胡子鲶致病性气单胞菌的分离鉴定及其致病力与毒力基因型相关性

为探讨广西南宁市、浦北县和玉林市暴发性死亡胡子鲶的病原菌及其所携带6种毒力基因对其致病力的影响,用常规方法从病鱼的心脏、肝脏等部位分离细菌,人工感染实验确定病原菌的致病性,以API 20NE生化鉴定和16S r RNA分子鉴定相结合的方法对病原菌进行鉴定,采用PCR扩增法检测病原菌的6种毒力基因携带情况。结果显示,从患病鱼中共分离到5株病原菌,其中嗜水气单胞菌3株,温和气单胞菌2株。3株嗜水气单胞菌与标准菌株Aeromonas hydrophila ATCC 7966(CP000462)的亲缘关系最近,相似性均为99.8%,2株温和气单胞菌与标准菌株Aeromonas sobria NO.106(AB472903.1)的亲缘关系最近,相似性均达99.9%。6种毒力基因在5株病原菌中的阳性检出率分别为Act和Aer基因100%,ahal、hly和Alt基因均为80%,ahp基因仅20%;毒力基因型共3种,在5株气单胞菌中分布情况为Act+ahal+hly+Alt+ahp-Aer+3株,占实验菌株的60%,为主要的毒力基因,Act+ahal+hly+Alt+ahp+Aer+和Act+ahal-hly-Alt-ahp-Aer+各1株,各占20%。携带全部所检6种毒力基因的菌株致病力最强,只携带Act和Aer 2种毒力基因的菌株致病力最弱。ahp基因在菌株的致病力中起重要作用,病原菌的致病力是多种毒力基因协同作用的结果。     

Identification of a novel cyprinid herpesvirus 3 genotype detected in koi from the East Asian and South‐East Asian Regions

Cyprinid herpesvirus 3 (CyHV‐3) is a highly contagious virus that causes significant morbidity and mortality in common carp Cyprinus carpio L. and considered to be one of the most important pathogens of koi and common carp worldwide. Cyprinid herpesvirus 3 infected consignments imported from East Asian and South‐East Asian regions were identified during quarantine period in Singapore, and virus from a 2005 consignment was successfully isolated in koi fin cells. A combination of sequence analyses and duplex PCR were used to characterize 15 CyHV‐3 isolates detected in koi consignments between 2005 and 2011. Sequence analyses of the enlarged 9/5, SphI‐5 and TK gene regions identified both the Asian 1 (n = 11) and European 4 (n = 4) genotypes. Duplex PCR analysis of two variable marker regions between ORF29 and ORF30 (marker I) as well as ORF133 and its upstream region (marker II) revealed viruses of genotypes J (I⁺⁺II⁺), U/I (I⁻⁻II⁻), an intermediate genotype (I⁺⁺II⁻) and a novel genotype, I⁺⁺II^+Δ, which was identified in viruses from seven different consignments. This novel genotype has a 13‐bp deletion in marker II, while maintaining the I⁺⁺ allele of marker I. The I⁺⁺II^+Δ genotype may have emerged from East Asian and South‐East Asian regions in recent years.     

怀头鲇体表溃烂症病原鉴定及致病性分析

为探讨黑龙江流域怀头鲇体表溃烂症的病因及防控措施,本研究采用常规方法从患病鱼的肝脏、脾脏和肾脏等部位分离病原菌,通过人工感染试验确定分离菌株的致病性,并对菌株的基本形态、理化特性、分子特征、毒力基因携带情况及耐药性等进行了系统研究。结果显示,从患病鱼体内分离得到3株病原菌,分别命名为NY-8、NY-9和NY-12;人工感染试验发现,NY-8和NY-9株对试验鱼有较强的致病力,NY-12株毒力较弱;3株细菌混合感染后,鱼体发病症状与临床自然发病症状一致,试验鱼死亡率高达到100%。综合理化特征和16S r RNA基因序列分析结果,确定NY-8、NY-9和NY-12株分别为气单胞菌属的维氏气单胞菌、杀鲑气单胞菌和嗜水气单胞菌。5种毒力基因在3株气单胞菌中的分布表现为两种基因型,h l y+/a e r+/a c t+/a l t+/G C A T+和h l y+/a e r-/act+/alt+/GCAT+,同时携带5种毒力基因的NY-8和NY-9分离株致病性显著高于NY-12株。3株细菌在耐药谱上有一定差异性,NY-8和NY-9株均对4种氟喹诺酮类药物敏感,对氨基糖苷类、呋喃类等药物耐药;NY-12株仅对左氧氟沙星和氟苯尼考等2种药物敏感。     

