Hydrophila Injection Research Articles

Immune response for acute Aeromonas hydrophila infection in two distinct color morphs of northern snakehead, Channa argus

To compare and analyze the differences in immunological response between the two color morphs of Channa argus, a fish cohort was divided into four groups: black C argus + PBS (B-PBS), black C argus + Aeromonas hydrophila (B-Ah), white C. argus + PBS (W-PBS), and white C. argus + A hydrophila (W-Ah). The B-PBS and W-PBS groups received 100 μL PBS, while the B-Ah and W-Ah groups received 3.6 × 105 CFU/mL A. hydrophila in the same volume. The death rate in each group was noted, changes in plasma biochemical indicators and the expression of liver immune-related genes were examined, and transcriptome techniques were used to compare the differences between the two colors of C. argus following stress. No mortality occurred in the B-PBS and W-PBS groups. Mortality in the W-Ah and B-Ah groups showed an upward and then downward trend after A. hydrophila injection. The highest mortality occurred within 24 h and was higher in the W-Ah group than in the B-Ah group. MDA levels in the B-Ah and W-Ah groups increased and then decreased, while SOD and T-AOC showed the reverse tendency. The W-Ah and W-PBS groups differed significantly in MDA at 3, 12, and 24 h, SOD from 6 to 96 h, and T-AOC between 6 and 48 h. Plasma MDA and T-AOC levels at 12 h and SOD levels at 24 and 48 h differed significantly between the B-PBS and B-Ah groups. In both the W-Ah and B-Ah groups, the expression levels of IL-1β and IL-8 in the liver showed a temporal pattern with an initial increase followed by a decrease, reaching peak levels after 24 h, while IL-10 showed the reverse pattern. Transcriptome analysis of the liver revealed significant differences between the two C. argus colors. Differential genes in black C. argus were mainly enriched in steroid biosynthesis, glycolysis/gluconeogenesis, and glutathione and propanoate metabolism pathways 24 h after infection. In contrast, differential genes in white C. argus were mainly enriched in pathways such as oxidative phosphorylation, pancreatic secretion, and protein digestion and absorption 24 h after infection. After A. hydrophila infection, white C. argus had higher mortality, more severe oxidative stress and inflammatory responses, and lower antioxidant capacity than black C. argus.

Beneficial effects of the butanoic acid derivative tributyrin on the growth, immunity and intestinal health of large mouth bass (Micropterus salmoides)

Tributyrin (TB) is a novel butanoic acid derivative with potent bioactive functions. In this study, an eight-week feeding trial aimed to investigate the effects of dietary TB on the growth, immunity and intestinal health of large mouth bass (Micropterus salmoides). Five experimental diets containing 0, 0.5, 1.0, 1.5 or 2.0 g/kg tributyrin were formulated and were regarded as TB0 (control), TB0.5, TB1.0, TB1.5 and TB2.0, respectively. Quadruplicate groups (four tanks per group, 30 fish per tank) of juvenile M. salmoides (initial weight: 20.00 ± 1.00 g) were fed to apparent satiation two times daily. After the feeding trial,Aeromonas hydrophila challenge tests were conducted and the A. hydrophila strains were subsequently sampled. As a result, the weight gain rate (WGR) was increased in the TB supplementation groups, and the TB1.5 group obtained the highest value. Furthermore, TB administration improved immune function. The content of serum complement 4 in the TB1.5 group was significantly higher than that in the control group. Increase of lysozyme activity and immunoglobulin M were significant in TB1.0 group than the control group. After A. hydrophila injection, TB1.0, TB1.5 and TB2.0 groups presented markedly higher survival rates than did the control group. Additionally, TB markedly enhanced the activities of amylase and lipase in the gut. Moreover, 16S rRNA sequencing revealed that TB could affect the intestinal microflora composition. Compared to those in the control group, the abundance of Fusobacteria were lower in the TB1.0 and TB1.5 groups. In conclusion, this study demonstrated that TB can improve the growth, immunity and intestinal health of M. salmoides, and the optimal dietary level of TB is determined to be 0.15%.

