Pseudomonas aeruginosa,a facultative pathogen of red palm weevil,Rhynchophorus ferrugineus

Pseudomonas aeruginosa was identified as a facultative pathogen of red palm weevil. Intra-haemocoelic injection of the pathogen within larvae and pre-pupae was more effective at killing the insects [with a median lethal dose (LD₅₀) of 9×10² to 2×10³ bacteria/insect] than inoculation by force feeding (LD₅₀ of 10⁵ to 4×10⁵ bacteria/insect) or by wading the insects in a suspension of the pathogen (LD₅₀ of 10⁵ to 2×10⁵ bacteria/insect). Injection of 3×10³ bacteria/insect killed 69% of larvae; small larvae were more susceptible (LD₅₀ of 9×10⁵ bacteria/larva) than either larger larvae (LD₅₀ of 10³ bacteria/larva) or pre-pupa. The median time to death of the small larvae following injection of P. aeruginosa was about 6 days but that following force feeding or wading was about 8 days. A secondary invader, Serratia marcescens, had no effect on the pathogenicity of P. aeruginosa but hastened death of larvae by about 3 days.A. Banerjee and T.K. Dangar were with the Central Plantation Crops Research Institute, Regional Station, Kayangulam 690 533, Kerala, India. They are now with the Central Rice Research Institute. Cuttack 753 006, Orissa, IndiaCPCRI research paper no. 870.     

Possible recovery from an acute envenomation in male rats with LD50 of Echis coloratus crude venom: I-A seven days hematological follow-up study

The effect of an acute LD₅₀ dose of Echis coloratus crude venom in male albino rats was tested on blood parameters: white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), also serum glucose, total protein, triglycerides with alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) enzyme activities. The effect of the LD₅₀ dose was monitored over a period of seven days, with time intervals of 1, 3, 6, 12, 24, 72 h. All of the tested parameters show fluctuations with time and with tendency to regain normal control level after 12 h. At 12–24 h it seems to be crucial for the process of physiological recovery, in spite of the irreversible damage and tissue distraction. The process of physiological adaptation and recovery from the lethal destructive venom effect seems to stabilize after one week, leaving the animal alive with several biochemical altered metabolisms and disturbed physiological profile.     

Heterorhabditis bacteriophora as a vector for introducing its associated bacterium into the hemocoel of Galleria mellonella larvae

The nematode Heterorhabditis bacteriophora serves as a vector enabling its bacterial associate to reach the hemocoel of its host, the seventh-instar larva of Galleria mellonella. At 28.5°C, the LD₅₀s of the orally introduced nematode-bacterial complex and the intrahemocoelically injected bacteria are three to six nematodes and one to two cells, respectively.     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.     

The effect of bursectomy and thymectomy on the course of avian influenza virus infection

The effect of hormonal bursectomy and neonatal surgical thymectomy on the course of an avian influenza virus infection in chickens was studied. Analysis of the immunologic status of the chickens prior to infection included assay of natural agglutinins to rabbit red blood cells and induced agglutinins to sheep red blood cells, serum immunoelectrophoresis patterns, and in vitro effects of phytohemagglutinin on lymphocyte transformation. At 6 wk of age the chickens received the influenza virus intratracheally. Daily temperatures and mortality were recorded and HAI antibody titers were measured 7 and 14 days later. Completely thymectomized chickens were characterized by a failure of lymphocyte transformation to take place in two successive studies and absence of thymic remnants at autopsy. Bursectomy was associated with a significantly higher occurrence of temperature elevation (P < 0.05) and mortality (P < 0.001). Thymectomy had no significant effect on the course of the virus infection. Preliminary findings with passive administration of serum from immune animals also suggested a role for antibody in host resistance. These studies suggest cell-mediated immunity is less important than humoral immunity in recovery from avian influenza virus infection.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.     

Bactericidal activity of hemolymph from the horseshoe crab, Limulus polyphemus

Bactericidal activity was found in Limulus serum, with great individual variation in titers toward different bacteria and also among individual horseshoe crabs toward the same bacterial species. These titers varied between monthly determinations of activity. There were crabs with zero activity toward each bacterial species tested. Although environmental factors are likely influences on the bactericidal activity of Limulus serum, the marked variability within similarly treated groups indicates large individual differences in the horseshoe crab population. The highest titers were recorded against those Gram-negative bacilli found normally in the environment. Lower titers were found against those species found normally in warmblooded animals and present in water as contaminants. The serum bactericidal factor is probably released from the circulating amoebocytes during clotting since there was no activity in the “plasma” portion of the blood. Exposure to heat (56°C, 30 min) destroyed the bactericidal activity.     

