Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Evaluation of Virulence Test Procedures for Yersinia enterocolitica Recovered from Foods

Recently a heat stable toxin (ST) and the vw factors of the plague bacteria were identified in Yersinia enterocolitica recovered from human infections. The vw factors were reported to be associated with a plasmid of 42-46 M Daltons in size and were essential for the expression of virulence. With this knowledge virulence tests were developed which allowed us to assess the virulence potential of food-borne Y. enterocolitica regardless of biotypes or serotypes. The tests evaluated were: (1) rapid presumptive test for the virulence plasmid; (2) gel electrophoretic confirmation of the virulence plasmid; (3) Laird's qualitative oral feeding test with thirst stressed mice; (4) quantitative LD₅₀ determination by i.p. injection of the mouse lethal ( i.e. serotype 0:8) strains in saline; (5) quantitative LD₅₀ determination of mouse non-lethal ( i.e. serotype 0:3) strains by i.p. injection of these strains suspended in 1 ml of 10% iron dextran saline solution for virulence enhancement. These tests were evaluated with the serotypes 0:3 and 0:8 strains associated with human infections with and without the virulence plasmid with reproducible results. Then the virulence tests procedures were applied to 79 food isolates. The virulence plasmid was detected only in the Nilehn biotype 2, 3 and 4 strains, but it was absent in Nilehn biotype 1 or the atypical strains that ferment rhamnose. The virulence of food and clinical isolates of Y. enterocolitica can be assayed fairly accurately with the above tests.     

Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates

Abstract Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified on the species level. Of 117 N. asteroides complex organisms, 34 (29%) were N. farcinica , 28 (24%) were N. nova , and 55 (47%) were N. asteroides sensu stricto . An analysis of the specimen sites from which the organisms were derived showed that isolates derived from blood, brain, or bone marrow were more likely to be N. farcinica than the other two species. A study of the virulence of ten strains of each species was undertaken, using a mouse model with intravenous inoculation. The 50% lethal doses (LD₅₀) for N. farcinica were significantly lower than those of the other two species. LD₅₀ values for N. nova and N. asteroides were not significantly different. The above data confirming the greater virulence of N. farcinica support the identification of species within the N. asteroides complex.     

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis

Abstract The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis . Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD₅₀ of 10³ colony forming units (CFU) or greater (low virulence) from those having an LD₅₀ of approximately 10¹ or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica , a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD₅₀ of 10² were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.     

Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon

P.-C. LIU, K.-K. LEE AND S.-N. CHEN. 1996. The pathogenicity of six Vibrio harveyi strains in tiger prawn, Penaeus monodon , was studied, using both live bacteria and extracellular products (ECP). The organisms originally isolated from diseased penaeids were more virulent using both live bacteria and ECP (LD₅₀, 4.87–8.65 times 10⁴colony-forming units (cfu) and 1.20–1.51 μg protein g^-1body weight) than the two reference strains originally isolated from either sea water (ATCC 25919; LD₅₀, 3.18 times 10⁶cfu and 2.70 μg protein g^-1body weight) or diseased Talorchestia sp. (ATCC 14126, 0.418 times 10⁶cfu and 2.34 μg protein g^-1body weight). Each strain was reisolated from the haemolymph and the hepatopancreas of moribund prawns following each bacterial challenge. Both the live bacteria and the ECPs of the penaeid isolates exhibited stronger proteolytic (caseinase), phospholipase and haemolytic activities than those of the reference strains. These results indicate that there are differences between penaeid and non-penaeid isolates of V. harveyi in pathogenicity and reveal that proteases, phospholipases, haemolysins or exotoxins might play leading roles in the pathogenicity of V. harveyi in the tiger prawn, Penaeus monodon .     

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.     

The effect of spray washing on the development of bacterial numbers and storage life of lamb carcases

Total bacterial numbers at the most highly contaminated sites on lamb carcases (in the crutch region and adjacent to the abdominal incision) were significantly reduced to log₁₀ 3.3/cm² when spray washed with unchlorinated water at 80°C and to log₁₀ 2.8 when water at 80°C containing 450 μg/ml chlorine was used, whereas numbers on carcases which were cloth cleaned or spray washed with water at 10°C remained at approximately log₁₀ 4.0. During refrigerated storage, however, carcases treated by all methods developed similar numbers of bacteria and had the same storage life, evidently because spray washing did not affect numbers of bacteria on the diaphragm. Although initial numbers of bacteria at this site were low (log₁₀ 2.9), their numbers, and also the amount of slime, increased more rapidly there than at other sites. In addition, spray washing did not significantly affect numbers of Pseudomonas spp or Brochothrix thermosphacta , which accounted for <1% of the microflora after slaughter at each site but whose numbers were between log₁₀ 6.1 and 7.5/cm² when carcases were rejected as spoiled.     

