猪瘟病毒野毒株TaqMan-MGB荧光定量PCR、鉴别方法的建立与应用

 【目的】建立一种快速、敏感、特异地检测猪瘟病毒野毒的一步TaqMan-MGB 荧光定量PCR方法,为临床鉴别猪瘟野毒和HCLV疫苗毒提供了准确可靠的工具。【方法】在猪瘟病毒基因组5′端非编码保守区设计一对猪瘟病毒通用引物和一条特异性MGB探针,通过优化,得到最佳反应体系和反应条件,进行特异性、灵敏度和临床样本符合检验。【结果】该方法在100～10-7范围内线性相关系数为0.998,检测极限达5.3×10-2pg 病毒核酸;对24个质控样本检测结果显示,该方法能检测除猪瘟兔化弱毒疫苗（HCLV）以外的猪瘟流野毒株,其它猪相关致病病原检测阴性;对122份疑似猪瘟样本检测的结果与本实验室建立的RT-nPCR方法的符合率为94.3％（115/122）,阳性和阴性符合率分别为86.4％（38/44）和98.7％（77/78）,Kappa值为0.87＞0.75。【结论】建立的一步TaqMan-MGB 荧光定量PCR方法能鉴别猪瘟野毒和HCLV疫苗毒,且特异性好、灵敏度高、与笔者先前建立的RT-nPCR方法对临床样本的检测结果具有高度的一致性。     

应用套式RT—PCR检测广西临床病料猪流感病毒

套式RT-PCR技术是在常规RT-PCR的基础上设计1对内套引物,对常规RT-PCR的产物再进行一次PCR扩增,该技术比常规RT-PCR技术的检测敏感度更高,更适于检测病原RNA含量极其少的临床病料。通过应用套式RT-PCR方法对来自广西7个市不同猪场的疑似猪流感的临床病料进行猪流感病毒检测,结果发现,被检的7个市均检出猪流感病毒,72份病料中31份为阳性,阳性率为43．06％,可见广西有呼吸道症状的病猪中有较高猪流感病毒感染率,必须重视和加强广西猪流感的监测与防控工作。     

氨胁迫对猪粪厌氧消化性能的影响

以猪粪为原料,采用批式试验方法,研究不同氨氮添加量(0、400、800、1600、2400、3200、4000 mg·L^-1)对厌氧消化产气效果的影响.结果表明:随着氨氮添加量的增加,总产气量和CH₄产率均呈现先升高后降低的变化趋势,氨氮添加量 ≥2400 mg·L^-1时,厌氧消化过程受到显著抑制;不同处理中猪粪挥发性固体(VS)的CH₄产率分别为328.5、338.1、323.2、304.9、276.2、124.9、56.1 mL·g^-1.氨氮添加量为0~800 mg·L^-1时,最大VS产CH₄速率分别为18.3、18.4、17.1 mL·g^-1·d^-1;氨氮添加量为2400 mg·L^-1时,产气高峰推迟,产CH₄速率明显降低;氨氮添加量 ≥400 mg·L^-1时,厌氧消化30 d底物的生物转化产CH₄效率随氨氮添加量的增加逐渐降低,分别为56.7%、54.5%、52.4%、30.6%、1.6%和1.3%;氨氮添加量为400~2400 mg·L^-1时,乙酸利用型产甲烷菌Methanosaeta的相对丰度总体随氨氮质量浓度的增加而降低,而氢利用型产甲烷菌Methanosarcina和Methanococcus具有相反的变化规律.     

猪源绿脓杆菌的分离鉴定及16S rRNA基因同源性分析

从解剖病变为心包炎、纤维素性肺炎、肺脓肿的病死仔猪体内分离到1株细菌,通过形态特征观察、生化试验,将分离菌株初步鉴定为绿脓杆菌。动物致病性试验结果显示,分离菌株对小鼠具有很强的致病性。药敏试验结果显示,分离菌株对氧氟沙星、恩诺沙星高度敏感,对头孢噻肟、庆大霉素中度敏感,对青霉素、氟苯尼考、氨苄西林、阿莫西林、强力霉素、卡那霉素等耐药。将分离菌株的16S rRNA基因序列与Gen Bank中发表的绿脓杆菌序列进行Blast比对,结果表明,分离菌株与绿脓杆菌不同菌株的同源性高达99%以上,进一步确定分离菌株为绿脓杆菌。基于16S rRNA基因序列,使用MEGA 5.05软件,构建系统进化树,结果表明,分离菌株与病人痰液和腹泻婴儿分离菌株的同源性最高,与未加工水果分离菌株同源性最低。     

