牛腺病毒3型的研究进展

牛腺病毒3型(bovine adenovirus type 3,BAdV-3)为腺病毒科(Adenoviridae)哺乳动物腺病毒属(Masta-denovirus)的成员,为无囊膜包裹的线性双链DNA病毒,是导致牛呼吸道疾病的重要病原之一.本文就BAdV-3的生物学特性、流行情况、致病性、检测技术等方面的研究进展进行...     

一起牦牛呼吸道疾病的病原学诊断

2022年2月,川西北某地牦牛暴发以呼吸道症状为突出特征的传染病。为确定病因,对发病牦牛进行了相关病原的病原学诊断。共采集13头发病牦牛的20份病料,采用PCR方法对牛病毒性腹泻病毒（BVDV）、牛副流感病毒3型（BPIV-3）、牛传染性鼻气管炎病毒（IBRV）、牛冠状病毒（BCoV）、牛呼吸道合胞体病毒（BRSV）、牛腺病毒3型（BAdV-3）以及多杀性巴氏杆菌（P. multocida,Pm）、溶血性曼氏杆菌（M. haemolytica,Mh）和牛支原体（M. bovis,Mb）等9种牛呼吸道病原进行检测。结果显示：在20份样本中共检测出IBRV、BAdV-3和Pm 3种病原,且存在混合感染,其他病原未检出;4份全血样本中检测出3份IBRV、1份BAdV-3阳性,1份胎盘组织样本中检出IBRV阳性,8份鼻拭子样本中检出3份IBRV、5份BAdV-3阳性,2份流产中阴道拭子中检出2份BAdV-3阳性,5份肺脏组织样本中检出4份Pm阳性;进一步对IBRV和Pm进行分型鉴定,确定该地牦牛感染的为1.2型IBRV和A型Pm。结果表明,引发该地牦牛呼吸道疫病的是1.2型IBRV、BAdV-...     

宁夏某肉牛场牛呼吸道疾病综合征相关病原检测及分型鉴定

2022年3月冬春换季期间，宁夏某肉牛场暴发牛呼吸道疾病综合征（bovine respiratory disease complex，BRDC）。为了解造成此次病情的病原，采集12份发病牛的鼻腔深部棉拭子样品，采用PCR和RT-PCR方法，分别对牛副流感病毒3型（BPIV-3）、牛冠状病毒（BCoV）、牛病毒性腹泻病毒（BVDV）、牛传染性鼻气管炎病毒（IBRV）、牛腺病毒3型病毒（BAdV-3）、牛呼吸道合胞体病毒（BRSV）、牛细小病毒（BPV）、多杀性巴氏杆菌（P. multocida）、溶血性曼氏杆菌（M. haemolytica，Mh）和牛支原体（M. bovis）共10种BRDC相关病原进行核酸检测。结果显示：共检出4种病原，其中BAdV-3和Mh的检出率均为100%，BCoV和BVDV检出率均为66.67%，其他病原均未检出；在12份牛鼻拭子样品中，存在BAdV-3+Mh、BAdV-3+Mh+BVDV、BAdV-3+Mh+BCoV和BAdV-3+Mh+BCoV+BVDV共4种不同组合形式的混合感染，混合感染率分别为16.67%、16.67%、25.00%和41.67%。对Mh和BVDV进行分型鉴定发现，该场流行的Mh为荚膜血清型A1型，BVDV为基因1a亚型。结果表明，该牛场发生的BRDC是由BAdV-3、BCoV、BVDV 1a亚型和Mh A1型等病原以不同组合形式混合感染所致。因此，牛场应加强生物安全管理和饲养管理，制定合理免疫程序，做好不同病原混合感染的综合防控。     