Baseline toxicity of emamectin and spinosad to Helicoverpa armigera (Lepidoptera: Noctuidae) for resistance monitoring

Acute toxicity studies of emamectin and spinosad against Helicoverpa armigera revealed that the pest is highly susceptible to both the insecticides. The median lethal dose (LD₅₀) of emamectin is 3.86 × 10⁻³ µg per larva. The median lethal concentrations (LC₅₀) of emamectin and spinosad were found to be 0.09 and 2.94 ppm, respectively. The discriminating doses were fixed based on the LC₉₅ of the susceptible population of H. armigera as 0.80 ppm for emamectin and 10 ppm for spinosad. Resistance was not observed when the discriminating doses of emamectin and spinosad were applied on field-collected populations of H. armigera from two intensive cotton growing areas, Coimbatore and Madurai, India.     

Characterization of Vibrio harveyi strains recovered from diseased farmed Senegalese sole (Solea senegalensis)

Aim:  To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole.
Materials and Results:  The Vibrio harveyi strains tested have broad phenotypic diversity based on their biochemical and exoenzymatic patterns, outer membrane proteins (OMP), extracellular product (ECP) patterns and presence of prophages. Lethal dose 50 (LD₅₀) of the strains and in vitro antagonism tests with two probiotic strains were also determined. The OMP analysis revealed three different patterns (A, M and V). The electrophoretic analysis of the ECP showed two different groups. All strains considered virulent based on their LD₅₀ exhibited the same protein pattern in their ECP (pattern I), while all nonvirulent strains showed a different profile (pattern II). About 32% of the tested strains were positive for prophages, although a clear relationship between virulence and the presence of prophages has not been established.
Conclusions:  The results obtained have shown differences between virulent and avirulent strains isolated from diseased farmed Senegalese sole based on the protein patterns of their ECP. However, a clear relationship between virulence and presence of prophages has not been established.
Significance and Impact of the Study:  The differences observed between virulent and nonvirulent strains could be used to design prophylactic strategies against diseases caused by V. harveyi in farmed Senegalese sole.     

Insecticide susceptibility and resistance of Blattella germanica (Blattaria: Blattellidae) in Seoul, Republic of Korea, 2007

The susceptibility of Blattella germanica (L.) in the Republic of Korea (ROK) to insecticides was evaluated under laboratory conditions using 12 insecticides currently used by the local public health centers and/or pest control operators in the ROK. The insecticides included seven pyrethroids and five organophosphates. Based on their LD₅₀ values, the order of susceptibility of B. germanica adults to the insecticides was chlorpyrifos-methyl, profenofos and chlorpyrifos with values of 0.07, 0.29 and 0.88 µg/♀, respectively. The least susceptibility was obtained with tetramethrin at LD₅₀ of 7.39 µg/♀. In the comparative resistance test, the resistance ratios (RR) of 12 insecticides were compared to each other using field-collected B. germanica adults in Seoul between 1993 and 2007. Blattella germanica demonstrated higher RRs to pyrethroids such as λ-cyhalothrin, and low RRs among the organophosphates. Among the pyrethroids, λ-cyhalothrin had the highest RRs of 111- and 129-fold differences at LD₅₀ and LD₉₀ values, respectively. Among the organophosphates, profenofos was observed to have the highest RRs of 4- and 15-fold differences at LD₅₀ and LD₉₀ values, respectively. However, there were no significant differences in susceptibility to tetramethrin, chlorpyrifos and fenitrothion. Blattella germanica was more susceptible to pyridafenthion showing a 0.7-fold difference in a resistance ratio (RRLD₅₀= LD₅₀ value of 2007/LD₅₀ value of 1993). Resistance ratio of tetramethrin was low, but susceptibility was also not high.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

The Effect of Temperature, Pressure and Chlorine Concentration of Spray Washing Water on Numbers of Bacteria on Lamb Carcases

Combined analysis of three experiments showed that when lamb carcases with initial bacterial numbers of between logi₁₀3.29 and 4.22/cm² were spray washed, statistically significant reductions in bacterial numbers of log₁₀O.5 were obtained when the spray wash water temperature was > 57°C, and reductions of log₁₀1.0 were obtained when the temperature was ≥ 80°C. Reductions at all temperatures were enhanced by log₁₀0.66 when the water contained 30 µg/ml chlorine, but increasing the concentration to 450 µg/ml reduced bacterial numbers only by a further log₁₀0–29. At highly contaminated sites increasing the duration of spraying from 30 to 120 s significantly increased the reductions obtained when water containing added chlorine was used. Reductions in bacterial numbers after spray washing with pressures of 3.5, 5.6. 7.7 kg/cm² were not significantly different.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

A desk study of the relationship between temperature and hatching time for the eggs of five species of salmonid fishes

SUMMARY. Information on temperature (T°C) and time from fertilization to 50% hatch ( D days) for five species of salmonid fishes has been used to assess several mathematical models relating D and T . No single equation gave the best fit to all five data sets. The power law with temperature correction (α), log₁₀₁ D = log₁₀ a + b log₁₀ ( T - α) and the quadratic, log₁₀ D = log₁₀ a + bT + b ₁ T ² (where a, b, b ₁, and α are constants), each accounted for over 97 % of the variance of D and were good fits to the observed data points for all five species. There was little difference between the predictions obtained from these two equations within the range of observed temperatures. Therefore, the simpler power-law model is preferred. However, there were substantial within-species differences between values of D predicted from extrapolations of the two models from 2 or 3°C down to 0°C. When more data for low temperatures become available it will be possible to make a more objective choice of model.     

