鳗弧菌、溶藻胶弧菌外膜蛋白的分离及特性

分别用SDS、Sarkosyl及PMSF法分离制备鳗弧菌（Vibrio anguillarum）、溶藻胶弧菌（Vibrio alginolyticus）主要外膜蛋白（MOMP），通过SDS-PAGE分析比较2种弧菌主要外膜蛋白的组成结构。结果表明，SDS、Sarkosyl和PMSF法分离提取的鳗弧菌和溶藻胶弧菌外膜蛋白中，SDS-PAGE电泳图谱不同，鳗弧菌外膜蛋白分子量为27-138kD；溶藻胶弧菌外膜蛋白分子量为27-104kD，其中，45kD、30kD和27kD外膜蛋白为鳗弧菌和溶藻胶弧菌所共有。经比较，Sarkosyl法对2种弧菌外膜蛋白的提取效果较好。对3种方法提取的2种弧菌的主要外膜蛋白进行SDS PAGE后，分别与吸附后的兔抗鳗弧菌、抗溶藻胶弧菌血清的Western-blotting印迹显示，兔抗鳗弧菌血清与其菌株的外膜蛋白主要有3条免疫反应带，其分子量分别为97kD、51kD和30kD，说明其可能与鳗弧菌抗原的特异性有关；兔抗溶藻胶弧菌血清与其菌株的外膜蛋白主要有2条免疫反应带，其分子量分别为51kD和27kD，说明可能与溶藻胶弧菌抗原的特异性有关，而51kD外膜蛋白可能是2种弧菌共有的特异性抗原。     

Diagnostic evaluation and short-term outcome as indicators of long-term prognosis in horses with findings suggestive of inflammatory bowel disease treated with corticosteroids and anthelmintics

Background

Recurrent colic and unexplained weight loss despite good appetite and adequate feeding and management practices are common conditions in the horse. However, little information has been published on the systematic diagnostic evaluation, response to treatment, prognostic factors or outcome of either presentation. The aims of this study were to 1) identify possible prognostic indicators and 2) report the short- and long-term response to treatment with corticosteroid therapy of a variety of horses with a presumptive diagnosis of inflammatory bowel disease (IBD).Thirty-six horses with a history of recurrent colic and/or unexplained weight loss were screened with a detailed clinical, clinicopathological and diagnostic imaging examination. Twenty horses were subsequently selected that had findings consistent with inflammatory bowel disease based on the fulfilment of one or more of the following additional inclusion criteria: hypoproteinaemia, hypoalbuminaemia, malabsorption, an increased intestinal wall thickness on ultrasonographic examination or histopathological changes in rectal biopsy. These 20 horses were treated with a standardized larvicidal anthelmintic regime and a minimum of three weeks of corticosteroid therapy.

Results

The initial response to treatment was good in 75% (15/20) of horses, with a 3-year survival rate of 65% (13/20). The overall 3-year survival in horses that responded to initial treatment (12/15) was significantly higher (P = 0.031) than in those that did not respond to initial treatment (1/5). The peak xylose concentration was significantly (P = 0.048) higher in survivors (1.36 ± 0.44 mmol/L) than non-survivors (0.94 ± 0.36 mmol/L).

Conclusions

The overall prognosis for long-term survival in horses with a presumptive diagnosis of IBD appears to be fair to moderate, and the initial response to anthelmintic and corticosteroid therapy could be a useful prognostic indicator. The findings of the present study suggest that a low peak xylose concentration in absorption testing is associated with a less favourable prognosis, supporting the use of this test.     

Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C)

Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n = 15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs.     

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.     

Serum Biomarkers of Clinical and Cytologic Response in Dogs with Idiopathic Immune‐Mediated Polyarthropathy

Background

Immune‐mediated polyarthopathy (IMPA) is common in dogs, and is monitored by serial arthrocenteses.

Hypothesis/Objectives

Plasma C‐reactive protein (CRP), interleukin‐6 (IL‐6), and CXCL8 (interleukin‐8) would serve as noninvasive markers of joint inflammation in IMPA.

Animals

Nine client‐owned dogs with idiopathic IMPA; 6 healthy controls.

Methods

Prospective study. Plasma CRP, IL‐6, and CXCL8 were measured by ELISA at baseline, 2, and 4 weeks during treatment with prednisone at 50 mg/m²/day. Arthrocenteses, the canine brief pain inventory (CBPI), and accelerometry collars were used to assess joint inflammation, lameness, and mobility at all 3 time points.

Results

C‐reactive protein concentrations were higher in IMPA dogs (median 91.1 μg/mL, range 76.7–195.0) compared with controls (median <6.3 μg/mL, <6.3–13.7; P = .0035), and were significantly lower at week 2 (10.6 μg/mL, <6.3–48.8) and week 4 (<6.3 μg/mL, <6.3–24.4; P < .001).C‐reactive protein was correlated with median CBPI scores (r = 0.68; P = .0004), joint cellularity (r = 0.49, P = .011), and mobility by accelerometry (r = −0.42, P = .048). Plasma IL‐6 concentrations were also higher in IMPA dogs (median 45.9 pg/mL), compared with controls (median <15.7 pg/mL; P = .0008). IL‐6 was lower in IMPA dogs by week 4 (<15.7 pg/mL; P = .0099), and was modestly correlated with CBPI scores (r = 0.47, P = .023). CXCL8 did not differ significantly between IMPA and healthy dogs.