Hepcidin from Onychostoma macrolepis: Response to Aeromonas hydrophila infection by down-regulating expression of inflammatory factor genes and regulating iron homeostasis potentially

Hepcidin, as an antimicrobial peptide, is associated with innate immunity and is considered a potential antibiotic substitute. In the present study, the hepcidin gene from the cavefish - Onychostoma macrolepis was identified and analyzed. The recombinant hepcidin protein (rOmhepc) was obtained by prokaryotic expression, evaluating the inhibitory effect of 5 pathogenic bacteria in vitro. Sixty O. macrolepis injected with 100 μL A. hydrophila (1.5 × 108 CFU/mL) were randomly divided into the therapeutic group and infection group, and therapeutic group was injected with 100 μL rOmhepc (100 μg/mL) at 6 and 18 h. The survival rates of O. macrolepis and bacterial load in liver were measured at 24 h. The liver tissues were collected at 0, 6, 12, and 24 h after A. hydrophila injection for investigating expression levels of immune-related, inflammatory factor genes and FPN1 gene. The results demonstrated that the hepcidin CDS contained 279 bp and encoded 93 aa. Hepcidin protein has a hydrophobic surface formed by multiple hydrophobic residues (CCGCCYC), and the theoretical pI was 7.53. Omhepc gene was expressed at varying levels in tested tissues, with the liver showing the highest expression, followed by the spleen. The expression of hepcidin gene following A. hydrophila infection was up-regulated and then down-regulated in liver, and the highest expression level was found at 12 h with a 10.93-fold. The rOmhepc remarkably inhibited the growth of A. hydrophila, Staphylococcus aureus, and Streptococcus agalactiae, with inhibition rates reaching 69.67 %, 42.97 %, and 65.74 % at 100 μg/mL. The mortality rates of O. macrolepis and bacterial load in liver were significantly decreased in the therapeutic group than that of infection group (p < 0.05). After the rOmhepc therapeutic, interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly down-regulated with 14.4-fold and 106.07-fold at 24 h. Furthermore, the expression of immune-related genes (C3, TNF-α, IFN-γ) and Ferroportin gene (FPN1) significantly decreased (p < 0.05).The integrated analyses indicated that the rOmhepc could significantly inhibit the growth of A. hydrophila both in vitro and in vivo, attenuating the over-expression of inflammatory factor, FPN1 and immune-related genes.

EFEKTIVITAS WADAH BUNDAR BERARUS TERHADAP PENINGKATAN RESPON IMUN BENIH IKAN MAS (Cyprinus carpio) YANG DIINFEKSI OLEH BAKTERI Aeromonas hydrophila

This research focused on determining the best survival performance of carp (C. carpio) fingerlings in circular containers combined with venturi aerators to generate water current, then challenged by A. hydrophila. The research used an experimental method of Completely Randomized Design (CRD) with four treatments and four replications. The treatment used was the use of venturi aerators on circular containers: without water current as a control (A), with a water current of 0.06 ms?¹ (B), 0.1 ms?¹ (C), and 0.14 ms?¹ (D). The study was initiated by rearing 5-7 cm fish fingerlings for 40 days and followed by a challenge test for 14 days. Carp fingerlings were challenged by A. hydrophila intraperitoneally at a density of 10? CFU ml?¹. Each tank consists of 15 fingerlings. Parameters observed included survival rate, the number of red blood cells and white blood cells, macroscopic symptoms, and water quality. The results showed survival rate in treatment (D) was the best (water current of 0.14 ms?¹), 98% before the challenge period and 95% after the challenge period. In addition, it can increase the hematological parameters as seen from the increase in the number of red and white blood cells after the challenge test, from 1.043 x 106 cells mm³?¹ to 2.36 x 106 cells mm³?¹ and 2.7 x 104 cells mm³?¹ to 9.3 x 104 cells mm³?¹ respectively. And it shows an increased immune response as seen from the recovery acceleration in macroscopic symptoms on the seventh day after A. hydrophila injection. While water quality did not differ between control and treatments.Keywords : current circular generating tank, carp fingerling, Aeromonas hydrophila, immune response

Dietary administration of Bacillus subtilis , inulin and its synbiotic combination improves growth and mitigates stress in experimentally infected Pseudoplatystoma reticulatum

