DNA Immunization Against Very Virulent Infectious Bursal Disease Virus with VP2‐4‐3 Gene and Chicken IL‐6 Gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2‐4‐3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin‐6 (ChIL‐6) genes were constructed and designated as pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 respectively. Several DNA vaccination experiments were performed: 1‐week‐old chickens were intramuscularly injected with only plasmid pcDNA3‐VP2, pALTER‐MAX‐VP2‐4‐3 or mixture with pALTER‐MAX‐ChIL‐6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 three times conferred protection for 90% of chickens. Enzyme‐linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER‐MAX‐ChIL‐6 were higher than those immunized simply with plasmid pcDNA3‐VP2 or pALTER‐MAX‐VP2‐4‐3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT‐PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2‐4‐3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL‐6 plasmid significantly increased the protection after challenge with the very virulent strain.     

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n = 53), 29% of foxes (n = 48), 29% of stone martens (n = 21) and in 25–50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.     

Effects of Exogenous Glycine Betaine on the Physiological Characteristics in Medicago sativa Seedlings under Low-temperature Stress北大核心CSCD

In this study,the 'Ganong 5th' alfalfa (Medicago sativa L.) was used as experimental material,and two application methods (leaf application,root application) and six exogenous betaine concentrations (0,10,20,30,40 and 50 mmol·L-1) were used to treat the seedlings of alfalfa. Plant growth and related physiological indexes were measured after 7 days of low temperature (4?) stress,and the physiological mechanism of exogenous betaine to alleviate the low temperature stress of alfalfa was explored. The results showed that exogenous GB alleviated the loss of plant height,root length,leaf dry weight,root dry weight and chlorophyll content,and increase the content of soluble sugar (SS) and free proline (Pro),and reduced the increase in leaf relative membrane permeability and MDA content. Exogenous GB increased antioxidant enzyme activities and non-enzymatic antioxidant content. And also,exogenous GB enhanced the osmotic adjustment capacity of the plant by increasing endogenous GB accumulation and BADH activity. These results indicated that exogenous GB enhanced the resistance of Medicago sativa to low-temperature stress under low temperature adjustment. The membership function analysis indicated that spraying 30 mmol·L-1 GB on the leaves and spraying 40 mmol·L-1 GB on the roots had the best effect on alleviating the low-temperature stress of alfalfa seedlings. © 2019 Ｊｏｕｒｎａｌ ｏｆ Ｒｅｆｒｉｇｅｒａｔｉｏｎ. All rights reserved.     

转基因家畜生物反应器浅谈（续）

转基因家畜生物反应器浅谈（续）陈庄，魏平华（广东省农科院畜牧研究所广州５１０６４０）２转基因家畜生物反应器研究的进展目前，转基因动物研究有三大焦点：一是转基因方法研究；二是利用转基因动物来研究动物基因的表达调控机制；三是利用转基因动物生产非常规畜牧产...     

CRISPR/Cas9介导的基因组编辑技术及应用研究进展

成簇的规律间隔的短回文重复序列及其相关蛋白系统(Clustered regularly interspaced short palindromic repeats,CRISPR/CRISPR-associated nuclease system,CRISPR/Cas)是广泛存在于细菌和古细菌的适应性免疫系统,其中由Ⅱ型CRISPR/Cas系统改造而来的第三代基因组编辑技术——CRISPR/Cas9系统已广泛应用于生命科学研究的多个领域。本文主要从CRISPR/Cas9系统的发现、作用原理、应用进展及与其他新兴的基因组编辑技术的对比等四个方面进行阐述,重点介绍其最新研究进展,并展望了CRISPR/Cas9系统的应用前景。     

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.     

Modulation of broilers’ caecal microflora and metabolites in response to a potential probiotic Bacillus amyloliquefaciens

