A replacement-type electrochemiluminescent aptasensor for lysozyme based on full-electric modification electrode coupled to silica-coated Ru(bpy)32+/silver nanospheres.

This research proposed a replacement-type electrochemiluminescent (ECL) aptasensor for lysozyme (LYZ) detection at trace levels based on a full-electric modification electrode (FEMG) coupled to silica-coated Ru(bpy)32+/silver nanospheres (Ru/SNs@SiO2). The multi-walled carbon nanotubes-doped-thionine (MWCNTs/PTn) electropolymerized modified electrode was decorated with electrodeposited gold nanoparticles (GNs) to form the FEMG. Then, the FEMG was utilized as sensing substrates for the immobilization of the anti-lysozyme aptamer (LA); the stability and number of LA attaching onto the FEMG were dramatically increased. The ECL measurement was used to evaluate the hybridization reaction of LA and the Ru/SNs@SiO2 marked DNA probe, and it was noted as Ia. After the combination of the LA with the LYZ, the target-triggered replacement of the DNA probe was actualized and the ECL measurement descended to Ib. The ECL difference (ΔIECL = Ia - Ib) before and after the replacement event was utilized for quantitation of LYZ. As a result, the fabricated aptasensor with great sensitivity and specificity achieved a wide linear range (10 fM-10 pM) and a low limit of detection (5 fM). It obtained satisfactory recovery for the detection of LYZ in human serum, and the results were identified with the LYZ ELISA kit. Therefore, the proposed ECL sensor is expected to become a promising approach in the field of biomolecule detection.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

Dietary supplementation of fermented lemon peel enhances lysozyme activity and susceptibility to Photobacterium damselae for orange-spotted grouper, Epinephelus coioides.

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Toxic effects of hexavalent chromium (Cr6+) on bioaccumulation, apoptosis, oxidative damage and inflammatory response in Channa asiatica.

The objective of this study was to evaluate the toxic effects of Cr6+ on bioaccumulation, digestion, immunity, oxidative stress, apoptosis and inflammation-related genes in Channa asiatica. The fish was exposed to waterborne Cr6+ concentrations (0, 0.5, 1.0 and 2.0 mg/L) for 28 and 56 days. Our results demonstrated that the accumulation of Cr6+ in tissues increased in a concentration-dependent manner, and the content in tissue was liver > gill > gut > muscle. Meanwhile, Cr6+ exposure led to a remarkable suppression of digestion, immunity and antioxidant capacity in C. asiatica. Inversely, MDA and PC content were positively correlated with Cr6+ exposure concentration. Furthermore, the expression of genes went up with the increase of waterborne Cr6+ concentration. Among them, HSP90, NF-κB and TNF-α have a sharp increase. These results elucidate that waterborne Cr6+ exposure may induce bioaccumulation, inhibit digestion and immunity, promote oxidative stress and up-regulate the expression of apoptosis and inflammation-related genes in C. asiatica.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

The moonlighting protein fructose 1,6-bisphosphate aldolase as a potential vaccine candidate against Photobacterium damselae subsp. piscicida in Asian sea bass (Lates calcarifer).

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (ß), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

Antioxidant enzyme activities and immune responses in rainbow trout (Onchorhynchus mykiss) juveniles fed diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts.

