Heat-killed Cells Research Articles

Enterobacter cloacae, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

Banana (Musa spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus Pseudocercospora fijiensis threatens fruit production. In this work, we examined the potential of the endophytes of banana plants Enterobacter cloacae and Klebsiella pneumoniae, as antagonists of P. fijiensis and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to P. fijiensis. To measure the effects on plant growth, the two plant bacteria and an E. coli strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (−30%) during the same period. Plants treated with bacteria showed good growth, but E. cloacae AC treated plants had significantly greater biomass than the E. cloacae HKC. After 60 days, plants inoculated with E. cloacae AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with 15NH4Cl or Na15NO3 as the nitrogen source. The 15N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers m/z 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired 15N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. E. cloacae might be a new alternative to promote growth and health of banana crops.

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold-starvation using propidium monoazide real-time polymerase chain reaction.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.

Towards application of water extract from heat-killed Lactococcus lactis H61 as a cosmetic ingredient.

We previously reported that oral administration of heat-killed Lactococcus lactis H61 improves certain human skin properties. For topical application of this strain, we reasoned that a bacterial cell extract obtained with an aqueous solvent could be readily formulated as a cosmetic ingredient. In the present study, we characterized the water extract from heat-killed H61. The extract had inhibitory activity for angiotensin-converting enzyme, which is known as suppression of inflammation of skin, and absorbed electromagnetic radiation in the UVB range. UVB-irradiated normal human epidermal keratinocytes (NHEKs) had lower viability than nonirradiated NHEKs. The NHEK survival rate was significantly higher in cells treated with the extract at 10mg dried cells per ml prior to UVB exposure than in untreated cells or cells treated with lower extract concentrations. At this concentration, the extract also inhibited the production of interleukin-8 induced by UVB. The extract did not protect against hydrogen peroxide-induced cell damage. These data indicate that topical application of the H61 extract alleviates UVB damage and reduces inflammation in skin cells. The present study expands the potential application of strain H61 to its use as a cosmetic ingredient in addition to its use in the food industry. SIGNIFICANCE AND IMPACT OF THE STUDY: In our previous report, oral administration of heat-killed Lactococcus lactis H61 improved certain human skin properties. This study aimed exploring the potential topical use of this strain. The water extract derived from heat-killed cells with angiotensin-converting enzyme inhibitory activity, which is known as suppression of inflammation of skin, could protect normal human epidermal keratinocytes (NHEKs) from damage caused by UVB. Higher interleukin-8 production by UVB-exposed NHEKs than nontreated cells was suppressed by addition of the extract. The extract absorbed electromagnetic radiation in the UVB range. This extract could help in the maintenance of skin health by suppressing inflammation.

Lactobacillus paracasei PS23 reduced early-life stress abnormalities in maternal separation mouse model

Maternal separation (MS) has been developed as a model for inducing stress and depression in studies using rodents. The concept of the gut-brain axis suggests that gut health is essential for brain health. Here, we present the effects of administration of a probiotic, Lactobacillus paracasei PS23 (PS23), to MS mice against psychological traits including anxiety and depression. The administration of live and heat-killed PS23 cells showed positive behavioural effects on MS animals, where exploratory tendencies and mobility were increased in behavioural tests, indicating reduced anxiety and depression compared to the negative control mice (P<0.05). Mice administered with both live and heat-killed PS23 cells also showed lower serum corticosterone levels accompanied by higher serum anti-inflammatory interleukin 10 (IL-10) levels, compared to MS separated mice (P<0.05), indicating a stress-elicited response affiliated with increased immunomodulatory properties. Assessment of neurotransmitters in the brain hippocampal region revealed that PS23 affected the concentrations of dopaminergic metabolites differently than the control, suggesting that PS23 may have improved MS-induced stress levels via neurotransmitter pathways, such as dopamine or other mechanisms not addressed in the current study. Our study illustrates the potential of a probiotic in reversing abnormalities induced by early life stress and could be an alternative for brain health along the gut-brain axis.

