Klebsiella pneumoniae is an opportunistic pathogen that causes serious infections in humans and animals. However, the availability of epidemiological information on clinical mastitis due to K. pneumoniae is limited. To acquire new information regarding K. pneumoniae mastitis, data were mined about K. pneumoniae strains on dairy cattle farms (farms A to H) in 7 Chinese provinces in 2021. Hypermucoviscous strains of K. pneumoniae were obtained by the string test. MICs of antimicrobial agents were determined via the broth microdilution method. Ten antimicrobial resistance genes and virulence genes were identified by PCR. The prevalence of K. pneumoniae was 35.91% (65/181), and 100% of the bacteria were sensitive to enrofloxacin. Nine antimicrobial resistance genes and virulence genes were identified and compared among farms. The hypermucoviscous phenotype was present in 94.44% of isolates from farm B, which may be a function of the rmpA virulence gene. Based on these data, the multidrug-resistant strains SD-14 and HB-21 were chosen and sequenced. Genotypes were assayed for K. pneumoniae isolates from different countries and different hosts using multilocus sequence typing (MLST). Ninety-four sequence types (STs) were found, and 6 STs present a risk for spreading in specific regions. Interestingly, ST43 was observed in bovine isolates for the first time. Our study partially reveals the current distribution characteristics of bovine K. pneumoniae in China and may provide a theoretical basis for the prevention and treatment of bovine K. pneumoniae mastitis. IMPORTANCE K. pneumonia is ubiquitous in nature and infects a wide range of hosts, including animals, and humans. It is one of the leading inducements of clinical mastitis (CM) in dairy cows, a prevalent and costly disease that is predominantly associated with bacterial infection. In general, CM caused by Gram-negative bacteria is more difficult to cure than that associated with Gram-positive pathogens, with an average cost per case of 211.03 U.S. dollars (USD) for Gram-negative bacterial infections compared with 133.73 USD for Gram-positive bacterial CM cases. After Escherichia coli, K. pneumoniae is the second most common Gram-negative cause of bovine CM, but it is the most detrimental in terms of decreased milk yield, discarded milk, treatment costs, death, and culling. In view of the economic implications of K. pneumoniae infection in dairy farming, research into population structure and antibiotic resistance is particularly important.

One strain of pigeon Newcastle disease virus(NDV) was isolated from a diseased racing pigeon loft suspected to be infected with NDV in Nanjing.This NDV isolate was named Pi/NJ/CH/4416/2016 (ND4416) and purified by using specific pathogen-free(SPF) eggs.Biological tests showed that this isolate showed high pathogenicity to pigeons,but lower to chickens.The resulting RNA samples were converted to cDNAs,followed by PCR amplification and sequencing of F and HNgenes.The cleavage site of the fusion protein(F) was 112RRQKRF117,a feature generally associated with virulent NDV strains.Phylogenetic analysis based on F and HNgene sequences showed that this strain belonged to Class Ⅱ,Ⅵb subgenotype.In addition,Pi/NJ/CH/4416/2016 and other strains prevailing in China were more closely related to the PPMV-1/Belgium/11-09620/2011(JX901124.1) strain.It supported that epidemic pigeon NDV isolates in China might originate from Belgium.Pi/NJ/CH/4416/2016 had a far genetic relationship from the classic vaccine strain La Sota (AF077761.1).Bioinformatics analysis revealed that a linear epitope tope consisting of amino acids 491-500 of HNprotein of this pigeon strain was different from La Sota.In addition,hemagglutination inhibition test revealed a noticeable antigenic difference between them.

江苏省某种鹅场15日龄雏鹅疑似感染鸭疫里氏杆菌,无菌采取脑、心脏、肝和脾等病料,进行病原分离纯化,通过细菌生化试验、血清型鉴定及设计特异性扩增鸭疫里氏杆菌外膜蛋白A(OmpA)基因的引物,进行PCR扩增,从8份病料中分离到2株血清2型鸭疫里氏杆菌.药物敏感性试验表明,2个分离株对红霉素和阿奇霉素的敏感性高于其他药物.对OmpA基因PCR产物进行基因序列测定,在NCBI上进行基因序列同源性比对,其基因序列与多株已发表的序列的同源性达到99％.进化分析表明,分离株均与D2、G2株具有很近的亲缘关系.