Conclusions

Plasma CRP and IL‐6 might be useful surrogate markers of synovial inflammation and disease activity in dogs with IMPA.     

Effect of feeding dried distillers' grains with solubles on milk yield and milk composition of cows in mid‐lactation and digestibility in sheep

We evaluated the effect of three sources of dried distillers' grains with solubles (DDGS) in diets of mid‐lactating dairy cows on milk production and milk composition and on digestibility in sheep. DDGS from wheat, corn and barley (DDGS₁), wheat and corn (DDGS₂) and wheat (DDGS₃) were studied and compared with a rapeseed meal (RSM). RSM and DDGS were characterized through in situ crude protein (CP) degradability. Nutrient digestibility was determined in sheep. Twenty‐four multiparous cows were used in a 4 × 4 Latin square design with 28‐day periods. Treatments included total mixed rations containing as primary protein sources RSM (control), DDGS₁ (D1), DDGS₂ (D2) or DDGS₃ (D3). RSM contained less rapidly degradable CP (fraction a), more potentially degradable CP (fraction b) and more rumen undegradable CP (UDP) than the three DDGS. In vivo digestibility of RSM organic matter was similar to DDGS. Calculated net energy for lactation (NE_L) was lower for RSM (7.4 MJ/kg DM) than for DDGS, which averaged 7.7 MJ/kg DM. Cows' dry matter intake did not differ between diets (21.7 kg/day). Cows fed D1 yielded more milk than those fed D3 (31.7 vs. 30.4 kg/day); no differences were found between control and DDGS diets (31.3 vs. 31.1 kg/day). Energy‐corrected milk was similar among diets (31.2 kg/day). Diets affected neither milk fat concentration (4.0%) nor milk fat yield (1.24 kg/day). Milk protein yield of control (1.12 kg/day) was significantly higher than D3 (1.06 kg/day) but not different form D1 and D2 (1.08 kg/day each). Feeding DDGS significantly increased milk lactose concentration (4.91%) in relation to control (4.81%). DDGS can be a suitable feed in relation to RSM and can be fed up to 4 kg dry matter per day in rations of dairy cows in mid‐lactation. However, high variation of protein and energy values of DDGS should be considered when included in diets of dairy cows.     

Genetic variation in the ovine uncoupling protein 1 gene: association with carcass traits in New Zealand (NZ) Romney sheep,but no association with growth traits in either NZ Romney or NZ Suffolk sheep

The uncoupling protein 1 (UCP1) plays an important role in the regulation of lipolysis and thermogenesis in adipose tissues. Genetic variation within three regions (the promoter, intron 2 and exon 5) of the ovine UCP1 gene (UCP1) was investigated using polymerase chain reaction‐single‐strand conformational polymorphism (PCR‐SSCP) analyses. These revealed three promoter variants (designated A, B and C) and two intron 2 variants (a and b). The association of this genetic variation with variation in lamb carcass traits and postweaning growth was investigated in New Zealand (NZ) Romney and Suffolk sheep. The presence of B in a lamb's genotype was associated with decreased subcutaneous carcass fat depth (V‐GR) (p = 0.004) and proportion of total lean meat yield of loin meat (p = 0.005), and an increased proportion of total lean meat yield of hind‐leg meat (p = 0.018). In contrast, having two copies of C was associated with increased V‐GR (p < 0.001) and proportion of total lean meat yield of shoulder meat (p = 0.009), and a decreased hind‐leg yield (p = 0.032). No associations were found with postweaning growth. These results suggest that ovine UCP1 is a potential gene marker for carcass traits.     

猪圆环病毒2型ORF2基因截断表达及ELISA抗体检测方法的初步建立

为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型（PCV2）ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光（IFA）的符合率为85.53%（130/152）,与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%（134/152）。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。     

副猪嗜血杆菌外膜蛋白P5的B细胞抗原表位鉴定

副猪嗜血杆菌外膜蛋白P5能诱导机体产生免疫保护反应,同时可以用于特异性的血清学诊断,本试验选取P5蛋白进行抗原表位鉴定。首先通过PCR扩增P5F1（1～204aa）,P5F2（170～296aa）及P5F3（280～371aa）3个片段,PCR产物分别定向克隆到表达载体pET32a（＋）中表达纯化。根据ELISA和Western blotting结果确定P5F3片段（280—371aa）是OmpP5的免疫优势决定区。为了进-步对该免疫优势决定区进行抗原表住鉴定,设计了-套11个部分重叠的短肽,这些短肽覆盖全部280～371aa片段。每-个短肽合成1对寡核苷酸链,退火后插入表达载体pGEX-6p-1,与GST进行融合表达。用HPS阳性血清进行ELISA和Western blotting扫描,鉴定出其表位位于”。TGNTCDAVKGRKALIT351。通过序列分析证实该抗原表位在不同的HPS菌株中高度保守。本试验确定了位于HPSOmpP5上的-个抗原表位,为建立-种方便、快捷、适用于现地大规模样品检测的鉴别诊断方法奠定了基础,同时也为HPS新型亚单位疫苗的研制,以及研究病原茵感染和机体免疫过程中P5蛋白与宿主体内相应分子之间的相互作用提供了有用信息。     

布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA—related融合蛋白,以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中,区分出s2疫苗接种牛与牛种布鲁菌感染牛。