Nutritional characters in Ramularia species

Groups of mice were neonatally thymectomized and treated with antithymocyte serum (ATS) prior to challenge infection with viable yeast phase (YP)Histoplasma capsulatum G-17M. Moderate leucocytosis and moderate lymphopenia were seen in immunodeficient animals after infection. Surviving immunodeficient mice exhibited low levels of migration, inhibition activity, while peritoneal exudate cells and spleen cells harvested from surviving infected and untreated normal mice showed significant migration inhibition in the presence of histoplasmin antigen.The LD₅₀ values for YP cells ofH. capsulatum were 1.1×10⁶ for normal untreated mice, 6.0×10⁵ for thymectomized mice, and 6.3×10⁵ for ATS-treated mice. Thymectomized mice that also received ATS treatment exhibited an LD₅₀ of 1.7×10⁵ and were 6.5 times more susceptible to infection then normal mice. Mice which were either thymectomized or treated with ATS were 1.7 times as susceptible as normal mice to infection withH. capsulatum. The criterion of susceptibility is a decrease in the LD₅₀ value.     

Comparison of lethalities in mouse versus goldfish for clinical and food isolates ofAeromonas hydrophila

Summary The lethality of 16 clinical or food isolates ofAeromonas hydrophila was assessed by determination of LD₅₀ (i.p.) in mice and goldfish. In mice LD₅₀ values for the variousA. hydrophila strains were similar, ranging from 1.2–21.0×10⁸ cells/animal. A wider range of LD₅₀ values, 0.03–11.8×10⁸ cells/animal, was observed with goldfish. Lethality was not correlated between the two test animals. Further, cytotoxic response in Y-1 adrenal cells did not correlate with lethality in either test animal. It appears that lethality is not a good measure of potential enterotoxigenicity, but may be useful in assessing the invasive character of isolates causing systemic infections in immunocompromised hosts.     

Common Duckweed (Lemna minor) Is a Versatile High-Throughput Infection Model For the Burkholderia cepacia Complex and Other Pathogenic Bacteria

Members of the Burkholderia cepacia complex (Bcc) have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF) patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed) is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth) larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD₅₀ values. A strong correlation (R² = 0.81) was found between the strains’ virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC) and five isogenic mutants with previously established LD₅₀ values in the larval model were tested against duckweed, and a strong correlation (R² = 0.93) was found between their raw LD₅₀ values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC) cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhB_Bc. Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial virulence factors and therapeutic strategies to combat them.     

Cytotoxic,larvicidal, nematicidal,and antifeedant activities of piperidin-connected 2-thioxoimidazolidin-4-one derivatives

The objective of this study was to investigate brine shrimp cytotoxicity, larvicidal, nematicidal, and antifeedant activities of novel piperidin-connected 2-thioxo-imidazolidin-4-one derivatives. The activities of target compounds were compared with some naturally occurring (?)-pinidinol, hydantocidin, and positive controls. Target compounds were synthesized via cyclocondensation method. The compounds were synthesized and then characterized by infrared spectroscopy, ¹H NMR, ¹³C NMR, mass spectral, and elemental analyses. Brine shrimp cytotoxicity assay was investigated using freshly hatched, free-swimming nauplii of Artemiasalina. Larvicidal screening was performed against urban mosquito larvae (Culex quinquefasciatus). Nematicidal activity was evaluated using juvenile nematodes of Meloidogyne javanica. Regarding antifeedant activity, marine-acclimated Oreochromis mossambicus fingerlings were used. Compounds 3a-c (piperidin-connected 2-thioxoimidazolidin-4-one) were found to be lethal to the second instar larvae of mosquito, which produced LD₅₀ values of 1.37, 6.66, 6.51 μg/mL, compared to compounds (?) pinidinol and hyantocidin LD₅₀ values of 18.28 and 22.11 μg/mL respectively. Compound 3a-c was found to kill 100% of fish fingerlings within 6 h at 20 µg/mL, with LD₅₀ values of 1.54, 1.79, 1.52 µg/mL, compared to compounds (?) pinidinol and hyantocidin with LD₅₀ values of 10.21 and 21.05 μg/mL respectively. Compound 3c with LD₅₀ value of 1.57 μg/mL demonstrated high nematicidal activity compared to compound 3a, 3b, (?) Pinidinol and Hyantocidin LD₅₀ values of 6.45, 2.42, 14.25, 26.30 μg/mL respectively. Therefore, the 2-thioxoimidazolidin-4-one with piperidin ring showed high potential cytotoxic, larvicidal, nematicidal, and antifeedent activities.     

Streptomyces sp. JS520 produces exceptionally high quantities of undecylprodigiosin with antibacterial, antioxidative, and UV-protective properties

A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to Streptomyces genus based on phenotypic and genetic analysis. Streptomyces sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93?%) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138?mg?l^?1 were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive Streptomyces genus. Purified pigment had antimicrobial properties against bacterial Bacillus and Micrococcus species (50?μg?ml^?1) and against Candida albicans species (100–200?μg?ml^?1 range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100?mM H₂O₂. Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15?min of exposure to ultraviolet light (254?nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8?%, respectively.     

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed₄₂, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed₄₂ accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed₄₂ signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed₄₂ signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed₄₂ signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed₄₂ signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed₄₂ variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.     