为构建新型H7亚型禽流感HA基因的原核表达载体，并在大肠杆菌中进行表达。从NCBI数据库下载H7亚型禽流感HA全基因序列，合成HA基因；定向克隆到原核表达载体pET－32a（＋）的多克隆位点中，构建重组原核表达质粒 pET －32a（＋）－HA；转化入大肠杆菌 BL21（DE3）中经 IPTG 诱导表达，表达产物经 SDS －PAGE 和Western－blot法鉴定。结果显示，重组质粒经双酶切及基因测序鉴定构建正确；重组的HA融合蛋白约为84 ku，大小与预期融合蛋白大小一致；重组HA融合蛋白可以与His标签单克隆抗体特异性结合，与H7阳性血清有较强的反应原性。成功构建了HA基因原核表达载体，并在大肠杆菌BL21（DE3）中获得重组HA融合蛋白表达。

To compare PCR and TaqMan real time PCR(FQ-PCR) for the detection of Mycoplasma haemosuis infection in pigs,the PCR method and FQ-PCR were set up with the primers targeting the 16S rRNA gene of M.haemosuis.Plasmid containing 16S rRNA gene was used as template to optimize the conditions of conventional PCR and FQ-PCR reactions.The results showed that these 2 assays have good sensitivity and specificity.The sensitivity of FQ-PCR was 1,000 times higher than conventional PCR.Twenty clinical samples were detected by these 2 assays,positive rate was 12/20 by conventional PCR,while 15/20 by FQ-PCR.In conclusion,these 2 assays can effectively detect the clinical infection of M.haemosuis and may provide useful tools in establishment of animal infection model.

The monoclonal antibodies(MAbs) were prepared by fusing mouse myloma cells(SP2/0) with spleen cells from BALB/c mice immunized with purified recombinant MSG1 protein of Mycoplasma suis.Two hybridoma cell lines secreting MAbs against MSG1 protein were screened by indirect enzyme-linked immunosorbent assay(ELISA) and named as 1A7 and 3G6,respectively.Western-blot results showed that the two MAbs reacted specifically with recombinant MSG1 protein.Their ELISA titers in supernatant and asicite were 1∶4096,1∶1024 and 1∶1638400,1∶51200,respectively.The isotypes of 1A7 and 3G6 both belong to IgG1/κ and the additive ELISA showed that the two MAbs reacted with the same antigen determinant.So,we concluded that the specific anti-MSG1 protein MAbs were developed and these MAbs may be useful in the development of detection methods and provide a basis for investigating the pathogenicity of M.suis.

In order to determine the immune regulatory effect of bursin on avian influenza (H9N2) vaccine, two hundred 13-day-old chicken were randomly divided into four groups. One was for the control, the others for vaccine, vaccine plus bursin and bursin treatments respectively. The change of average index of bursa of fabricius and spleen and serum antibody titer in chicken were examined by means of hemagglutination inhibition test every 7 days from the 13th day after treatment. The results showed that bursin could enhance chicken serum antibody titer on avian influenza (H9N2) oil adjuvant inactivated vaccine and develop bursa of fabricius significantly. Bursin also could prolong the maintenance of high serum antibody titer in chicken. It was suggested that bursin could improve immune effects of avian influenza (H9N2) oil adjuvant inactivated vaccine in chicken

为了确定抗体水平对疫苗免疫的影响,本试验对规模化猪场不同日龄仔猪和母猪产前产后的猪瘟疫苗和伪狂犬疫苗抗体水平进行监测,并将抗体水平的变化和疫苗的免疫结合起来.结果表明,母猪产前产后的高水平猪瘟和伪狂犬疫苗抗体使仔猪具有较高的母源抗体水平,在一定时间接种猪瘟疫苗和伪狂犬疫苗后,仔猪体内的抗体水平迅速下降至较低水平并持续很长时间,而且下降后的抗体水平上升非常缓慢,使免疫猪产生一个免疫间隙期.该研究证明了仔猪体内高水平母源抗体对免疫的疫苗产生了中和作用,使抗体效价迅速降低和疫苗有效作用成分减少.因此,制定合理的免疫程序非常重要.

