利用CRISPR/Cas9系统构建的转基因家蚕对核型多角体病毒的抑制

CRISPR/Cas9介导的基因编辑已经被证明可以通过破坏病原体的基因来有效地抑制感染。西南大学的DONG Z等学者最近构建了表达CRISPR/Cas9的家蚕转基因系和以家蚕核型多角体病毒(BmNPV)为目标的sgRNA,以调节家蚕早期基因的表达。通过G,代杂交获得4个转基因杂交系:Cas9(-)/sgRNA(-),Cas9(C)/sgRNA(-),Cas9(-)/sgRNA(C)和Cas9(C)/sgRNA(C)。研究证明了Cas9(C)/sgRNA(C)转基因系有效地编辑了家蚕核型多角体病毒(BmNPV)基因组的目标位点,并在BmNPV感染后观察到大片段缺失。研究者进一步对Cas9(C)/sgRNA(C)转基因系的抗病毒分析表明,半致死剂量(LD_(50))比正常接种包涵体后的值高出1 000倍。作者分析了Cas9(C)/sgRNA(C)转基因杂交系的经济性状和脱靶效率,与正常水平无显著差异。作者的研究表明CRISPR/Cas9介导的基因编辑可以更有效地靶向家蚕核型多角体病毒(BmNPV)基因组,并可作为一种昆虫抗病毒的方法。     

家蚕组蛋白甲基转移酶BmAsh2对蚕蛹的性别决定至关重要

性别决定是一个分级管理的过程,包括昆虫在内的不同生物具有高度的多样性。在鳞翅目昆虫家蚕中,由W染色体衍生的FempiRNA被鉴定为主要的性别决定因子,它揭示了一个独特的piRNA介导的性别决定途径。然而,家蚕性别决定的综合机制目前尚不清楚。中国科学院昆虫发展与进化生物学研究中心的LI Z等研究发现蚕蛹蛋白BmSiwi是决定家蚕性别的关键,而不是BmAgo3。CRISPR/Cas9介导的BmSiwi的敲除导致发育中性别阻滞和部分雌性性别逆转,而BmAgo3耗竭只影响卵子发生。研究者鉴定了3种组蛋白甲基转移酶(HMTs),它们在BmSiwi突变蛾中显著下调。其中一种HMT是BmAsh2,引起了piRNAs和转座因子(TEs)的失调,支持了它在piRNA信号通路中的作用。更重要的是,研究发现BmAsh2突变导致了性别发育阻滞和部分雌性对雄性的性别逆转,以及性别决定基因Bmdsx和BmMasc的失调。其他两种HMT是BmSETD2和BmEggless,它们的突变并不影响piRNA介导的性别决定。组织学分析和免疫沉淀结果支持BmAsh2和BmSiwi蛋白之间的功能相互作用。试验数据首次提供了证据,证明组蛋白甲基转移酶(HMT)BmAsh2在piRNA介导的蚕蛹性别决定中起关键作用。     

Outbreak of clinical listeriosis in sheep: evaluation from possible contamination routes from feed to raw produce and humans

We report the results of clinical and microbiological investigations on Listeria monocytogenes infections in a flock of 55 sheep and describe the implications for the safety of the raw milk and raw-milk cheeses produced in the on-farm dairy. The outbreak was caused by feeding grass silage, which was contaminated with 5 log10 CFU L. monocytogenes/g. Clinically, although having been fed from the same batch of silage, abortive (nine ewes), encephalitic (one ewe) and septicaemic (four ewes) forms of listeriosis were observed during the outbreak phase. As the starting point of feeding the contaminated silage was known we could calculate an incubation period of 18+/-2 and 26 days for the abortive and the encephalitic form of listeriosis, respectively. Pathologically, the septicaemic cases suffered from Listeria accumulation at comparable numbers in visceral organs but not in the brain. Only a single ewe developed central nervous symptoms and a rhomb-encephalitis was immunohistologically confirmed. In this case the infection proceeded from the nasal mucosa into the brain, with no infections of the liver, spleen and other visceral organs. Sampling of the cheese production chain, the farm environment and the persons living at the farm revealed the exposure of a farm-worker to an isolate genetically indistinguishable from the outbreak clone, obviously through the consumption of faecally contaminated bovine raw milk. The cheese under processing was free of Listeria because, as a result of intensive consultations, the farmer ensured a proper acidification of the cheese. The epidemiological findings suggest that food safety matters should be assessed in any case where infection of food-producing animals with potential human pathogens is observed.     

Field detection of resistance to isometamidium chloride and diminazene aceturate in Trypanosoma vivax from the region of the Boucle du Mouhoun in Burkina Faso

A longitudinal study assessed the chemoresistance to isometamidium chloride (ISM) and diminazene aceturate (DA) in the region of the Boucle du Mouhoun in Burkina Faso. A preliminary cross-sectional survey allowed the identification of the 10 villages with the highest parasitological prevalences (from 2.1% to 16.1%). In each of these 10 villages, two herds of approximately 50 bovines were selected, one being treated with ISM (1mg/kg b.w.) and the other remaining untreated as control group. All animals (treated and untreated herds) becoming infected were treated with DA (3.5mg/kg b.w.). In total, 978 head of cattle were followed up. Fortnightly controls of the parasitaemia and PCV were carried out during 8 weeks. The main trypanosome species was Trypanosoma vivax (83.6%) followed by Trypanosoma congolense (16.4%). In two villages, less than 25% of the control untreated cattle became positive indicating no need to use prophylactic treatment. These two villages were not further studied. Resistance to ISM was observed in 5 of the remaining 8 villages (Débé, Bendougou, Kangotenga, Mou and Laro) where the relative risk (control/treated hazard ratios) of becoming infected was lower than 2 i.e. between 0.89 (95% CI: 0.43-2.74) and 1.75 (95% CI: 0.57-5.37). In contrast, this study did not show evidence of resistance to DA in the surveyed villages with only 8.6% (n=93) of the cattle relapsing after treatment. Our results suggest that because of the low prevalence of multiple resistances in the area a meticulous use of the sanative pair system would constitute the best option to delay as much as possible the spread of chemoresistance till complete eradication of the disease by vector control operations.     

