犬细小病毒Taq Man荧光定量PCR检测方法的建立与初步应用

根据GenBank中CPV VP2蛋白基因序列,选择CPV VP2基因保守序列,分别设计1对特异性引物和TaqMan探针,建立了CPV Taq Man荧光定量PCR检测方法。对CPV Taq Man荧光定量PCR的反应体系和反应条件进行优化,并对其特异性和敏感性进行了测定,建立标准曲线。结果,CPV Taq Man荧光定量PCR对CPV检测阳性,并能对10拷贝/μL的CPV模板检测阳性,标准曲线的相关系数为0.9992,但对非CPV模板检测阴性。结果表明,所建立的CPV Taq Man荧光定量PCR具有很好的特异性和敏感性,标准曲线具有很强的实用性。同时对CPV Taq Man荧光定量PCR与CPV PCR进行了比较试验,结果前者比后者的敏感性高100倍。采用CPVHA、CPV PCR与CPV Taq Man荧光定量PCR对55份临床样品进行检测,CPV HA对30份样品检测阳性,CPVPCR对40份样品检测阳性,CPV Taq Man荧光定量PCR对47份样品检测阳性。这表明,CPV Taq Man荧光定量PCR对样品的检出率最高,具有特异、敏感、快速的特点,适用临床样品的检测。     

牛传染性鼻气管炎gB基因TaqMan探针及SYBR Green 1荧光定量PCR方法的建立与比较

为建立牛传染性鼻气管炎病毒(IBRV)荧光定量PCR检测方法,本研究利用IBRV g B基因序列设计引物及相应探针,分别建立Taq Man探针与SYBR Green 1荧光定量PCR方法,并对两种方法的敏感性等综合比较。结果显示,SYBR Green 1荧光定量PCR方法的相关系数(R2)为0.998,扩增效率(E)为95.7%;Taq Man探针荧光定量PCR方法的R2为0.999,E为108.3%;两种方法的敏感性均为0.2 TCID50,变异系数均小于3%。综合结果显示,两种检测方法无显著差异。最后,本研究成功建立了检测IBRV的荧光定量PCR,将为IBRV的临床检测提供方法。     

传染性胰脏坏死病毒实时荧光定量RT-PCR检测方法的建立及应用

传染性胰脏坏死病毒(infectious pancreatic necrosis virus,IPNV)是一种严重危害鲑鳟鱼类的重要传染性病原。为建立IPNV快速检测方法，根据IPNV VP2蛋白编码基因保守序列，设计并筛选出特异性引物和Taq Man探针，并优化筛选引物以及探针检测浓度、退火温度和退火时间，建立了一种实时荧光定量RT-PCR检测方法，并对方法进行了评价。结果显示：该方法灵敏度高，对IPNV的检测限为5 copies/μL；特异性强，对传染性造血器官坏死病毒、鲤春病毒血症病毒、病毒性出血性败血症病毒、草鱼呼肠孤病毒、流行性造血器官坏死病毒等其他病毒核酸扩增均呈阴性；重复性良好，组内与组间重复平均变异系数均小于3%；用本研究建立的实时荧光定量RT-PCR方法，对40份经病毒分离鉴定的样品进行IPNV检测，符合率为100%。结果表明，本研究建立的方法具有快速、特异、敏感等优点，可检测病毒含量较低的组织样品，对于IPNV的快速检测更具优势。     

山羊伪结核棒状杆菌TaqMan荧光定量PCR检测方法的建立和初步应用

为了快速检测无临床症状的山羊是否感染山羊伪结核棒状杆菌(Corynebacterium pseudotuberculosis),根据Gen Bank中伪结核棒状杆菌磷脂酶D(phospholipase D,PLD)基因序列,分别设计1对特异性引物和Taq Man探针,经过反应条件和体系优化,测定特异性、敏感性和稳定性,建立山羊伪结核病Taq Man荧光定量PCR检测方法,并对54份临床样品进行检测。结果显示:建立的山羊伪结核棒状杆菌方法具有很好的特异性,在20μL扩增体系中,引物浓度为0.20μmol/m L,探针浓度为0.45μmol/m L,退火温度59℃时最佳;最低可检测出4.6×102个拷贝数的细菌DNA,标准曲线相关系数是0.996。同时,对Taq Man荧光定量PCR和常规PCR进行了比较,前者的敏感性是后者的1 000倍;对54份临床样品进行检测,Taq Man荧光定量PCR检出的阳性率为68.5%(37/54),常规PCR检出的阳性率为53.7%(29/54)。研究表明:Taq Man荧光定量PCR法检出率较高,有特异、敏感且快速的特点,适用于临床样品的检测。     

