Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Plasmids of Staphylococcus aureus associated with live and processed poultry

A series of 23 strains of Staphylococcus aureus originally isolated from processed poultry was screened for the presence of plasmids. Plasmids were more common in strains of Staph. aureus characteristically associated with live poultry than with strains endemic in poultry plants and strains of human origin. Two plasmids with sizes of 1·65 and 18·2 kilobase pairs (kBp) were present in three strains considered typical of Staph. aureus forma specialis 'altilis' and two plasmids with sizes of 1·65 and 17 kBp were present in three of four strains of Staph. aureus var. gallinae. A 1·65 kBp plasmid was present in all seven strains of these poultry biotypes and in three of 14 'endemic' strains. All the 1·65 kBp plasmids were shown by blot hybridization to share sequence homology. There was also some sequence homology between the 18·2 kBp and 17 kBp plasmids. These results were supported by restriction enzyme digest analyses. A study of cured derivatives of strain PS221 f.sp. 'altilis' suggested that the 18·2 kBp plasmid encoded the genetic determinant(s) responsible for caseolysis. Both the 1·65 and the 18·2 kBp plasmids also exerted an effect on the production of acid from lactose. In no other characteristic did cured strains resemble the plasmid-free 'endemic' strains. This was therefore consistent with the notion that the genetic determinants associated with the cultural characteristics of endemic strains are chromosomally located.     

Biotypes and DNA ribopatterns of thermophilic campylobacters from faeces and seawater in Eastern Spain

Twelve strains of thermophilic campylobacters isolated in Valencia from faeces of infants and young children with diarrhoea, and from seawater, were biotyped and examined by analysis of chromosomal DNA Hae III ribopatterns. The strains were identified as Campylobacter jejuni and C. coli , and all had different ribopatterns except for three identical faecal isolates of C. jejuni biotype I. No correlation was found between Lior biotype and Hae III ribopattern but several bands within ribopatterns were identified as possible species markers for use in diagnostic and epidemiological studies.     

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.     

Delta-endotoxin proteins associated with spherical parasporal inclusions of the four Lepidoptera-specific Bacillus thuringiensis strains

Four Lepidoptera-specific reference strains of Bacillus thuringiensis , belonging to serovars sumiyoshiensis (H3a:3d), fukuokaensis (H3a:3d:3e), darmstadiensis (H10a:10b) and japonensis (H23), which produce spherical parasporal inclusions, were examined for comparative characterization of δ-endotoxins. SDS-PAGE profiles of the alkali-solubilized parasporal inclusions revealed the presence of single major protein bands of 130 kDa in the four strains. Chymotrypsin and trypsin treatment of the proteins gave profiles different from those of the strains HD-1 (serovar kurstaki , H3a:3b:3c) and T84 A1 (serovar sotto , H4a:4b). Also, minor variations were observed in proteolysis profiles among the four strains. The LC₅₀ values of purified parasporal inclusions for the silkworm ( Bombyx mori ) larvae were 7·35, 6·45, 3·08 and 2·63 μg g⁻¹ diet, respectively, showing that their toxicity levels were 5–15 times lower than that of the strain HD-1 (0·49 μg g⁻¹ diet). Analysis by immunodiffusion and immunoblotting with polyclonal antisera revealed that parasporal inclusion proteins of the four strains are highly related, whereas they shared few or no common antigens with those of the strains HD-1, T84 A1 and Buibui (serovar japonensis ).     

Large plasmids in ruminal strains of Selenomonas ruminantium

The plasmid content of six different isolates of Selenomonas ruminantium from the rumen of sheep, cows or goats was examined by electron microscopy. In addition to small plasmids (< 12 kb) studied previously, all six strains contained at least one plasmid larger than 20 kb. Plasmid sizes of 1·4, 2·1, 2·4, 5·0, 6·2, 20·4, 20·8, 22·7, 23·3, 29·3, 30·7, 34·4 and 42·6 kb were estimated from contour length measurements. DNA-DNA hybridization experiments revealed homology among the large plasmids from five strains, while the 20·8 kb plasmid from a sixth isolate showed no apparent relationship with the plasmids of the other strains.     