Juveniles of Pseudoplatystoma reticulatum (36.25 g and 19.32 cm) were randomly distributed in 16 tanks with 80 L of water. The fish were fed with diets containing probiotic (2%), prebiotic (0.5%), synbiotic (2% probiotic +0.5% prebiotic) and diet without additives (control). After 60 days of feeding, the fish were challenged with inoculation of the Aeromonas hydrophila (0.9 × 106 CFU/mL). Growth performance, physiological, immunological and haematological analyses were evaluated. Fish fed with probiotic, inulin and synbiotic diet showed greater (p = 0.018) weight gain when compared to control fish, and the condition factor was better (p < 0.001) in fish fed with probiotic. Cortisol concentrations increased in all treatments after challenge (p = 0.27), however, after A. hydrophila injection of 6 h, the cortisol levels increased significantly in fish that received control diet. The haematocrit (p < 0.001) and lymphocytes percentages (p < 0.001) decreased after A. hydrophila injection of 3 and 6 h, and neutrophils percentages increased (p < 0.001), without difference among treatments. The not supplemented and supplemented with prebiotic and synbiotic fish groups had decreased haemoglobin concentration (p = 0.01) at after 3 and 6 h. The juvenile catfish fed with probiotic (2% Bacillus subtilis), prebiotic (0.05% inulin) and the synbiotic combination for 60 days had better productive performance, ensuring greater weight gain and growth. The supplemented diets inhibited the cortisol increase when fish are challenged with A. hydrophila, but it does not inhibited the effects of the stress imposed by the bacterial infection.

Molecular identification and functional characterization of IRF4 from common carp (Cyprinus carpio. L) in immune response: a negative regulator in the IFN and NF-\u03baB signalling pathways

BackgroundThe interferon (IFN) regulatory factors (IRFs) were originally identified as transcription factors playing critical roles in the regulation of IFN-related genes in the signal pathway. In mammals, IRF4 plays a vital role in both the innate and adaptive immune system. This study aims to reveal the molecular characterization, phylogenetic analysis, expression profiles and the regulatory role in the IFN and NF-κB signalling pathways of IRF4 in common carp (Cyprinus carpio. L) (abbreviation, ccIRF4).ResultsHere, ccIRF4 was identified and characterized, it contained a DNA binding domain (DBD) which possess five tryptophans and an IRF-associated domain (IAD). The predicted protein sequence of the ccIRF4 showed higher identities with grass carp (Ctenopharyngodon idella) and zebrafish (Danio rerio). Phylogenetic analysis suggested that ccIRF4 has the closest relationship with zebrafish IRF4. Quantitative real-time PCR analysis showed that ccIRF4 was constitutively expressed in all investigated tissues with the highest expression level in the gonad. Polyinosinic:polycytidylic acid (poly I:C) stimulation up-regulated the ccIRF4 expressions in the liver, spleen, head kidney, skin, foregut and hindgut. Upon Aeromonas hydrophila injection, the expression level of ccIRF4 was up-regulated in all tissues with the exception of spleen. In addition, ccIRF4 was induced by lipopolysaccharide (LPS), peptidoglycan (PGN) and Flagellin in head kidney leukocytes (HKLs). Overexpression of the ccIRF4 gene in epithelioma papulosum cyprini cells (EPC) down regulated the expressions of IFN-related genes and proinflammatory factors. Dual-luciferase reporter assay revealed that ccIRF4 decreased the activation of NF-κB through MyD88.ConclusionsThese results indicate that ccIRF4 participates in both antiviral and antibacterial immune response and negatively regulates the IFN and NF-κB response. Overall, our study on ccIRF4 provides more new insights into the innate immune system of common carp as well as a theoretical basis for investigating the pathogenesis and prevention of fish disease.

Effect of infection with Aeromonas hydrophila on antioxidant capacity, inflammation response, and apoptosis proteins in Chinese mitten crab (Eriocheir sinensis)

