without TBT; T2 - water without and food with TBT; and T3 - water and food with TBT. To determine the depuration time, the crabs were collected in a contaminated area, maintained in the laboratory with clean water, and removed every 15 days for 120 days. The concentrations of TBT and DBT (dibutyltin) were determined by chromatographic analysis. The TBT was taken up by the crabs mainly via food, and the presence of DBT in crab tissues was hypothesized to result from internal TBT degradation. TBT (as well as DBT) was depurated rapidly by C. vittatus. After approximately 30 days, the initial concentration of 111 ± 36 ng Sn g⁻¹ w. w. decreased to 3 ± 3 ng Sn g⁻¹ w. w., and after 75 days the TBT concentration was below the detection limit. The same pattern was recorded for DBT, which showed a higher depuration rate than TBT. The rapid TBT and DBT depuration is useful information, since C. vittatus and possibly other hermit crabs may be used as indicators of recent or recycled environmental contamination.     

Susceptibility and resistance of the rock crab,Cancer irroratus,to natural and experimental bacterial infection

The incidence of naturally occuring bacterial infection in a Connecticut population of rock crab, Cancer irroratus, varied between 10 and 60% of the population. The fluctuation in incidence was correlated significantly with hemocyte concentrations. Twelve percent of the crabs sampled during Jenuary were found to be infected with a previously undescribed Vibrio. The Vibrio was demonstrated as pathogenic over a wide range of temperatures. High mortalities in experimentally infected crabs appear to result from hemocyte clumping and extensive intravascular clotting triggered by an endotoxin. The lobster pathogen, Aerococcus viridans var. homari, was found to be pathogenic at 25°C to C. irroratus, a prey species of the lobster. Results suggest that this species can act as a reservior host for A. viridans var. homari.     

Type and nature of cues used by Nucella lapillus to evaluate predation risk

The ability of prey to detect and adequately respond to predation risk influences immediate survival and overall fitness. Chemical cues are commonly used by prey to evaluate risk, and the purpose of this study was to elicit the nature of cues used by prey hunted by generalist predators. Nucella lapillus are common, predatory, intertidal snails that evaluate predatory risk using chemical cues. Using Nucella and a suite of its potential predators as a model system, we explored how (1) predator type, (2) predator diet, and (3) injured conspecifics and heterospecifics influence Nucella behavior. Using laboratory flumes, we determined that Nucella responded only to the invasive green crab (Carcinus maenas), the predator it most frequently encounters. Nucella did not respond to rock crabs (Cancer irroratus) or Jonah crabs (Cancer borealis), which are sympatric predators but do not frequently encounter Nucella because these crabs are primarily subtidal. Predator diet did not affect Nucella responses to risk, although starved predator response was not significantly different from controls. Since green crabs are generalist predators, diet cues do not reflect predation risk, and thus altering behavior as a function of predator diet would not likely benefit Nucella. Nucella did, however, react to injured conspecifics, a strategy that may allow them to recognize threats when predators are difficult to detect. Nucella did not react to injured heterospecifics including mussels (Mytilus edulis) and herbivorous snails Littorina littorea, suggesting that they are responding to chemical cues unique to their species. The nature of cues used by Nucella allows them to minimize costs associated with predator avoidance.     

Endogenous rhythms of haemolymph flow and cardiac performance in the crab Cancer magister

Heart rate and arterial haemolymph flow rates were measured in freshly trapped Dungeness crabs, Cancer magister, using a pulsed-Doppler flowmeter. In the laboratory, freshly collected subtidal crabs exhibited endogenous rhythms in both cardiac function and haemolymph flow through one or more arterial systems, of both tidal and diurnal periodicity. The strongest tidal rhythms were recorded in the sternal and paired anterolateral arteries. These endogenous rhythms of selective tissue perfusion are related to an underlying locomotor activity, but may also be involved with hormonal transport or feeding. Changes in both heart rate and stroke volume were responsible for the increases in haemolymph flow rates. These rhythms were not entrained by aerial exposure, since confinement of arhythmic crabs in intertidal cages did not re-entrain an endogenous tidal rhythm. Endogenous locomotory rhythms are known to be controlled by neurohormones released in cycles from the sinus gland on the eyestalk. These hormones may also control the endogenous cardiovascular rhythms, since these were abolished after eyestalk ablation in freshly collected Cancer magister. These results suggest that hormones synthesized and released by the X-organ/sinus gland complex may, together with pericardial hormones, play a role in modulation of crustacean cardiovascular function.     