Listeria monocytogenes cells injured by high hydrostatic pressure and their recovery in nutrient-rich or -free medium during cold storage

ABSTRACTCells of Listeria monocytogenes suspended in phosphate-buffered saline (PBS) were treated by high hydrostatic pressure (HHP; 500 MPa, 25°C, 10 min), diluted by ten folds using trypticase soy broth (TSB) or PBS, and stored at cold temperatures of 0–15°C. Viable cell count in TSB increased logarithmically close to the initial count at each storage temperature, while that in PBS increased temporarily and subsequently decreased to almost nondetectable level except the case at 15°C, where it showed logarithmic increase thereafter. Based on proliferation experiments where their healthy cells were inoculated to TSB or to PBS containing their heat-killed dead cells, it was suggested that increase in the viable count of HHP-treated cells in TSB and PBS could be ascribed to the recovery of colony forming ability and/or proliferation depending on the cold storage temperature.

Pretreatment with a Heat-Killed Probiotic Modulates the NLRP3 Inflammasome and Attenuates Colitis-Associated Colorectal Cancer in Mice.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Inflammation contributes to cancer development and inflammatory bowel disease is an important risk factor for CRC. The aim of this study is to assess whether a widely used probiotic Enterococcus faecalis can modulate the NLRP3 inflammasome and protect against colitis and colitis-associated CRC. We studied the effect of heat-killed cells of E. faecalis on NLRP3 inflammasome activation in THP-1-derived macrophages. Pretreatment of E. faecalis or NLRP3 siRNA can inhibit NLRP3 inflammasome activation in macrophages in response to fecal content or commensal microbes, P. mirabilis or E. coli, according to the reduction of caspase-1 activation and IL-1β maturation. Mechanistically, E. faecalis attenuates the phagocytosis that is required for the full activation of the NLRP3 inflammasome. In in vivo mouse experiments, E. faecalis can ameliorate the severity of intestinal inflammation and thereby protect mice from dextran sodium sulfate (DSS)-induced colitis and the formation of CRC in wild type mice. On the other hand, E. faecalis cannot prevent DSS-induced colitis in NLRP3 knockout mice. Our findings indicate that application of the inactivated probiotic, E. faecalis, may be a useful and safe strategy for attenuation of NLRP3-mediated colitis and inflammation-associated colon carcinogenesis.

Optimisation of a propidium monoazide based method to determine the viability of microbes in faecal slurries for transplantation

The efficacy of faecal microbiota transplantation (FMT) as a therapeutic intervention may depend on the viability of the microorganisms in faecal slurries (FS) prepared from donor stool. However, determining the viability of these organisms is challenging. Most microorganisms in stool are refractory to culture using standard techniques, and culture-independent PCR-based methods derive signal from both viable and non-viable cells. Propidium monoazide (PMA) treatment has been shown to be effective in preventing PCR amplification of DNA from non-viable bacteria in a range of contexts. However, this methodology can be sensitive to factors such as bacterial load and sample turbidity. We describe the optimisation of a PMA treatment methodology for FS that restricts quantitative PCR-based bacterial enumeration to viable cells. When applied to concentrated FS (10–25% stool content), PMA treatment at 100 μM concentration was ineffective in preventing DNA amplification from heat-killed cells. Efficacy was not significantly improved by doubling the PMA concentration. However, PMA treatment efficacy was improved markedly following 10-fold sample dilution, and was found to be optimal at 100-fold dilution. Substantial reductions in viable bacterial load could be observed following both freeze-thaw and heat-treatment of FS. This method successfully prevented DNA amplification of heat-killed Pseudomonas and Staphylococcus spiked into stool and could reliably determine the proportion of live bacteria and viable E. coli counts present in fresh and heat-treated stool. With appropriate sample dilution, PMA treatment excluded >97% of non-viable cells from amplification in all assays, without significantly affecting the amplification of DNA from viable cells. This method can be applied to optimise sample processing of FMT donor material, and to characterise bacterial viability within faecal samples more widely.

