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.     

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。     

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.     

Immunohistochemical studies on the splenic lobe of the pancreas in young Japanese quails (Coturnix c. japonica)

The splenic lobe (Lobus splenicus) of the pancreas of young meat-type quails (Coturnix c. japonica) was examined by immunohistochemical and light microscopic methods. The endocrine cells are mainly grouped as alpha, beta and mixed islets. A large region consisting of alpha cells is located in the central region of the splenic lobe whereas numerous beta islets are detected in the periphery of the splenic lobe. Alpha islets are in the majority composed of toluidine blue positive A cells and a few toluidine blue negative D and / or avian pancreatic polypeptide (APP) endocrine cells. Beta islets contain only a few toluidine blue negative B and a few D cells. Immunohistochemical staining of the splenic lobe reveal in the centre of beta islets numerous insulin immunoreactive cells and scarcely in alpha islets, exocrine tissue and / or among acinar cells. Somatostatin immune-reactive cells form a circular layer in the periphery of beta islets whereas these cells are uniformly distributed throughout the alpha islet parenchyma and exocrine tissue. In conclusion, the morphology but also the endo- and exocrine functions of the splenic lobe of quails are similar to observations in other avian species such as chicken, duck, goose and pigeon.     

Histologic characteristics and local cellular immunity of the gland of the third eyelid after topical ophthalmic administration of 2% cyclosporine for treatment of dogs with keratoconjunctivitis sicca

OBJECTIVE: To evaluate the efficacy of topical administration of a 2% solution of cyclosporine (CsA) for treatment of dogs with keratoconjunctivitis sicca (KCS) and to correlate results with histopathologic characteristics and local cellular immunity of the gland of the third eyelid. ANIMALS: 24 dogs with bilateral KCS. PROCEDURE: Lacrimal secretion was measured, using Schirmer tear test (STT) strips. Leukocyte and T-lymphocyte subsets were determined in blood samples. Histopathologic changes as well as CD4+, CD8+, and alpha-naphthyl-acetate esterase-positive (ANAE+) lymphocytes were evaluated. RESULTS: Clinical signs resolved at the end of 1 month in conjunction with significantly increased STT values, compared with baseline values. Fifteen and 30 days after discontinuation of CsA treatment, a decrease was observed in STT values in both eyes; however, only values for the right eye were significantly different. There was a significant decrease in the number of lymphocytes and ANAE+ lymphocytes 15 and 30 days after discontinuation of CsA treatment, compared with baseline values. Differences were not observed in number of CD4+ lymphocytes among treatment groups. However, there was a significant decrease in number of CD8+ lymphocytes with reversal of the CD4+:CD8+ in both eyes after CsA treatment for 30 days, compared with the control group. Increased secretory activity and decreased lymphocyte infiltration were characteristic histopathologic findings. CONCLUSIONS AND CLINICAL RELEVANCE: Topical administration of a 2% solution of CsA was effective for the treatment of dogs with KCS. Strict follow-up monitoring is required after the cessation of treatment because of the possibility of recurrence of KCS.     

Echinococcosis/hydatidosis in western Iran

In the present study, 115 stray dogs (56 males and 59 females, mixed breed), 86 golden jackal (Canis aureus, 42 males and 44 females), 60 red foxes (Vulpes vulpes, 33 males and 27 females), and three female wolves (Canis lupus) were examined for Echinococcus granulosus infection, as well as, 32,898 sheep, 10,691 goats, 15,779 cattle and 659 buffaloes for hydatid infection from five provinces in western Iran during 3 years (1997-2000). Meanwhile fertility rates of different types and forms of cysts isolated from infected animals and the viability of protoscolices were also determined. Results indicated that 19.1% of the dogs, 2.3% of the golden jackals and 5% of the red foxes were infected with Echinococcus granulosus. 11.1% of the sheep, 6.3% of the goats, 16.4% of the cattle and 12.4% of the buffaloes were also found to be infected with hydatid cyst. The cysts isolated from liver and lungs of the sheep show higher fertility rate than the cysts of liver and lungs of goats, cattle and buffaloes.