The effects of interspecific competition and prey odor on foraging behavior in the rock crab, Cancer irroratus (Say)

The effects of competitor pressure and prey odor on foraging behavior of the rock crab, Cancer irroratus (Say), were investigated. The Jonah crab, Cancer borealis (Stimpson), was chosen as the interspecific competitor because it shares resources with C. irroratus. Four treatments were tested for their effect on foraging: the presence or absence of a competitor and two types of prey odor; body odor (living mussel) and tissue extract (dead mussel tissue). The presence of Jonah crabs did not influence location time, search time, prey size selected, or handling time of the rock crabs. However, rock crabs responded differently to the presence of body odor and tissue extract cues. The presence of extract odor decreased the time to locate prey while increasing the number of prey manipulated and prey size selected. When prey body odor was present, rock crabs displayed less investigative behaviors than in the presence of extract odor, illustrated by reduced location time. Extract odor provided a stronger and more attractive cue than body odor, but increased prey manipulation and search time. Extract odor induced increases in manipulation and searching for prey but canceled out the benefits of decreased location time, resulting in crabs from both treatments displaying similar search times. These elevated behaviors may be associated with foraging for injured and cracked prey or may indicate an area of conspecific feeding.     

Preference Alters Consumptive Effects of Predators: Top-Down Effects of a Native Crab on a System of Native and Introduced Prey

Top-down effects of predators in systems depend on the rate at which predators consume prey, and on predator preferences among available prey. In invaded communities, these parameters might be difficult to predict because ecological relationships are typically evolutionarily novel. We examined feeding rates and preferences of a crab native to the Pacific Northwest, Cancer productus, among four prey items: two invasive species of oyster drill (the marine whelks Urosalpinx cinerea and Ocenebra inornata) and two species of oyster (Crassostrea gigas and Ostrea lurida) that are also consumed by U. cinerea and O. inornata. This system is also characterized by intraguild predation because crabs are predators of drills and compete with them for prey (oysters). When only the oysters were offered, crabs did not express a preference and consumed approximately 9 juvenile oysters crab⁻¹ day⁻¹. We then tested whether crabs preferred adult drills of either U. cinerea or O. inornata, or juvenile oysters (C. gigas). While crabs consumed drills and oysters at approximately the same rate when only one type of prey was offered, they expressed a strong preference for juvenile oysters over drills when they were allowed to choose among the three prey items. This preference for oysters might negate the positive indirect effects that crabs have on oysters by crabs consuming drills (trophic cascade) because crabs have a large negative direct effect on oysters when crabs, oysters, and drills co-occur.     

Effect of Extracellular Products of Pseudoalteromonas atlantica on the Edible Crab Cancer pagurus

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100°C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 μg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 μg g of body weight⁻¹) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.     

Ventilatory and cardiovascular modulation associated with burying behaviour in two sympatric crab species, Cancer magister and Cancer productus

Physiological parameters associated with burying were investigated in the Dungeness crab, Cancer magister, and the red rock crab, Cancer productus. Ventilation frequency of the branchial chambers increased while the crabs were burying, this was associated with the greater oxygen demand of the tissues. The number of ventilatory reversals in C. magister increased in number as well as in duration and magnitude when the crabs were buried, which functioned to clear the branchial chambers of sediment. In contrast, the number of ventilatory reversals in C. productus decreased. On the surface of the sand, cardiac parameters (heart rate, stroke volume, cardiac output) of both species remained stable. During the burial process, there was a large increase in cardiac output which was afforded primarily by an increased stroke volume of the heart. Once buried, cardiac output declined in both species; this was due to a decrease in stroke volume in C. productus, but a decreased heart rate in C. magister. There were also differences in haemolymph flows through each arterial system. During the burying process, both species increased haemolymph flow to the muscles of the limbs via the sternal artery. Once buried haemolymph flows to the limbs decreased, and increased flow to eyestalks and antennae via the anterior aorta occurred. Perfusion of the digestive organs via the anterolateral and hepatic arteries did not change when the crabs were buried. There was an increase in flow through the posterior aorta, of C. magister, but flow through this artery did not change in C. productus. Periods of spontaneous cardiac arrest were observed in both species while resting on the surface. These increased in duration in C. productus when buried, but there was no change in C. magister. Changes in ventilatory and cardiac variables were closely linked on the surface, but tended to uncouple when the animals were buried. The physiological responses of C. magister resembled those of true sand-dwelling crabs, whereas the responses of C. productus paralleled those of crabs that only bury occasionally in the substrate. Although these two species often occur sympatrically, they employ different physiological mechanisms when buried in the sediment.     

