Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk*

Aims:  To determine the inactivation effect of X-ray treatments on Cronobacter ( E. sakazakii ) in tryptic soy broth (TSB), skim milk (0% fat), low-fat milk (1% and 2%) and whole-fat milk (3·5%).
Methods and Results:  X-rays were produced using the RS 2400 generator system (Rad Source Technologies Inc.). Cronobacter (in TSB), inoculated skim milk (0% fat), low-fat milk (1% and 2% fat) and whole-fat milk (3·5% fat) were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 2·0, 3·0, 4·0, 5·0 and 6·0 kGy X-ray doses. Surviving bacteria in the TSB and inoculated milk, before and after treatment, were enumerated using plating method onto trypticase soy agar. Greater than 7·0-log CFU reduction in Cronobacter population was observed with 4·0, 5·0, 6·0, 6·0 and 6·0 kGy X-ray in the TSB, skim milk, 1% fat milk, 2% fat milk and 3·5% fat milk, respectively.
Conclusions:  Treatment with X-rays significantly ( P  <   0·05) reduced Cronobacter to less than detectable limits (<1 log CFU ml⁻¹) in skim milk at 5·0 kGy and milk with 1% fat content and greater at 6·0 kGy dose levels. The D-value for Cronobacter in TSB was significantly ( P  <   0·05) lower than those in milk samples.
Significance and Impact of the Study:  Treatment with X-rays could be an effective and safe alternative technology to control pathogenic bacteria ( Cronobacter ) in the dairy industry.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Effects of malachite green on leucocyte abundance in rainbow trout, Salmo gairdneri (Richardson)

Blood counts from more than 1000 young-of-the-year rainbow trout, Salmo gairdneri (Richardson), were examined to determine if static exposure to malachite green at 1·35,13·5, or 21·0 mg/1 for 25–30 min or 42·0 or 72·0 mg/1 for 5 min caused chronic leucopaenia. The major changes in fish exposed for 25–30 min came during the first 24 h. After an initial lag of 3·4 h, total leucocyte-thrombocyte counts in both treated and control fish rapidly declined. Recovery was essentially complete 1·4 days after exposure, and no leucopaenia was noted after 14 or 28 days. Thrombocytosis developed during the first 24 h in fish exposed to the higher concentrations. Lymphopaenia and neutrophilia also developed, but abated after the 4th post-treatment day. Leucocyte numbers in control and exposed groups were virtually the same by the 14th day. The total leucocyte-thrombocyte counts in fish exposed for 5 min declined after 24 h, but counts were not as depressed as those in fish exposed for 30 min.
Because leucocyte changes similar to those in exposed fish were evident in the controls in both experiments, we believe that the leucocyte changes in rainbow trout exposed to malachite green were a result of a nonspecific vertebrate stress syndrome, rather than of specific leucocytotoxic effect of this chemical.     

Microsatellite variation in marine, freshwater and anadromous fishes compared with other animals

From a total of 524 microsatellite loci considered in nearly 40 000 individuals of 78 species, freshwater fish displayed levels of population genetic variation (mean heterozygosity, h=0·46, and mean numbers of alleles per locus, a=7·5) roughly similar to those of non-piscine animals (h=0·58 and a=7·1). In contrast, local population samples of marine fish displayed on average significantly higher heterozygosities (h=0·79) and nearly three times the number of alleles per locus (a=20·6). Anadromous fish were intermediate to marine and freshwater fish (h=0·68 and a=11·3). Results parallel earlier comparative summaries of allozyme variation in marine, anadromous, and freshwater fishes and probably are attributable in part to differences in evolutionarily effective population sizes typifying species inhabiting these realms.     

Changes in the acidity and lactic acid content of 'Nono', a Nigerian cultured milk product

Changes in the pH value and lactic acid content of the uninoculated, cultured Nigerian whole milk product, 'Nono', during incubation at room temperature (27°± 2°C) for 120 h were monitored. The pH decreased from 6·78 ± 0·19 to 4·30 ± 0·11 while the lactic acid content increased from 0·32 ± 0·04% to 2·50 ± 0·02% (w/v). This was accompanied by souring and curdling of the milk particles.     