Aerobic Bacterial Flora of Common Carp (Cyprinus carpio L.) Cultured in Earthen Ponds in Saudi Arabia

ABSTRACT

Quantitative and qualitative estimation of bacterial flora present in pond water, sediments, gills, and intestine of healthy common carp Cyprinus carpio cultured in Saudi Arabia were performed and identified to species level where possible. Mean total viable bacterial counts in pond water ranged from 1.2?±?2.9?×?10⁴ to 2.5?±?3.5?×?10⁵ cfu/mL; in sediments, 9.3?±?2.1?×?10⁷ to 2.7?±?3.5?×?10⁹ cfu/g; in gills filaments, 4.3?±?2.9?×?10⁶ to 1.6?±?3.9?×?10⁷ cfu/g; and in intestine, 8.7?±?4.1?×?10⁹ to 5.4?±?3.2?×?10¹⁰ cfu/g. Gram-negative rod-shaped bacteria dominated (76%) the populations. In total, 12 bacterial genera and 15 species were identified. Pond water and sediment bacteria had the reflection on bacterial composition of gills and intestine of carp. Intestinal bacteria showed more diversification in contrast to gill bacteria. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Corynebacterium urealyticum, Vibrio vulnificus, Vibrio sp., and Staphylococcus sp. were the common bacteria in all the populations. In pond water and carp intestine, A. hydrophila, S. putrefaciens, V. cholerae, and C. urealyticum were the most dominant bacteria (prevalence ≥ 10%) where pond sediments and the carp gills experienced with more one dominant bacterium V. vulnificus. Only the A. hydrophila covered one fourth (25%) of the total bacterial populations.     

Enhancement of the innate immune system and disease‐resistant activity in Cyprinus carpio by oral administration of β‐glucan and whole cell yeast

The effects of dietary β‐ (1,3) glucan and whole cell yeast (Sacharomyces uvarum) on the immune response and disease resistance to Aeromonas hydrophila were investigated in Cyprinus carpio. β‐(1,3) glucan was extracted from the yeast. Both β‐(1,3) glucan and whole yeast were incorporated into the diet at 1% level and fed to common carp C. carpio for a period of 60 days. Control and treated fish were exposed to A. hydrophila on the 30th and the 60th day of the experimental period. Dietary supplementation of glucan significantly increased the white blood cell count in fish on the 60th day (2.91±0.04 × 10⁴), and the highest nuetrophil nitro blue tetrazolium (NBT) activity was also observed in glucan‐fed fish (30th day). A consistent increase in neutrophil (NBT) activity was also observed in whole cell fed fish until the end of the experiment. Similarly, β‐(1,3) glucan and whole cell yeast enhanced the serum lysozyme activity from the 15th day onwards but higher activity was reported on the 30th day in glucan and the 60th day in whole cell yeast‐fed fish. Suplementation of β‐(1,3) glucan protected the fish from A. hydrophila infection. Nearly 75–80% of the fish survived pathogen exposure (relative percentage survival). However, only 54–60% survival was observed in the whole cell‐fed fish. β‐(1,3) glucan and whole cell yeast protect the fish from pathogens by enhancing the cellular and humoral immune response in C. carpio.     