Aeromonas hydrophila (A. hydrophila) as a serious bacterial disease endangering aquaculture and the Chinese mitten crabs (Eriocheir sinensis) industry. The present study was conducted to investigate the effects of A. hydrophila on the antioxidant, inflammation, immunity and apoptosis of the E. sinensis. The E. sinensis (female: 150 crabs and male: 150 crabs; 67.11 ± 0.76 g) were randomly divided into the control group (Foot injection with 200 μl PBS) and infection group (Foot injection with 200 μl A. hydrophila of 106 cfu/mL). The hepatopancreas and serum was collected to detect the related indicators after injection 24 h. The results showed that A. hydrophila significantly reduced the malondialdehyde (MDA) level and gamma-glutamyl-cysteine synthetase (γ-GCS) activity in the hepatopancreas of male and female crabs (P < 0.05). A.hydrophila also significantly decreased the total-superoxide dismutase (T-SOD) activity while the levels of total antioxidant capacity (T-AOC) and total glutathione (T-GSH) were significantly increased in the hepatopancreas and serum of male crabs (P < 0.05). At the transcriptional level, the expression of catalase (CAT) and glutathione peroxidases (GPx), Glutathione S-transferase (GST) in the hepatopancreas of male and female crabs was significantly reduced compared to the control group (P < 0.05). However, A. hydrophila could not significantly change the Kelch-like ECH-associated protein 1 (Keap1) gene expression level in both of male and female carbs. A. hydrophila injection for 24 h, the lysozyme (LZM) and phenoloxidase (PO) activity was significantly increased in the hepatopancreas and serum of the male and female crabs (P < 0.05). Simultaneous increase of immune-related enzyme activity (acid phosphatase and alkaline phosphatase) was found in the serum of male and female crabs (P < 0.05). However, the acid phosphatase (ACP) and alkaline phosphatase (ALP) activity was significantly decreased in the hepatopancreas of male and female crabs (P < 0.05). Meanwhile, the LZM mRNA level was significantly decreased in the hepatopancreas of E. sinensis (P < 0.05). Furthermore, A. hydrophila significantly inhibited the mRNA expression of immune regulated factors (Interleukin enhancer binding factor 2: ILF2, interleukin-16: IL-16, Toll-like receptor: TLR) in the male and female crabs. The levels of inflammatory cytokines (interleukin-1β: IL-1β, interleukin-6: IL-6, interleukin-8: IL-8, interleukin-10: IL-10) were significantly increased in the hepatopancreas of male and female crabs. Moreover, A.hydrophila increased the mRNA expression of apoptosis - related genes in male crabs (p38 mitogen-activated protein kinase: p38, adamalysin 17: ADAM17, Cysteine-aspartic acid protease 3: Caspase 3, and Bcl-2-associated X: BAX), but reduced the expression of p38, ADAM17, Caspase 3 and BAX genes in female crabs. In conclusion, A. hydrophila could induce oxidative stress and the response of inflammation and immunity, and also trigger the mRNA expression changes of apoptosis related-genes in E. sinensis. This study provides a theoretical basis for the study of E. sinensis diseases.

Identification and functional characterization of a fish-specific tlr19 in common carp (Cyprinus carpio L.) that recruits TRIF as an adaptor and induces ifn expression during the immune response

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp (Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence (Cctlr19) was identified in common carp. Phylogenetic analysis revealed that CcTlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that CcTlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that CcTlr19 recruited TRIF as an adaptor. Furthermore, CcTlr19 can activate the expression of ifn-1 and viperin. Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19.

RNase1 can modulate gut microbiota and metabolome after Aeromonas hydrophila infection in blunt snout bream.

Pancreatic ribonuclease (RNase1) of Megalobrama amblycephala exhibits both antimicrobial and digestive activity. The gut microbiome improve the digestion and metabolic capacity and enhance the functioning of the immune system of the host against pathogenic bacteria. In this study, we aimed to assess the protective effect of RNase1 on Aeromonas hydrophila-induced inflammation and intestinal microbial metabolism. Megalobrama amblycephala were randomly divided into three groups: control (injected PBS), infection (A. hydrophila-injected), and treatment group (RNase1 pretreatment 24 h before the A. hydrophila injection). The morphological symptoms were significantly alleviated by RNase1. RNase1 reshaped the perturbed gut microbiota by upregulating Proteobacteria and Vibrio richness and downregulating Firmicutes, Chlamydiae, Bacillus, and Gemmobacter richness. The lysophosphatidylcholine, (±) 17 HETE, D- (+) -cellobiose, and PC (20:5) in the treatment group were restored by RNase 1 protein treatment to the level of the control group. In the treatment group, phospholipid metabolism, fatty acid metabolism, glucose metabolism and lipid metabolism were different from the control and infection groups. The proinflammatory factors concentration in intestinal samples significantly increased after A. hydrophila infection. Our results revealed that RNase1 plays an important role in resistance to pathogen invasion, reducing inflammation, and improving intestinal function, thus inhibiting the occurrence of disease.

The Effect of Adding Bacillus NP5 to Feed on Growth, Survival Rate, and Protection Against Aeromonas hydrophila of Catfish (Clarias sp.)