牛副流感3型病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体多重PCR检测方法的建立

为建立检测牛副流感3型病毒(BPIV3)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)和牛支原体(M.bovis)的多重PCR检测方法,本研究根据GenBank中登录的BPIV3的HN基因、IBRV的g C基因、BVDV的5'UTR和M.bovis的oppD/F基因序列设计特异性引物,通过优化反应条件建立了一种可以同时检测以上4种病原的多重PCR方法。结果显示,该方法对牛呼吸道合胞体病毒、小反刍兽疫病毒、牛布鲁氏菌、羊布鲁氏菌、牛源多杀巴氏杆菌A型和B型无特异性扩增,对BPIV3和BVDV的cDNA最低检测量分别为100 pg和1 ng,对IBRV和M.bovis的DNA最低检测量分别为10 pg和100 pg,具有较高的特异性和灵敏性。对临床33份鼻拭子样品和12份牛肺样品的检测结果与已发表文献中PCR方法的检测结果一致。本研究建立的多重PCR检测方法可同时对BPIV3、IBRV、BVDV和M.bovis进行检测,为这4种病原的诊断和检测提供了一种实用、便捷的方法。     

牛轮状病毒VP7基因荧光定量RT-PCR检测方法的建立

为建立快速特异的定量检测牛轮状病毒(BRV) VP7基因的荧光定量PCR检测方法,本研究设计扩增BRV VP7基因的特异性引物,将扩增的VP7基因片段(342 bp)克隆于pMD 18-T载体中(pMD-VP7),作为重组质粒标准品,建立EvaGreen荧光定量RT-PCR(qRT-PCR)检测方法.经反应条件优化,结果表明该方法的最低检测量为8.03拷贝/μL.该方法对牛病毒性腹泻病毒、牛冠状病毒、猪流行性腹泻病毒及牛结核分枝杆菌的检测结果均为阴性,表明其具有良好的特异性.批内和批间重复变异系数均小于3％.采用该方法对12份ELISA阳性样本检测出10份阳性,符合率83.33％.本研究建立的BVR VP7 qRT-PCR检测方法具有特异、灵敏、快速和对标本的检测能力较强等特点,可以用于临床诊断和流行病学调查.     

牛轮状病毒与病毒性腹泻病毒的双重RT-PCR检测方法的建立

建立了能够同时检测牛轮状病毒(BRV)与牛病毒性腹泻病毒(BVDV)的双重RT-PCR方法.应用两对特异性引物进行了双重RT-PCR扩增,这两对引物分别对应于BRV的VP7基因和BVDV的5-UTR中的部分编码序列,其扩增产物分别为342bp和196bp.说明该方法的特异性强、敏感性高,可检测到1pg的病毒RNA,可应用于临床诊断和流行病学研究.     

猪源牛病毒性腹泻病毒一步法RT-PCR快速检测方法的建立及应用

为检测猪源牛病毒性腹泻病毒,根据牛病毒性腹泻病毒5′端非编码区基因保守序列设计引物,建立了检测牛病毒性腹泻病毒一步法反转录-聚合酶链(RT-PCR)方法,并对其特异性、敏感性进行了研究。该一步法RT-PCR对牛病毒性腹泻病毒扩增结果为阳性,对照毒株扩增结果均为阴性,对牛病毒性腹泻病毒检测的灵敏性为1 pg总RNA量,应用该方法检测临床病料和猪瘟细胞毒活疫苗样品结果显示,该一步法RT-PCR方法检测速度快、特异性强、敏感性高,可用于猪瘟细胞毒活疫苗中污染的牛病毒性腹泻病毒(BVDV)外源病毒检测。     

猫疱疹病毒Ⅰ型PCR检测方法的建立与初步应用

建立猫疱疹病毒Ⅰ型(FHV-1)PCR检测方法,应用于临床样品中FHV-1的快速检测。根据猫疱疹病毒Ⅰ型的gI基因序列设计了一对特异性引物,以疑似猫疱疹病毒Ⅰ型感染猫的眼鼻分泌物总DNA为模板,建立了PCR检测方法,对所建立的方法进行特异性、敏感性和重复性验证,并对26份临床样本进行检测。结果表明,所建立的PCR诊断方法与犬细小病毒(CPV)、犬瘟热病毒(CDV)、绵羊肺炎支原体(M.ovis)、牛支原体(M.bovis)、牛疱疹病毒Ⅰ型(BHV-1)均无交叉性反应,最低检测DNA模板浓度为3.23×10~3 copies/μL。对26份临床病例进行检测,阳性率61.54%。说明建立的FHV-1PCR检测方法具有特异性强、敏感度高、方便快捷等优点,适合于临床FHV-1感染的快速检测。     