In the present study, antioxidant activity, immune responses, and growth performance of rainbow trout (Onchorhynchus mykiss) juveniles fed with diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts were assessed. Four different concentrations of aqueous methanolic extract of the plants (0% (control), 0.1%, 0.5%, and 1% (D, dandelion; L, lichen) were added to the diets, and fish were fed for 75 days. On the 15th, 45th, and 75th day of the study, liver antioxidant enzyme activities were determined, and immune responses were determined every 15th day. The results showed that SOD activity increased in the fish group of 0.1% D on the 15th and 45th day compared to control; however, it was lower in all the lichen extract-treated groups than in control at almost all sampling times, except on the 15th day in the 0.1% L group. CAT activity showed an increased value (P < 0.05) in 0.5% L and 1% L treated fish groups on the 15th day, in fish of 1% D and 1% L groups on 45th and on 75th day in 0.1% D group. GPX activity increased on the 15th day of the study in fish of 0.1% D group, on the 45th day in 1% D and 1% L groups and on the 75th day in fish of 0.5% D, 0.1% D, and 0.5% L groups (P < 0.05). G6PDH enhanced in all treatment groups compared to control on the 15th day, except in 0.1% L and 0.5% L groups. An elevated G6PDH activity was also observed on the 75th day of the study in 0.5% D, 1% D, and 0.5% L fish groups. An increase on lipid peroxidation (LP) was observed in all L groups on the 45th day of the study. Lysozyme activity was determined to be the highest in 0.5% and 1% L on the 45th day, in 0.1% L on the 60th day and in the 0.5% L fish group on the 75th day compared to control (P < 0.05). Myeloperoxidase was found to be the highest at the end of the study in 1% L fish group compared to the control (P < 0.05). In conclusion, we suggest the use of dandelion to combat oxidative stress and to lower FCR and the use of lichen to modulate the immune response in rainbow trout. The use of such products will be economical for aquaculture and harmless for the environment.

Label-free fluorescent sensor for one-step lysozyme detection via positively charged gold nanorods.

In the article, a simple and label-free strategy was proposed for the sensitive detection of lysozyme based on the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) to DNA-templated silver nanoclusters (DNA/AgNCs). To construct the sensor, a DNA template was designed with a C-rich sequence at the 5'-terminal for the synthesis of AgNCs, while a lysozyme binding aptamer (LBA) at the 3'-terminal for the recognition of lysozyme, and such DNA/AgNCs was used as the fluorescence probe. Meantime, the fluorescence signal of such DNA/AgNCs can be quenched based on the electrostatic adsorption of them with (+)AuNRs, due to the surface energy transfer. In the presence of lysozyme, the specific binding happened between the LBA section of DNA/AgNCs and lysozyme, inducing the reduction of the total charge of DNA/AgNCs and weakening the adsorption of them with (+)AuNRs, which directly resulting in the recovery of the fluorescence signal. Besides, the fluorescence signal recovery of DNA/AgNCs has a linear positive proportional relationship with lysozyme concentration in the range of 10 pM-2.0 nM under the optimal conditions. Moreover, a satisfactory recovery (99.6-107.2%) was obtained while detecting lysozyme in human serum samples. Graphical abstract A simple and label-free strategy was proposed for the sensitive detection of lysozyme based on the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) to DNA-templated silver nanoclusters (DNA/AgNCs).

Human milk immunomodulatory proteins are related to development of infant body composition during the first year of lactation.

BACKGROUND: To investigate relationships between infant body composition (BC) and human milk (HM) immunomodulatory proteins (IMPs) during the first 12 months of lactation. METHODS: BC of breastfeeding dyads (n = 20) was measured with ultrasound skinfolds (infants) and bioimpedance spectroscopy (infants/mothers) at 2, 5, 9, and/or 12 months post partum. Breastfeeding frequency, 24-h milk intake, and IMP concentrations (lactoferrin, lysozyme, secretory immunoglobulin A (sIgA)) were measured, and calculated daily intakes (CDIs) were determined. We used linear regression/mixed-effects models and adjusted results for multiple comparisons. RESULTS: No associations were seen between maternal characteristics and IMP concentrations/CDIs or between IMP concentrations and infant BC. Lactoferrin CDI was negatively associated with infant fat-free mass index (P = 0.002); lysozyme CDI was positively associated with infant fat mass (P = 0.004) and fat mass index (P = 0.004) measured with ultrasound skinfolds. CONCLUSION: In this small cohort of infants breastfed on demand during first year of life, we report differential associations of HM IMPs with infant BC, showing that in addition to their critical role in shaping infant immunity, lactoferrin, and lysozyme also influence development of infant BC, highlighting the importance of breastfeeding for 12 months and beyond. IMPACT: HM IMPs (concentrations and, most importantly, daily intakes) time-dependently and differentially associate with development of infant lean mass and adiposity during first 12 months of lactation. There is no information on how intakes and concentrations of these components affect development of infant BC. HM contains IMPs-lactoferrin, lysozyme, and sIgA, which not only play a critical role in shaping infant's immunity, but also influence infant growth and development of BC, highlighting the importance of breastfeeding for 12 months and beyond and warranting careful consideration of the dose effects of supplemented formula.