Probiotics are useful organisms in preventing the spread of disease in aquaculture. Therefore this study aims to evaluate the effect of administering Bacillus NP5 on the growth, survival rate, and histology of catfish with Aeromonas hydrophila infection. To achieve this, catfish (7.75 ± 0.02 g) were reared for 45 days with a density of 20 per tank. Furthermore, the study consisted of 4 treatments and 3 replications, namely K+: 0% Bacillus NP5+0.1 mL A. hydrophila injection, K-: 0% Bacillus NP5 + without A. hydrophila injection, A: 0.3% Bacillus NP5, and B: 0.8% Bacillus NP5. The results showed that the addition of probiotics significantly increased the growth and survival rate of catfish (P <0.05) compared to K+. The higher value of specific growth rate was found in treatments A and B (0.22 ± 0.03 and 0.23 ± 0.03% day-1, respectively) and the lowest in K+ (0.14 ± 0.03% day-1). Also, the tissue damage in K+ was higher than in the probiotic treatment. Besides, hyperplasia, congestion, and secondary lamella fusion in the gill tissue were found in the K+, while other treatments were found only in hyperplasia. Melanomacrophages occurred in catfish kidney tissue for all treatments, however, hydropic degeneration was found in the control treatment. The addition of Bacillus NP5 in feed resulted in higher growth, survival, and tissue damage compared to K+. Also, the addition of 0.8% Bacillus NP5 resulted in less catfish tissue damage.

Secretory immunoglobulin M (sIgM) heavy chain gene in Siniperca kneri Garman: Molecular characterization and expression analysis in response to Aeromonas hydrophila challenge

Immunoglobulin M (IgM) is widely recognized as the major isotype of teleost immunoglobulins. Its role in protecting fish against a number of pathogens is critical. In the current study, the IgM heavy chain gene of Siniperca kneri Garman was cloned for the first time. Its IgM heavy chain cDNA is 1952 bp in length and encodes a polypeptide of 583 amino acid residues, which includes a signal peptide, a variable region (VH) and four constant regions (CH1, CH2, CH3 and CH4). This confirms the secretory pattern of IgM (sIgM). The sIgM amino acid sequence shares high identity with those of Siniperca chuatsi (88.57%), Lateolabrax japonicus (65.73%) and Latris lineata (64.92%). Analysis of phylogenetic trees shows that the sIgM of S. kneri Garman is clustered with perciformes fish homologues and constructed as one clade. QRT-PCR analysis found the highest sIgM expression in the head kidney, with higher expressions in the spleen and liver than in the brain, intestine, mesonephros, gill, skin and heart. After Aeromonas hydrophila injection, the sIgM expression pattern was up-regulated and peaks occurred in the head kidney, spleen, intestine and gill on days 5, 7 and 21 post-challenge. Changes in the peak expression of sIgM compared to controls were most significant in the head kidney (38.59-fold) and spleen (23.75-fold), with smaller differences in the gill (9.54-fold) and intestine (7.75-fold). This suggests that it is possible that sIgM is of greater importance to systemic immunity than mucosal immunity. Its importance to the adaptive immune response is thereby confirmed.

Combination of Moringa Leaf Meal and Probiotics in Feed for Tilapia (Oreochromis niloticus) Seeds Survival and Immune System

Prevention of disease in tilapia seeds can be done by increasing non-specific defense systems by improving and supplementing the nutritional content of the feed. This study aims to determine the effect of adding Moringa leaf meal combined with probiotics to feed on the survival rate of tilapia seeds and the number of seed blood cells that have been infected with Aeromonas hydrophila. The method used was an experimental method using a Completely Randomized Design (CRD) with 5 treatments and 3 replications. Treatment A = addition of 4% moringa leaf meal + 6 ml / kg of probiotic feed + A. hydrophila injection; Treatment B = addition of 6% moringa leaf meal + 6 ml / kg of probiotic feed + A. hydrophila injection; Treatment C = addition of 8% moringa leaf meal + 6 ml / kg of probiotic feed + A. hydrophila injection; Treatment D = feed + probiotic 6 ml / kg of feed (positive control) + injection of A. hydrophila; Treatment E = feed + probiotic 6 ml / kg of feed (negative control) + injection of physiological solution. The results showed the addition of Moringa leaf meal combined with probiotics got the best results in treatment C with a survival value of 86.67%, hemoglobin levels of 2.3 g%, erythrocytes of 2.20 × 106 cells, leukocytes of 11.5 × 104 cells, and the total number of intestinal bacteria was 10.34 × 106 cfu / ml.