Effects of daily consumption of the probiotic Bifidobacterium animalis subsp. lactis CECT 8145 on anthropometric adiposity biomarkers in abdominally obese subjects: a randomized controlled trial

BackgroundThe effects of probiotic Bifidobacterium animalis subsp. lactis CECT 8145 (Ba8145) and those of its heat-killed form (h-k Ba8145) on human anthropometric adiposity biomarkers are unknown.ObjectiveTo assess the effect of Ba8145 and h-k Ba8145 ingestion on anthropometric adiposity biomarkers.DesignRandomized, parallel, double-blind, placebo-controlled trial with abdominally obese individuals. Participants (n = 135) consumed 1 capsule/day containing 1010 colony forming unit (CFU) of Ba8145, 1010 CFU of h-k Ba8145, or placebo (maltodextrin) for 3 months.ResultsBa8145 ingestion decreased waist circumference, waist circumference/height ratio, and Conicity index (P < 0.05) versus its baseline. Changes versus the placebo group reached significance (P < 0.05) after the h-k Ba8145 treatment. Ba8145 decreased the body mass index compared with baseline and placebo group (P < 0.05). The decrease in visceral fat area after Ba8145 treatments reached significance (P < 0.05) only after h-k Ba8145. When analyses by gender were performed, significance remained only for women. Diastolic blood pressure and HOMA index decreased (P < 0.05) after h-k Ba8145. Gut microbiome analyses showed an increase in Akkermansia spp. after Ba8145 treatment, particularly in the live form, which was inversely related to weight (P = 0.003).ConclusionsIn abdominally obese individuals, consumption of Ba8145, both as viable and mainly as heat-killed cells, improves anthropometric adiposity biomarkers, particularly in women. An increase in the gut Akkermansia genus appears as a possible mechanism involved. Our results support Ba8145 probiotic as a complementary strategy in obesity management.

Escherichia coli Cells Exposed to Lethal Doses of Electron Beam Irradiation Retain Their Ability to Propagate Bacteriophages and Are Metabolically Active.

Reports in the literature suggest that bacteria exposed to lethal doses of ionizing radiation, i.e., electron beams, are unable to replicate yet they remain metabolically active. To investigate this phenomenon further, we electron beam irradiated Escherichia coli cells to a lethal dose and measured their membrane integrity, metabolic activity, ATP levels and overall cellular functionality via bacteriophage infection. We also visualized the DNA double-strand breaks in the cells. We used non-irradiated (live) and heat-killed cells as positive and negative controls, respectively. Our results show that the membrane integrity of E. coli cells is maintained and that the cells remain metabolically active up to 9 days post-irradiation when stored at 4°C. The ATP levels in lethally irradiated cells are similar to non-irradiated control cells. We also visualized extensive DNA damage within the cells and confirmed their cellular functionality based on their ability to propagate bacteriophages for up to 9 days post-irradiation. Overall, our findings indicate that lethally irradiated E. coli cells resemble live non-irradiated cells more closely than heat-killed (dead) cells.

Effects of 12-wk Lactobacillus plantarum OLL2712 treatment on glucose metabolism and chronic inflammation in prediabetic individuals: A single-arm pilot study.