山瑞鳖细菌性败血症病原菌的分离鉴定及其毒力基因检测

查明广西南宁、贵港和桂平养殖山瑞鳖细菌性败血症的病原菌及其6种毒力基因的携带情况,为有效防控山瑞鳖细菌性败血症提供参考。本研究以常规方法从患病山瑞鳖的心脏和肝脏取样、分离细菌,人工感染方法确定分离菌株的致病性,细菌鉴定采用API 20NE生化鉴定和16S rRNA分子鉴定相结合的方法进行,PCR扩增法对菌株的溶血素基因(hemolysin gene,hly)、气溶素基因(aerolysin gene,Aer)、细胞兴奋性肠毒素基因(cytotonic enterotoxin gene,Alt)、细胞毒性肠毒素基因(cytotoxic enterotoxin gene,Act)、黏附素基因(major adhesin gene,ahal)和丝氨酸蛋白酶基因(serine protease gene,ahp)6种毒力基因进行检测。结果显示,从患病山瑞鳖心脏和肝脏中共分离到4株优势菌SRB125、SRB142、SRB143和SRB345,对健康山瑞鳖的平均致死率为97.50%~100.00%,是引起山瑞鳖细菌性败血症的病原菌;生化和分子鉴定结果显示,4株分离菌均为嗜水气单胞菌(Aeromonas hydrophila),与A.hydrophila L3-5(KP716701)菌株的亲缘关系最近,同源相似性均达到99.9%;6种毒力基因共包含2种毒力基因型,在4株菌株中的分布为hly~+Aer~+Alt~+Act~+ahal~+ahp~+和hly~+Aer~+Alt~+Act~+ahal~+ahp~–各2株,来源于南宁的SRB143和桂平的SRB345菌株均缺失ahp基因。     

Inhibitory activity of probiotic Enterococcus faecium MC13 against Aeromonashydrophila confers protection against hemorrhagic septicemia in common carp Cyprinuscarpio

The present study involved the control of Aeromonas hydrophila in Cyprinus carpio by bacteria Enterococcus faecium MC13 isolated from fish Mugil cephalus intestine. Antagonistic effects were confirmed against Aeromonas hydrophila by cross-streaking and the agar spot method. Probiotic effects of the isolate were confirmed by injection and oral administration to check for nonpathogenicity to fish. All the two probiotics were orally administered to fingerlings of Cyprinus carpio for 60 days through feed. The intestinal load of bacteria and NBT assay were analyzed on 7, 15, 30, and 60th day of treatment. Experimental fish were challenged with Aeromonas hydrophila on 30 and 60th day. The neutrophil activity was higher in E. faecium MC13 fed fish on day 60 (1.525 ± 0.379 OD). In respect of relative percentage survival (RPS), the higher protection of 75 and 77.8% was observed in E. faecium MC13 fed fish on 30 and 60th day of challenge with Aeromonas hydrophila This study identified the bacterial probiont MC13 (Enterococcus faecium) effectively controlled the Aeromonas hydrophila infection in Cyprinus carpio.     

银离子对几种细菌的杀菌效应

由抗生素导致的耐药性问题日益严重,因此新型抗菌药物的研发迫在眉睫。银离子因具有抗菌作用,以及安全、无耐药性、稳定性高等优点而备受关注。为了探究银离子的抗菌机制,本实验选择几种常见水生细菌,研究其对银离子的耐受性和细菌种内、种间的"僵尸效应"(被银离子杀死的细菌可以杀死其他新鲜的细菌),以进一步明确银离子长效杀菌的机理。结果显示,银离子对5种细菌都表现出了明显的生长抑制。同种细菌和异种细菌之间都表现出明显的"僵尸效应",并且随着银离子浓度的提高,"僵尸效应"的效果越明显。为进一步研究银离子杀菌机制,通过透射电镜观察银离子处理后的嗜水气单胞菌和无乳链球菌,结果显示银离子处理后的细菌出现胞质皱缩,细胞膜呈弥散状态,甚至破裂,细胞内容物外流,最终致使细菌死亡。研究表明,银离子对5种细菌都有明显的生长抑制效应,并发现细菌之间存在"僵尸效应",可为研发新型的抗菌药物提供参考。     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.     

