越南水稻黄矮病毒多克隆血清抗体的制备

[目的]制备用于检测水稻黄矮病毒(RYSV)的多克隆抗体,为水稻和黑尾叶蝉上的病毒诊断提供技术支持.[方法]利用两种不同来源的RYSV抗原免疫家兔,一种是从受感染的水稻病叶组织纯化获得RYSV病毒粒子;另一种是从越南RYSV分离株克隆出完整N蛋白编码基因,然后将其连接至pET-28a载体上,在大肠杆菌Rosetta(DE3)菌株中进行诱导表达获得N蛋白抗原.其中,N蛋白抗原又以两种形式(洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆)对家兔进行免疫.最后,采用PTA-ELISA分析评估家兔抗体血清(多克隆抗体)对水稻叶片和黑尾叶蝉RYSV的特异性和敏感性.[结果]分离自越南RYSV分离株的N基因由1542个核苷酸组成,与来自我国和日本RYS分离株N基因序列进行比对,其核苷酸序列同源性分别为98.1%和97.9%,对应的推导氨基酸序列同源性为83.4%和99.4%.以RYSV病毒粒子、洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆3种抗原免疫家兔获得的多克隆抗体均能有效检测出水稻植株中的RYSV,其中,感病植株的OD405分别为1.449、2.337和1.649,健康植株的OD405分别为0.375、0.294和0.283.PTA-ELISA检测结果表明,获得的多克隆抗体能从单个带病毒黑尾叶蝉中检测出RYSV,且该结论在RT-PCR检测中得到进一步验证.[结论]制备获得的多克隆抗体对RYSV具有较高特异性和敏感性,可用于水稻和黑尾叶蝉上的病毒诊断,同时表明以含有病毒植物蛋白的聚丙烯酰胺切片直接注射免疫模型动物制备多克隆抗体具有可行性.     

A comparative evaluation of parasitological tests and a PCR for Trypanosoma evansi diagnosis in experimentally infected water buffaloes

In this study five parasitological methods and a polymerase chain reaction (PCR) were compared for the diagnostic sensitivity for Trypanosoma evansi in experimentally infected water buffaloes over a period of 15 weeks. The combined estimates of sensitivity (CE(se)) of the PCR proved to be highest at 78.2%, closely followed by the mouse inoculation (MI), the micro-haematocrite centrifugation technique (MHCT) and the mini-anion-exchange centrifugation technique (MAECT) with CE(se) of, respectively, 74.0, 69.6 and 62.4%. The CE(se) of the buffy-coat technique (BCT) at 38.6% and the sodium dodecyl sulfate (SDS) clarification technique at 25.1% were considerably lower. PCR detected consistently all buffaloes infected from week 3 post-infection (PI) onwards. For MI this occurred after 5 weeks PI while for MHCT and MAECT these sustainable high levels were reached in the 7th week PI. BCT and SDS never detected all buffaloes infected. The influence of time and temperature on the viability of T. evansi in heparinized blood from water buffalo was also studied. In general we observed that the survival time tends to be longer when blood is kept at 4 degrees C. In samples kept in direct sunlight parasites became undetectable with the MHCT after 30min. After treatment of the water buffaloes with diminazene aceturate, the PCR signal disappeared within 24h.     

Characteristics and Physicochemical Properties of Gelatin Extracted from Scales of Seabass (Lates calcarifer) and Grey Mullet (Mugil cephalus) in Vietnam

In this study, gelatin was obtained from the scales of seabass (Lates calcarifer) and grey mullet (Mugil cephalus) collected in Vietnam by using Alcalase® 2.4 L FG for pretreatment and electrochemically activated water with pH 2.0–2.5 for extraction. The characteristics and physicochemical properties of gelatin were investigated. The results indicated that the gelatin had a high protein content (88.6–90.0%) with ash (1.43–1.55%) and no fat. The gelatin was identified as type A due to its pH value. The yields of gelatin were 14.1–15.2%. The gel strength and melting point were 270.3 g and 26.7°C, respectively, for gelatin from seabass scales and 249.1 g and 25.9°C, respectively, for samples from grey mullet scales. In contrast, the viscosities of gelatin were found to be 6.97 cP for seabass and 8.73 cP for grey mullet. Both gelatins contained α-chain and β-chain as the major components. Circular dichroism spectra indicated that the triple helical structure in collagen was almost transformed to the random coil structure in gelatin. The proportion of imino acids was high (197–205 residues/1,000 residues). So, gelatin from seabass and grey mullet scales could be used as a potential replacement for mammalian gelatin.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.     

