Farmer reported prevalence and factors associated with contagious ovine digital dermatitis in Wales: A questionnaire of 511 sheep farmers

In 2012, 2000 questionnaires were sent to a random sample of Welsh sheep farmers. The questionnaire investigated farmers’ knowledge and views on contagious ovine digital dermatitis (CODD) – an emerging disease of sheep responsible for causing severe lameness, welfare and production problems. The overall response rate was 28.3% with a usable response rate of 25.6%. The between farm prevalence of CODD was 35.0% and the median farmer estimated prevalence of CODD was 2.0%. The disease now appears endemic and widespread in Wales. Furthermore, there has been a rapid increase in reports of CODD arriving on farms since the year 2000. Risk factors for CODD identified in this study include the presence of bovine digital dermatitis (BDD) in cattle on the farm and larger flocks. Farmers also consider concurrent footrot/interdigital dermatitis, buying in sheep, adult sheep, time of year and housing to be associated with CODD. Further experimental research is necessary to establish whether these observations are true associations.     

羊传染性脓疱病毒PCR检测方法的建立及应用

根据NC-005336 ORFV全基因中gORF011设计合成一对引物。建立用于检测羊传染性脓疱病毒的PCR方法。此方法检测羊传染性脓疱病毒结果与病毒分离培养,电镜检查结果一致。经过对扩增产物进行序列分析,与NC-005336 ORFV的核苷酸序列同源性高达97.48％。通过特异性,敏感性及临床应用实验证明,此方法可以检测10^4ICID50浓度病毒;临床应用检出率为94．7％,显著高于病毒分离培养36．8％的检出率;并能与羊痘病毒,口蹄疫病毒鉴别;此方法用于检测羊传染性脓疱病毒是特异的、敏感的、可行的。     

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.     

羊传染性脓疱病毒感染山羊皮肤成纤维上皮细胞差异表达miRNA分析

旨在研究羊传染性脓疱病毒（orf virus，orfv）感染山羊皮肤成纤维细胞（goat skin fibroblasts cell，GSF）对GSF细胞microRNA（miRNA）表达谱影响，探究miRNA在orfv感染过程中的作用及调控机制。分别提取感染和未感染orfv的GSF细胞总RNA，构建miRNA文库，利用高通量测序技术进行miRNA差异表达分析，对差异表达miRNA靶基因进行预测，并进行GO和KEGG分析，随机选取10个差异miRNA进行RT-qPCR验证。结果显示，orfv感染组和未感染GSF细胞组相比共有678个显著差异表达的miRNA（fold change≥1.5），其中，上调表达miRNA有509个，下调表达miRNA有169个，uniq_miRNA的Venn图分析显示，感染组和对照组共有的miRNA仅占8.21%；GO和KEGG分析显示，差异表达miRNA主要参与脂质代谢、受体及细胞因子信号转导等细胞生物学过程，RT-qPCR验证结果与高通量测序结果一致。本研究结果表明，orfv感染GSF细胞对其编码的miRNA有显著影响，获得大量GSF细胞编码的与orfv感染相关的差异miRNA，为进一步从宿主miRNA层面揭示orfv感染和致病机制提供了参考依据。     

传染性支气管炎病毒山东及北京分离株血清型及RFLP基因分型的比较研究

分离并鉴定出山东省不同地区的IBV分离株A、B、C、D和北京分离株F，利用中和试验分别对各分离株及标准株M41、T等进行血清分型。设计一对此物，分别对各毒株的S1基因进行RT－PCR扩增，扩增片段长约1700bp，利用其PCR产物进行HaeⅢ酶切，根据酶切图谱，进行基因分型。结果表明：A、C、D和M41酶切图谱相同，属Mass基因型，分别为900、400和300bp左右的片段；北京F株有4条酶切条带，与4／91基因型不同，分别为1000、300、200和小于100bp左右的片段；B株有4条酶切条带，理论推断与T株相似，分别为700、500、300和180bp左右的片段，可能为同一基因型。血清学分型结果与基因分型结果不符。     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.     

Molecular typing of Staphylococcus aureus isolated from cows, goats and sheep with intramammary infections on the basis of gene polymorphisms and toxins genes

We investigated 116 Staphylococcus aureus isolates from cows, goats and sheep with intramammary infections (IMI) in Italy to provide information about the spread of enterotoxigenic strains and to compare strains isolated from different ruminant species. The isolates were typed by restriction fragment length polymorphism (RFLP) analysis of the coagulase (coa) gene, by analysis of polymorphisms of the X region of protein A (spa) gene and by detection of genes encoding enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej and sel). Seven different coa types and 12 different spa types were distinguished. On the basis of polymerase chain reaction-RFLP, 29 different coa subtypes were identified. Two different coa subtypes accounted for 49% and 67% of bovine and ovine isolates respectively. Only seven coa subtypes were observed in isolates from more than one host species and no coa subtype was present in isolates from all three ruminant species. Furthermore, 85 of the isolates (73%) harboured at least one enterotoxin gene (se) with a predominance of sea, sed and sej among isolates from bovine IMI, and sec and sel among isolates from caprine and ovine IMI. Comparing the S. aureus isolates on the basis of gene polymorphisms and presence of se genes, significant differences were found in distributions of genotypes among isolates from cows, goats and sheep.     

Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe

Until recently, Toxoplasma gondii was considered to be clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known of the genetics of T. gondii strains from Africa. In this study, we genotyped 19 T. gondii isolates from chickens from six African countries (Egypt, Kenya, Nigeria, Congo, Mali, and Burkina Fasco) using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed four genotypes. Thirteen isolates belong to the Type III lineage, five isolates have Type II alleles at all loci except apico and they belong to the Type II lineage. One isolate from Nigeria had atypical genotype. In general, these isolates were mostly clonal Type III and II strains that predominate in North American and European. DNA sequencing at several loci for representative isolates confirmed the results of PCR-RFLP genotyping. Taken together with recent studies of T. gondii isolates from Africa, it is clear that the three clonal lineages (Types I, II and III) predominate not only in North America and Europe, but also in Africa.     

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.     

Use of serology and polymerase chain reaction for the rapid eradication of small ruminant lentivirus infections from a sheep flock: A case report

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected.     

猪繁殖与呼吸综合征病毒GP5、M蛋白基因的克隆及其基因疫苗构建

参照GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲型代表株VR2332 GP5和M蛋白基因序列,设计并合成2对引物,用RT-PCR方法分别扩增出PRRSV野毒株GP5和M蛋白基因603,525bp片段,并将其分别克隆到pMD18-T载体。测序正确后,将GP5和M蛋白基因分别克隆到真核表达载体pEGFP-C1上,成功构建基因疫苗表达载体pEGP5-C1和pEM-C1。小鼠免疫试验证实,这些基因疫苗质粒可以诱导小鼠产生特异性抗体,并在二免后1周开始检测到特异性淋巴细胞增殖反应。