Transcriptome analysis of the immunomodulation by Arctium lappa L. polysaccharides in the Chinese mitten crab Eriocheir sinensis against Aeromonas hydrophila

Arctium lappa L. polysaccharide (designated ALP) has been used as an immunomodulator in animals. Immunomodulation by ALP in the Chinese mitten crab Eriocheir sinensis was detected after Aeromonas hydrophila treatment (1.0 × 107 colony-forming units) in this study. ALP significantly inhibited the decrease in the total hemocyte count (THC) (P < 0.05). Twenty transcriptome libraries were constructed from the hemocytes of crabs, and in total, 600,827,724 reads and 794,113 transcripts were obtained and clustered into 337,358 unigenes, which were annotated in the CDD, KOG, NR, NT, PFAM, TrEMBL, GO and KEGG databases. The differentially expressed gene (DEG) analysis indicated that 97,800 genes were upregulated and 103,745 genes were downregulated, among which 2616 and 932 immune-related DEGs were enriched according to the GO and KEGG annotations at 12 h, respectively (Q value<0.05). EsDorsal, EsPI3K, EsLectin and EsLGBP mRNA were upregulated, and STAT mRNA was downregulated significantly after A. hydrophila injection. ALP treatment inhibited the increase in EsDorsal and EsPI3K expression and promoted the increase in EsLectin and EsLGBP expression. The alkaline phosphatase (AP) and glutamyl transpeptidase (GT) activities in the supernatant of the hemolymph increased significantly after ALP treatment, and the immunofluorescence assay indicated that ALP could inhibit the upregulation of TNF-α and IL-6 in hemocytes of E. sinensis challenged by A. hydrophila at 12 h. Therefore, it was concluded that ALP played an important role in effective immunomodulation in Chinese mitten crabs (E. sinensis) during A. hydrophila infection.

The growth performance, antioxidant capacity, immunological responses, and the resistance against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus) fed Pistacia vera hulls derived polysaccharide

The present study aimed at evaluating the supplementation of dietary Pistacia vera hulls derived polysaccharide (PHDP) at varying levels (0, 2.5, 5, and 10 g/kg diet) on the growth rate, digestive enzyme activity, immune response, and antioxidative capacity of Nile tilapia. After the feeding trial (60 days), fish were injected intraperitoneally with Aeromonas hydrophila, and the cumulative mortality was calculated for 10 days. The final body weight, weight gain, specific growth rate, survival rate, amylase activity, red blood cells, hemoglobin, serum total protein, and serum lysozyme activity (LZM) were significantly higher in fish fed PHDP at 5 and 10 g/kg than fish fed 0 and 2.5 g/kg diet (P ≤ 0.05). However, the feed conversion ratio and glucose levels were significantly decreased by 5 and 10 g PHDP/kg diet. Further, the protease digestive activity, serum alkaline phosphatase, and serum alternative complement (ACH50) had higher values in fish fed 5 g PHDP/kg diet than tilapia fed 0 g PHDP/kg diet (P ≤ 0.05). The serum protease and immunoglobulin (Ig) presented the highest values in fish fed 10 g PHDP/kg diet followed by fish fed 5 g PHDP/kg diet (P ≤ 0.05). The LZM, ACH50, and Ig in the mucus samples were significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 0 g PHDP/kg diet (P ≤ 0.05). The levels of superoxide dismutase and catalase in the liver tissue was significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 2.5 g PHDP/kg diet (P ≤ 0.05). On the other hand, malondialdehyde concentration was significantly lower in tilapia fed 5 and 10 g PHDP/kg diet than tilapia fed 0 and 2.5 g PHDP/kg diet (P ≤ 0.05). The lowest mortality rate and the highest relative percentage survival were in fish fed 5 g/kg followed by 10 g level after 10 days of A. hydrophila injection. Accordingly, dietary PHDP at 5–10 g/kg is recommended to improve the growth performance, antioxidative capacity, immune response, and resistance against A. hydrophila in Nile tilapia.

Comprehensive assessment of benthic diatom ( Amphora coffeaeformis ) as a feed additive in Nile tilapia ( Oreochromis niloticus ) diet