Previously, we demonstrated that the administration of heat-killed OLL2712 cells suppressed chronic inflammation and improved hyperglycemia in a mouse model of obesity and diabetes. The aim of this study was to preliminarily examine the effect of OLL2712 supplementation on glucose metabolism and chronic inflammation in prediabetic subjects. This study was a prospective, 12-wk, single-arm, open trial, followed by a 4-wk posttreatment period. Inclusion criteria were fasting plasma glucose levels of 105 to 130 mg/dL in an age range of 35 to 65 y. Thirty individuals consumed a dairy beverage containing ∼1 × 1010 heat-killed OLL2712 cells for 12 wk. The ingestion of the OLL2712 beverage significantly improved fasting plasma glucose levels, serum glycoalbumin levels, and insulin resistance indexes compared with baseline levels. The intervention also suppressed serum monocyte chemotactic protein-1 and interleukin-6 levels, which are proinflammatory cytokines involved in the development of insulin resistance and hyperglycemia. Furthermore, stratified analysis by these proinflammatory cytokine levels revealed that the beneficial effects of OLL2712 beverage were observed particularly in individuals with chronic inflammation at baseline. The results of this study suggested that heat-killed OLL2712 cells have the potential to improve insulin resistance and glucose metabolism by suppressing chronic inflammation.

Induction of gut Lactobacillus reuteri in normal ICR mice by oral administration of L. plantarum AN1

To clarify the existence of indigenous intestinal gut bacteria sensitive to orally administered probiotic strains of lactic acid bacteria, the effects of live and heat-killed Lactobacillus plantarum AN1 cells, administered to normal ICR mice via drinking water were observed. Amplicon sequencing of the 16S rDNA (V4) of caecal contents revealed that the live AN1 cells increased the abundance of an indigenous sensitive gut bacterium (SGB) Lactobacillus reuteri. From faecal samples, five typical dominant colonies were detected on BL agar. Among the typical colonies, smooth brown colonies were detected only in mice fed live AN1 cells, and small brown colonies with a black centre were detected in mice fed live and heat-killed AN1 cells. These two colony types were determined to be L. plantarum and L. reuteri, respectively. Three other typical dominant colonies were detected in all mouse groups, and were determined to be Lactobacillus intestinalis, Lactobacillus gasseri, and L. reuteri, respectively. These results suggest that non-dominant L. reuteri is an L. plantarum-SGB in ICR mice. Practical applications The non-dominant L. reuteri is an L. plantarum-SGB in ICR mice, and it is possible to demonstrate functionality with orally administered probiotics. Isolation and application of human or livestock SGB is considered promising for the development of new functional foods.

Rhizobia promote the growth of rice shoots by targeting cell signaling, division and expansion.

The growth-promotion of rice seedling following inoculation with Sinorhizobium meliloti 1021 was a cumulative outcome of elevated expression of genes that function in accelerating cell division and enhancing cell expansion. Various endophytic rhizobacteria promote the growth of cereal crops. To achieve a better understanding of the cellular and molecular bases of beneficial cereal-rhizobia interactions, we performed computer-assisted microscopy and transcriptomic analyses of rice seedling shoots (Oryza sativa) during early stages of endophytic colonization by the plant growth-promoting Sinorhizobium meliloti 1021. Phenotypic analyses revealed that plants inoculated with live rhizobia had increased shoot height and dry weight compared to control plants inoculated with heat-killed cells of the same microbe. At 6days after inoculation (DAI) with live cells, the fourth-leaf sheaths showed significant cytological differences including their enlargement of parenchyma cells and reduction in shape complexity. Transcriptomic analysis of shoots identified 2,414 differentially-expressed genes (DEGs) at 1, 2, 5 and 8 DAI: 195, 1390, 1025 and 533, respectively. Among these, 46 DEGs encoding cell-cycle functions were up-regulated at least 3 days before the rhizobia ascended from the roots to the shoots, suggesting that rhizobia are engaged in long-distance signaling events during early stages of this plant-microbe interaction. DEGs involved in phytohormone production, photosynthetic efficiency, carbohydrate metabolism, cell division and wall expansion were significantly elevated at 5 and 8 DAI, consistent with the observed phenotypic changes in rice cell morphology and shoot growth-promotion. Correlation analysis identified 104 height-related DEGs and 120 dry-weight-related DEGs that represent known quantitative-trait loci for seedling vigor and increased plant height. These findings provide multiple evidences of plant-microbe interplay that give insight into the growth-promotion processes associated with this rhizobia-rice beneficial association.