Diversity among lactococci isolated from ewes' raw milk and cheese

P. GAYA, M. BABÍN, M. MEDINA and M. NUÑEZ.1999.The technological and genetic characteristics of lactococci present in ewes' raw milk and 1-d-old ewes' raw milk cheeses sampled over a 1-year period were investigated. The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1·25 units after 6 h incubation at 30 °C reached 14·5% in spring vs 10·7% in summer, 8·3% in autumn and 3·0% in winter. In 1-d-old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1·25 units increased up to 32·3% in spring vs 23·4% in summer, 8·0% in autumn and 10·3% in winter. Fast acid-producing lactic acid bacteria mainly belonged to the genus Lactococcus . Using polymerase chain reaction protocols, fast acid-producing lactococci were grouped as 61  Lactococcus lactis subsp. lactis , 13  L. lactis subsp. cremoris and 14  L. lactis subsp. lactis biovar diacetylactis. Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid-producing lactococci, using two primers, resulted in 21 different RAPD patterns for L. lactis subsp. lactis isolates, nine RAPD patterns for L. lactis subsp. cremoris isolates and three RAPD patterns for L. lactis subsp. lactis biovar diacetylactis isolates. Up to 19 different RAPD patterns were found for L. lactis isolates from cheeses made in a particular month.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

A blood-free enrichment medium for growing Campylobacter spp. under aerobic conditions

Detection limits for Campylobacter jejuni strains JH93 and ATCC 29428 in a new blood-free enrichment broth (BFEB) were investigated under aerobic conditions. Cultures of Camp. jejuni were inoculated into 50 ml BFEB containing 10% food homogenate in 50 ml screw-cap tubes. After 24 h enrichment under aerobic conditions, Camp. jejuni were isolated on four selective agar media. The least squares means of the detection limit 50% endpoint (DL₅₀) values were 0·4 (plain BFEB), 0·9 (crabmeat), 1·7 (mushroom), 1·7 (raw milk) and 2·1 (oyster) colony forming units (cfu) 5 g⁻¹ food. The efficiency of the BFEB was significantly affected ( P < 0·05) by food type and bacterial strain. Overall, the BFEB enrichment compared favourably with the existing US Food and Drug Administration method under modified atmosphere. In addition, the BFEB method did not require the use of blood, special equipment or Oxyrase^® to reduce oxygen tensions.     

Food consumption and growth of two piscivorous fishes, the mandarin fish and the Chinese snakehead

Rates of maximum food consumption and growth were determined for immature mandarin fish Siniperca chuatsi (47·2—540·2 g) and Chinese snakehead Channa argus (45·0—546·2 g) at 10, 15, 20, 25, 30 and 35) C. The relationship between maximum rate of food consumption ( C max), body weight ( W ) and temperature ( T ) was described by the multiple regression equations: In C max=−4·880+0·597 In W +0·284 T −0·0048 T ² for the mandarin fish, and In C max=−6·718+ 0·522 In W +0·440 T −0·0077 T ² for the Chinese snakehead. The optimum temperature for consumption was 29·6) C for the mandarin fish and 28·6) C for the Chinese snakehead. The relationship between growth rate ( G ), body weight and temperature was ln( G +0·25)=−0·439−0·500 ln W +0·270 T −0·0046 T ² for the mandarin fish, and ln( G +0·25)=−6·150+ (0·175−0·026 T ) ln W +0·571 T −0·0078 T ² for the Chinese snakehead. The weight exponent in the growth–weight relationship was −0·83 for the mandarin fish, but decreased with increasing temperature for the Chinese snakehead. The optimum temperature for growth was 29·3) C for the mandarin fish, but tended to decrease with increasing weight for the Chinese snakehead, being 30·3) C for a 45-g fish, and 26·1°C for a 550-g fish.     

Survival and reproductive performance of the desert pupfish, Cyprinodon n. nevadensis (Eigenmann and Eigenmann), in acid waters

Desert pupfish, Cyprinodon n. nevadensis , were exposed to pH levels of 8·3 (control), 7·0, 6·5, 6·0, 5·5 and 5·0 to determine the effects of acidity on reproduction. Egg production was significantly reduced at every pH level tested below the control. Egg-laying virtually ceased at pH 5·0, while egg viability was reduced to less than 50% of the control value at pH 6·5, when eggs were tested at the same pH at which they were laid. The 96 h LC₅₀ for this species was pH 4·56, considerably below that for successful reproduction. Larvae were less tolerant to acid stress than were adults.
Acclimation of reproductive performance to increased acid levels did not occur. Reproductive performance did not fully recover to control levels when the fish were placed in more favourable conditions after prolonged exposure to low pH.
Reduction in the number and development of oocytes was observed in the ovaries of acid stressed fish resulting in the decreased reproductive potential.     

