Development of an Immunobinding Assay with Monoclonal Antibodies to Diagnose Mycoplasma bovis in Semen

Veterinary Research Communications -     

Doppler evaluation of maternal and fetal vessels during normal gestation in the bitch

The aim of this work is to evaluate the haemodynamic characteristics of maternal and fetal vessels during normal pregnancy in bitches, using Colour and Pulsed wave Doppler ultrasonography, in order to obtain more information about maternal and fetal circulation. The blood waveforms of the uteroplacental arteries, aorta, caudal cava vein and umbilical cord of the fetuses were recorded weekly in 16 pregnant bitches. Also, the measurements of Peak Systolic, End Diastolic Velocities, Resistance and Pulsatility Indices were carried out. Uteroplacental blood flow was biphasic while the ones of the umbilical artery and aorta were first systolic and then diastolic. The cava showed a typical waveform of venous vessels. During gestation the EDV and PSV of fetal vessels increased (alpha<0.05) while the PI and RI of all vessels examined decreased (alpha<0.05) except for the IP of the Aorta. The Doppler ultrasonography was used to study the characteristics of maternal and fetal vessel flow and their progressive changes during pregnancy. This study can be considered a further contribution in diagnosing and monitoring high-risk pregnancies in Veterinary Medicine.     

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

Effect of a difficult calving on the vigour of the calf, the onset of maternal behaviour, and some behavioural indicators of pain in the dam

The neonate's development and survival is dependent upon being vigorous at birth and receiving appropriate maternal care. However, difficulty at delivery can result in less vigorous offspring and maternal care can be altered, probably as a consequence of exhaustion, pain and human intervention. The first 3 h after expulsion of the calf were observed continuously from videos following twelve natural calvings and sixteen calvings assisted by farm staff (including four malpresentations) from Holstein cows. Calvings were balanced within groups for parity of the dam, genetic group, sex and birth weight of the calf, calving pen and calving season. Assisted calves were less vigorous with higher latencies to attempt to stand, achieve standing, walk and reach the udder than unassisted calves (P < 0.05). Furthermore, assisted calves also tended to be less likely to stand and walk within the first 3 h after birth (P < 0.1), spent more time lying on their flank (P = 0.019) and had more frequent bouts of this behaviour (P = 0.033). Assisted dams did not take longer to lick the calf and performed as much licking as unassisted dams (P > 0.05), indicating no delayed onset or impaired expression of maternal behaviour in dams given assistance at delivery. Study of potential pain-related behaviours revealed that assisted dams spent less time self-grooming (P = 0.033) than dams delivering naturally, which could suggest greater pain. However, there were no significant differences in any of the other pain-related behaviours. Our results suggest that, although maternal behaviour was unaffected by a difficult delivery, dairy calves born following difficult calvings have lower vigour in the first 3 h after birth than unassisted calves. This might have longer-term effects on the health and survival of the calves.     

抗ICAM-1单克隆抗体的生物学功能研究

本研究通过体外细胞试验和体内动物试验,对已制备的抗细胞间粘附分子1(ICAM-1)单克隆抗体(MAb)4F1和2C5的生物学活性进行检测.间接免疫荧光试验表明:这两株ICAM-1 MAb可以与人血管内皮细胞ECV304细胞中的ICAM-1结合.以乳酸脱氢酶释放法检测ICAM-1 MAb抑制单核细胞与血管内皮细胞的黏附作用.4F1和2C5 MAb分别可以使细胞黏附降低34.4％和26.9％.二甲苯致小鼠耳廓肿胀试验和毛细血管通透性试验表明:ICAM-1 MAb能够抑制小鼠耳廓肿胀度,使伊文思蓝渗出量降低.本研究证明所制备的MAb均具有阻断ICAM-1及抑制炎症反应的功能,其中4F1生物学活性高于2C5.     

牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备

为制备牛病毒性腹泻病毒(BVDV)的Core蛋白及其多克隆抗体,根据Core蛋白的编码基因序列,设计合成一对特异性引物,利用RT-PCR扩增BVDV Core基因并定向插入Pet30a载体中,构建重组质粒Pet30a-Core,经酶切鉴定后转化大肠埃希菌TOP 10感受态细胞,筛选获得阳性重组菌,以IPTG进行诱导后成功表达出分子质量为20 ku的重组Core蛋白。将诱导表达的蛋白产物经融合蛋白的Ni柱亲和法进行纯化,利用纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,并利用间接ELISA法测出多克隆抗体效价达到1∶512 000。蛋白质印迹法(Western blot)和间接免疫荧光试验(IFA)证实,多克隆抗体可与细胞中的BVDV抗原发生反应,具有良好的免疫原性和特异性。本实验成功制备了BVDV重组Core蛋白的兔源多克隆抗体,为BVDV的检测及其Core蛋白功能研究奠定了基础。     

隐孢子虫病抗体治疗的研究进展

从超免疫初乳、超免疫血清、超免疫卵黄和单克隆抗体几个方面概述了国内外隐孢子虫病抗体治疗的研究现状,并介绍了预防隐孢子虫病疫苗的研究成果,为进一步防治该病提供了参考。     

Prokaryotic Expression and Polyclonal Antibody Preparation of African Swine Fever Virus Georgia 2007/1 Strain CD2v Protein

The study was aimed to express the EP402R gene of African swine fever virus (ASFV) Georgia 2007/1 strain via prokaryotic expression system,obtain the recombinant CD2v protein,and prepare polyclonal antibodies against the purified recombinant CD2v protein.After codon optimization,ASFV EP402R full-length gene was linked into pET-28a(+) expression vector to construct prokaryotic recombinant expression plasmid.After induction by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant CD2v protein was used as immunogen to prepare mouse anti-CD2v polyclonal antibodies.The antibody titer was measured by indirect ELISA and the specificity was further analyzed by indirect immunofluorescence assay (IFA) and Western blotting.The results showed that ASFV EP402R gene was successfully cloned into pET-28a(+),and pET-28a-EP402R was obtained.The recombinant plasmid was transformed into E.coli BL21(DE3) for expression,the recombinant protein was expressed mainly in the form of inclusion bodies,with molecular mass at about 47 ku,while some of the recombinant protein could also exist in a soluble form.Western blotting results showed that the purified protein had good immunoreactivity.The indirect ELISA result showed that the polyclonal antibodies had a high titer of 1:512 000,IFA and Western blotting results indicated that it could specifically recognize recombinant CD2v protein.These results confirmed the recombinant CD2v protein expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This research provided technical support for further study of ASFV EP402R biological function,as well as its gene-deletion based vaccine development.