Four hundred and fifty Nile tilapia (Oreochromis niloticus), each weighing ca. 4 g, were randomly allocated into five equal groups in triplicate. The first group acts as control (A0). A1, A2, A3 and A4 groups were fed on rations supplemented with diatom, Amphora coffeaeformis, at 2.5, 5.0, 7.5 and 10%, respectively, for 63 days. Assessment of fish growth, biochemical composition, erythrocyte alterations, gonadal maturation, serum biomarkers, and liver and intestinal pathogens was carried out to identify fish welfare. Fish were challenged by Aeromonas hydrophila injection for an additional 14 days; then, mortality rate and some blood parameters were monitored. Results demonstrated that Amphora sp. insignificantly impacted fish growth, but it induced significant depletion in lipid content. Blood smear investigation indicated erythrocyte alterations in A4 group compared with A0 group. Male gonadosomatic index and relative fecundity demonstrated an increment in A3 and A4 groups respectively. A boost in fish immunity of A3 group was demonstrated through serum biomarkers and pathogenic bacterial identification in liver and intestine compared with other groups. Cumulative mortality of stressed fish was 90% in the A0 group and decreased to 30% in the A3 group. Blood analysis of stressed fish indicated higher tolerance in the A3 group. In conclusion, Amphora sp. can be used as an immunostimulant for increasing disease resistance under stress conditions.

Pelodiscus sinensis embryos infected with Aeromonas hydrophila show higher survival rates than infected Mauremys reevesiis embryos

Emydidae and Trionychidae used different strategies to adapt to the environmental condition. However, the differences in the immune responses are not yet fully understood. In this study, Pelodiscus sinensis (P. sinensis) embryos infected with Aeromonas hydrophila (A. hydrophila) showed significantly higher survival rates than infected Mauremys reevesiis (M. reevesiis) embryos. Transcriptomic profiles of infected and non-infected M. reevesiis and P. sinensis embryos were explored. In total, 8,358 differentially expressed unigenes were detected, of which 3,294 and 5,064 genes were considered to be up- and down-regulated in P. sinensis compared to those in M. reevesiis. Six differentially expressed genes (DEGs) were randomly selected for further validation by qRT-PCR, and the results agreed with the RNA-seq data. Analysing the transcriptome data, eight significantly up/down-regulated immune genes (basic transcription factor 3 (BTF3); patched domain containing 1 (PTCHD1); Sep (O-phosphoserine) tRNA: Sec (selenocysteine) tRNA synthase (SEPSECS); zinc finger FYVE-type containing 16 (ZFYVE16); EF-hand domain family member D2 (EFHD2); zinc finger MYM-type containing 4 (ZMYM4); histone deacetylase 9 (HDAC9); interleukin 10 receptor subunit beta (IL-10RB)) were found. Expression patterns of eight selected genes after A. hydrophila injection were detected via qRT-PCR. In the early developmental stages of infected embryos, HDAC9 and IL-10RB significantly up-regulated which play a key role in the activation of NK cells and Th cells in P. sinensis, while M. reevesiis up-regulated BTF3 and ZFYVE16 first to start proliferation of B-lymphoid cells. We provide new insights into the immunity difference between Emydidae and Trionychidae, facilitating the development of new methods to treat bacterial infections in turtles.

THE BLOOD PICTURE OF HARD-LIPPED BARB [Osteochilus hasselti CV] THAT INFECTED BY Aeromonas hydrophila BACTERIA

The experimental research was carried out on March to August, 2018, at The Fisheries Laboratory, Djuanda University, Bogor. The research is aimed to know and analyze the blood picture of hard-lipped barb that infected by Aeromonas hydrophila. The experimental research used complete randomized design with 7 treatments and each 2 replications. The treatments are Control (without A. hydrophila injection), A (with 104 cfu/mL injection), B (with 105 cfu/mL injection), C (with 106 cfu/mL injection), D (with 107 cfu/mL injection), E (with 108 cfu/mL injection), and F (with 109 cfu/mL injection). The other than Control treatment, each fish in the experimental research was injected via intramuscular with a virulen strain Aeromonas hydrophila bacteria according to the dose of treatment as much as 0.1 mL. The results of research showed that total erythrocytes 1,01 x 106 sel/mm3-1,31 x 106 sel/mm3 and the hemoglobin 2,3 g%-5,2 g% levels have experienced decrease, while total leucocytes 4,32 x 104 sel/mm3-7,53 x 104 sel/mm3 have experienced increase and hematocrite 19,2%-21,4% levels were stable.

Dietary L-Tryptophan Modulates the Hematological Immune and Antibacterial Ability of the Chinese Mitten Crab, Eriocheir sinensis, Under Cheliped Autotomy Stress.

