动物产品中牛、羊源性成分多重PCR检测方法的建立

以肉骨粉、鱼粉、猪肉干和鱼肉干为研究对象,异硫氰酸胍法提取总DNA,18S rDNA片段的扩增结果表明提取到的DNA中不存在抑制PCR的物质。应用梯度PCR技术对牛、羊源性成分检测的退火温度进行了优化,在单一PCR检测技术的基础上分别进行了18S rDNA片段和牛、羊源性成分的多重PCR分析,得到了预期的结果。试验表明,本文建立的多重PCR方法具有快速、简便、准确等特点,对动物产品牛、羊源性成分检测具有重要意义。     

Influence of communal alpine pasturing on the spread of pestiviruses among sheep and goats in Austria: first identification of border disease virus in Austria

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.     

Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology

The polymerase chain reaction has become an important diagnostic tool for the veterinary virologist. Conventional methods for detecting viral diseases can be laborious or ineffective. In many cases PCR can provide a rapid and accurate test. In this article we explain the basic principles of PCR and supply a reference list of its uses in diagnostic veterinary virology.Abbreviations BLV bovine leukaemia virus - BVDV bovine viral diarrhoea virus - DNA deoxyribonucleic acid - ds double-stranded - ELISA enzyme-linked immunosorbent assay - PCR polymerase chain reaction - RNA ribonucleic acid - ss single-stranded - TK thymidine kinase     

Demonstration of Generalized Infection with Caprine Herpesvirus 1 Diagnosed in an Aborted Caprine Fetus by PCR

Veterinary Research Communications -     

A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD=1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD=126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Comparison of invasive and oscillometric blood pressure measurement techniques in anesthetized sheep,goats, and cattle

ObjectiveTo determine the level of agreement between an oscillometric (O-NIBP) and an invasive method (IBP) of monitoring arterial blood pressure (ABP) in anesthetized sheep, goats, and cattle.Study designProspective clinical study.AnimalsTwenty sheep and goats, 20 cattle weighing <150 kg body weight, and 20 cattle weighing >150 kg body weight.MethodsAnimals were anesthetized and systolic ABP (SABP), mean ABP (MABP), and diastolic ABP (DABP) were measured using IBP and O-NIBP. Differences between IBP and O-NIBP, and 95% limits of agreement (LOA) between SABP, MABP, and DABP values were assessed by the Bland–Altman method.ResultsMean difference ± standard deviation (range) between SABP, DABP, and MABP measurements in sheep and goats was 0 ± 16 (-57 to 38) mmHg, 13 ± 16 (-37 to 70) mmHg, and 8 ± 13 (-34 to 54) mmHg, respectively. Mean difference between SABP, DABP, and MABP measurements in small cattle was 0 ± 19 (-37 to 37) mmHg, 6 ± 18 (-77 to 48) mmHg, and 4 ± 16 (-73 to 48) mmHg, respectively. Mean difference between SABP, DABP, and MABP measurements in large cattle was -18 ± 32 (-107 to 71) mmHg, 7 ± 29 (-112 to 63) mmHg, and -5 ± 28 (-110 to 60) mmHg, respectively. The 95% LOAs for SABP, DABP, and MABP were -31 to +31, -19 to +44, and -19 to +34 mmHg, respectively in sheep and goats; were -37 to +37, -19 to +44, and -19 to +34 mmHg, respectively in small cattle; and were -81 to +45, -50 to +63, and -59 to +50 mmHg, respectively in large cattle.ConclusionsAgreement was poor between O-NIBP and IBP monitoring techniques.Clinical relevanceArterial BP should be monitored in anesthetized sheep, goats, and cattle using IBP.     

鸡毒支原体PCR检测试剂盒的研制与应用

根据基因库中鸡毒支原体 1 6SrRNA的序列研制PCR检测试剂盒 ,用于检测鸡毒支原体 (MG)。结果表明该MG PCR检测试剂盒对不同MG参考菌株和地方分离株均能特异性地扩增出 732bp条带 ,而对其他禽支原体和禽病病原体的扩增结果为阴性。该MG PCR试剂盒最低能检测出 1 0 0fg的MGDNA模板。保存期测定结果表明 ,该MG PCR试剂盒在 - 2 0℃条件下保存至 1 ,3 ,6和 9个月时 ,其敏感性无明显变化 ,仍能检测到 1 0 0fg至 1pg的MGDNA模板。保存至 1 2个月时其敏感度虽降低了 1个滴度 ,但仍能 1 0 0 %检出人工感染鸡的临床样品     

PCR技术检测猪伪狂犬病毒及其潜伏感染部位的研究

本研究合成了伪狂犬毒糖蛋白ｇｐ５０基因引笺，该引物能够扩增糖蛋白ｇｐ５０基因中４３４－６５１之间的２１７ｂｐＤＮＡ片段，该片段含 ＳａｌＩ酶切位点，应用引物对几种不同的伪狂犬病毒株多聚酶链式反应扩增结果全为阳性。     