本研究将附红细胞体的MSG1蛋白基因按大肠杆菌密码子偏嗜性改造后进行全基因人工合成,合成的基因连接入pBAD/HisB载体后导入大肠杆菌Top10感受态细胞中,进行MSG1诱导表达,确定诱导表达的最佳时间和最佳诱导剂浓度。对表达的蛋白应用His单抗进行了特异性鉴定,对鉴定后的蛋白应用Ni-NTA纯化试剂进行了不同条件下的蛋白纯化。试验结果表明,诱导蛋白的分子量在45 ku左右,加入0.2%的L-阿拉伯糖,诱导2 h后所表达的蛋白量最高。经Western-blot鉴定,该蛋白可以被His单抗特异性识别,经纯化后可以得到较高纯度的MSG1蛋白。表达的MSG1蛋白经纯化后免疫家兔制备多克隆抗体,Western blot证明制备的抗体具有高度的特异性。MSG1蛋白的表达和多抗的制备可以为后续研究附红细胞体的吸附机制、致病机理,以及附红细胞体病的治疗等提供理论基础。

To study the genotype of Haemophilus parasuis and its distribution in China,57 Heamophilus parasuis isolates along with 15 reference strains of all known serovars were subjected to be analyzed using PCR-FRLP(restriction fragment length polymorphism) based on tbpA gene.The analysis of the 1.9-kb tbpA amplicon using Taq I,Ava I and Afa I endonucleases showed 9 RFLP patterns of the15 reference strains and 15 patterns of the 57 field isolates.Moreover,the first 3 genotypes of most prevalence in China were DBN(38%),ABN(18%) and DBP(12%).Our results confirmed that Haemophilus parasuis existed widely in China with numerous genotypes and also provided significant basis for the prevention and control of the disease in China.

From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR–FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes.

The complete open reading frame 5 (ORF5) sequences of 34 field porcine reproductive and respiratory syndrome virus (PRRSV) isolates from China in 2002–2007 were detected and compared with the different variable Chinese isolates S1, CH-1a, HB-1, HB-2 and JXA1. The results showed that all isolates were of type 2 PRRSV and could be assigned to two clusters. The isolates in cluster sg1 was high similar with the highly pathogenic PRRSV strain JXA1, while sg2 clustered with type 2 PRRSV isolate VR2332. It was interesting that the isolate SH02 which was isolated from Shanghai in 2002 has 98.8% identity with JXA1 emerged in 2006. And the ZJJ07 isolate was found to be a natural recombinant between a Chinese highly pathogenic SY0608 isolate and a VR-2332 derivative NH04 isolate. Analysis of the potential glycosylation sites indicated that they were frequently mutated and formed five putative N-linked glycosylation (NGS) sites patterns based on N30, 33–35, 44 and 51 in those isolates. It indicated that the highly variable PRRSV strain with different NGS patterns spread widely in China. The great genetic diversity could be taken into consideration for the control and prevention of this disease.

Extensive genetic variations have been found among porcine reproductive and respiratory syndrome virus(PRRSV) isolates.The variations could lead to the emergence of more virulent strains of PRRSV.In order to determine the evolutionary trend and the causes for increasing virulence of PRRSV,nucleotide sequences and derived amino acids sequences of GP5 proteins from 14 strains were analyzed and phylogenetic tree was constructed.The results showed that the glycosylation sites at amino acid 26 to 39 among the isolates from 2006 were changed and the isolates formed a new minor branch in phylogenetic tree.Phylogenetic analysis indicated the isolates may be originated from CH-1a,which was isolated from China in 1995.

为了解江苏高邮市农户散养健康仔猪群猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)感染和带毒情况,本研究自2003年1月至2008年9月,随机选择并采集68个农户105窝母猪的372头2～5周龄仔猪血清,分别用ELISA和PCR方法检测PCV2和PRRSV抗体和病毒核酸,结果为,PCV2和1PRRsV感染率分别为17.5%(65/372)和32.3%(120/372),其中Pcv2和PRRSV混合感染阳性率为12.9%(48/372),占PCV2感染猪的73.9%(48/65),而PRRSV感染率逐年提高,2008年达43.3%,从而为该病防制提供了理论依据.