Effects of graded levels of dietary myo‐inositol on non‐specific immune and specific immune parameters in juvenile Jian carp (Cyprinus carpio var. Jian)

The aim of this study was to evaluate the effects of myo‐inositol (MI) on non‐specific immune and specific immune defence in fish. A total of 1050 Jian carp (Cyprinus carpio var. Jian) (22.28±0.07 g) were randomly distributed into seven groups, of three replicates each, of feeding diets containing graded levels of MI (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg MI kg^?1 diet). After a 60‐day growth trial, an infectious trial was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that the red blood cell (RBC) and the white blood cell count were significantly increased with increasing MI levels up to 535.8 mg kg^?1 diet (P<0.05). The spleen index showed a tendency similar to RBC, whereas the head kidney index showed the inverse pattern (P<0.05). The phagocytic activity of leucocytes, haemagglutination titre, lysozyme activity, anti‐A. hydrophila antibody titre and immunoglobulin M, after being injected with A. hydrophila, were all improved with an increase in the MI levels up to 232.7–687.3 mg kg^?1 diet respectively (P<0.05). Myo‐inositol did not influence serum acid phosphatase activity and total iron‐binding capacity (P>0.05). These results suggested that MI could enhance non‐specific immune and specific immune responses in fish.     

Environmental factors on virulence of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

Aeromonas hydrophila are known for being opportunistic pathogens, harboring various virulence factors and triggering lesions and death in fish. The disease caused by bacteria can make fish inappropriate for human consumption, besides representing a risk to public health. The pathogenesis can be influenced by environmental variables, affecting fish productivity and mortality. The present study aimed to determine whether A. hydrophila harbor the virulence genes aerolysin, hydrolipase, elastase, lipase, cytotonic enterotoxin (ast), lateral flagellum (laf), and polar flagellum (fla) and to evaluate the influence of environmental variables on in vitro growth, in vivo virulence and expression of some of these genes. Polymerase chain reaction (PCR)-based screening for the presence of these virulence genes was performed on 35 isolates. Six isolates containing different profiles of virulence genes were tested for in vitro growth under different conditions of pH, temperature, and ammonia and for in vivo virulence under these same environmental conditions. RT-qPCR was used to quantify the expression of aerolysin, lipase, and fla genes. All the tested environmental factors influenced the growth of A. hydrophila, while pH and ammonia concentrations influenced the bacterial virulence. The expression of the fla gene increased when bacteria were grown in higher ammonia concentration. The mortality established by Aeromonas is influenced by several environmental factors pinpointing the importance of its control in fish farming to avoid higher economic loses associated to bacterial disease outbreaks.     

Supplementation of potassium, magnesium and sodium chloride in practical diets for the Pacific white shrimp, Litopenaeus vannamei, reared in low salinity waters

The culture of Litopenaeus vannamei in inland low salinity waters is currently being practiced in various countries around the world. These environments are often deficient in key ions essential for normal physiological function, including potassium (K⁺) and magnesium (Mg²⁺). Farmers have sometimes been able to counteract ionic deficiencies in the water profile by adding mineral salts containing sources of K⁺ and Mg²⁺. The purpose of this study was to explore the possibility of correcting deficiencies of K⁺ and Mg²⁺ in the water profile with dietary supplementation of these minerals. Two separate 7‐week experiments were conducted in 4.0 g⁻¹ artificial low salinity water to evaluate the effects of mineral supplements (K⁺, Mg²⁺ and NaCl) to diets of L. vannamei reared in low salinity waters. In trial 1 seven diets were formulated (10 g NaCl kg⁻¹, 20 g NaCl kg⁻¹, 150 mg kg⁻¹ Mg²⁺, 300 mg kg⁻¹ Mg²⁺, 5 g K⁺ kg⁻¹, 10 g K⁺ kg⁻¹, and a basal diet to serve as a control). Minerals were added in the form of purified potassium chloride (KCl), magnesium chloride (MgCl₂·6H₂O) and NaCl. Trial 2 evaluated the use of a coating agent for the Mg²⁺ and NaCl treatments, while a K⁺ amino acid complex was utilized in the K⁺ treatments to reduce mineral leaching. Trial 2 was performed using similar treatment levels as trial 1. Shrimp survival and growth were assessed in both experiments. Results from trial 1 indicated no significant differences in survival, growth or percent weight gain. Results from trial 2 revealed no significant differences in survival and growth in the NaCl and Mg²⁺ treatments. However, significant differences in growth (P < 0.05) were observed when using the 10 g K⁺ kg⁻¹ treatment, suggesting that dietary supplementation of a K⁺ amino acid complex may help improve growth of the species in low salinity waters.     

