3株鸭疫里默氏杆菌的分离鉴定和耐药性分析

鸭疫里默氏杆菌病是由鸭疫里默氏杆菌(RA)引起的一种鸭急性传染病。2021年4月,山东省菏泽市某养鸭场21日龄肉鸭突然出现食欲减退、共济失调等症状,剖检可见明显纤维素性心包炎和肝周炎,为确定发病原因,本试验采集了病鸭的心脏、肝脏和大脑等组织,对病鸭的心脏和肝脏组织进行病理学观察,从病鸭大脑和肝脏组织中分离细菌,通过革兰染色、血清型分析和PCR方法对分离细菌进行鉴定,并对分离菌株进行耐药性和耐药基因分析。结果显示,病鸭心脏血管高度扩张,充满红细胞,心包膜外有明显的纤维素渗出物,肝细胞脂肪变性;通过细菌分离鉴定共获得了3株鸭疫里默氏杆菌(分别命名为RA-HZ01、RA-HZ02和RA-HZ03),其中血清型6型1株、7型2株。药敏试验结果显示,RA-HZ01和RA-HZ03对头孢噻肟、万古霉素、美罗培南和恩诺沙星敏感,对克林霉素中介,对卡那霉素、多西环素、红霉素、复方新诺明和多黏菌素耐药,而RA-HZ02对美罗培南、恩诺沙星和多黏菌素敏感,对头孢噻肟和卡那霉素中介,对其他抗菌药耐药。耐药基因检测结果显示,RA-HZ01和RA-HZ03携带氨基糖苷类耐药基因aac(6′)-I、aph(3′)-VI和aac(6′)-Ib,RA-HZ02携带氨基糖苷类耐药基因aac(6′)-I和aph(3′)-VI、喹诺酮类耐药基因qnrS以及消毒剂耐药基因qacE△1。结果表明,引起该鸭场此次发病的病原主要是鸭疫里默氏杆菌,且其具有多重耐药表型。本试验结果对本地区鸭场鸭疫里默氏杆菌病的诊断和防治具有一定的指导意义。

Phylogenetic and epidemiological characteristics of H9N2 avian influenza viruses in Shandong Province, China from 2019 to 2021

H9N2 avian influenza virus(AIV) has widely circulated in poultry worldwide and sporadic infections in humans and mammals. During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2AIVs. Phylogenetic analyses showed that the eight gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades(HA and NA genes), G1-like clades(PB2 and M genes), and SH/F/98-like clades(PB1, PA, NP and NS genes). The isolates showed mutation sites that preferentially bind to humanlike receptors(HA) and mammalian fitness sites(PB2, PB1 and PA), as well as mutations in antigen and drug resistance sites. Moreover, studies with mice revealed four isolates with varying levels of pathogenicity. The average antibody titer of the H9N2 AIVs was 8.60 log2. Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

Identification of an H1N1 subtype of swine influenza virus and serological analysis

To investigate the epizootic of swine influenza virus(SIV),60 nasal swabs were collected from a clinical cases of pig farm in Tai’an City,Shandong Province of China in April 2017.SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced.An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1).Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch,seven genome segments belonged to avian-like H1N1 influenza virus lineage.The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G,which is a typical molecular biological characteristic.Five potential N-glycosylation sites(N14,N26,N277,N484 and N543) were found in the HA gene.To further investigate the epidemiology of SIV in this farm,the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens.The results showed that the total positive rate was 65.43%.The positive rates of single virus infection detected by EAH1N1,2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35,30.85 and 7.47%,respectively.The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm.These data provide evidence of an epizootic of SIV.

St udy on Hsp90 Expression in Different Tissues and Its Antibody in Serum of Chickens Infected with Marek's Diseases

To investigate the dynamic change of heat shock protein 90 （Hsp90） in the genesis and development of tumor, we successfully established tumor animal model using Marek’s disease and then determined the location of Hsp90 in the tumor tissue using immunohistochemistry method, the antibody titer level of Hsp90 in the serum and the expression level in the tissue using enzyme-linked immunosorbent assay （ELISA） method. Our result showed that Hsp90 location in the tumor tissue was signiifcantly associated with the tumor cell and most in the cytoplasm of the tumor cell, and Hsp90 expression level in the tissue and the antibody titer level in the serum was most signiifcantly increased with the development of tumor. This is the ifrst report to show the presence of Hsp90 in tumor tissues induced by the Marek’s disease, with its expression correlated to the tumoral grading. These data may also be valuable for developing new molecular anti-cancer therapies.

动物干扰素研究概况

随着我国养殖业的迅猛发展,各种疾病的发生和危害日益加重,特别是病毒性疫病,已成为传染病的主体。对于病毒性疾病的治疗仍然缺乏有效的药物,且大多数尚无效果可靠的疫苗,再加上新发和再发病不断出现,使得病毒病危害尤为突出,急待寻找提高机体免疫、抵抗病毒感染的替代药物。干扰素(IFN)作为一种细胞因子具有广谱抗病毒、抗肿瘤和免疫调节活性,还可以促进机体免疫反应,提高机体抗病毒感染能力,使病毒在机体存活和复制的几率降到最低。目前发现Ⅰ型、Ⅱ型和Ⅲ型3种类型的IFN,利用3种类型的IFN的抗病毒和调节免疫的功能,研究抑制病毒复制的新型生物制剂,成为人们关注和研究的热点,为病毒性疫病,特别是新发或突发的重大疫病防控提供参考。

Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS 1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction （RT-PCR） and cloned into a prokaryotic expression vector, pET-28a（＋）, and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay （ELISA） was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

Relationship between Tumor Lesion of Kidney Tissue and Expression of HSP60 in Chickens Infected by MD

[ Objective] The paper was to investigate the relationship between pathological lesion and transcription, expression and distribution of heat shock protein 60 （HSP60） in kidney of chickens infected by Marek＇s disease virus （MDV）. [ Method] Tumor animal model was successfully established with chickens infected by MDV. Real-time RT-PCR, histopathological and immunohistochemistrical methods were used to detect the pathological lesion, transcription level, expression level and distribution of HSP60 in the kidney. [ Result] Obvious pathological changes appeared in kidney tissue of chicken after injection of MDV for 21 d. HSP60 was mainly distributed in the cytoplasm of tumor cell and interstitial macrophages. The expression of HSP60 in challenge group was always higher than blank control group and vaccine control group, and the difference reached extremely significant level from 28 to 86 day-ages （ P 〈 0.01 ） ; the expression of HSP60 in challenge group at 28 day-age was about 8.608 and 12.752 times of blank control group and vaccine control group, respectively. The mRNA transcription levels of HSP60 in challenge group showed a downward parabola during 7 to 42 day-ages; the mRNA transcription level of HSP60 in challenge group was rising in early stage （7 -21 d）, and then gradually dropped; it reached the peak value at 21 d, being 1.222 and 1.179 times of blank control group and vaccine control group, respectively. The mRNA transcription level of HSP60 was higher than two control groups throughout the entire experiment, and the difference reached extremely significant level from 14 to 28 day-agas （P 〈0.01 ）. [ Conclusion] HSP60 expression in kidney tissue and its mRNA transcription level can reflect MDV infection, tumor formation and development progress, with close relationship with pathogenesis of tumor diseases, which is expected to be used as one of the determination indicators for diagnosis and morbidity process of tumor diseases in chicken.