The effects of body mass and feeding on metabolic rate in small juvenile Atlantic cod

Apparent specific dynamic action (SDA) amplitude in young juvenile Atlantic cod Gadus morhua (1 to 8 g wet mass), fed a standardized meal and then exercised in a circular swimming respirometer at a constant swimming speed of 0·5 ± 0·3 body lengths s^-1, occurred within l h after feeding in all juveniles. SDA amplitude were 1·4 to 1·8 times higher in fed fish compared to unfed fish, and rates of oxygen consumption decreased as body mass increased. SDA duration had a tendency to decrease with increasing body mass and was shortest, at 6 h, in the smallest fish (1–1·5 g), but increased to 10–11 h in the largest fish. Apparent SDA in fed fish ( R _r) scaled with a mass exponent of 0·89, while maximum metabolic rate ( R _max) determined by chasing fish to exhaustion and then measuring oxygen consumption for 12 h, and unfed routine metabolic rate (R_r) scaled with a mass exponent of 0·79 and 0·76 respectively. Relative aerobic scope ( R _max– unfed R _r) did not appear to vary over the 1 to 8 g increase in wet mass. These results show that as body mass increased in young juvenile Atlantic cod: (1) apparent SDA ( R _f) increased more rapidly than R _max, and (2) apparent SDA took up >98% of the relative aerobic scope and that young Atlantic cod allocated most of the energy to growth, and left little for other metabolic activities.     

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4°C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0·21–0·24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8°C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1·87, 1·54 and 1·11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1·99, 2·10 and 1·06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

Movements of adult Atlantic salmon through a reservoir above a hydroelectric dam: Loch Faskally

Movements of adult Atlantic salmon were determined as they migrated through Loch Faskally, a 4-km long hydroelectric reservoir in North-east Scotland. The horizontal and vertical movements of four salmon were monitored for periods of 4–7 days using depth-sensing acoustic transmitters in June–July 1995. Each fish began sustained directed upstream movements within 5·5 h after release at swimming speeds of 0·15–0·40 bl s⁻¹. Three fish reached the head of the loch after 7·25–17 h, but then returned downstream. The four fish remained in the upper half of the loch for 15–51 days, making localized movements. Mean depths of fish were 3·7–4·0 m (max 20·7 m). Two fish were recorded at significantly shallower depths at night than during the day. Departure from the loch coincided with periods of high water flow into the reservoir. In May–July 1996, 17 radio-tagged salmon entered Loch Faskally and reached the head of the loch in 3 h–5·8 days (mean 39 h). The durations of stay in the loch varied from 3 h 50 min to 67·4 days (mean 10·9 days). Only two radio-tagged salmon left the loch under conditions of high water flow into the loch.     

Water flux and mass gain during lactation in free-living ringed seal (Phoca hispida) pups

Three ringed seal ( Phoca hispida ) pups were used to calculate water flux and milk intake, based on mass increase and dilution of injected tritiated water. Biological half life of tritium in ringed seal pups was 130 ± 17 h (mean ± S.D.). The plateau level of the isotope indicating equilibrium with the body water was, in all cases (N = 8), reached before the first blood sample was collected after 0·5 h. Daily water flux in the pups was 62·9 ± 21·5 ml kg^-1. Estimated daily milk intake of 1103 ± 388 ml milk resulted in a mass increase of 386 ± 104 g. Neonates had a fat content of 4·75%, and a water content of 70·1% (N = 3). Corresponding figures for a pup close to weaning were 36·5% and 46·3%. Isotope dilution calculations of body water gave an overestimate of 1·6% compared with body water content from desiccation. Ringed seal milk was found to contain 38·1% fat, 9·9% protein, 2·3% lactose and 1% ash.     

Autotriploid tench Tinca tinca (L.) larvae obtained by fertilization of eggs previously subjected to postovulatory ageing in vitro and in vivo

Eggs of diploid tench Tinca tinca were half-stripped out and stored for 0 (control batch), 1, 3 and 5 h at mean ± s . d . 17·0 ± 0·4 and 21·9 ± 0·5° C or for 0, 1, 2, 3, 4 and 5 h at 24·0 ± 0·0° C in vitro prior to fertilization. The eggs remaining in vivo in the fish kept at 17·0 ± 0·4 and 21·9 ± 0·5° C were collected and fertilized in the same time intervals. Fertilization rate and larval yield mostly decreased after 3–5 h storage of eggs both in vitro and in vivo and only the diploid larvae were found in all control batches. Triploid larval yields increased to a maximum 5·26% after 5 h in vitro storage at 24·0° C and 1·07 and 1·60% after 3 h in vitro storage at 21·9 and 17·0° C, respectively. Triploid larval yield during in vivo storage at 21·9° C reached a maximum 0·91% after 5 h. As the spontaneous autotriploid larvae arose solely from fertilized eggs previously subjected to postovulatory egg ageing by means of prolongated storage, the autotriploidy was probably caused by failure of extrusion of the second polar body.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.