Demonstration and Isolation of Clostridium botulinum Types from Whitefish Chubs Collected at Fish Smoking Plants of the Milwaukee Area

A total of 1,071 whitefish chub samples were examined at eight stages of processing, including sampling aboard ship, various processing steps in the smoking plant, and display in retail cases. The frequency of Clostridium botulinum contamination of freshly caught and eviscerated chubs was approximately 13 to 14%. The highest percentage of contamination (20%) was noted among chubs sampled at the brining step of processing. The prevalence of contamination among chubs sampled at other processing stages prior to the smoking operation ranged from 6 to 14%. Of 858 freshly smoked chubs that had been processed at 180 F for 30 min (internal temperature of loin muscle), 10 were contaminated with C. botulinum (1 Type B and 9 Type E). The use of heat-shocked (60 C for 15 min) and nonheat-shocked enrichment cultures in combination yielded a greater number of positive samples than either method yielded when used alone. Each toxic enrichment culture obtained was subcultured to obtain isolation of the toxigenic organism. Toxigenic pure cultures of C. botulinum were obtained from 80% of the fish samples observed to produce toxic enrichment cultures.     

The Growth and Toxin Production of Clostridium botulinum Type E in Certain Vacuum Packed Fish

The growth and toxin production of Clostridium botulinum type E in various types of vacuum packed fish products was tested, with particular reference to the time/temperature relationship of storage. Toxin production was most rapid in herring although smoking retarded its development. With as small an inoculum as 10² spores/pack, fresh herring became toxic after storage for 15 days at 5°. Irradiation of three species of fish after inoculation with Cl. botulinum type E showed that spores surviving radiation germinated and produced toxin more rapidly than an equivalent concentration of spores in nonirradiated fish.     

Interrelationship of Heat and Relative Humidity in the Destruction of Clostridium botulinum Type E Spores on Whitefish Chubs

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.     

Sensitivity of an Enrichment Culture Procedure for Detection of Clostridium botulinum Type E in Raw and Smoked Whitefish Chubs

The sensitivity of an enrichment culture procedure for detecting Clostridium botulinum type E in whitefish chubs (Leucichthys sp.) was assayed. Data demonstrated that fish inoculated with 10 or more viable C. botulinum spores regularly develop specifically neutralizable enrichment cultures. Mild heat treatment (60 C, 15 min) substantially reduced the sensitivity of enrichment culturing. This effect was particularly noticeable in the culturing of fish which harbored fewer than 10 spores each. Evidence is presented which indicates that sensitivity of enrichment, without heat, approaches the level of one spore per fish. Smoked whitefish chubs, containing from one to several hundred spores each, were examined for toxin content after storage at 5, 10, 15, and 28 C for as long as 32 days. The lowest temperature at which detectable toxin was produced was 15 C. This occurred in 1 of 10 fish incubated for 14 days. C. botulinum was regularly recovered, by enrichment culture, from fish inoculated with small numbers of spores, even though toxin was not detected by direct extraction of incubated fish. Persistence of C. botulinum type E spores was observed to decline with an increase in the temperature and time at which inoculated fish were stored.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

Heat Injury of Bacillus subtilis Spores at Ultrahigh Temperatures

The following three criteria indicated that Bacillus subtilis A spores were injured, but not completely inactivated, by ultrahigh temperature treatment. (i) Significant reductions in survivors were observed when spores were enumerated with a standard medium but not when the medium contained added CaCl(2) and sodium dipicolinate. (ii) After a damaging heat treatment, more survivors were enumerated with the standard medium after incubation at 32 C than at 45 C, which was opposite to the result with untreated or slightly heated spores. (iii) Apparent numbers of survivors increased during the initial period of 3 C storage when enumerated with the standard medium at 45 C. No injury was evident when survivors were enumerated at either incubation temperature with the medium containing added CaCl(2) and sodium dipicolinate. Heat activation of the spores did not significantly influence the appearance of heat injury. The data suggested that the heat injury occurred in a germination system which was required in the absence of CaCl(2) and sodium dipicolinate.     

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores

The mechanism(s) of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms by base-exchange technique and these spores were heated at 95°C. Of the four ionic forms, i.e. Ca²⁺, Na⁺, H⁺ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

Requirement for and Sensitivity to Lysozyme by Clostridium perfringens Spores Heated at Ultrahigh Temperatures

The requirement of ultrahigh temperature (UHT)-treated Clostridium perfringens spores for lysozyme and the sensitivity of heated and unheated spores to lysozyme were studied. The UHT-treated spores requiring lysozyme for germination and colony formation originated from only a small portion of the non-UHT-treated spore population. This raised a question of whether the requirement for lysozyme was natural to the spores or was induced by the UHT treatments. However, these spores did not require lysozyme for germination before UHT treatment, which confirmed that the requirement for lysozyme had been induced by the UHT treatment. Only 1 to 2% of the spores were naturally sensitive to lysozyme; therefore, the mere addition of lysozyme to the plating medium did not permit the enumeration of all survivors. Treatment of UHT-treated spores with ethylenediaminetetraacetate (EDTA) sensitized the spores to lysozyme and increased by 10- to 100-fold the number of survivors that were detected on a medium containing lysozyme. Under the heating conditions used, spores that were naturally sensitive to lysozyme and spores that required EDTA treatment were equally heat resistant.     

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

The effect of procaine on the partitioning of plasmid pSC101

Abstract An examination of samples obtained from a commercial fish smoker, using seawater agar with incubation at 4°, 15° and 37°C for up to 28 days, revealed the presence of large bacterial populations in smoked fish. However, initially only low bacterial numbers, i.e., 2 × 10³/g, were present in the muscle of fresh, whole haddock ( Melanogrammus aeglefinus ). With filleting, there was a sudden increase in numbers to 9.2 × 10⁵/g. Yet immediately after smoking, the bacterial populations decreased (5 × 10⁵/g), followed by a gradual increase with storage (e.g., 2 × 10⁶/g after 24 h). Representative colonies were presumptively identified as Acinetobacter, Alcaligenes , coryneforms, Pseudomonas and Vibrio spp.     

Laboratory and field studies on Glugea stephani (Hagenmuller), a microsporidan parasite of pleuronectid flatfishes.

The microsporidan Glugea stephani is a common parasite of juvenile English sole (Parophrys vetulus) in Yaquina Bay, Oregon. Field observations indicated that fish became infected only in the upper estuary where summer temperatures were above 15C and the incidence of infection reached 79.8% in the late fall. Laboratory infections developed and parasite growth occurred only at or above 15C. The parasite was successfully transmitted to juvenile English sole by brine shrimp (Artemia salina) and amphipod (Corophium spinocorne) vectors as well as by direct ingestion of spores by the host. Infections that resulted from ingestion of spore-carrying vectors were much heavier than those resulting from the direct ingestion of spores. The speckled sanddab (Citharichthys stigmaeus), a nonpleuronectid flatfish, and chum salmon (Oncorhynchus keta) were refractory to G. stephani infection in the laboratory.