鸡传染性法氏囊病病毒实时荧光定量RT—PCR检测方法的建立

本试验根据GenBank中鸡传染性法氏囊病病毒(IBDV)基因组设计一对针对病毒vp5基因的引物和一条特异性Taq Man探针,建立了一种快速检测IBDV核酸载量的Taq Man荧光定量RT-PCR方法.通过对反应条件和反应体系的优化,使得该方法在108拷贝/μL～101拷贝/μL范围内具有良好的线性关系.能够灵敏地检测初始模板中30个拷贝的病毒核酸,其灵敏度是常规RT-PCR检测方法的100倍.该方法不与其它的禽源病毒发生非特异性反应,并且具有良好的重复性,为IBDV的定性定量检测提供了有效的工具.     

A型猪塞内卡病毒TaqMan荧光定量PCR方法的建立

本研究针对A型猪塞内卡病毒(Senecavirus A, SVA)基因组保守区域设计了特异性引物及探针,建立了快速检测SVA的Taq Man荧光定量PCR方法。结果显示,以构建的重组质粒为标准品建立的Taq Man荧光定量PCR方法,标准曲线具有良好的线性关系,线性相关系数达0.9974;特异性良好,与口蹄疫病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒、猪伪狂犬病毒不存在交叉反应;对SVA核酸最低检测下限为3.75 copies/μL,而普通PCR最低检测下限为3.75×103 copies/μL;批内和批间的变异系数为均小于5%,重复性良好。本研究建立的SVA TaqMan荧光定量PCR方法为检测猪水疱性疾病病原提供了一种快速、灵敏的检测方法,为开展SVA流行病学调查提供了技术支持。     

H7亚型禽流感病毒核酸标准品制备及双探针荧光RT-PCR检测研究

从禽流感病毒A/Chicken/1/2002(H7N1)核酸中扩增了完整HA的基因编码序列,克隆测序后,采用体外转录方法制备c RNA。用RNA保存液稀释至含量约108Copies/μL。分装后进行均匀性和稳定性检验,通过8家实验室联合定值,取平均值作为定值结果;经统计分析计算了该标准品的不确定度。此外,采用Taq Man方法,根据禽流感病毒H7亚型的血凝素基因的末端,采用1对引物和2条MGB探针,建立了禽流感病毒H7亚型Taq Man荧光RT-PCR快速检测技术。应用建立的方法对研制的标准品进行检测,检测限可达10基因拷贝;对新城疫病毒及其他亚型的流感病毒核酸进行检测,结果表明,建立的方法特异性良好,而且双探针的设计思路更能保证病毒核酸的有效检测,防止漏检。     

猪博卡病毒实时荧光定量PCR检测方法的建立

根据GenBank公布的猪博卡病毒(PBoV)序列,通过VP1/2基因设计引物和Taq Man探针建立实时荧光定量PCR检测方法。建立的方法与PPV、PRRSV及PCV均无交叉反应,具有较高特异性,在107 copies/mL～101 copies/mL模板范围内具有良好的线性关系,所制作的标准曲线相关系数为0.997,最低可检测到101copies/mL的阳性质粒。说明所建立的PBoV实时荧光定量PCR检测方法具有灵敏度高、特异性好和精确性高等优点。     

水疱性口炎病毒荧光RT-PCR鉴别检测方法的建立与应用

本研究建立荧光定量RT-PCR鉴别检测水疱性口炎病毒的方法。针对Gen Bank登录的水疱性口炎两血清型-新泽西型(VSV-NJ)和印第安纳型(VSV-IND)的L基因保守区,设计1对引物和2条探针。通过对荧光定量RT-PCR反应条件的优化,建立了Taq Man荧光定量RT-PCR快速检测VSV的方法。与常规RT-PCR试验比较表明,所建立的荧光RT-PCR检测技术快速、敏感,检测时限3 h以内,具有很好的特异性和重复性,可以实现水疱性口炎病毒的快速检测分型。通过对342份临床样品进行检测,结果表明,所建立的检测方法适用于样品中水疱性口炎病毒的直接检测。     