The dynamic role of pH in microbial reduction of uranium(VI) in the presence of bicarbonate

The negative effect of carbonate on the rate and extent of bioreduction of aqueous U(VI) has been commonly reported. The solution pH is a key chemical factor controlling U(VI)aq species and the Gibbs free energy of reaction. Therefore, it is interesting to study whether the negative effect can be diminished under specific pH conditions. Experiments were conducted using Shewanella putrefaciens under anaerobic conditions with varying pH values (4–9) and bicarbonate concentrations ([CO32−]T, 0–50 mmol/L). The results showed a clear correlation between the pH-bioreduction edges of U(VI)aq and the [CO32−]T. The specific pH at which the maximum bioreduction occurred (pHmbr) shifted from slightly basic to acidic pH (∼7.5–∼6.0) as the [CO32−]T increased (2–50 mmol/L). At [CO32−]T = 0, however, no pHmbr was observed in terms of increasing bioreduction with pH (∼100%, pH > 7). In the presence of [CO32−]T, significant bioreduction was observed at pHmbr (∼100% at 2–30 mmol/L [CO32−]T, 93.7% at 50 mmol/L [CO32−]T), which is in contrast to the previously reported infeasibility of bioreduction at high [CO32−]T. The pH-bioreduction edges were almost comparable to the pH-biosorption edges of U(VI)aq on heat-killed cells, revealing that biosorption is favorable for bioreduction. The end product of U(VI)aq bioreduction was characterized as insoluble nanobiogenic uraninite by HRTEM. The redox potentials of the master complex species of U(VI)aq, such as (UO2)4(OH)7+, (UO2)2CO3(OH)3−, and UO2(CO3)34−, were calculated to obtain insights into the thermodynamic reduction mechanism. The observed dynamic role of pH in bioreduction suggests the potential for bioremediation of uranium-contaminated groundwater containing high carbonate concentrations.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

Antioxidant effects of live and heat-killed probiotic Lactobacillus plantarum Ln1 isolated from kimchi.

The aim of this study was to evaluate the probiotic properties of Lactobacillus plantarum Ln1 isolated from kimchi and the antioxidant activities of live and heat-killed cells. L. plantarum KCTC 3108, which has been used as a commercial probiotic strain, was used as a control. L. plantarum strains (Ln1 and KCTC 3108) can survive under artificial gastric conditions (pH 2.5 in 0.3% pepsin for 3h and 0.3% oxgall for 24h), and adhere strongly to HT-29 cells. In addition, L. plantarum Ln1 did not produce carcinogenic β-glucuronidase, whereas it showed a higher β-galactosidase production of 3067.42mU/mL. The antioxidant activity of L. plantarum Ln1 was assessed using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, β-carotene and linoleic acid inhibition, and reducing power assays. In all these methods, live L. plantarum Ln1 showed a higher antioxidant activity than the control strain. In heat-killed cells of L. plantarum Ln1, β-carotene bleaching inhibitory activity and reducing power was higher than DPPH and ABTS radical scavenging activity. These results suggested that live or heat-killed L. plantarum Ln1 isolated from kimchi might be useful as an antioxidant probiotic.

Ochratoxin A reduction ability of biocontrol agent Bacillus subtilis isolated from Korean traditional fermented food Kimchi