Tattoo skin reaction as a skin manifestation of systemic sarcoidosis.

A 41-year-old man presented with itching of the skin surrounding his tattoos, blurred vision, fever, general fatigue, and arthralgia. Physical examination revealed skin bulges confined to the tattoo ink lines. Histological analyses of the skin revealed non-caseating granulomas surrounding the tattoo inks. Together with other clinical manifestations including uveitis, lymph nodes swelling, and elevated serum angiotensin-converting enzyme and lysozyme, he was diagnosed with systemic sarcoidosis. The administration of prednisolone alleviated the sarcoidosis-related symptoms, including skin changes. This case illustrates that skin changes on tattoos can be a presenting manifestation of systemic sarcoidosis and that skin biopsy is useful in early diagnosis.

Lysozyme is a component of the innate immune system linked to obesity associated-chronic low-grade inflammation and altered glucose tolerance.

BACKGROUND & AIMS: Several proteins of the innate immune system are known to be deregulated with insulin resistance. We here aimed to investigate the relationship among circulating lysozyme (both plasma concentration and activity) and obesity-associated metabolic disturbances. METHODS: Plasma lysozyme concentration was determined cross-sectionally in a discovery (Cohort 1, n = 137) and in a replication cohort (Cohort 2, n = 181), in which plasma lysozyme activity was also analyzed. Plasma lysozyme was also evaluated longitudinally in participants from the replication cohort (n = 93). Leukocyte lysozyme expression (LYZ mRNA) were also investigated in an independent cohort (Cohort 3, n = 76), and adipose tissue (AT) LYZ mRNA (n = 25) and plasma peptidoglycan levels (n = 61) in subcohorts from discovery cohort. RESULTS: Translocation of peptidoglycan (as inferred from its increased circulating levels) was linked to plasma lysozyme, hyperinsulinemia and dyslipidemia in obese subjects. In both discovery and replication cohorts, plasma lysozyme levels and activity were significantly increased in obesity in direct association with obesity-associated metabolic disturbances and inflammatory parameters, being circulating lysozyme negatively correlated with fasting glucose, HbA1c and insulin resistance (HOMA-IR) in obese subjects. Of note, total cholesterol (p < 0.0001) and LDL cholesterol (p = 0.003) contributed independently to age-, gender- and BMI adjusted plasma lysozyme activity. Longitudinally, changes in HbA1c levels and serum LDL cholesterol were negatively associated with circulating lysozyme antimicrobial activity. On the contrary, the change in glucose infusion rate during the clamp (insulin sensitivity) was positively associated with lysozyme concentration. CONCLUSIONS: Increased plasma lysozyme levels and activity are found in obese subjects. The longitudinal findings suggest that plasma lysozyme might be protective on the development of obesity-associated metabolic disturbances.

Efficacy of mozuku fucoidan in alternative protein-based diet to improve growth, health performance, and stress resistance of juvenile red sea bream, Pagrus major.