检测和鉴别犬瘟热病毒强、弱毒株的复合反转录-套式聚合酶链式反应(RT-nPCR)的建立及初步应用

根据GenBank上登录的犬瘟热病毒（Canine distemper virus，CDV）基因组全序列，选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4，并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3，用引物P1／P4进行RT—PCR，然后用引物P2／P3／P4进行复合套式PCR，建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应（RT—nPCR）的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段，从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段，与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT—nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明，有15份类似CDV强毒，5份类似CDV弱毒。本研究建立的复合RT—nPCR可以有效检测CDV感染，能够将强、弱毒株区分开，可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。     

荧光定量PCR快速检测食品中沙门氏菌方法的建立及初步应用

根据编码鼠伤寒沙门氏菌肠毒素stn基因的核苷酸序列设计1对引物和荧光探针，通过对荧光定量PCR反应体系和反应条件的摸索，建立了检测沙门氏菌的核酸荧光定量PCR方法。对该方法的特异性与敏感性研究结果显示，该方法检测沙门氏菌结果均为阳性，而非沙门氏菌均为阴性；对带有沙门氏菌肠毒素stn基因的阳性质粒的检测敏感性为4个／μL。用该方法对人工污染沙门氏菌的鲜猪肉和鲜鸡蛋进行检测，当检样中沙门氏菌初始含菌量分别为1CFU／g（鲜猪肉）和1CFU／g（鲜鸡蛋），经过12h的增菌后，检测结果均为阳性。该方法具有简便、快速、特异性强、敏感性高等特点。此研究为食品中沙门氏菌快速检测试剂盒的研制打下了良好的基础。     

Urinary excretion of purine derivatives and plasma allantoin level in sheep and goats during fasting

The relationship between blood plasma level and urinary excretion of allantoin (AN) was examined in sheep and goats during fasting to investigate the possible use of purine derivatives (PD) in urine and/or plasma for estimating the microbial protein production in the rumen, and the further digestion in the lower guts of ruminants. Urinary AN excretion decreased markedly during fasting (0.13 mmol/kgW^0.75 per day), although urinary levels of other PD, hypoxanthine + xanthine and uric acid did not differ irrespective of the feeding condition, that is, feeding, fasting and refeeding in both species. The AN concentration in blood plasma also decreased drastically in the starvation period, and was suddenly increased on refeeding in sheep and goats, and these phenomena were very similar to those of urinary AN excretion. Therefore, there was a high positive correlation between plasma AN level and urinary AN excretion, and the coefficient of correlation was statistically significant (P < 0.01). These results clearly indicate that changes in urinary AN reflect change in plasma AN, which is induced by the catabolism of purine base in the body.     

A comparison of plasma metabolite levels in goats and sheep during continuous low-level administration of fenbendazole

Plasma levels of fenbendazole (FBZ) and its sulphoxide (OFZ) and sulphone (FBZ.SO₂) metabolites were measured in goats and sheep during low-level administration of FBZ given by intraruminal infusion or formulated into a urea-molasses feed supplement block (UMB). In experiment 1, 6 goats and 6 sheep were offered UMB containing 0.5 g FBZ/kg (MUMB) and individual block consumption was measured daily for 18 days. In experiment 2, some of the same animals (n=4 for each species) received FBZ by intraruminal infusion at 1, 1.5 and 3 mg/kg liveweight per day for 7 days at each dosage. FBZ, OFZ and FBZ.SO levels were determined in plasma collected every 3 days in experiment 1 and on days 4, 5 and²6 of each infusion period in experiment 2. In both experiments, higher equilibrium levels were observed for the three metabolites in sheep than in goats. Significant linear relationships were observed between the daily FBZ dosages and the plasma levels of the three metabolites in both species. The regression coefficients were significantly higher in sheep than in goats for FBZ and OFZ but not for FBZ.SO₂, and they were also significantly higher during MUMB administration than during infusion for all three metabolites in both species. FBZ is a suitable anthelmintic for incorporation into a MUMB formulation for use in livestock production systems where responses to molasses urea supplementation have been demonstrated and gastrointestinal parasitism impairs productivity. The results indicate that target dose rates for goats should be 0.75 mg/kg per day compared with 0.5 mg/kg per day for sheep.Abbreviations ANOVA analysis of variance - FBZ fenbendazole - FBZ.SO₂ fenbendazole sulphone - HPLC high-performance liquid chromatography - MUMB urea-molasses feed supplement block containing 0.5 g fenbendazole/kg - OFZ fenbendazole sulphoxide - UMB urea-molasses feed supplment block     

Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

Detection of BHV-1 in a Naturally Infected Bovine Fetus by a Nested PCR assay

Bovine herpesvirus 1 (BHV-1) is frequently associated with abortion in naturally and experimentally infected cattle. Most of the virus isolation and immunofluorescent antibody protocols described in the literature for detecting BHV-1 in bovine foetuses are rather laborious, costly and time-consuming. The detection is described of BHV-1 in the tissues of a naturally aborted bovine foetus by a nested PCR assay with no further hybridization procedures.Optimal results were achieved by filtering the foetal tissues on a chromatography column before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels.This nested PCR was faster and easier to perform than the virus isolation test. To our knowledge, this is the first time that BHV-1 has been detected in the tissues of a naturally infected bovine foetus by means of a nested PCR. The test seems to be a practical alternative for rapid detection of BHV-1 in bovine foetus.     