In pond cultures of juvenile Eriocheir sinensis, limb autotomy stress seriously affects and restricts the quality and economic benefits of aquaculture. This study was designed to evaluate the effects of dietary supplementation of L-tryptophan on E. sinensis under the cheliped autotomy stress. In the present study, 252 crabs were divided into four groups: dietary L-trp supplementation with 0.28, 0.40, 0.53, and 0.70%, and their hematological immunity, antioxidant capacity, anti-stress, and antibacterial ability were evaluated after 14 days of using biochemical analysis, flow cytometry, and molecular biology techniques. First, we counted the mortality after 14 days of feeding and found that compared with other treatments, dietary supplementation of 0.53 and 0.70% L-trp significantly lowered the mortality of E. sinensis. Moreover, the total hemocyte count (THC), hemocyanin, and glutathione (GSH) content, and glutathione peroxidase (GSH-Px) activity significantly increased at 7 and 14 d with dietary supplementation of 0.53 and 0.70% L-trp, in contrast with the significant decrease in malondialdehyde (MDA) content at 14 d in the same dietary groups (P<0.05). Next, the bacterial challenge test after 14 days of feeding showed that the THC levels, phagocytic rate, and acid phosphatase (ACP) and alkaline phosphatase (ALP) activity were significantly higher with dietary supplementation of 0.53 and 0.70% L-trp after 12 and 24 h of Aeromonas hydrophila injection, along with a significant improvement in the antioxidant capacity (P<0.05). Further, we measured the expression of antibacterial-related protein genes (EslecB and HSP 90) and found that they were significant up-regulated in the hepatopancreas, hemocytes, intestine, and gill in the groups with dietary supplementation of 0.53% and 0.70% L-trp after 12 h or 24 h of A. hydrophila injection (P<0.05). Taken together, the observations in this study indicate that dietary supplementation of L-trp can enhance the antioxidant capacity and improve the hematological immune status and antibacterial ability of E. sinensis under the cheliped autotomy stress, thereby increasing the survival rate of E. sinensis under cheliped autotomy stress.

RNase1 alleviates the Aeromonas hydrophila-induced oxidative stress in blunt snout bream

RNase1 is an enzyme important in host defense in vertebrates where it degrades the RNA of bacteria and viruses. We evaluated the effect of RNase1 on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into four groups: a blank group (none-treated M. amblycephala), a control group (injected PBS), a challenge group (A. hydrophila-injected) and a treatment group (pre-treated with RNase1 24 h before the A. hydrophila injection), and we collected five tissues of each group. Then we recorded changes in the levels of glutathione (GSH), oxidized glutathione (GSSG), hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and lysozyme; and the relative mRNA expression of catalase (CAT), selenium-dependent glutathione peroxidase (GPx), Cu/Superoxide dismutase (Cu/Zn-SOD), glutamate-cysteine ligase (GCLC), glutathione reductase (GR) and nuclear factor erythroid 2-related factor 2 (Nrf2) for four groups. The expression of six genes was highest in liver and blood of the blank group. It was significantly higher in the gut of the treatment group (compared to control and challenge groups) 12 h after the infection. The treatment group exhibited a significant increase in GSH, SOD and CAT activity, and a decrease in GSSG, MDA and lysozyme content (compared to the control and challenge groups) 6 and 12 h after infection. These results suggest that supplementation with RNase1 protein can enhance resistance against A. hydrophila infections in M. amblycephala.

Molecular cloning, expression and antibacterial activity of goose-type lysozyme gene in Microptenus salmoides

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a g-type lysozyme gene was cloned from Microptenus salmoides. The g-type sequence consisted of 582 bp, which translated into a 193 amino acid (AA) protein (GenBank accession no: MH087462). The predicted molecular weight and theoretical isoelectric point were 21.36 kDa and 6.91 respectively and no signal peptide was observed. The qRT-PCR analysis showed that the g-type lysozyme gene was differentially expressed in various tissues under normal conditions and the highest g-type lysozyme level was observed in liver, gill and spleen while there seemed to be low expression in the muscle, heart and head-kidney. The expression of g-type lysozyme was differentially upregulated in the spleen, gill and intestine after stimulation with heat stress and Aeromonas hydrophila (A. hydrophila). Under heat stress and A. hydrophila injection, the g-type lysozyme mRNA levels all in spleens, gill and intestine tissues increased significantly (P < 0.05), with the maximum levels attained at 12 h, 24 h (or 12 h) and 24 h. Thereafter, they all decreased significantly (P < 0.01) and the expression in gill returned to nearly the basal value within 72 h. Those results suggested that g-type lysozyme was involved in the immune response to heat stress and bacterial challenge. The cloning and expression analysis of the g-type lysozyme provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides. The g-type lysozyme gene perhaps also played an important role in the immune responses against bacterial invasion.