Efficacy of florfenicol for control of mortality associated with Francisella noatunensis subsp. orientalis in Nile tilapia,Oreochromis niloticus (L.)

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram‐negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg^?1 body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg^?1 bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol‐treated groups 4 days after treatment. The mean LOG of CFU Fno mg^?1 spleen was 3–5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg^?1 bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia.     

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 10⁶ CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.     

Dynamics of piscine francisellosis differs amongst tilapia species (Oreochromis spp.) in a controlled challenge with Francisella noatunensis subsp. orientalis

A 25‐week immersion challenge was conducted exposing Oreochromis mossambicus, Oreochromis aureus and Oreochromis urolepis hornorum to Francisella noatunensis subsp. orientalis (Fno). Two populations were compared for each fish species; ‘resident fish’ were defined as fish maintained in tanks since week 0 of challenge, whereas ‘naïve fish’ were defined as fish added to tanks once temperature in water reached <26 °C at 21 weeks post‐challenge. Fno genome equivalents (GEs) in water were similar in all treatments 1 h post‐challenge; however, significantly lower Fno GEs were detected 2 weeks post‐challenge in all tanks, and the only treatment with detectable Fno GE after 4 weeks of challenge were the O. mossambicus tanks. Twenty‐one weeks post‐challenge, naïve fish were stocked with ‘resident’ cohorts. Over a 4‐week period, mortalities occurred consistently only in O. mossambicus naïve cohorts. Overall presence of granulomas in spleen of survivors was similar (>55%) in all resident populations; however, in naïve populations, only O. mossambicus presented granulomas. Similarly, only O. mossambicus presented viable Fno in the spleen of survivors, and Fno GEs were only detected in O. mossambicus, and in resident O. aureus. In conclusion, the results of this study suggest different susceptibility of tilapia species to piscine francisellosis.     

Genotypic diversity,and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%–100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.     

Differential pathogenicity of five Streptococcus agalactiae isolates of diverse geographic origin in Nile tilapia (Oreochromis niloticus L.)

Streptococcus agalactiae is an emerging pathogen of fish and has caused significant morbidity and mortality worldwide. The main objective of this study is to assess whether pathogenic differences exist among isolates from different geographic locations. Nile tilapia (Oreochromis niloticus L.) were administered an intraperitoneal injection of suspension containing USA, Brazil, Honduras, Israel, or Kuwait S. agalactiae isolates at concentrations ranging from 10² to 10⁷ cfu mL^?1. The LD₅₀ values 7 days after challenge were as follows: USA (1.0 × 10² cfu mL^?1), Brazil (1.5 × 10³ cfu mL^?1), Honduras (6.8 × 10³ cfu mL^?1), Israel (1.0 × 10⁴ cfu mL^?1) and Kuwait (7.2 × 10⁵ cfu mL^?1). Fish from all groups exhibited lethargy, anorexia, exophthalmia, corneal opacity, erratic swimming, petechiae and mortality. Opercular clearing and ascites were only found after infection with certain geographic isolates. The findings in this study indicate that S. agalactiae isolates of different geographic origin can cause significant mortalities after experimental challenge and can have different pathogenic capacities. Isolates from the Americas (USA, Brazil and Honduras) were more pathogenic to Nile tilapia than isolates from the Middle East/Asia (Israel and Kuwait).     

Francisella sp., an emerging pathogen of tilapia, Oreochromis niloticus (L.), in Costa Rica

Francisella sp. is an emergent bacterial pathogen that causes acute to chronic disease in warm and cold water cultured and wild fish species. During the past 3 years, the bacterium has been detected in tilapia, Oreochromis niloticus , cultured in Costa Rica. Infected fish presented non-specific clinical signs, such as erratic swimming, anorexia, anaemia, exophthalmia and high mortality. Upon macroscopic and microscopic examination, several internal organs (mainly spleen and kidney) were enlarged and contained white nodules. Histological examination revealed the presence of multifocal granulomatous lesions, with the presence of numerous small, pleomorphic, cocco-bacilli. The bacteria were isolated from infected tilapia on selective media and grown on several media with and without antibiotics. Specific PCR primers to the Francisella genus were used to confirm the preliminary diagnoses. In comparison with several bacterial 16S rRNA sequences, our isolate was found to share 99% identity with other Fransicella spp. isolated from fish, and more than 97% identity to the human pathogen Francisella tularensis . Koch's postulates were fulfilled after experimental intraperitoneal and gill exposure challenges.     