猪传染性胃肠炎药物治疗研究进展

猪传染性胃肠炎对养猪业的危害很大,对于该病除采用疫苗预防之外,进行临床上的药物防控也不容忽视,因为目前疫苗预防方面也存在着一定的问题.临床上对猪传染性胃肠炎的治疗有较多的报道,有中药复方制剂治疗方面的尝试,也有根据临床腹泻症状进行西药治疗,还有中西药相结合进行治疗.这对于临床防控猪传染性胃肠炎不仅提出了许多可以借鉴的方法,也为临床防治猪传染性胃肠炎提供了思路.     

基于全卷积神经网络的猪背膘厚快速准确测定

为了有效地弥补猪B超图像人工手绘背膘厚的不足,为生猪育种工作提供更精准和稳定的背膘厚测定新方法。该研究将全卷积神经网络（Fully Convolutional Networks,FCN）模型应用于猪B超图像的背膘分割和背膘厚测定上,开发出一套使用Python调用FCN模型对猪B超图像背膘厚进行自动测定的系统。通过开展验证集验证试验、屠宰比对试验和人员比对试验,发现模型测定结果和标注结果之间差异不显著（P>0.05）,两者相关系数达0.92(P<0.01);B超标准测定背膘厚和FCN分割测定背膘厚的相关系数达到0.97(P<0.01);专家组组内标准差为0.17 mm（最小）,行业外组组内标准差为1.67 mm（最大）,而FCN分割结果稳定性强,不受人员因素的影响。因此,该方法可以实现对外种猪B超背膘厚的精准、快速、稳定测量,减少猪场对专业人员的依赖,降低测定人员培训成本,减少工作人员工作量。     

Above and Below Ground Measurements of Greenhouse Gases from Swine Effluent Amended Soil

Abstract

As a means of economic disposal and to reduce need for chemical fertilizer, waste generated from swine production is often applied to agricultural land. However, there remain many environmental concerns about this practice. Two such concerns, contribution to the greenhouse effect and stratospheric ozone depletion by gases emitted from waste‐amended soils, have not been thoroughly investigated. An intact core study at Auburn University (32 36′N, 85 36′W) was conducted to determine the source‐sink relationship of three greenhouse gases in three Alabama soils (Black Belt, Coastal Plain, and Appalachian Plateau regions) amended with swine waste effluent. Soil cores were arranged in a completely random design, and treatments used for each soil type consisted of a control, a swine effluent amendment (112 kg N ha^?1), and an ammonium nitrate (NH₄NO₃) fertilizer amendment (112 kg N ha^?1). During a 2‐year period, a closed‐chamber technique was used to determine rates of emission of nitrous oxide (N₂O)–nitrogen (N), carbon dioxide (CO₂)–carbon (C), and methane (CH₄)–C from the soil surface. Gas probes inserted into the soil cores were used to determine concentrations of N₂O‐N and CO₂‐C from depths of 5, 15, and 25 cm. Soil water was collected from each depth using microlysimeters at the time of gas collection to determine soil‐solution N status. Application of swine effluent had an immediate effect on emissions of N₂O‐N, CO₂‐C, and CH₄‐C from all soil textures. However, greatest cumulative emissions and highest peak rates of emission of all three trace gases, directly following effluent applications, were most commonly observed from sandier textured Coastal Plain and Appalachian Plateau soils, as compared to heavier textured Black Belt soil. When considering greenhouse gas emission potential, soil type should be a determining factor for selection of swine effluent waste disposal sites in Alabama.     