We evaluated the efficacy of mozuku fucoidan supplementation to alternative dietary proteins used in fish meal (FM) replacement to enhance growth, immunity, and stress resistance of Pagrus major. Seven isonitrogenous (45% protein) experimental diets were formulated where diet 1 (D1) was FM-based control diet. Diets 2 to 7 were formulated by replacing 25, 50, and 75% of FM protein with soy protein isolate (SPI) protein, and each replacement level was supplemented without or with fucoidan at 0.4% for diet groups D2 (FM25), D3 (FM25Fu), D4 (FM50), D5 (FM50Fu), D6 (FM75), and D7(FM75Fu), respectively. Each diet was randomly allocated to triplicate groups of fish (4.1 g) for 56 days. Significantly higher weight gain and specific growth rate were observed in fish fed FM50Fu diet group, and it was not differed (P > 0.05) with fish fed FM25Fu diet group. FM-based control diet showed intermediate value, and it was not differed (P > 0.05) with or without fucoidan-supplemented ≤ 50% FM replacement groups and FM75Fu diet group. Significantly lower growth performances were observed in FM75 diet group. At each replacement level, fucoidan-supplemented groups showed nonsignificant improvement of feed utilization performances. Fish fed fucoidan-supplemented diets showed best condition of oxidative and freshwater stress resistance. Lysozyme activity, NBT, and peroxidase activity showed higher (P > 0.05) values in fucoidan-supplemented groups compared with the non-supplemented groups. Catalase activity was significantly lower in FM75Fu diet group. Catalase activity is significantly influenced by the interaction effects of fucoidan and FM replacement level. In conclusion, fucoidan supplementation could increase the efficiency of utilizing SPI (≥ 75%) without any adverse effects on red sea bream performance.

Effect of photoperiod and temperature on indicators of immunity and wellbeing of endangered golden mahseer (Tor putitora) broodstock.

Recently, we reported the possibility of successfully inducing captive maturation and spawning in golden mahseer through photothermal manipulation. Subsequently, we felt that it was imperative to understand the impact of these environmental manipulations on immunity, stress response, antioxidant potential, and general well-being of adult mahseer to develop a healthy broodstock. For this purpose, two experiments were carried out with changes in photoperiod (experiment I) and temperature (experiment II). In experiment I, random groups of adult female and male Tor putitora were subjected to three photoperiods (8L:16D, 12L:12D and 16L:8D) for 100 days. Decreasing levels of plasma melatonin with increasing photoperiod confirmed the physiological significance of different light-dark conditions in mahseer brooders. In terms of stress, plasma cortisol levels showed a linear increase with decreasing light duration in both males and females. Similarly, the level of thiobarbituric acid reactive substances was also significantly higher in males kept at 8L:16D. Plasma concentration of total immunoglobulins was found reduced in female brooders at 8L:16D, but this was not evident in males. In females, total antioxidants were found significantly elevated at 12L:12D. On the contrary, superoxide dismutase activity was lower at 12L:12D in females. The photoperiod has substantially influenced the plasma total protein and albumin levels in males. In experiment II, random groups of adult T. putitora were reared at ambient (21.2 ± 1.4 °C) or elevated temperature (23.7 ± 1.3 °C) groups for 121 days. The higher temperature was found to significantly decrease lysozyme, myeloperoxidase, and anti-protease activities in female mahseer brooders. However, total immunoglobulin levels were reduced significantly at elevated temperature both in males and females. No other temperature-related significant changes were observed in antioxidant potential, anti-oxidative enzymes or well-being related indices.

Immunomodulation, antioxidant enhancement and immune genes up-regulation in rainbow trout (Oncorhynchus mykiss) fed on seaweeds included diets.

This study investigated the stimulatory effects of dietary inclusion of Gracilariopsis persica (GP), Hypnea flagelliformis (HF) and Sargassum boveanum (SB) on immune indices, antioxidant capability and immune related genes expression of rainbow trout (Oncorhynchus mykiss). Seven iso-nitrogenous and iso-caloric diets with 0, 5 and 10% of each macroalgae were prepared and fed to rainbow trout juveniles for 83 days. Serum lysozyme (Lyz) and respiratory burst activity (NBT) along with activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and expression of LyzII, TNFα and IL-1ß genes in head kidney samples were determined by days 47 and 83. Our results revealed that dietary inclusion of seaweeds improved fish immune status. Long term feeding of fish on seaweed contained diets (except for GP10) improved serum Lyz activity in comparison to control group. Similarly, extended feeding on GP5 and HF10 and HF10 included diets improved SOD and POD levels, respectively. Genes expression studies revealed that seaweeds contained diets noticeably enhanced expression of LyzII, TNFα and IL-1ß in comparison to control fish. However, results revealed that such stimulatory effects were more evident at lower dietary inclusion level and shorter feeding time. In conclusion, the results depicted that dietary inclusion of the seaweeds effectively improved serum immune indices and head kidney antioxidant status and immune related genes expression in a time and dose dependent manner.