罗非鱼谷胱甘肽过氧化物酶1基因的克隆与分析

采用cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术,克隆了罗非鱼（Oreochromis niloticus）谷胱甘肽过氧化物酶1（glutathione pemxidasel,GPx1）基因完整的编码序列（complete coding sequence,CDS）。GPx1基因全长984bp,5’uTR56bp,CDS576bp,3’UTR352bp,PolyA20bp;第791—885位碱基（位于3’UTR）形成1个硒半胱氨酸插入序列（selenocysteine insertion sequence,SECIS）,协助174～176位密码子TGA（UGA）编码1个硒半胱氨酸（Sec）。GPx1包含191个氨基酸,分子量21．8kDa,等电点8．04,无信号肽和潜在的N．糖基化位点。蛋白结构分析表明该基因编码蛋白为非跨膜蛋白。序列比对显示,GPx1单体具有Sec、Trp、Gln和Asn构成的催化四联体。罗非鱼GPx1与其他脊椎动物GPx1相比较,核苷酸序列相似性为43．2％-58．2％,氨基酸序列相似性为58．1％～80．6％。进化分析显示,处在分类学上不同纲的脊椎动物GPx1分别占据了不同分支。利用Swiss-Model预测了罗非鱼GPx1的3D结构,序列分析显示,GPx1可以形成1个同源四聚体。     

患聚缩虫病的中国对虾同工酶及蛋白的初步研究

本文报道由聚缩虫(Zoothamnium sp.)附着所引起的中国对虾(Penaeus orientatis)的生化病理变化。与健康虾相比,病虾肉中乳酸脱氢酶的平均绝对活性减少至41.7%;苹果酸脱氢酶的平均绝对活性减少至70.1%;超氧物歧化酶的平均绝对活性也有减弱;肌蛋白的平均绝对活性减少至65.0%。     

Growth, feed utilization and body composition of tilapia (Oreochromis sp.) fed with cottonseed meal-based diets in a recirculating system

The suitability of cottonseed meal (CSM) as a major source of plant protein in feeds for tilapia ( Oreochromis sp.) was tested by examining growth and feed intake, feed digestibility, liver gossypol concentrations, feed utilization, and body mineral composition. Juvenile tilapia at an initial average size of 11.8 ± 1.6 g were divided into triplicate groups per dietary treatment and offered five different formulated diets. In these feeds fish meal (FM) protein was gradually replaced by protein from CSM (0, 25, 50, 75, and 100%; diets 1, 2, 3, 4 and 5, respectively). The experiments were conducted in a recirculation system at a water temperature of 27 ± 1°C in glass aquaria for 16 weeks. Tilapia growth did not differ significantly ( P  > 0.05) with up to 50% substitution of FM with CSM. Fish meal replacement above 50% resulted in significant growth decline with time. Fish fed with 100% FM and diets including 50% CSM had significantly better daily weight gain, daily feed intake and feed efficiency ratio than those fed with 100% CSM. Fish fed with 75% CSM and above had lower concentrations of body iron, calcium and phosphorus than controls (100% FM). Concentrations of total gossypol in diets (ranging from 0.11 to 0.44% in diets 2–5) resulted in proportional increase of total gossypol in fish liver (32.3, 72.3, 99.4 and 132.1 μg g⁻¹ wet weight) in groups fed with diet 2, 3, 4, and 5, respectively. We concluded that CSM can partially replace FM as a main source of protein in feed for tilapia at not more than 50%. The presence of gossypol in CSM was identified as the major limiting factor for acceptance and utilization of CSM-based diets in tilapia farming.     

罗非鱼谷胱甘肽过氧化物酶1的克隆与分析

采用cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术,克隆了罗非鱼（Oreochromis niloticus）谷胱甘肽过氧化物酶1（glutathione peroxidase1,GPx1）基因完整的编码序列（complete coding sequence,CDS）。GPx1基因全长984 bp,5UTR 56 bp,CDS 576 bp,3UTR 352 bp,PolyA 20 bp;第791~885位碱基（位于3UTR）形成1个硒半胱氨酸插入序列（selenocysteine insertion sequence,SECIS）,协助174~176位密码子TGA（UGA）编码1个硒半胱氨酸（Sec）。GPx1包含191个氨基酸,分子量21.8 kDa,等电点8.04,无信号肽和潜在的N-糖基化位点。蛋白结构分析表明该基因编码蛋白为非跨膜蛋白。序列比对显示,GPx1单体具有Sec、Trp、Gln和Asn构成的催化四联体。罗非鱼GPx1与其他脊椎动物GPx1相比较,核苷酸序列相似性为43.2%~58.2%,氨基酸序列相似性为58.1%~80.6%。进化分析显示,处在分类学上不同纲的脊椎动物GPx1分别占据了不同分支。利用Swiss-Model预测了罗非鱼GPx1的3D结构,序列分析显示,GPx1可以形成1个同源四聚体。

     

尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定

采用Sephacryl S-300过滤层析和DEAE-Sepharose Fast Flow离子交换层析相结合的方法从尼罗罗非鱼(Oreochromis niloticus)成熟卵子匀浆液中分离纯化出了一种高分子量的蛋白。该蛋白能被Schiff试剂、甲基绿和苏丹黑B着色,Western blot显示能被金鱼卵黄脂磷蛋白(lipovitellin,Lv)多克隆抗血清特异性识别,在非变性条件下分子量约为560 k D,在SDS变性条件下分子量约为112 k D,结果表明分离纯化的蛋白是一种含有糖、磷、脂基团的蛋白,符合鱼类Lv的性质,且与金鱼Lv有免疫交叉反应,从蛋白的性质和免疫原性以及分子量大小等角度判断,本研究获得的高纯度蛋白为尼罗罗非鱼卵黄脂磷蛋白;纯化的罗非鱼Lv在反复冻融、37℃及60℃处理条件下均未出现降解,表明罗非鱼Lv比鱼类卵黄原蛋白(Vitellogenin,Vtg)更为稳定。研究结果为罗非鱼Lv抗体的制备奠定了基础。     

Phenotypic and genotypic characterization of Streptococcus spp. isolated from tilapia (Oreochromis spp.) cultured in river-based cage and earthen ponds in Northern Thailand

Streptococcus spp. are major pathogenic bacteria associated with massive mortality in tilapia. This study investigated the phenotypic and genotypic characterization of Streptococcus agalactiae (GBS) and Streptococcus iniae (S. iniae) isolated from tilapia in river-based floating cage and earthen pond farms in northern Thailand. Isolates were identified by biochemical and molecular analyses. Capsular typing, enterobacterial repetitive intergenic consensus polymerase chain reaction and multilocus sequence typing were performed to investigate the genetic relatedness. Six and one isolates were confirmed as GBS and S. iniae, respectively. All Streptococcus spp. isolates were obtained from 4 river-based cage farms (4/33), while samples collected from earthen pond farms (N = 28) were negative for streptococcosis. All GBS with serotype Ⅲ and sequence type (ST) 283 was observed. The β-haemolytic GBS isolates were resistant to five antimicrobials, while the S. iniae was susceptible to all antimicrobials. This study indicates both GBS and S. iniae are the major bacterial pathogens responsible for streptococcosis infection in farmed tilapia of northern Thailand with GBS as dominant species. This survey highlights that the river-based cage farms seriously impact on the healthy development of the tilapia industry.     

Nile tilapia (Oreochromis niloticus) show high digestibility of lipid and fatty acids from marine Schizochytrium sp. and of protein and essential amino acids from freshwater Spirulina sp. feed ingredients

We determined apparent digestibility coefficients (ADCs) of macronutrients, amino acids and fatty acids for freshwater (Spirulina, SPI; Chlorella, CHL) and marine (Schizochytrium, SCI) microalgal ingredients in Nile tilapia via four diets: reference (Ref containing fish and plant feedstuffs) and SPI, CHL and SCI (Ref/dried algal cells at 7:3). ADCs of crude protein were significantly higher in SPI than in CHL, comparing well with literature values for fishmeal and plant feedstuffs. ADCs of all essential amino acids were generally higher in SPI. Very high lysine and methionine ADCs in SPI and SCI were similar to or higher than reported value for plants. SCI had 5–10 times richest lipid and n?3 PUFA contents, significantly highest ADCs for lipid, n?3 and n?6 PUFA; and highest content and digestibility of DHA (undetected in other microalgae). Crude fibre ADCs were significantly higher in Ref and SPI than in SCI and CHL. CHL's highest fibre content (79 g kg^?1) possibly depressed its ADCs of five essential amino acids and n?3 PUFA. Results suggest that SPI is a good alternative protein and SCI a quality fish oil substitute or long‐chain PUFA supplement for tilapia diets.     

Occurrence of multiple antibiotic resistance and genotypic characterization in Edwardsiella tarda isolated from cage‐cultured hybrid red tilapia (Oreochromis sp.) in the Ping River,Northern Thailand

Edwardsiella tarda is a pathogen that causes edwardsiellosis in aquatic animals. The emergence of multiple antibiotic‐resistant strains makes antibiotic treatment difficult. This study aimed to investigate the antibiotic susceptibility patterns and the genotypic characterization of E. tarda isolated from cage‐cultured red tilapia in Thailand. A total of 30 isolates were identified as E. tarda using biochemical and molecular analysis. The disc diffusion method for testing antibiotic susceptibility showed all the isolates were resistant to colistin sulphate and oxolinic acid. High levels of resistance to amoxicillin, ampicillin, ceftazidime, oxytetracycline and sulphamethoxazole/trimethoprim were observed as well. The multiple antibiotic resistance index ranged from 0.25 to 0.92, indicating that these isolates had been exposed to high risk sources of contamination where antibiotics were commonly used. All the isolates carried the bla_TEM gene based on polymerase chain reaction (PCR). The tetA and sul3 genes were detected in 90% (27/30) and 26.7% (8/30) of the isolates respectively. Nine different genetic groups of isolates were obtained using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC‐PCR). A correlation between genetic types and multiple antibiotic‐resistant patterns was found. These results highlight the potential risks of multiple antibiotic‐resistant isolates for humans and the environment.