Generation of mouse monoclonal antibodies specific to tilapia immunoglobulin using fish immunoglobulin/BSA complex for monitoring of the immune response in Nile tilapia Oreochromis niloticus

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.     

常用饲草饲料瘤胃降解规律的研究

本文探讨了贵州常用饲草饲料在山羊瘤胃的降解规律,旨在为研究科学配制山羊日粮提供依据。试验选用2只装瘤胃瘘管的山羊,采用瘤胃尼龙袋法研究3类10种常用饲草饲料的干物质（DM）、粗蛋白（CP）、有机物（OM）瘤胃降解率。结果表明：3类饲草饲料的DM降解率由高到低依次为：精饲料（70.75%）﹥单一牧草（68.26%）﹥混合牧草66.59%（P﹤0.05）;单一牧草的CP降解率分别比精饲料和混合牧草高4.5%（P﹤0.05）和6.05%（P﹤0.05）;单一牧草的OM降解率分别比精饲料和混合牧草高1.2%（P﹥0.05）和6.38%（P﹤0.05）。由此可见,不同饲草饲料在瘤胃内的降解规律不同,青绿饲料影响饲草饲料的瘤胃降解率。     

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP_AHPND), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb–protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10⁷ cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP_AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.     

干旱季节日粮中添加益生菌和过瘤胃脂肪酸对饲喂玉米青贮料生长山羊平均日增重和瘤胃代谢的影响

本试验目的在于调查添加益生菌、棕榈油过瘤胃脂肪酸(POBF)和益生菌 POBF对饲喂玉米青贮料的生长山羊平均日增重和瘤胃代谢影响.试验选用了27只约5月龄、体重(13.4±1.3)kg杂交生长山羊(Thai native×Anglo-Nubian),根据完全随机因子试验方法分成9组,进行10周饲养试验.POBF包含0、20和40 g/(goat·d)3个水平,益生菌包含0、2.5和5.0 g/(goat·d)3个水平.结果表明:POBF显著地降低日均干物质采食量(DMI)(P<0.05),益生菌提高DMI(P<0.05),20 g/(goat·d)POBF 益生菌[2.5和5.0 g/(goat·d)]不影响DMI(P>0.05).益生菌和益生菌 POBF显著提高平均日增重(ADG)(P<0.05).益生菌极显著地提高DM和OM消化率(P<0.01),POBF 益生菌显著提高DM和OM消化率(P<0.05).POBF、益生菌、POBF 益生菌均提高CP消化率(P>0.05).益生菌和POBF 益生菌显著提高NDF消化率(P<0.05).益生菌、POBF和益生菌 POBF极显著提高挥发性脂肪酸总量(P<0.01)和丙酸百分比例(P<0.05),降低乙酸百分比例(P>0.05).本试验结果证明:益生菌和POBF都能提高饲喂玉米青贮料的生长山羊ADG和瘤胃代谢水平,添加2.5 g/(goat·d)益生菌和20 g/(goat·d)POBF效果较好,同时添加2.5 g/(goat·d)益生菌和20 g/(goat·d)POBF效果最佳.     

ORIGINAL ARTICLE: Effects of supplementing rice straw with Leucaena (Leucaena leucocephala) and Madras thorn (Pithecellobium dulce) foliages on digestibility,microbial N supply and nitrogen balance of growing goats

A total of 12 crossbred (Boer × Anglo‐Nubian) goats were chosen from a commercial farm on the basis of similar body weight (25.0 ± 3.1 kg). The goats were housed in individual pens and allowed 3 weeks to adapt to experimental conditions. The goats were randomly allocated to three treatments in a 3 × 3 Latin square experiment (replicated four times). Within each period, each goat was given rice straw as roughage plus the respective treatment diet. The diets were iso‐nitrogenous and iso‐energetic containing cassava pulp, molasses, urea and commercial mineral and vitamin mix. The experimental treatments were (i) soybean meal (SBM), (ii) partial substitution of SBM with Leucaena (Leucaena leucocephala) foliage and (iii) partial substitution of SBM with Madras thorn (Pithecellobium dulce) foliage. Nutrient intakes, ruminal characteristics (pH, ammonia nitrogen and volatile fatty acids), nitrogen balances, plasma urea nitrogen and microbial N supply were not significantly different among treatments. The results of this study indicate that protein foliages from locally grown shrubs and trees can substitute imported feedstuff concentrates (e.g. SBM) as protein supplement for goat production.     

Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene

Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus–specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate–labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP–LFD technique for V. alginolyticus was 1.8 × 10² CFU/mL while that of PCR was 1.8 × 10³ CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10³ CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP–LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP–LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.

Received November 11, 2014; accepted March 29, 2015     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS‐PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross‐reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody‐based assays.