牛呼吸道合胞体病毒RT-LAMP检测方法的建立

牛呼吸道合胞体病毒(BRSV)是引起牛呼吸道疾病的一种病毒性病原体。本研究建立一种利用环介导等温扩增技术(RT-LAMP)快速检测BRSV的新的方法,针对BRSV N基因(GeneBank:AF054668.1)设计4对特异性LAMP引物,每组引物特异性识别靶基因序列上4个独立区域,利用扩增靶DNA的Bst2.0 DNA聚合酶,63℃恒温水浴50 min完成病毒检测,使用钙黄绿素检测荧光目视和琼脂糖凝胶电泳方法,同时对牛的其他主要病毒进行特异性检测,结果证明特异性好。该RT-LAMP检测方法的灵敏度达到BRSV模板稀释的10~(-6)倍,可成功检测到临床样本中的BRSV基因组,为BRSV的临床检测提供了一种快速的试验手段,更适合BRS的诊断和临床现场快速检测。     

一步法RT-PCR快速检测猪瘟活疫苗中BVDV外源病毒方法的建立

为检测商品化猪瘟细胞毒活疫苗中牛病毒性腹泻病毒污染,根据牛病毒性腹泻病毒5′端非编码区基因保守序列设计引物,建立了检测牛病毒性腹泻病毒一步法反转录—聚合酶链(RT-PCR)方法,并对其特异性、敏感性进行了研究。该一步法RT-PCR对牛病毒性腹泻病毒扩增结果为阳性,对照毒株扩增结果均为阴性,对牛病毒性腹泻病毒检测的灵敏性为1pg总RNA量,应用该方法,检测了32批猪瘟细胞毒活疫苗样品,以上结果表明该一步法RT-PCR方法检测速度快、特异性强、敏感性高,可用于猪瘟细胞毒活疫苗中污染的牛病毒性腹泻病毒外源病毒检测。     

Development and Application of Sandwich ELISA for Diagnosis of Fascioliasis gigantica in Early Stage

To establish a method for the diagnosis of Fascioliasis gigantica in early stage, five hybridoma cell lines were recovered and used for preparation of monoclonal antibodies.7D2 was used as a capture antibody and 7D1 as detection antibody.Coating antibody dilution was 1:6 400 (0.208 μg/mL), 5% nonfat milk was used as blocking solution and detection antibody dilution was 1:10 000 (0.200 μg/mL).The detection limit of the sandwich ELISA was 1:3 200 (0.156 μg/mL), with good specificity and stability, and there was no cross reaction with other kinds of parasite antigen.The results showed that the method provided the important conditions and theoretical basis for the diagnosis of Fascioliasis gigantica in early stage. This would save unnecessary economic losses to the livestock, and it had clinical application value.     

大片形吸虫病早期诊断夹心ELISA方法的建立及应用

为建立一种早期诊断大片形吸虫病的试验方法,本试验对5株分泌抗大片形吸虫分泌排泄抗原(ES)单克隆抗体的杂交瘤细胞进行复苏、制备单克隆抗体,配对筛选出7D1、7D2单克隆抗体,将7D2作为包被抗体,7D1作为酶标抗体,通过条件优化,建立早期诊断大片形吸虫病的夹心ELISA方法。结果显示,当包被抗体稀释度为1:6 400 (0.208 μg/mL)、采用5%脱脂奶粉封闭、酶标记抗体稀释度为1:10 000 (0.200 μg/mL)时,最早可检测到感染此病第7天小鼠的血清循环抗原,其敏感性可达到1:3 200 (0.156 μg/mL),与其他几种寄生虫抗原均无交叉反应,具有较高的特异性和稳定性。本试验成功建立了早期检测大片形吸虫病的夹心ELISA方法,为大片形吸虫病的诊治及开发早期诊断大片形吸虫病的试剂盒奠定了基础,具有临床应用价值。     