Bacterial Flora of the Hemolymph of the Blue Crab, Callinectes sapidus: Numerical Taxonomy

Bacteria isolated from the hemolymph of normal blue crabs were found to be predominantly Vibrio sp., Pseudomonas sp., Acinetobacter sp., Bacillus sp., Flavobacterium sp., and coliforms. Vibrio parahaemolyticus, a cosmopolitan facultative pathogen widely implicated in outbreaks of gastroenteritis related to the consumption of improperly processed seafood, was present in crabs collected between the months of May to November and was identified in up to 21% of the hemolymphs sampled.     

Prevalence and level of Listeria monocytogenes and other Listeria sp. in ready-to-eat minimally processed and refrigerated vegetables

Minimally processed and refrigerated vegetables can be contaminated with Listeria species bacteria including Listeria monocytogenes due to extensive handling during processing or by cross contamination from the processing environment. The objective of this study was to examine the microbiological quality of ready-to-eat minimally processed and refrigerated vegetables from supermarkets in Osijek, Croatia. 100 samples of ready-to-eat vegetables collected from different supermarkets in Osijek, Croatia, were analyzed for presence of Listeria species and Listeria monocytogenes. The collected samples were cut iceberg lettuces (24 samples), other leafy vegetables (11 samples), delicatessen salads (23 samples), cabbage salads (19 samples), salads from mixed (17 samples) and root vegetables (6 samples). Listeria species was found in 20 samples (20 %) and Listeria monocytogenes was detected in only 1 sample (1 %) of cut red cabbage (less than 100 CFU/g). According to Croatian and EU microbiological criteria these results are satisfactory. However, the presence of Listeria species and Listeria monocytogenes indicates poor hygiene quality. The study showed that these products are often improperly labeled, since 24 % of analyzed samples lacked information about shelf life, and 60 % of samples lacked information about storage conditions. With regard to these facts, cold chain abruption with extended use after expiration date is a probable scenario. Therefore, the microbiological risk for consumers of ready-to-eat minimally processed and refrigerated vegetables is not completely eliminated.     

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nⁱⁿ-C^out topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N^out-Cⁱⁿ) of the coliphage lambda and S68 (2 TMDs; Nⁱⁿ-Cⁱⁿ) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.     

Two novel non-destructive biomarkers to assess PAH-induced oxidative stress and porphyrinogenic effects in crabs

Two novel, non-destructive assays were developed to evaluate contaminant-induced lipid peroxidation (thiobarbituric acid-reacting substances, TBARS, levels) and haem biosynthesis disruption (porphyrin excretion) in decapod crabs. A laboratory experiment was conducted whereby pie-crust crabs (Cancer novaezelandiae) were fed cockles (Austrovenus stutchburyi) collected from a contaminated and reference site and TBARS levels and porphyrin excretion determined using fluorometric analysis in urine samples. Pyrene metabolite levels were also measured in the same urine samples to assess polycyclic aromatic hydrocarbon (PAH) exposure. Contaminant-exposed crabs exhibited elevated urinary TBARS and porphyrin levels and a strong correlation was found between these two assays and the urinary pyrene metabolite concentrations. However, there was large within-treatment variability, which precluded a clear separation between the control and the impacted group. Nevertheless, consistency in the direction of the response shows that the biomarkers reflect pollutant levels and validates the use of these simple techniques from human medicine for environmental assessments.     

Bacteriophage-resistant industrial fermentation strains: from the cradle to CRISPR/Cas9

Bacteriophage contamination and cell lysis have been recurring issues with some actinomycetes used in the pharmaceutical fermentation industry since the commercialization of streptomycin in the 1940s. In the early years, spontaneous phage-resistant mutants or lysogens were isolated to address the problem. In some cases, multiple phages were isolated from different contaminated fermentors, so strains resistant to multiple phages were isolated to stabilize the fermentation processes. With the advent of recombinant DNA technology, the early scaleup of the Escherichia coli fermentation process for the production of human insulin A and B chains encountered contamination with multiple coliphages. A genetic engineering solution was to clone and express a potent restriction/modification system in the production strains. Very recently, an E. coli fermentation of 1,3-propanediol was contaminated by a coliphage related to T1. CRISPR/Cas9 technology was applied to block future contamination by targeting seven different phage genes for double-strand cleavage. These approaches employing spontaneous mutation, genetic engineering, and synthetic biology can be applied to many current and future microorganisms used in the biotechnology industry.     

Survival of Virus in Chilled, Frozen, and Processed Oysters

Samples of whole and shucked Pacific and Olympia oysters, contaminated with 10(4)-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 10(4) PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area.