In the present study, a new biocontrol strain, Bacillus subtilis KU-153, was isolated from the Korean traditional fermented food Kimchi and evaluated for its ability to reduce the ochratoxin A (OTA) content in culture medium. A 16 S rRNA gene sequencing analysis revealed the identity of newly isolated strain KU-153 as B. subtilis. The growth kinetic study of B. subtilis KU-153, in terms of the OTA reduction in culture medium, confirmed its biocontrol efficacy. To verify its ability to reduce the OTA content in culture medium, bacterial extracts (intracellular and extracellular) of B. subtilis were separated and compared with whole B. subtilis cells (viable and heat-killed). No reduction in the OTA content was observed in culture medium with extracellular and intracellular extracts, while viable and heat-killed cells of B. subtilis showed significant levels (p < 0.05) of OTA reduction in culture medium. Interestingly, B. subtilis heat-treated cells showed a higher OTA reduction (45%) than viable cells (22%). Further, B. subtilis heat-treated cells were assessed for their ability to reduce OTA levels in artificially contaminated red wine samples that resulted in an OTA reduction of approximately 90%, suggesting the biocontrol potential of the newly isolated strain B. subtilis KU-153 on OTA reduction.

Development of anti-Aeromonas hydrophila serum from Rabbits

An experiment was conducted to prepare anti-Aeromonas hydrophila serum in two rabbits (Oryctolagus cuniculus). Formalin killed cell (FKC) and heat killed cell (HKC) were prepared by adding 0.5% formalin and 2.5 h heat treatment at 60°C of freshly cultured Aeromonas hydrophila (AQC810) isolated from naturally infected koi fish kidney respectively. Slide agglutination tests were performed for the determination of the presence of specific antibody against Aeromonas hydrophila in the serum collected from the blood of pre-immune rabbits or post immune rabbits. No agglutination of pre-immune serum with the homologous antigen proved that the rabbits had no previous Aeromonas contamination. Rabbits were injected subcutaneously with the FKC added with same amount of Freund’s complete adjuvant. Three booster doses were given without the adjuvant at weekly intervals after second week of first injection. Agglutination titration of anti-Aeromonas hydrophila rabbit serum against HKC and FKC of homologous bacteria showed that the titers increased with the post inoculation days which rose at their peak during the fifth week of immunization and after three consecutive booster doses at weekly intervals starting from second week of first immunization. Rabbits were sacrificed after six weeks of immunization, whole blood, collected and centrifuged to accumulate the serum that were complementinactivated by heating to 58ºC for 30 min and stored at -20°C in appendorph tubes, with the aim of rapid diagnosis of Aeromonas infections in fishes. Result of this study would initiate the way of vaccine development against MAS (motile Aeromonas septicemia) in the aquaculture fishes of Bangladesh.Res. Agric., Livest. Fish.5(1): 137-145, April 2018

Ghrelin Attenuates Muscle Atrophy In Tumor-bearing Mice

Cachexia is a multi-organ syndrome characterized by muscle and fat wasting. Ghrelin is a hormone known to release growth hormone and to stimulate appetite by binding to its receptor growth hormone secretagogue receptor (GHSR)-1a. However, recent data suggest that not all effects of ghrelin are mediated through this receptor. PURPOSE: To investigate if ghrelin attenuates cancer-induced muscle atrophy in a GHSR-1a-independent manner. METHODS: 5-6-month-old male C57BL/6J GHSR-1a wildtype (WT) and knockout (KO) mice were inoculated with 1x106 heat-killed (HK, control) or live Lewis Lung Carcinoma (LLC) cells in the right flank. When the tumor was palpable (1 wk), tumor-bearing mice were injected with vehicle (saline solution, TV) or ghrelin (0.8 mg/kg, TG), IP twice a day, while HK mice were injected with vehicle (n = 8-10). Body weight, lean body mass and fat mass were measured by NMR before tumor implantation and 2 weeks after tumor noted. Gastrocnemius (GAS) muscles were harvested for analysis 2 weeks after tumor noted. RESULTS: Tumor implantation induced a significant decline in body weight and fat mass in both strains. As expected, ghrelin attenuated the fat loss in WT (TV vs. TG: -52% vs. -23% from baseline, p = .009) but not in KO (TV vs. TG: -56% vs. -60%, p = .85). Tumor-induced muscle loss was attenuated by ghrelin from 16% to 8% in WT (p = .004) and from 25% to 19% in KO (trend in significance, p = .06). Similarly, the decrease in fiber cross-sectional area of GAS muscles was prevented by ghrelin in WT (TV vs. TG: p = .008; HK vs. TG: p = .472) but this difference did not reach significance in KO. In addition, the mRNA levels of ‘atrogenes’ atrogin-1 and MuRF-1 in GAS muscle significantly increased in response to tumor implantation in both strains. With ghrelin treatment, the increase of atrogin-1 and MuRF-1 in WT was decreased by 5-fold (p = .022) and 7-fold (trend, p = .058), respectively. In KO tumor-bearing mice, the attenuation of atrogin-1 and MuRF-1 by ghrelin was 5-fold (p = .008) and 6-fold (trend, p = .07), respectively. CONCLUSION: Ghrelin mitigates cancer-induced muscle atrophy at least in part through GHSR-1a-independent mechanisms. This work was funded by the U.S. Dept of Veterans Affairs (MERIT grants BX002807 and CX000174) and NIH Grant AG040583 to JMG.