Effects of dietary supplementation with probiotic live yeast Debaryomyces hansenii on the immune and antioxidant systems of leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila

Two trials were conducted to determine the efficacy of fish fed live yeast Debaryomyces hansenii strain CBS 8339 on immune and antioxidant systems in leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila. Juveniles (12±0.5 g) were fed with a control diet or a D. hansenii‐supplemented diet (10⁶ colony‐forming units per gram) for 5 weeks. The live weight of fish was registered on a weekly basis. After 4 weeks, fish from each treatment were immunocompromised with pathogenic A. hydrophila and further fed for 1 week in order to evaluate the effect on immunological and antioxidant parameters. Generally, the results showed enhanced growth performance in fish fed the diet containing yeast compared with the control. Addition of live yeast had no significant effect on the immunological parameters after 4 weeks of feeding. However, post infection with A. hydrophila fish fed the yeast‐supplemented diet resulted in a significant increase in the levels of plasmatic immunoglobulin M. Superoxide dismutase and catalase (CAT) activities were significantly higher in the yeast group. In this fish, CAT and heat shock protein 70 genes were up‐regulated before and after infection of A. hydrophila. The present study is the first one reporting that yeast (D. hansenii) can enhance immunity and resistance against A. hydrophila.     

Antibacterial efficacy and pharmacokinetic evaluation of sanguinarine in common carp (Cyprinus carpio) following a single intraperitoneal administration

Sanguinarine (SA), with antimicrobial and antiparasitic activities against fish pathogens, exhibits great potential commercial use in aquaculture. However, little information on pharmacokinetics of SA restricts further application in aquaculture. In this study, pharmacokinetics of SA in common carp (Cyprinus carpio) following a single intraperitoneal administration [10 mg kg^?1 BW (body weight)] was evaluated by high‐performance liquid chromatography (HPLC). The peak concentration (C_max) of SA in kidney was 11.8 μg g^?1, which was higher than in other tissues and plasma. The terminal half‐life in fish tissue and plasma was as follows: 42.3 h (kidney) > 37.2 h (liver) > 20.1 h (gill) > 18.8 h (muscle) > 10.9 h (spleen) > 10.0 h (plasma). Additionally, we determined the bacterial loads in tissues of common carp infected with Aeromonas hydrophila after i.p. administration of SA at 0, 5, 10 and 20 mg kg^?1 BW. The results showed that i.p. administration of SA at 10 mg kg^?1 BW significantly enhanced antibacterial efficacy against A. hydrophila, where the antibacterial ratio in the gill, kidney, spleen and liver on day 5 was 95.13%, 93.33%, 90.09% and 92.82%, respectively. Overall, these results suggested the potential of SA to treat A. hydrophila infection in common carp farming industry.     

Effects of potential probiotic Bacillus subtilis KADR1 and its subcellular components on immune responses and disease resistance in Labeo rohita