猪圆环病毒Ⅱ型TaqMan荧光定量PCR检测方法的建立及应用

为了快速准确检测猪圆环病毒Ⅱ型(PCV-2)并对病毒拷贝数进行定量,试验根据Gen Bank中PCV-2保守序列(登录号为FJ644559.1)设计1对特异性引物和Taq Man探针,制备标准品,建立PCV-2的荧光定量检测方法,并对临床样品进行检测。结果表明:该方法在1×101~1×108拷贝/μL的模板范围内具有良好的线性关系,相关系数可达0.999;敏感性是常规PCR方法的100倍;对猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)均为阴性,没有交叉反应;批内、批间重复试验变异系数均小于2.50%;在临床检测中,比常规PCR方法检测PCV-2的阳性检出率高出38%。说明试验成功建立了PCV-2 Taq Man荧光定量PCR检测方法,可用于临床检测PCV-2感染及对其拷贝数进行定量。     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

A brief history of the prevention of infectious diseases by immunisations

Infectious diseases have always been a terrible scourge for humans. The appearance of these plagues, as they were called without distinction, was generally connected to various conditions: asters, climatic changes or religious reasons. The concept of contagious, and then infectious, diseases came slowly. Variolation, i.e. transmission of ‘virulent’ matter to induce a natural disease and the immunity against it, was brought from Constantinople to England by Lady Montague, in 1721. This ‘variolation’ technique was also often performed in veterinary medicine against diseases like sheep-pox or pleuropneumonia. As ‘vaccination’ is the term generally accepted for ‘immunisation’, variolation can be the word designating such a technique. The second period of the history of immunisation began, in 1880, with the studies of Pasteur and his collaborators. A great number of bacterial vaccines were developed: dead, live but attenuated or only parts of pathogens. The viruses were produced in animals, then in eggs and at last, in tissue cultures. Second generation vaccines appeared with genetic engineering: recombinant vaccines, vector vaccines, nucleic acids vaccines, and markers vaccines, among others. These novel technologies can permit the development of new ones and improve the quality of the vaccines already existing.     

Micronutrient Content of Certain Tropical Conventional and Unconventional Feed Resources of Southern India

The ash, silica and certain important micronutrients were estimated in conventional and unconventional feed and fodder resources available in Southern India. Commonly used dry roughages, such as paddy straw, ragi straw, maize kadbi/stalk, jowar kadbi/stalk, bajra stalk and wheat straw, were high in ash (9.9% +/- 0.77%) and silica (6.4% +/- 0.65%) and low in most of the other micronutrients, except iron, with paddy straw containing most silica (>9%). Cultivated non-leguminous (maize, jowar) and improved green crops (hybrid napier, guinea, green panic, NB-21, CO-1) were also high in ash (10.5% +/- 0.60% and 12.5% +/- 0.51%) but were moderate sources of P, Mg and Cu and good sources of Zn (98 +/- 13.8 ppm and 55 +/- 6.7 ppm). Leguminous green fodders (stylosanthus, lucerne, cow pea, soyabean) were excellent sources of Ca (1.9% +/- 0.16%), Mg (0.40% +/- 0.05%), Cu (30 +/- 5.2 ppm), Zn (121 +/- 14.7 ppm) and Fe (1234 +/- 166 ppm) and moderate sources of P. Mixed local grasses and weeds were high in silica (6.9% +/- 1.00%) but were good sources of Cu, Zn and Fe. Cereal grains (maize, wheat, rice, ragi) were low in ash (2.9% +/- 0.33%) and were relatively poor sources of Ca (0.22% +/- 0.03%), Mg (0.19% +/- 0.03%) and Cu (13 +/- 3.1 ppm). Pulses were low to medium sources of most minerals and good sources of Fe (1230 +/- 293 ppm). Oil seed cake/extractions (groundnut cake, cotton seed cake, soyabean meal, sunflower cake, safflower cake) and cereal by-products (rice polish, rice bran, wheat bran) were excellent sources of P (1.1% +/- 0.47% and 2.3% +/- 0.19%) and good sources of Zn (65 +/- 3.9 ppm and 66 +/- 10.7 ppm) and Fe (938 +/- 130 ppm and 662 +/- 126 ppm). Among the unconventional feeds screened, orange peel, sunflower heads, meat meal, rubber seed cake, spirulina algae and sea weeds contained plentiful Ca, Zn and Fe: tree leaves/top feeds (mulberry, erythrina, glyricidia, banana, subabul, groundnut haulms) were excellent sources of Ca (1.5% +/- 0.13%), Zn (120 +/- 22.9 ppm) and Fe (1033 +/- 133 ppm) but relatively poor sources of P. Soyabean husk, cocoa seed husk, rubber seed cake and meat meal were moderate to good sources of P (1.0% and 0.33%). The high Zn and Fe values of most feeds/fodders were probably due to soil contamination. This account of the micronutrient content of feed/fodder resources should help in strategic supplementation intended to alleviate local deficiencies.     