GPV野毒株的分离及PCR检测方法的应用

在本实验中，用根据CPVHI标准毒核苷酸VP1-VP3和VP3两个区段基因序列设计的2特异性引物CRP1/CRP2和CF1/CF2，用该2对引物对从鹅细小病毒野毒感染的病鹅分离的GPV野毒株进行PCR检波，结果2对引物CRP1/CRP2和CF1/CF2，均能特异性地扩增出与预期片段大小相符的0.6bp和1.6bp两个片段，回归试验的结果表明，该CFV野毒株的感染潜伏期为8d，病程为1～3d，致死率达100%。     

宁夏羊胃肠道线虫对现行驱虫药的抗药性调查

采用粪便虫卵减少试验对宁夏地区所属灵武、贺兰、盐池、吴忠、中宁、中卫、永宁和银川市郊8个县（市）的12个绵羊场、6个山羊场进行了丙硫苯咪唑和阿维菌素抗药性的随机调查。结果表明：在用丙硫苯咪唑调查的10个绵羊场和6个山羊场中，虫卵减少率在95％以下和95％的置信域下限在90％以下的有山羊场2个、绵羊场2个，证明对丙硫苯咪唑有抗药性；1个绵羊场和1个山羊场的虫卵减少率是96．3％、95．9％，但95％置信域的下限在90％以下，具有抗药性可疑；山羊群中丙硫苯咪唑的抗药性为33．3％，绵羊群为20．0％。用同样的方法调查了1个山羊场和5个绵羊场（其中有4个羊场曾执行了丙硫苯咪唑的试验），查出1个山羊场和1个绵羊场对阿维菌素具有抗药性可疑，其虫卵减少率分别为97．3％和95．5％，置信域下限在90％以下。揭示了宁夏地区羊消化道线虫对现行驱虫药的抗药状态，为今后防治提供了依据。     

副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻弧菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测。结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2 cfu和3 cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱弧菌、麦氏弧菌无交叉反应。研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用。     

Haematological and lymphocyte subset analyses in sheep inoculated with bovine immunodeficiency-like virus

Bovine immunodeficiency-like virus (BIV) was passagedin vivo by intraperitoneal transfusion of ovine whole blood. Prior to transfusion, the recipient sheep were given sodium thioglycolate intraperitoneally to induce mild non-suppurative inflammation. The anti-BIV antibody response, haematology, and peripheral blood lymphocyte subsets (B, , CD2⁺, CD4⁺ and CD8⁺) of recipient sheep were assessed for one year following transfusion. Passaging was successful since serum anti-BIV antibody responses were detected in 5 of the 6 recipient sheep; 1 of the 5 remained seropositive throughout the study. Lentivirus was not isolated from the recipient sheep, but provirus was detected by the polymerase chain reaction in DNA from peripheral blood leukocytes in 3 of the 5 sheep that seroconverted. In the BIV-inoculated sheep, neutrophils and eosinophils were significantly increased (p0.05) at 3 months and between 6 and 8 months postinoculation, respectively. B, CD2⁺ and CD4⁺ cells and the CD4⁺/CD8⁺ ratios were significantly increased (p0.05) 2 months postinoculation. Mild, transient haematological changes occurred in BIV-exposed sheep, but illness was not detected in the year.     

Development of a PCR to diagnose BLV genome in frozen semen samples

The sanitary and economic impact of BLV infection is associated with the interference in the international movement of cattle and their germ plasm. Although experimental data support the improbability that semen from BLV-positive bulls could infect recipient cows, restriction for commercialization of semen from infected animals is still present. The objective of this work was to standardize a PCR assay to diagnose the presence of BLV genome in frozen semen samples. The developed methodology involves the amplification of an internal fragment of gag gene. The limit of detection of this technique was six viral particles, using gag-PCR followed by hybridization analysis. Frozen semen samples from seropositive bulls were analyzed. It was possible to detect proviral DNA in 9 out of 173 samples. Additionally, a biological test in susceptible sheep was performed in order to evaluate the transmission of BLV genome by semen from seropositive animals. This data strongly suggest that semen from seropositive bulls that resulted negative by PCR can be used for artificial insemination (AI), accompanied by proper collection protocols. The development of this PCR assay constitutes a valuable diagnostic tool to determine the BLV-free status of frozen semen samples used for AI.