Effects of dietary fern (Adiantum capillus-veneris) leaves powder on serum and mucus antioxidant defence, immunological responses, antimicrobial activity and growth performance of common carp (Cyprinus carpio) juveniles.

A 56-day research was performed to examine the influence of graded levels (0 (control), 0.5, 1 and 2%) of Fern (Adiantum capillus-veneris) leaves powder (FLP) in diet on immune competence and growth of common carp (Cyprinus carpio, initial weight = 20 g). The serum total immunoglobulins content and lysozyme activity in the 1 and 2% FLP groups remarkably increased compared to the other groups (P < 0.05). The skin mucosal lysozyme activity enhanced with increasing dietary FLP level in a dose-response manner. Fish fed on the FLP-supplemented diets had higher skin-mucosal superoxide dismutase activity than the control (P < 0.05). However, serum antioxidant enzymes were not affected by dietary fern (P > 0.05). The serum bactericidal activity against human and fish pathogens increased with enhancing the FLP level in diet against Staphylococcus aureus, Escherichia coli (EHEC ATCC 43895), Escherichia coli (CI), Pseudomonas aeruginosa, Klebsiella pneumonia and Aeromonas hydrophila. The serum antibacterial activity against Yersinia ruckeri in the 2% FLP group was higher than the other treatments. Furthermore, the serum bactericidal activity against P. aeruginosa (ATCC 27853) only observed in fish fed on the 1 and 2% FLP-supplemented diets. The skin mucosal bactericidal activity and inhibitory effects increased with enhancing the FLP level in diet against E. coli, K. pneumonia, Y. ruckeri and A. hydrophila in a dose response manner. Moreover, the skin mucosal bactericidal activity against S. aureus only observed in fish fed on 1 and 2% FLP-supplemented diets. The weight gain values in the 1 and 2% FLP groups were higher than the other treatments (P < 0.05). Feed conversion ratio (FCR) improved with increasing FLP level in diet in a dose-response manner (P < 0.05). By considering serum and mucosal bactericidal activities against different pathogenic bacteria, the supplementation of 2% FLP in diet is recommended for C. carpio during grow-out phase.

Photoluminescent hydrophilic cyclodextrin-stabilized cysteine-protected copper nanoclusters for detecting lysozyme.

Lysozyme (LYZ) sensors have attracted increased attention because rapid and sensitive detection of LYZ is highly desirable for various diseases associated with humans. In this research, we designed L-cysteine-protected ultra small photoluminescent (PL) copper nanoclusters (CuNCs) conjugated with ß-cyclodextrin (ß-CD) for rapid detection of LYZ in human serum samples at room temperature. The proposed ß-CD-CuNCs exhibited excellent water solubility, ultrafine size, good dispersion, bright photoluminescence, and good photostability. The ß-CD-CuNCs exhibit an excitation and emission maximum at 370 nm and 492 nm, respectively, with an absolute quantum yield (QY) of 54.6%. ß-CD-CuNCs showed a fluorescence lifetime of 12.7 ns. The addition of LYZ would result in PL quenching from ß-CD-CuNCs. The lowest detectable LYZ concentration was 50 nM for the naked eye and the limit of detection (LOD) and limit of quantification (LOQ) were 0.36 nM and 1.21 nM, respectively, by emission measurement observed in the LYZ concentration range from 30 to 100 nM. It is important to note that the PL ß-CD-CuNC strategy possessed great selectivity toward LYZ relative to other biomolecules. The proposed nanosensor was efficiently applied to determine the LYZ level in human serum sample average recoveries from 96.15 to 104.05% and relative standard deviation (RSD) values lower than 3.0%. Moreover, the proposed sensing system showed many advantages, including high speed, high sensitivity, high selectivity, low cost, and simple preparation.