Feline intracranial neoplasia: retrospective review of 160 cases (1985-2001)

The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.     

Bovine Mastitis in Selected Areas of Southern Ethiopia

A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia. A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian, were examined by clinical examination and the California mastitis (CMT) test. Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively. Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.2%) clinically and 129 (60.8%) subclinically. The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season. Mastitis increased with parity number (R = 0.9). Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative. Of the 199 positive samples, Staphylococcus accounted for 39.2%, Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0%. It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.     

不同日粮氮水平对山羊氮代谢和微生物蛋白质合成的影响

本试验旨在探讨不同日粮氮水平对内蒙古白绒山羊氮代谢和微生物蛋白质合成(MCP)的影响,以达到提高氮利用率,减少环境污染和饲料资源的浪费。试验选用9只体况良好、体重为(43.83±2.95)kg,装有瘤胃瘘管的内蒙古白绒山羊,按体重随机分为3组,每组3只。日粮分为低氮7.5%、中氮10.5%、高氮13.5%3个氮水平。试验使用常规试验方法分析测定粪尿血液等生化指标,通过全收尿法和尿嘌呤衍生物法估测MCP。结果表明:随着氮水平增加,氨氮(NH3-N)浓度、氮摄入量、尿氮排出、尿中尿素氮(UUN)和MCP显著增加(P0.05),干物质采食量(DMI)显著降低(P0.01);沉积氮占总摄入氮的比例在中氮组最高,MCP在高氮组最高。综上所述,适当降低日粮氮水平可减少粪尿氮排放,提高反刍动物氮利用率。     

Microsporidial keratopathy in two dogs

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin‐fixed, paraffin‐embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow‐up are discussed.     

优质肉鸡S3系体重与体尺性状指标的主成分分析

为研究地方优质肉鸡S3系体重和体尺性状之间的内在相关性,12周龄时选取同一饲养条件下的150只鸡(公母各半)进行体重和体尺性状等7个指标的测定。结果表明:S3系公鸡的体重、体斜长、龙骨长、胸角均显著高于母鸡(P0.05);公母鸡体重、体斜长、胸宽、胫围这4项指标变异系数较大,有较大选育空间;体重与体斜长、龙骨长、胸宽、胸角、胫围呈极显著正相关(P0.01),相关系数分别为0.57、0.52、0.51、0.49和0.47,与胸深、胫长呈显著相关(P0.05),相关系数分别为0.44和0.39;其他各体尺指标间也存在不同程度的正负相关性(P0.05或P0.01);体重和7个体尺性能指标综合成5个复合指标,累计贡献率达92.6355%,其中第l、2、3、4、5主成分分别解释总变异的36.5353%、22.8642%、13.3115%、10.4535%和8.4710%;各主成分的特征根分布较广,经统计计算入选的5个主成分所包含信息的侧重点各有相同,分别都在一定程度上反映了S3鸡的体型特征和生长发育规律,为今后选育出更加符合市场需求的屠宰加工优质型肉鸡指明了的方向。