γ射线防治美洲幼虫腐臭病在商业上的应用

引言美洲幼虫腐臭病(AFB)是一种蜜蜂幼虫病,可造成蜂蜜生产、蜂数和蜂群的重大损失。此病系幼虫芽孢杆菌(Bacilluas larvae)引起,可由受该菌孢子污染的蜂具在箱、群之间传播。用γ射线照射受污染的蜂具,可杀死上面的孢子,对养蜂业极为有益。为此,本文特谈谈这种技术。     

Recombinant pseudorabies virus expressing P12A and 3C of FMDV can partially protect piglets against FMDV challenge

One of the crucial factors for evaluation of an effective genetically engineered vaccine is whether susceptible animals are protected from virus challenge after vaccination. In this study, a recombinant pseudorabies virus (PRV-P12A3C) that expressed capsid precursor polypeptide P12A and nonstructural protein 3C of foot-and-mouth disease virus (FMDV) was used as a vaccine. The expression of P12A3C and its immunogenicity and protective efficacy against FMDV challenge were measured. Humoral and cellular immune responses were evaluated after each immunization. Subsequently, each piglet was challenged with 1000 ID₅₀ (50% infection dose) FMDV serotype O, named OR/80, which is used to produce vaccine in China. PRV-P12A3C induced a high level of neutralizing antibody and FMDV-specific lymphocytes. Inactivated vaccine provided 100% protection, and the vector strain (TK⁻/gE⁻/gI⁻) showed no protection. PRV-P12A3C induced 60% protection, compared with piglets that were vaccinated with TK⁻/gE⁻/gI⁻. The severity of clinical signs for the remaining two piglets was lighter and the appearance of vesicles was delayed.     

Impacts of a freedom farrowing pen design on sow behaviours and performance

The limited space in farrowing crate imposes many challenges, such as prolonged farrowing duration and high piglet stillbirth rate. Although the features of farrowing pens compensate for the drawbacks of farrowing crates, they are associated with high piglet crushing mortality caused by the greater space afforded to sows and their rolling-over behaviour. Therefore, a freedom farrowing pen was designed to overcome the drawbacks of both farrowing crates and farrowing pens. The main features of the freedom farrowing pen are its left anti-crushing bar and detachable right anti-crushing bar on the sides of the sow lying area. It also has a 10 cm-high anti-crushing bar in the non-lying area. Eighteen healthy, multiparous Yorkshire sows (3-7 parity) were averaged and randomly assigned to farrowing crates, farrowing pens, and freedom farrowing pens to compare the effects of the farrowing systems on sow behaviour and performance. Results showed that the farrowing duration and the mean piglet birth intervals were longer for the sows in farrowing crates than for those in farrowing pens and freedom farrowing pens (P<0.05), but there was no difference between the sows in farrowing pens and those in freedom farrowing pens (P>0.05). The piglet stillbirth rate was higher for the sows in farrowing crates than for those in farrowing pens and freedom farrowing pens (P<0.001). Crushing mortality was higher among piglets in farrowing pens (P<0.001), but there was no difference between piglets in freedom farrowing pens and those in farrowing crates (P>0.05). The freedom farrowing pen and the farrowing pen allowed sows to turn around and move freely, but because of the different structures of their anti-crushing bars, the increase in sow movement did not cause higher piglet crushing mortality (P>0.05). Sows in freedom farrowing pens were found to be more protective of their piglets.     

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.     

Seasonal and photoperiodic regulation of secretion of hormones associated with reproduction in Magang goose ganders

This study examined the reproductive endocrine profile under natural and artificial photoperiods in Magang goose ganders. Group 1 ganders (n=8) served as non-treated controls and were exposed to natural photoperiod throughout the experiment from 13th January to 17th December 2004. Group 2 ganders (n=8) were exposed to 18 h long daily photoperiod for 60 days from 13 January till 15 March 2004 and again to 16 h photoperiod for 75 days till 10th October 2004, and the 11h short photoperiod in the remainder periods of the experiment. In control ganders, plasma LH concentrations were high in normal breeding seasons (August-March) and decreased to low levels in non-breeding season from April to July. Testosterone concentrations changed similarly to that of LH throughout the seasons. Seasonal pattern of PRL concentrations was opposite to those of LH and testosterone, with low values in breeding season and high values in non-breeding season. In artificial photoperiod treated ganders, increasing photoperiod increased PRL and decreased LH and testosterone concentrations, while decreasing photoperiod reversed these changes. There were no seasonal or photoperiod caused changes in plasma T3 concentrations in both control ganders and artificial photoperiod treated ganders. These results demonstrated that in Magang goose ganders that long photoperiod stimulates PRL secretion and decreases LH secretion, which terminates reproductive season in spring and early summer, and short photoperiod stimulates LH secretion and inhibits PRL secretion rendering ganders enter into reproductive season.