高效液相色谱-质谱法测定猪组织中硝基咪唑类药物残留量

建立了一种高灵敏度和高选择性的检测猪组织中硝基咪唑类药物残留的高效液相色谱-大气压化学电离质谱(APCI)法,可同时进行猪组织中3种硝基咪唑类药物残留的确证分析.本方法检测猪肌肉、肝脏、肾脏和皮下组织中硝基咪唑类药物的检测限为0.5～1μg/kg;空白组织添加硝基咪唑类药物水平为0.5、1、2或4μg/kg时,测得回收率:肌肉为61.1%～85.4%,肝脏为62.3%～80.4%,肾脏为61.6%～82.0%,皮下组织为63.6%～84.1%.     

The 1997-1998 classical swine fever epidemic in The Netherlands--a survival analysis

The aim of this analysis was to characterise the temporal pattern of infection during the 1997/98 classical swine fever (CSF) epidemic in The Netherlands and hence identify and quantify risk factors for infection in different enterprise types and areas. Survival analysis and Cox proportional hazards regression were used to describe the epidemic. Substantial differences in temporal survival patterns (herd breakdown rate) were found between areas where different control policies operated. Factors with a significant influence on the infection hazard of individual herds included: sow numbers as a percentage of total sows and fatteners (HR = 3.38 for mixed herds (0.1–60% sows) vs. fattening herds (0% sows) and HR = 2.74 for breeding herds (60–100% sows) vs. fattening herds), the number of ‘transport contacts per month’ (>0.3 vs. <0.3; HR = 4.11), pig density (pigs/km²) in the area (HR_{1000 pigs} 1.48) and herd size (HR_{100 pigs} = 1.01).

Pre-emptive slaughter in an area appeared to be associated with lower subsequent disease levels. Higher frequency of transport contacts for welfare slaughter during the epidemic, however, well regulated and controlled, was associated with a substantially higher risk of becoming infected. The positive association of a higher pig density with CSF indicates the potential importance of local spread as a factor in disease transmission and emphasizes that dilution of the pig population can contribute to reduction in CSF occurrence. This analysis suggests however, that if pre-emptive slaughter can promptly be applied effectively in an area after initial diagnosis, pig density is then not a significant factor. Mixed and breeding herds had a higher probability of becoming infected than fattening herds, possibly due to different types and frequencies of inter-herd contacts. These contacts continue to some extent during the epidemic, despite the standstill of animal movements.     

Experimental Pathological Study of Acute African Swine Fever

To study the clinicopathology and histopathology of African swine fever (ASF), and to explore the internal relationship between pathological changes and disease occurrence and development and its pathological mechanism, 13 Landrace pigs with bodyweight about 20 kg were intramuscular injected with African swine fever virus (ASFV), strain Pig/HLJ/18 at a dose of 10²HAD₅₀·mL^-1. During the experiment, all the dead pigs were systematically dissected and sampled, paraffin sections were produced, and haematoxylin-eosin staining was performed. Clinicopathological evaluation standards for acute ASF were established, then pathological lesions (classification variables) were expressed by counting frequency and percentage, and the lesion degree (continuous variables) was graded and scored according to different pathological changes of various tissues and organs. The results showed that all infected pigs were in line with the clinical characteristics of ASF, including acute, febrile and highly infectious, with a 100% incidence rate and 100% mortality. The dead pigs showed typical characteristics of septicemia, cadavers prone to corruption, blood clotting adverse or hemolysis, rigor mortis incomplete. The main pathological lesions were hemorrhagic necrotizing lymphadenitis, acute inflammatory splenomegaly (septic spleen), cerebral edema, pulmonary edema and lung consolidation et al. The spleen and lymphonodus are the target organs attacked by ASFV, with the most significant lesions, the earliest occurrence time, the longest duration and the highest frequency. The most prominent pathological changes are blood circulation disorders, including multiple pathological manifestations such as edema, hyperemia, congestion, hemorrhage, infarction, disseminated intravascular coagulation (DIC), and the most important characteristics are hemorrhagic lesions. The inflammatory reaction of lymphocytic exudation caused by ASFV runs through the whole process, especially in the middle and later stages of the course. The results suggest that the main pathological process of acute African swine fever is a typical immune/inflammatory cascade reaction and severe systemic blood circulation disorder, which resulted in the high incidence rate and high mortality rate of acute ASF.