Studies have found that a dietary supplement of Bacillus amyloliquefaciens improved the growth performance, increased the nutrient digestibility of hosts and modulated the intestinal microflora. A total of 360 1‐day‐old Ross broilers were randomly divided into three treatments: a control group with a basal diet, an antibiotic group with a basal diet and added colistin sulphate, and a probiotics group with a basal diet and added Bacillus amyloliquefaciens. The HiSeq high‐throughput sequencing analysis of 16S rRNA was used to investigate the differences in birds’ caecal microflora, and metabolomics was used to analyse changes in caecal metabolites. Results showed that the supplementation of Bacillus amyloliquefaciens significantly improved the BW and ADG compared with the control birds. Results of sequencing indicated that (i) 645, 670, 596 unique operational taxonomic units (OTUs) were found in birds supplemented with Bacillus amyloliquefaciens on day 7, 21 and 42, separately, (ii) due to the diversity and relative abundance of the birds’ caecal microflora, the OTUs of the caecal microflora clustered according to age and treatment, except on day 42, (iii) among the six predominate families (Ruminococcaceae, Lachnospiraceae, Enterobacteriaceae, Erysipelotrichaceae, Lactobacillaceae and Rikenellaceae), the supplementation of Bacillus amyloliquefaciens significantly increased Enterobacteriaceae on day 42, (iv) Bacillus amyloliquefaciens increased the relative abundance of Faecalibacterium and Ruminococcus on day 21, increased the Faecalibacterium and Blautia and decreased the Ruminococcus on day 42. The metabolomics of caecal metabolites showed that the dietary Bacillus amyloliquefaciens changed the caecal metabolites involved of amino acid metabolism and glyceride metabolism, and the antibiotics changed the caecal metabolites that were related to carbohydrates and amino acid metabolism on day 21.     

Immunogenic properties of a bovine herpesvirus-1 (BHV-1) recombinant expressing major pseudorabies virus (PrV) glycoproteins in combination

A recombinant bovine herpesvirus type 1 (BHV-1), designated BHV-1/TF17-1, which expresses pseudorabies virus (PrV) glycoproteins gB, gC, gD, gE and gI in combination was constructed. To test the protective immunity, 10 mice were inoculated with BHV-1/TF17-1 and three weeks later 10 mice were intraperitoneally (i.p.) challenged with 20 LD50 virulent PrV (YS-81). BHV-1/TF17-1 protected all the mice from the PrV lethal challenge while all the control mice died in around 3 days. Mice vaccinated with BHV-1/TF17-1 acquired high PrV-neutralizing antibody titers and demonstrated strong delayed type hypersensitivity responses and moderate in vitro lymphocyte proliferative responses to PrV antigen. Since the major PrV glycoproteins were integrated into virions (probably into viral envelope), BHV-1/17-1 was neutralized with anti-PrV antiserum. However, the susceptibility of BHV-1/TF17-1 to anti-PrV antiserum is 2- to 4-fold lower than that of PrV vaccine lines. Our results demonstrated the possibility of BHV-1/17-1 as a vaccine to protect piglets from Audjesky's disease where maternal antibodies against PrV interfere attenuated live PrV vaccines.     

银杏-杜仲叶发酵物对脂多糖应激肉仔鸡的肠道功能和免疫反应的影响

本试验旨在研究饲粮中添加银杏-杜仲叶发酵物（FGEL）对脂多糖（LPS）应激肉仔鸡的生长性能、肠道结构、消化吸收功能和免疫反应的影响,并与未发酵银杏-杜仲叶（GEL）进行比较。采用2×3双因子试验设计,即2种免疫应激处理（腹腔注射LPS或注射等量生理盐水）和3种饲粮处理（基础饲粮及在基础饲粮中添加0.40%GEL或0.40%FGEL的试验饲粮）。选取1日龄健康爱拔益加（AA）肉仔鸡360只,随机分为6个处理,每个处理均设6个重复,每个重复10只鸡。试验期为21 d。在试验第13、15、17、19和21天,3个应激处理的试鸡腹腔注射500μg/kg BW的LPS,其他3个处理注射等量的生理盐水。在试验第21天最后1次注射后2～4 h进行屠宰取样。结果表明：1）与对照饲粮相比,饲粮添加FGEL的肉仔鸡料重比（F/G）有显著改善（P=0.024）,饲粮添加FGEL显著缓解了LPS应激肉仔鸡平均日增重（ADG）(P=0.023)和血清D-木糖的含量（P=0.011）的降低。2）与对照饲粮相比,饲粮添加FGEL显著缓解了LPS应激肉仔鸡十二指肠（P=0.011）和空肠（P=0.030）的相对重量...     

Surgical management of a malacic corneal ulcer in a greater one-horned Asian rhinoceros (Rhinoceros unicornis) using a free island tarsoconjunctival graft

A greater one-horned Asian rhinoceros (Rhinoceros unicornis) presented for presumed ocular trauma to the left eye, with secondary bacterial infection, resulting in severe and progressive corneal ulceration. Following a poor response to medical therapy, the animal was anesthetized for further examination, and a bulbar conjunctival pedicle graft performed. This graft failed by 48-h postsurgery as a result of self-trauma. The animal was re-anesthetized, and a free island tarsoconjunctival graft performed. This second procedure was successful, resulting in globe preservation, cosmesis, and functional vision in the affected eye.