Effects of suckling intensity on milk yield and piglet growth from lactation-enhanced gilts

The effects of suckling intensity on milk yield and piglet growth were determined when lactation capacity of the sow was enhanced through overexpression of a mammary-specific transgene, bovine alpha-lactalbumin. Lactational response to increased suckling stimulation was determined by fostering litters of the same age (d 1) or 7 d older (d 7) than the day of lactation to sows nontransgenic (control) or transgenic (TG) for bovine alpha-lactalbumin. Twenty first-parity gilts were allocated to 4 treatments dependent on gilt genotype and age of litter fostered (control d 1, control d 7, TG d 1, and TG d 7). Litters were standardized to 10 piglets within 24 h postpartum, and nonbirth piglets were fostered to gilts with an equal litter BW within age groups at 36 h postpartum. Milk yield was determined by the weigh-suckle-weigh method on d 6, 9, 12, 15, and 18 of lactation. Mean daily milk yield was greater (P = 0.031) for TG gilts compared with control gilts and tended to be greater (P = 0.056) for all gilts with d-7 piglets compared with those with d-1 piglets. Daily milk yield of TG d 7 gilts increased rapidly to peak at d 9 and was greater than milk yield of all control gilts at d 9 (P < 0.01), 12 (P < 0.02), and 15 (P < 0.02). Mean daily milk yield of TG d 7 gilts was 2.1 kg greater (P = 0.002) than for control d 7 gilts and 2.0 kg greater (P = 0.004) than for TG d 1 gilts. Daily milk yield of control d 1 gilts was not different from that of TG d 1 gilts (P = 0.49) or control d 7 gilts (P = 0.63). Piglet BW gain between d 3 and 6 was greater (P < 0.01) in the TG d 7 group than for all other groups and was greater (P < 0.05) than the control groups between d 6 and 9. No difference was found when comparing accumulated BW gain of the piglets between the day of age at foster (d 1 vs. 7; P = 0.606) or between the control d 1 and control d 7 groups (P = 0.759). Accumulated BW gain of piglets suckling TG d 7 gilts from d 3 through 9 was greater (P < 0.02) than that of the other groups and continued to be greater (P < 0.05) than that of either of the control groups through d 15. However, by d 15, accumulated BW gain of piglets suckling TG d 1 gilts was no longer different (P = 0.40) from that of the TG d 7 group and was greater (P < 0.05) than that of the control d 1 group. The enhanced lactation potential of these TG gilts synergized with suckling intensity to stimulate increased milk production during early lactation, resulting in increased piglet growth.     

Comparison of plasma progesterone, transrectal ultrasound and pregnancy specific proteins (PSPB) used for pregnancy diagnosis in reindeer

The study aimed to compare plasma progesterone concentrations, rectal ultrasonography and plasma concentrations of pregnancy-specific protein B (PSPB) used for pregnancy diagnosis in reindeer. A total of 1,595 blood plasma samples were collected between 1991 and 1996 from 3 semidomestic reindeer (Rangifer tarandus tarandus) herds on the Norwegian mainland (Mager?y, S?r?y, Filefjell) and from 92 wild Svalbard reindeer (Rangifer tarandus platyrhynchus). Samples were collected between January and late April. Plasma levels of progesterone and PSPB were measured and used as indicators of pregnancy. In addition, animals from the Filefjell herd and the Svalbard reindeer were investigated using transrectal ultrasound. The results showed that plasma progesterone lower than 7 nmol l-1 rarely occurs in females diagnosed pregnant either by ultrasound or by observing a calf at foot 7 months after blood sampling. A very good agreement was found between plasma progesterone and PSPB when used for pregnancy diagnosis. On the Norwegian mainland, but not to the same extent on Svalbard, a high proportion of females with a high progesterone concentration was diagnosed not pregnant by ultrasound. This probably reflects a high rate of false negative diagnoses by the ultrasound method rather than false positives in the progesterone analysis.     