EFFECTS OF A MULTISTRAIN BIOFERTILIZER AND PHOSPHORUS RATES ON NUTRITION AND GRAIN YIELD OF PADDY RICE ON A SANDY SOIL IN SOUTHERN VIETNAM

Field experiments during two successive rainy seasons were conducted in southern Vietnam to evaluate the effects of a commercial inoculant biofertilizer (‘BioGro’) and fused magnesium phosphate (FMP) fertilizer on yield and nitrogen (N) and phosphorus (P) nutrition of rice. Inoculation with BioGro containing a pseudomonad, two bacilli and a soil yeast significantly increased grain yield in the second season and straw yield in both seasons by 3–5%. The FMP fertilizer significantly increased grain yield from 1.72–2.33 t ha^?1 to 2.99–3.58 t ha^?1 along with total N and P accumulation at all rates in both cropping seasons. In the first season the difference in grain yield between BioGro treated and untreated plots was marginal but in the second season BioGro out-yielded the control at all the rates of added P. Overall, BioGro application did not compensate for low P fertilizer application to the same extent previously demonstrated for low N fertilizer applications.     

Formation of molecular chlorine from the photolysis of ozone and aqueous sea-salt particles

Halogen atoms from the reactions of sea-salt particles may play a significant role in the marine boundary layer. Reactions of sodium chloride, the major component of sea-salt particles, with nitrogen oxides generate chlorine atom precursors. However, recent studies suggest there is an additional source of chlorine in the marine troposphere. This study shows that molecular chlorine is generated from the photolysis of ozone in the presence of sea-salt particles above their deliquescence point; this process may also occur in the ocean surface layer. Given the global distribution of ozone, this process may provide a global source of chlorine.     

Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2–1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density‐gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral‐like particles (~100–180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%–99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow‐out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.     

Effects of dietary arachidonic acid on final maturation,spawning and composition of gonad of black sea urchin Diadema setosum (Leske, 1778)

This study investigated the effect of dietary arachidonic acid (ARA, 20:4n‐6) on final maturation, spawning performance and composition of the gonad of black sea urchin (Diadema setosum). Four different dietary ARA levels of 0.3 g/kg (Control diet), 1.4 g/kg (ARA 1.4), 2.5 g/kg (ARA 2.5) and 3.7 g/kg (ARA 3.7) were prepared and tested in this feeding trial for 90 days. The result showed that growth in terms of weight was not relatively affected by dietary ARA. Gonadosomatic Index (GSI) of animals were gradually increased in all the diets throughout the trial. The highest GSI level (25.7 ± 3.16%) was observed in animals fed with the diet ARA 1.4. ARA content in the gonad proportionally increased with dietary ARA levels. The highest larval survival rate (85.5 ± 5.67%) was found in diet ARA 2.5. The results of this study indicate that ARA plays an important role in the reproduction functions of black sea urchin and supplementing an appropriate level of ARA in the broodstock diet could induce the final maturation and improve reproduction of black sea urchin, but a higher level of ARA (3.7 g/kg) seems to have a negative effect on both growth rate and GSI.     

Genetic variation in root morphology and microsatellite DNA loci in upland rice (Oryza sativa L.) from Vietnam

Drought is a major constraint to the productivity of rice in upland ecosystems. The rice root system plays an important role in the regulation of water uptake and extraction from deep soil layers. The aim of this research was to study the variation in root morphology and the genetic diversity in upland rice accessions. Thirty-three upland rice accessions originated from Vietnam along with 13 selected upland rice lines from several other countries were used in this study. Variation in root morphology was observed in most of the investigated root traits such as maximum root length, total root dry weight, deep root to shoot ratio, and total root to shoot ratio. Most of the traits showed significant correlation and appeared interrelated. Genetic diversity among upland rice accessions was studied with microsatellite markers. Forty-one alleles were detected with 14 rice microsatellite primer pairs among all the rice accessions. Two dendrograms have been created based on 35 microsatellite alleles and 10 morphological traits data for 38 accessions and compared. These results provided useful information for the selection of suitable cross combinations for mapping quantitative trait loci (QTLs) related to drought resistance in upland rice.