Migration flexibility between freshwater and marine habitats of the pond smelt Hypomesus nipponensis

The life histories of the pond smelt Hypomesus nipponensis collected from Japanese fresh waters and brackish (sea) waters were studied by examining the strontium (Sr) and calcium (Ca) concentrations in their otoliths. The Sr:Ca ratios in the otoliths changed with the salinity of the habitat. The pond smelt living in a freshwater environment showed consistently low Sr:Ca ratios throughout the otolith, averaging 1·2–1·3 × 10⁻³. These samples were identified as a standard freshwater type. In contrast, fish collected from the intertidal zone showed higher otolith Sr:Ca ratios than those in the standard freshwater type, and the ratios fluctuated along the growth phase. In addition to the two representative life‐history types of H. nipponensis , i. e . freshwater and anadromous life‐history types, other pond smelts were found to have an estuarine resident life history‐type with no freshwater phase, indicating that the pond smelt has a flexible migration strategy with a high degree of behavioural plasticity and an ability to utilize the full range of salinity in its life history.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Application of Fluorescent Antibody for Detecting Capsular Substances in Staphylococcus aureus

A fluorescent antibody technique was developed for the determination of the capsular-type of strains of Staphylococcus aureus. It compared favourably with the method using serum-soft agar (Yoshida 1972). With the new technique, many populations of encapsulated and unencapsulated strains were investigated. Of 1421 fresh isolates of Staph. aureus , 54 were encapsulated and among these 54·8% and 48·1% were mono- and polyvalent, respectively. Capsular-type antigens A and B were found in 92·5% and 44·4% of strains respectively; capsular-types C and D were found relatively infrequently. In the other group, of unencapsulated strains, capsular-type antigen production was demonstrated in 125 out of 163 strains examined. Mono- and polyvalent capsular-types (A and B antigen producing strains) comprised 77·6% and 22·4%. respectively. In these capsular-types A and B were found in 54·4% and 62·4%, respectively: capsular-type antigen C and D producing strains were again infrequent. These results indicate that a majority of ordinary Staph . aureus strains produce capsular-type antigens although isolation of the encapsulated strains is infrequent.     

Inactivation of Strains of Salmonella senftenberg by Gamma Irradiation

S ummary : Strains of Salmonella senftenberg isolated from Norwegian herring meal and strain 775W were exposed to gamma radiation from a ⁶⁰Co source. When they were irradiated in phosphate buffered saline solution, the average D₁₀ values for all the strains was 19·3 krad. On irradiating strains 56 and 775W in herring meal the D₁₀ values were 192·1 and 188·5 krad, respectively, thus indicating that the suspending medium had a great effect on the radiation resistance of the organisms. Radiation doses of the order of 0·8–1·3 Mrad are recommended for the decontamination of herring meal.     

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5

Aims:  The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle.
Methods and Results:  106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2·0, pH 4·0, 0·15% and 0·3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 ( n  = 3) and F5 ( n  = 1). While no LAB grew at pH 2·0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7·6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6·6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici.
Conclusion:  Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth.
Significance and Impact of the Study:  Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5·5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5·5, 225 mg/1 of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6·5, 225 mg/1 of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5·5, but the addition of sorbate (225 mg/1 undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.     

Retrospective approach to investigating the early life history of a diadromous fish: allis shad Alosa alosa (L.) in the Gironde–Garonne–Dordogne watershed

In this study, the identification of major ontogenetic events and the duration of the fresh water and estuarine phases before the seaward exit of young allis shad Alosa alosa were investigated retrospectively, using the otoliths of spawners. Daily growth increment periodicity in the otoliths was validated with an accuracy of 4 days (9·4%) for fish >30 days. The specific limits of the Sr:Ca ratios in the otoliths, indicating fish movements between salinity 'compartments', were up to 1·1 ×10⁻³ for the freshwater phase, 1·1 to 1·9 × 10⁻³ for the estuarine phase and 1·9 × 10⁻³ for the marine phase. In early life history, a major event occured at 22 days, which was marked on the otoliths by a change in the direction of the otolith growth axis. This event was related to a change in larval swimming behaviour. Juvenile freshwater residence time was between 58 and 123 days, with a median duration of 88 days. Contrary to what has been estimated in previous studies, the present study showed that the estuarine phase lasted from 4 to 36 days, with a median value of 11 days. Seaward exit occurred in fish aged from 63 to 150 days, with a median age of 99 days. These results have important implications for the sustainable management of this species.     

A two-year study of the distribution of ''thermophilic'' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype

The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period. Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples. Sewage was almost always contaminated with campylobacters (96·6% of samples) and of the food samples both poultry (55·5%) and offal (47·0%) were commonly contaminated. Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample. Serotype Pen 2 was the commonest type found in human faeces (18·9%) and it was also commonest in offal (21·3%), beef (40·0%), sewage (17·7%) and was the third commonest type in poultry. A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42°C was suitable for both toxins. Detection of cytotoxic activity was most sensitive using HeLa cells. The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable. Not all strains isolated from cases of enteritis in human beings produced toxin; 23·1% produced cytotonic enterotoxin and 17·5% produced cytotoxin. There was no correlation between serotype and toxin production. The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.     