Research advancement on rabies was introduced in this paper,and the epidemic of the disease in the world,especially among human,dog,cat,deer and fur animal such as fox,wolf etc in our country was elucidated.Lastly,some problems were pointed out and some suggestions were put forward on increasing public will,consummating laws and statutes,compulsory immunity and improving the quality of vaccine.

The S1 and SY0608 strains of porcine reproductive and respiratory syndrome virus (PRRSV) were individually isolated and had different pathogenicity in pigs in 1997 and 2006. In order to understand their genomic characteristics, the full-length genome of S1 and SY0608 isolates were sequenced and analyzed. The results indicated that their genome composition differed significantly and shared only 88.5% nucleotide identity with each other. The genetic variation and amino acid substitutions were not randomly distributed in the genome, and mainly focused on ORF1a, ORF3 and ORF5. The SY0608 strain, with high pathogenicity, had a 30-amino-acid deletion at amino acid positions 480 and 532-560 in comparison with the S1 strain. The alignment of amino acid sequence of Nsp1-Nsp8, GP2-GP5, M and N of S1 and SY0608 with other PRRSV isolates demonstrated that variation was mainly found in the Nsp2, GP3 and GP5 proteins. In comparison with the S1 strain, the SY0608 strain showed some potential glycosylation site mutations in GP5 at amino acid positions between 26 and 39, which might be associated with viral antigenicity. Phylogenetic analysis showed that the two strains belonged to two different branches that do not indicate differences in pathogenicity. Interestingly, the deletion strains isolated recently in China formed a new minor branch, revealing the same evolutionary trend.

The paper focused on the development of antiserum,yolk antibodies and monoclonal antibodies used for animals in our country,and give good examples to demonstrate the theory,technique and application of theses products.

文中首先简要地介绍了RV研究进展,然后对狂犬病在全世界的流行作了阐述,特别对我国大陆地区人、犬、猫、鹿和毛皮兽中的狐、貉、獾狂犬病的流行状况与特点进行了评说。最后对我国在狂犬病防制中存在的问题,提出了一些诸如加强宣传、提高民众的公益意识、完善法律法规、实施对犬猫强制免疫以提高免疫率和切实提高疫苗质量等狂犬病防制的建议。

<正>文中从发生在毛皮兽养殖业发展比较集中地区的一起犬瘟热暴发流行和一些种狐诉讼咨询为实例评述我国目前毛皮兽(貂狐貉)养殖发展中存在的问题,诸如部门管理错位与失误,长期来无发展规划与无种兽、饲养、环境标准和疾病防控规范;长期来存在品种持续退化及产仔率、成活率和毛皮质量低下,经济效益和发展波动不稳;由于不规范的分散饲养导致了生态环境污染、生物安全失控和疫病扩散频发等等。同时,提出了一些相应的建议与对策。

Efficacy of immunoprotection against coccidiosis was investigated by in ovo inoculation with the oocysts of Eimeria tenella and/or E.acervulina.The fertiled Lingnanhuang broiler chicken eggs were injected through amniotic sac on 18 d of incubation with the oocysts of E.tenella and / or E.acervulina.The hatched chickens were reared in wire-cages and oocysts outputs were detected using OPG(oocysts per gram of feces) during day 3~10 post-hatched.The vaccinated chickens were challenged with a high dose of the homologous oocysts of E.tenella and / or E.acervulina at 14 d posthatch,and the immunity efficacy against homologous Eimeria oocyst challenge was evaluated by relative weight gain(RWG),feed conversion rate(RCR) and relative oocysts production(ROP).The chickens inoculated in ovo with oocysts had significantly reduced oocysts output(ROPs were in range of 4.7%~23.5%), meanwhile,with significant increase in RWG(reached 76.5%~83.6%) and better FCR(ranged in from 2.72 to 2.96) compared to the nonimmunized counterparts.The results suggested the feasibility of immunizing chicken by in ovo injection with two different Eimeria species of oocysts.