The present study was conducted to determine the effects of Bacillus subtilisKADR1 and its subcellular components on immunity and disease resistance in Labeo rohita against Aeromonas hydrophila infection. Fish were fed diet containing different concentrations of live bacterial cells (DI‐10⁶, DII‐10⁸ and DIII‐10¹⁰ CFU/g) and another group of fish were immunized intraperitoneally with cellular components (WCPs, CWPs and ECPs) of Bacillus subtilisKADR1. After 4 weeks of trial, fish were challenged intraperitoneally with Aeromonas hydrophila cell suspension and survival percentage was recorded. Significantly higher post‐challenge survivability was recorded in fish groups fed 10⁸ CFU/g of KADR1 (80.24%; RPS = 75.76%) or immunized with WCPs (77.77%; RPS = 72.73%), compared with the control (18.51%). Analysis of immunological parameters viz. serum lysozyme, phagocytosis, serum total protein, respiratory burst, serum IgM levels, superoxide dismutase and alternative complement pathway activity reflected significant enhancement (p < .05) of immune response in fish fed 10⁶, 10⁸ and 10¹⁰ CFU/g of live cells, or immunized with cellular components of Bacillus subtilisKADR1, with the highest values were observed DII fed group, followed by the group immunized with WCPs. Our results suggest that dietary administration of Bacillus subtilisKADR1 at 10⁸ CFU/g can effectively enhance the immune responses and disease resistance in Labeo rohita against Aeromonas hydrophila.     

Immune response,disease resistance and intestinal microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of pantothenic acid

This study was to investigate the effect of dietary pantothenic acid (PA) on the disease resistance, immune response and intestinal microflora on juvenile Jian carp (Cyprinus carpio var. Jian). Seven diets (4.0, 15.5, 25.6, 36.1, 45.9, 56.1 and 65.9 mg PA kg^?1) were fed to Jian carp (12.95 ± 0.03 g) for 9 weeks. After 9‐week feeding trial, the challenge experiment with Aeromonas hydrophila was conducted to determine the impact of PA on fish disease resistance. Survival rate after challenge was promoted with the increasing PA levels (P < 0.05). Blood counts also significantly increased up to the dietary PA level of 25.6 mg PA kg^?1 (P < 0.05). Leucocyte phagocytic activity, lectin potency, lysozyme and acid phosphatase activity, and total iron‐binding capacity were improved with increasing PA levels (P < 0.05). Serum immunoglobulin M level and agglutination antibody titre to A. hydrophila were increased (P < 0.05) in fish fed the diets with the dietary PA levels between 56.1 and 65.9 mg kg^?1. PA also promoted the growth and reproduction of Lactobacillus and depressed Escherichia coli and A. hydrophila (P < 0.05). These results suggested that pantothenic acid could improve disease resistance, immune response, and the balance of intestinal microflora in juvenile Jian carp.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon‐tagged A. hydrophila strains using miniTn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10^?3 and 10^?6 and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 × 10⁴ CFU mL^?1. Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.     

Lemon,Citrus aurantifolia,peel and Bacillus licheniformis protected common carp,Cyprinus carpio,from Aeromonas hydrophila infection by improving the humoral and skin mucosal immunity,and antioxidative responses

The role of dietary lemon peel (LM) and/or Bacillus licheniformis (BL) on the growth, immunity, and resistance against Aeromonas hydrophila in common carp, Cyprinus carpio was investigated in this study. LM and BL were included in diets at 0% (T0), 10⁸ CFU/g BL (T1), 1.5% LM and 10⁸ CFU/g BL (T2), and 3% LM and 10⁸ CFU/g BL (T3). Fish fed with T1, T2, or T3 had higher weight gain, specific growth rate, white blood cells count, and blood total protein with lower feed conversion ratio than T0 group (p < .05). The albumin increased significantly (p < .05) in fish fed both BL and LM (T3). The serum superoxide dismutase (SOD), catalase, lysozyme, and bactericidal activities were significantly increased in fish fed both BL and LM (T2 and T3), while serum glutathione peroxidase increased in fish fed BL (T2) (p < .05). Fish fed T1, T2, and T3 diets displayed higher SOD and lower malondialdehyde than fish fed T0 (p < .05). After the A. hydrophila challenge, the mortality rate was significantly lower in T1, T2, and T3 groups than the T0 group (p < .05). The obtained results revealed that LM and BL could be used to increase resistance against A. hydrophila infection in carp. However, further field studies should be performed to confirm the obtained results.