Effects of ammonium sulfate on stress physiology and innate immunity of Western mosquitofish (Gambusia affinis).

Fertilizers increase yield of crops but may have unintended negative effects on fish as a byproduct of runoff into bodies of freshwater. The objective of this study was to determine if environmentally relevant concentrations of an ammonium fertilizer impacts stress and innate immunity in Western mosquitofish (Gambusia affinis). The mosquitofish were exposed to different concentrations of ammonium sulfate fertilizer: 0 ppm, 40 ppm, and 80 ppm. To test the effects of ammonium sulfate on stress physiology, cortisol released into water by individual fish was collected after 1 week of exposure and again after 2 weeks of exposure and quantified with an enzyme immunoassay. Cortisol levels in the 0-ppm group were not significantly different over the course of the study, but we found a significant increase in cortisol levels in the fish exposed to 40 ppm and 80 ppm. We found reduced survival in fish from the 40 ppm and 80 ppm of ammonium sulfate groups compared with the 0-ppm group. We also used blood samples to complete a lysozyme assay as a measure of innate immune defense. Higher concentrations of ammonium sulfate correlated with significantly lower lysozyme activity in the fish. Overall, our results suggest that relatively low amounts of ammonium sulfate runoff into bodies of water are likely to have negative sublethal and lethal effects on small fishes.

Ziziphus mauritiana supplementation of Nile tilapia (Oreochromis niloticus) diet for improvement of immune response to Aeromonas hydrophila infection.

This study evaluated the effectiveness of dietary Ziziphus mauritiana leaf powder (ZLP) to control Aeromonas hydrophila infection in Nile tilapia and reduce damage to vital immune organs. Four experimental groups were fed a diet supplemented with ZLP at concentrations of 0, 5, 10, and 20 g/kg (w/w) for 6 weeks. At the end of the feeding trial, all groups were intraperitoneally injected with pathogenic A. hydrophila. It was found that Z. mauritiana significantly (P < 0.05) upregulated (lysozyme, interleukin 1 beta) and superoxide dismutase gene expressions as well as improved the activity of serum lysozyme and liver antioxidant enzymes. The fish that were fed a ZLP-supplemented diet also exhibited significantly higher survival rates after A. hydrophila challenge than those that were fed a ZLP-free diet (P < 0.05). Supplementation of 10 g/kg ZLP most effectively reduced the histopathological alterations caused by A. hydrophila challenge in the liver, spleen, kidney, and muscle of the fish. In conclusion, ZLP can be effective in controlling A. hydrophila infection in Nile tilapia (particularly at a concentration of 10 g/kg) through enhancement of its immune and antioxidant status.

Effect of acclimated temperature on thermal tolerance, immune response and expression of HSP genes in Labeo rohita, Catla catla and their intergeneric hybrids.

The ability of a species and population to respond to a decrease or an increase in temperature depends on their adaptive potential. Here, the critical thermal tolerance (CTmax and CTmin) of four populations: Labeo rohita, Catla catla, and their reciprocal hybrids L. rohitaâ™€× C. catla♂ (RC) and C. catla♀ × L. rohita♂ (CR) being acclimatized at four acclimation temperatures (22, 26, 30 and 34 °C) were determined. All populations indicated substantial variations (P < 0.05) in CTmax and CTmin values. L. rohita displayed, comparatively the highest CTmax with largest total and intrinsic polygon zones as well as the upper and lower acquired thermal tolerance zones followed by RC and CR hybrids, while C. catla showed significantly the highest CTmin value and the smallest intrinsic and acquired thermal tolerance zones. Both hybrids illustrated low parent heterosis (≤11%). Additionally, the highest expression of Hsp70 and Hsp90 (heat shock proteins) genes, serum lysozyme level, respiratory burst activity and lowest lipid peroxidation level under lower and higher temperature shock further illustrated strong physiological mechanism of L. rohita in contrast to C. catla, to deal with acute temperature, while hybrids, especially F1 RC hybrid appeared as a good option to replace C. catla in relatively higher and lower temperature areas.