The Resistance of Spores of Clostridium botulinum Type E to Heat and Radiation

Spores of Clostridium botulinum type E have been screened for resistance to heating in aqueous suspension at 60, 70 and 80°. D ¹⁷⁶ ranged from <0·33 min to 1·25 min. z values of eight strains were between 9·0 and 10·7, and for one strain was 7·4.
Radiation survivor curves have been plotted for nine strains, and a further nine strains screened. The "shoulder" extends to about 0·4 megarad, whereafter the curve becomes exponential within a D value from 0·065 to 0·16 megarad.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Freshwater adaptation during larval, juvenile and immature periods of starry flounder Platichthys stellatus, stone flounder Kareius bicoloratus and their reciprocal hybrids

The freshwater tolerance of starry flounder Platichthys stellatus , stone flounder Kareius bicoloratus and their reciprocal hybrids, produced by artificial insemination, were examined in the larval, juvenile and immature phases. Survival rate on being transferred to fresh water in the pre-settlement phase was 0% in stone flounder and hybrids and 16·7% in starry flounder. This rate in the post-settlement phase was elevated to >50% in starry flounder and hybrids but was still 0% in stone flounder and similarly in the immature period starry flounder and hybrids survived in fresh water, although stone flounder did not. The lamella chloride cells of the gill epithelium increased in starry flounder and hybrids in fresh water in all periods. Densities of lamella chloride cells increased from 1·6 ± 0·4 (mean ± s . e . number of cells per 1 mm filament) before the transferral (day 0) to 60·3 ± 6·2 on 14 days after the transfer to fresh water (day 14) in starry flounder in the immature period. These densities in hybrids were 0·6 ± 0·3 and 1·0 ± 0·3 on day 0, and, 35·3 ± 2·8 and 23·2 ± 4·6 on day 14, respectively. Stone flounder did not show a substantial change in chloride cell densities throughout the experimental period. These results suggest that low salinity tolerance was well developed in the settlement period in starry flounder and hybrids, and hybrids were also adapted to fresh water sufficiently regardless of the cross type.     

The relationship of oocyte diameter and incubation temperature to incubation time in temperate freshwater fish species

Based on the analysis of six egg variables and incubation temperature of 65 temperate freshwater fish species, the possible relationships between oocyte diameter, incubation time and incubation temperature were reassessed and compared to the results obtained from marine fishes. Most freshwater species have eggs (mean ± s . d . 2·19 ± 1·52 mm) larger than marine species, that are chiefly demersal and develop stuck to various substrata, such as plants or rocks. A strong negative relationship was found between incubation time ( t , days) and incubation temperature ( T , ° C): t = 186·23e^{−0·197 T} ( r ²= 0·87). A strong dependence of incubation time on oocyte diameter ( Ø , mm) and incubation temperature was also found and was defined as: log₁₀ t = 3·002 + 0·599 log₁₀ Ø − 1·91 log₁₀ ( T + 2), which explained 92% of the variance of the data set. Five major groups of species were defined based on the principal component analysis (PCA) of four quantitative variables. There were two distinct groups of salmonids, displaying demersal and non-adhesive eggs with a long incubation time at low temperature, the eggs of which required a high number of degree-days. There was a large group of species possessing small, mostly demersal and adhesive eggs developing at high temperature during a short period of time, and requiring a low number of degree-days. Between these two extremes, there was a fourth group displaying intermediate values and a fifth group including three species with large, adhesive and demersal eggs incubating at high temperatures during a short period of time. The burbot Lota lota displayed an unusual combination of variables compared to the remaining species in the data set.     

Migratory patterns and contribution of stocking to the population of European eel in Lithuanian waters as indicated by otolith Sr:Ca ratios

Otolith Sr:Ca ratios were examined to evaluate the contribution of the stocked eel Anguilla anguilla elvers, which have been stocked in Lithuanian waters and mixed with naturally recruited eels for several decades, to the native eel population. Stocked eels were identified by the freshwater signature (Sr:Ca ratios <2·24 × 10⁻³) on the otolith after the glass eel stage. Naturally recruited eels, that had migrated through the North and Baltic Seas, were characterized by an extended seawater and brackish-water signature (Sr:Ca ratios >3·23 × 10⁻³) after the glass eel stage. Of 108 eels analysed, 21 eels had otolith Sr:Ca ratio profiles consistent with stocking while 87 showed patterns of natural recruitment. The ages of naturally recruited eels arriving in Lithuanian fresh waters varied from 1 to 10 years, with a mean ±  s.d . age of 5·2 ± 2·1 years. Eels from the inland Lake Baluošai were all freshwater residents of stocked origin. Stocked eels, however, accounted for only 20% of the eels from the Curonian Lagoon and 2% of eels sampled in Baltic coastal waters. This finding does not support the hypothesis that the eel fishery in the Curonian Lagoon depends mostly on stocking.