Prevalence of Chlamydophila felis and feline herpesvirus 1 in cats with conjunctivitis in northern Italy

The prevalence of Chlamydophila felis and feline herpesvirus 1 (FHV-1) infection in cats with conjunctivitis in northern Italy was investigated by conventional polymerase chain reaction (PCR) testing. In cats with conjunctivitis, C felis and FHV-1 were detected in 14 of 70 (20%) and in 23 of 70 (33%) animals, respectively. None of the 35 control cats were positive for C felis, whereas 7 (20%) of these cats were positive for FHV-1. Mixed infections were present in 5 of 70 cats (7%). Cats positive for C felis were significantly younger than control animals (P = .02), whereas no significant age differences were observed between FHV-1-positive cats and control cats (P = .41) or between FHV-1-positive animals and C felis-positive animals (P = .16). Cats sampled during acute-phase conjunctivitis were also investigated for the presence of C felis by conjunctival scrapings. In this acute phase, substantial agreement was found when comparing the results of the 2 methods (K = .80). The association between PCR results and conjunctivitis was evaluated for the 2 pathogens. The presence of C felis was significantly associated with conjunctivitis (P = .004), whereas the detection of FHV-1 did not significantly correlate with the clinical sign (P = .25), suggesting that, by itself. PCR is not suitable for the diagnosis of FHV-1-related conjunctivitis.     

口蹄疫病毒RT-PCR检测和分型方法的建立及初步应用

参考GenBank中各个血清型口蹄疫病毒3D、vp1、2A基因的标准序列，设计引物P1／P2和S1／S2。建立用于检测口蹄疫病毒及其利用引物S1／S2克隆片段同源性比较而确定血清型的RT-PCR方法。通过敏感性试验检测，2对引物均可以检测到10TCID50的病毒量；特异性试验的检测，2对引物对正常细胞、牛黏膜病病毒、猪瘟病毒、水疱性口炎病毒、牛传染性鼻气管炎病毒的检测结果均为阴性。利用该方法对病牛的流涎液体、水疱液体、舌皮组织、感染犊牛心脏等组织进行检测初步结果显示：该方法可以对O型和AsiaⅠ型口蹄疫病毒进行特异性检测，能够用于口蹄疫急性及亚临床感染的诊断及流行病学调查。     

生长猪饲粮磷水平对磷酸氢钙中磷真消化率评定的影响

本试验旨在研究生长猪饲粮磷水平对磷酸氢钙(DCP)中磷的全肠道真消化率评定的影响。选用56头体重为(25.90±0.25)kg的杜×长×大杂交去势公猪,根据体重随机分为7个处理组,每组8个重复,每个重复1头猪。对照组饲喂未添加DCP的基础饲粮,6个处理组在基础饲粮中分别添加0.64%、1.28%、1.92%、2.56%、3.20%、3.84%DCP。使用全收粪法测定磷的表观全肠道消化率。结果表明:生长猪总磷的表观全肠道消化率随饲粮DCP水平提高呈线性和二次增加(P0.05);随着DCP添加水平的提高,由差量法测得无机磷的全肠道真消化率呈二次上升的趋势(P0.1),具体表现为无机磷的全肠道真消化率随饲粮DCP水平提高呈现出先增加后降低的趋势;通过线性回归法发现,生长猪饲喂低DCP饲粮后测得磷的全肠道真消化率显著高于饲喂高DCP饲粮(P0.05)。由此可见,饲粮磷水平显著影响差量法和线性回归法测得DCP的全肠道真消化率;使用差量法测定饲粮原料磷的全肠道真消化率时应将饲粮磷水平维持在接近磷需要量的水平,使用线性回归法测定磷的全肠道真消化率时应将饲粮磷水平控制在低于磷需要量的范围内。     