Interactions between the Newly Described Small- and Fast-Swimming Mixotrophic Dinoflagellate Yihiella yeosuensis and Common Heterotrophic Protists.

The mixotroph Yihiella yeosuensis is a small- and fast-swimming dinoflagellate. To investigate its protistan predators, interactions between Y.yeosuensis and 11 heterotrophic protists were explored. No potential predators were able to feed on actively swimming Y. yeosuensis cells, which escaped via rapid jumps, whereas Aduncodinium glandula, Oxyrrhis marina, and Strombidinopsis sp. (approximately 150μm in cell length) were able to feed on weakly swimming cells that could not jump. Furthermore, Gyrodinium dominans, Luciella masanensis, and Pfiesteria piscicida were able to feed on heat-killed Yihiella cells, whereas Gyrodinium moestrupii, Noctiluca scintillans, Oblea rotunda, Polykrikos kofoidii, and Strombidium sp. (20μm) did not feed on them. Thus, the jumping behavior of Y.yeosuensis might be primarily responsible for the observed lack of predation. With increasing Yihiella concentration, the growth rate of O.marina decreased, whereas that of Strombidinopsis did not change. However, with increasing Yihiella concentration (up to 530ng C/ml), the ingestion rate of Strombidinopsis on Yihiella increased linearly. The highest ingestion rate was 24.1ng C per predator per d. The low daily carbon acquisition from Yihiella relative to the body carbon content of Strombidinopsis might be responsible for its negligible growth. Thus, Y.yeosuensis might have an advantage over its competitors due to its low mortality rate.

Lactobacillus plantarum AN1 cells increase caecal L. reuteri in an ICR mouse model of dextran sodium sulphate-induced inflammatory bowel disease

To clarify the different effects of live and heat-killed probiotics on inflammatory bowel disease (IBD), the anti-inflammatory and protective effects of Lactobacillus plantarum AN1 cells isolated from the fermented fish aji-narezushi on murine macrophage RAW264.7 cells and in ICR mice with dextran sodium sulphate (DSS)-induced IBD were determined. L. plantarum AN1 cells showed anti-inflammatory activities in vitro, indicated by secretion of nitric oxide (NO) from RAW264.7 cells in the presence and absence of Escherichia coli 0111 lipopolysaccharide (LPS). L. plantarum AN1 cells also protected RAW264.7 cells against hydrogen peroxide toxicity. There was no difference between these effects in heat-killed and ultraviolet irradiation-killed cells. In the murine IBD model, both live and heat-killed L. plantarum AN1 cells via drinking water tended to ameliorate atrophy of colon length, mucosal tissue damage, and spleen enlargement. Amplicon sequencing of 16S rDNA (V4) revealed that both live and heat-killed AN1 cells increased abundance of indigenous lactic acid bacteria, particularly Lactobacillus reuteri. The results suggest that increased indigenous lactic acid bacterial abundance and feeding with L. plantarum AN1 cells synergistically improve effects against IBD.