Estimation of the growth rates of epiphytic bacteria and Lemna minor in a river

SUMMARY. Growth of Lemna minor fronds in the River Frome during summer was found to be logarithmic with time and the growth rate (log₁₀) was 0.066 day⁻¹. This is equivalent to a doubling time of 4.5 days. The life expectancy of the fronds was 34 days.
The net change in the density of bacteria epiphytic on the lower surface of Lemna fronds in the R. Frome was monitored using a direct microscopic technique. The observed increase in bacterial numbers has been partitioned into the components of attachment and growth, assuming that attachment occurred at a constant rate and that the bacterial population grew logarithmically. The line which fitted the data best gave an attachment rate of 5.7 × 10⁵ bacteria cm⁻² day⁻¹ and a growth rate (log₁₀) for the bacteria of 0.044 day⁻¹ which is equivalent to a doubling time of 164 h.
Estimates of the rate of detachment of bacteria from Lemna plants were obtained from a laboratory experiment which assumed negligible growth of bacteria in 1 h. The number of bacteria which detached per hour and the sizes of the bacterial populations on the plants before and after detachment were estimated using a plating technique. Different detachment rates were monitored. The detachment rate (analogous to growth rate) which is judged to be most similar to an in situ value was 0.0031 h⁻¹ (0.074 day⁻¹). This rate added to the specific growth rate given above resulted in a corrected growth rate of 0.118 day⁻¹ equivalent to a doubling time of 61 h.     

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

Abstract The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 × LD₅₀) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10–20 fold) more immunogenic than the alternative forms of the protein.     

RAPID PROCEDURE FOR DETECTING CIGUATOXINS IN FISH

Flesh and viscera/gill tissues of six amberjacks (Seriola dumerilii), suspected positive for ciguatoxins, were each extracted and the toxins partially purified. Both flesh and viscera/gill of only five fish were toxic to mice exhibiting ciguatoxins (CTX) symptoms. The methanol extracts of the five fish were pooled and concentrated, the volume of flesh extract was 50.0 mL (129.4 mg toxins/mL) and viscera/gill had 25.0 mL (25.5 mg toxins/mL). Pooled extracts exhibited CTX symptoms in mice but only flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh was 198.17 g fish, equivalent to 58.3 mouse unit. An efficient fractionation and purification procedure was developed for the extracts using an HPTLC and silica gel 60 plate with a chromatographic solvent mixture of chloroform:methanol:water (60:35:8, v/v). The system yielded 10 fractions for flesh and 9 for viscera/gill. Scanned plates were subdivided into three equal zones, each scraped, methanol extracted and tested in mice. The 2nd zone (R_f fractions between 0.40 and 0.66) was very toxic to mice compared to 1st or 3rd zones and the mice had CTX symptoms. The scanner for this 2nd zone had a cluster of minor peaks on both sides of the major one with a sum total area of 62.47% indicating multiplicity of CTX in amber-jack fish. The major peak, at retention time of 1.48 s and a single area of 43.28%, is believed to be the main ciguatoxins present. The HPTLC is a rapid and sensitive procedure for ciguatoxins in fish flesh extracts with a detection limit of 40.0 ± 1.9 picogram toxins.     

Bacteria in supragingival plaque samples can be killed by low-power laser light in the presence of a photosensitizer

The purpose of this study was to determine whether bacteria in supragingival plaque samples could be killed by low-power laser light in the presence of a suitable photosensitizer. Plaque samples were obtained from 10 volunteers, treated with either toluidine blue O (TBO) or aluminium disulphonated phthalocyanine (AlPcS₂), and then exposed to light from a helium/neon (HeNe) or gallium aluminium arsenide (GaAs) laser respectively. Following irradiation, substantial reductions were achieved in the total anaerobic count as well as in the number of viable streptococci and actinomyces present in the samples. In the absence of laser light, the sensitizers themselves had little effect on the viability of the bacteria in the plaque samples. The HeNe/TBO combination appeared to be more effective than the GaAs/AlPcS₂ combination, achieving log₁₀ reductions of 2·95, 5·40 and 3·34 in the total anaerobic count, streptococci and actinomyces respectively with a light energy dose of 1·31 J. If effective in vivo , lethal photosensitization may be useful as a means of eliminating plaque bacteria from a carious lesion prior to its restoration.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

Dopamine Neurotoxicity: Inhibition of Mitochondrial Respiration

Abstract: Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD₅₀ = 90 µg. Norepinephrine was less effective with LD₅₀ = 141 µg. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC₅₀ = 8 µ M , and that of norepinephrine was twice as much (IC₅₀ = 15 µ M ). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.