Primed Immune Responses to Gram-negative Peptidoglycans Confer Infection Resistance in Silkworms

A heightened immune response, in which immune responses are primed by repeated exposure to a pathogen, is an important characteristic of vertebrate adaptive immunity. In the present study, we examined whether invertebrate animals also exhibit a primed immune response. The LD₅₀ of Gram-negative enterohemorrhagic Escherichia coli O157:H7 Sakai in silkworms was increased 100-fold by pre-injection of heat-killed Sakai cells. Silkworms pre-injected with heat-killed cells of a Gram-positive bacterium, Staphylococcus aureus, did not have resistance to Sakai. Silkworms preinjected with enterohemorrhagic E. coli peptidoglycans, cell surface components of bacteria, were resistant to Sakai infection. Silkworms preinjected with S. aureus peptidoglycans, however, were not resistant to Sakai. Silkworms preinjected with heat-killed Sakai cells showed persistent resistance to Sakai infection even after pupation. Repeated injection of heat-killed Sakai cells into the silkworms induced earlier and greater production of antimicrobial peptides than a single injection of heat-killed Sakai cells. These findings suggest that silkworm recognition of Gram-negative peptidoglycans leads to a primed immune reaction and increased resistance to a second round of bacterial infection.     

The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD₅₀ of 2 × 10² CFU, whereas the LD₅₀ for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.     

Salt resistance in two cashew species is associated with accumulation of organic and inorganic solutes

This study establishes relationships between salt resistance and solute accumulation in roots and leaves of two contrasting cashew species. The sensitive (Anacardium microcarpum) and resistant (A. occidentale) species showed maximum root LD₅₀ values (the external NaCl concentration required for a 50% reduction in dry weight) of 63 and 128?mM NaCl, whereas the shoot LD₅₀ values were 90 and 132?mM, respectively. The salt sensitivity was directly associated with Na⁺ accumulation and especially with the Cl^? content in leaves and to a minor extent in roots. The accumulation of saline ions was associated with higher net uptake rates by roots and transport rates from root to shoot in the sensitive cashew species. The K⁺/Na⁺ ratios were not associated with salt resistance either in roots or leaves. Proline and free amino acid concentrations were strongly increased by salinity, especially in the leaves of the resistant species. The soluble sugar concentrations were not influenced by NaCl treatments in leaves of both species. In contrast, the root soluble sugar content was significantly decreased by salinity in the sensitive species only. In conclusion, the higher salt sensitivity of A. microcarpum is associated to an inefficient salt exclusion system of the leaves, especially for Cl^?. On the other hand, the resistant species displays higher concentrations of organic solutes especially a salt-induced accumulation of proline and free amino acids in leaves.     

Trypanosoma evansi: Effect of experimental infection on the osmotic fragility, lipid peroxidation and calcium-ATPase activity of rat red blood cells

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells.     

Evaluation of <Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae as an in vivo model for assessing the relative toxicity of food preservative agents

Larvae of Galleria mellonella are widely used for evaluating the virulence of microbial pathogens and for measuring the efficacy of anti-microbial agents and produce results comparable to those that can be obtained using mammals. In this work, the suitability of using G. mellonella larvae to measure the relative toxicity of a variety of food preservatives was evaluated. The response of larvae to eight commonly used food preservatives (potassium nitrate, potassium nitrite, potassium sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium nitrite and sodium acetate) administered by feeding or by intra-haemocoel injection was measured. A significant correlation between the LD₅₀ (R ²?=?0.8766, p?=?0.0006) and LD₈₀ (R ²?=?0.7629, p?=?0.0046) values obtained due to oral or intra-haemocoel administration of compounds was established. The response of HEp-2 cells to the food preservatives was determined, and a significant correlation (R ²?=?0.7217, p?=?0.0076) between the LD₅₀ values of the compounds administered by feeding in larvae with the IC₅₀ values of the compounds in HEp-2 cells was established. A strong correlation between the LD₅₀ values of the eight food preservatives in G. mellonella larvae and rats (R ²?=?0.6506, p?=?0.0156) was demonstrated. The results presented here indicate that G. mellonella larvae may be used as a model to evaluate the relative toxicity of food preservatives, and the results show a strong positive correlation to those obtained using established cell culture and mammalian models.     

The comparative susceptibilities of Pieris brassicae and P. rapae to a granulosis virus from P. brassicae

A comparison was made of the dosage-mortality responses of larvae of Pieris brassicae and P. rapae to infection by P. brassicae granulosis virus (GV). Bioassays with first, second, third, and fourth-instar larvae of both species revealed a marked difference in susceptibility between instars and between species. Median lethal dosages (LD₅₀s) for P. rapae larvae ranged from five capsules for the first instar to 662 capsules for the fourth instar. With P. brassicae, this range extended from 66 capsules to 2.3 × 10⁷ capsules. The time-mortality responses of the two species were similar when fed virus dosages equivalent to an LD₉₀. Median lethal times (LT₅₀s) ranged from 5 days for first-instar larvae to 7–8 days for fourth-instar larvae. A comparison between a long-established laboratory stock of P. brassicae and a stock recently acquired from the field showed no significant difference in their susceptibility to GV. The implications of the pronounced species differences in susceptibility to GV infection are discussed in relation to the potential field control of P. rapae and P. brassicae.     

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10² to 5.6 × 10² bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10⁴), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10⁴ bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10⁵ bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10⁷ was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.