寡核苷酸探针原位检测石蜡切片中PRRSV核酸

根据GenBank收录的美洲型猪繁殖与呼吸综合征病毒（PRRSV）ATCCVR-2332株ORF6和ORF7基因序列，用O1igo软件设计并合成大小为37bp的寡核苷酸探针，经生物素标记后，成功建立了原住检测石蜡组织切片中PRRSV核酸的方法。该探针能检测到56PgPRRSV核酸的RT—PCR产物DNA，能特异检测出PRRSV核酸及其PCR产物，而对猪瘟病毒（HCV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪乙脑病毒（JEV）的核酸呈阴性反应。应用该方法检测PRRSVSC-1株人工感染的28日龄仔猪，在感染后7d即可在肺脏、肾脏、扁桃体、胸腺、肺门淋巴结、十二指肠和大脑检测到PRRSV核酸。该法可用于仔猪PRRSV感染的诊断和组织中核酸的定位及分布研究，也可用于甲醛固定组织的回顾性诊断。     

Urinary tract manifestations of protothecosis in dogs

Records of 13 dogs with systemic infection with Prototheca sp. from 3 veterinary teaching hospitals were reviewed. Acute renal failure secondary to disseminated infection with Prototheca zopfii was diagnosed in 2 dogs. In 1 dog, acute renal failure developed during administration of immunosuppressive drugs for treatment of anterior uveitis. During diagnostic evaluation of this dog, Prototheca sp. organisms were noted in urine sediment and renal biopsy specimens. In the 2nd dog, acute renal failure was diagnosed after treatment for bacterial cystitis. After diagnosis of protothecosis, organisms were successfully isolated by aerobic urine culture. Both dogs with acute renal failure did not respond to conventional medical therapy. In total, Prototheca sp. was noted in urine sediment in 4 of 8 dogs and successfully cultured from urine in 5 of 7 dogs. Four of 5 dogs had organisms noted in the kidneys on histopathologic examination. In all dogs, the species identified was P zopfii. Sensitivity testing of 3 isolates revealed wide differences in in vitro drug resistance. Examination and culture of urine is recommended as a practical method for diagnosis of systemic infection with Prototheca sp.     

Use of a Compounded Long-Acting Progesterone Formulation for Equine Pregnancy Maintenance

The objective of this study was to test the efficacy of a compounded long-acting progesterone formulation (BioRelease P4 LA 150; BETPHARM, Lexington, KY) containing 150 mg progesterone/ml for pregnancy maintenance in mares after prostaglandin (PG) F_2α-induced luteolysis. On day 18 of gestation, mares were randomly assigned to one of four groups (n = 7/group): (1) saline-treated control (Saline); (2) PGF_2α-treated control (PGF); (3) PGF_2α- and Regu-Mate-treated (Regu-Mate); and (4) PGF_2α- and BioRelease P4 LA 150-treated (BioRelease). On day 18, Saline mares received 1 ml sterile saline IM, whereas PGF, Regu-Mate, and BioRelease mares received 250 μg cloprostenol IM. Beginning on day 18, Regu-Mate mares received 10 ml Regu-Mate orally once daily and BioRelease mares received 10 ml BioRelease P4 LA 150 containing 150 mg/ml progesterone IM once every 7 days; treatments were continued until day 45 or until pregnancy loss occurred. Pregnancy diagnosis was performed every 3 days between days 18 and 45 (or until pregnancy loss). Pregnancy loss was defined as complete absence of a discernible embryonic vesicle as determined with transrectal ultrasonography. Pregnancy loss rates between days 18 and 45 were: Saline, 1/7; PGF, 7/7; Regu-Mate, 1/7; and BioRelease, 0/7. The pregnancy loss rate was higher (P < .01) in PGF_2α-treated control mares compared with the other groups. There were no differences (P > .1) in pregnancy loss rates among the saline-treated control, Regu-Mate-treated, and BioRelease P4 LA 150-treated mares. These results indicate that intramuscular administration of BioRelease P4 LA 150 containing a total of 1.5 g progesterone every 7 days provided a sufficient level of progesterone to maintain pregnancy between days 18 and 45 of gestation in mares that lacked an endogenous source of progesterone; therefore, this long-acting formulation of progesterone appears to be an efficacious and suitable alternative to currently available progesterone formulations that require daily administration.