Construction of Mutation Library in Haemophilus parasuis by Inserting Tn5 Transposon and the Screening of Attenuated Strain

[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.

Cloning and Expression of pilA Gene of Outer Membrane Protein of Haemophilus parasuis

[Objective] The aim was to clone and express the pilA gene of outer membrane protein of Haemophilus parasuis.[Method] The published pilA gene sequence of HPS was analyzed for primer synthesis,and the genome of serotype 5-type of HPS was used as template for PCR amplification of the pilA gene of HPS;the recombinant expression plasmid was constructed and transformed into E.coli BL21（DE3）after induced by IPTG.SDS-PAGE and Western blot analysis were then carried out.[Result] The molecular weight of expressed protein was consistent with the expected（43 kD）.[Conclusion] The results provided a foundation for the preparation of subunit vaccine and diagnostic reagents.

Detection and Characterization of β-Lactam Resistance in Haemophilus parasuis Strains from Pigs in South China

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis （PFGE）. Overall, the commonly detected resistance phenotypes were resistant to ampicillin （26.09%）, penicillin （22.61%）, amoxicillin （21.74%）, cefazolin （14.78%）, cefaclor （12.17%）, and cefotaxime （6.96%）. These strains showed high minimal inhibitory concentration （MICs） to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase （ESBL） phenotype. Moreover, 19 strains harboured bla genes including TEM-1 （n=5）, TEM-116 （n=10）, and ROB-1 （n=5）. Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime （MIC=8 mg L-1）. The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

Association between the Phenotypes and Genotypes of Antimicrobial Resistance in Haemophilus parasuis Isolates from Swine in Quang Binh and Thua Thien Hue Provinces, Vietnam

Haemophilus parasuis (H. parasuis) is one of the bacterial pathogens of great concern as it causes huge economic losses to the swine industry worldwide. One of the reasons why the control of H. parasuis has failed is the increase in antimicrobial resistance (AMR). The country of Vietnam has the second-largest pig production in Asia. However, there is still a lack of data about the AMR prevalence of H. parasuis in Vietnam.The purpose of this study is to investigate the prevalence of AMR and analyze the association between AMR and AMR genes (ARGs). The H. parasuis strains used in this research were isolated from swine in the Quang Binh and Thua Thien Hue Provinces, Central Vietnam, as reported in our previous study. All of the strains were tested for AMR against 25 antibacterial agents using the broth microdilution method and for the presence of ARGs using the polymerase chain reaction (PCR) method. The tested strains were shown to have a high frequency of resistance to trimethoprim/sulfamethoxazole (94.6%), followed by resistance to colistin, chloramphenicol, gentamicin, penicillin, lincomycin, and amoxicillin. The most prevalent ARGs in these strains were blaTEM-1 (94.6%), int (76.8%), gyrA (58.9%), and rmtD (50.0%). Cefuroxime, chloramphenicol, and tobramycin resistances were strongly correlated with the presence of the ARGs blaROB-1 (odds ratio (OR) = 26.3, 95% confidence interval (CI) 2.7–255.7, p = 0.002), catl (OR = 25.1, 95% CI 2.4–258.9, p = 0.004), and strB (OR = 23.5, 95% CI 2.6–212.6, p = 0.001), respectively.This study reveals for the first time the current situation of H. parasuis AMR in Central Vietnam, which is helpful for the clinical control of this disease, as well as for the development of policies and clinical practice guidelines to reduce AMR in swine production in Central Vietnam.

Serologic Investigation on Pig Haemophilus parasuis in Some Regions of Sichuan Province

To investigate the infection of Haemophilus parasuislin some regions of Sichuan Province, indirect hemagglutination from 2006 to 2008 was tested by Haemophilus parasuis antibody, and 1 062 serum samples from 10 different regions and sources in Sichuan Province were also examined. The results showed that the positive rate ranged from 2% to 30% with average of 10%. The positive rate varied with pigs at different ages, and especially that of 30-day-old to 70-day-old weaned piglets reached 25%. Thus, there were various infections of HaemophUus parasuis in some regions of Sichuan Province, and the statistic data provided a reference for effective prevention and control of Haemophilus parasuis in Sichuan Province.

Establishment and Application of an Indirect ELISA for Detection on Antibody of Haemophilus parasuis

[Objective]The aim was to establish an indirect ELISA for detection on antibody of Hps. [Method] The optimal conditions of indirect ELISA were selected and determined based on heat-resistant serotype 4 and 5 of Hps; specific,repeating and sensitive tests were conducted and 200 serums were detected. [Result]The optimal conditions were as follows： coating concentration of antigen at 10 μg /ml,and coating for 2 h at 37 °C; PBST containing 20 g /L of skim milk powder as blocking fluid for 30 min; serum dilution at 1∶ 80; reaction time of antigen for 45 min; dilution of secondary antibody at 1∶ 12 000 and effecting for 30 min; color development reaction for 15 min. [Conclusion] The established indirect ELISA is good in specificity and repetitiveness with higher sensitivity than that of indirect hemagglutination test; the results of clinic samples （ negative /positive serums） were in consistent with those detected with foreign ELISA kits. The established method can be made use of in serum antibody of Hps de- tection and seroepidemiology study.

Comparison of Two Construction Methods of Targeting Gene of Haemophilus parasuis

Haemophilus parasuis is one kind of constant bacteria in porcine upper respiratory tract, and it can cause multiple serositis, arthritis and other diseases under certain conditions. Due to lack of efficient genetic operating system, its pathogenic mechanism is not very clear. Ligation with DNA ligase and fusion PCR were used to construct targeting hhdA gene of Haemophilus parasuis, respectively. The fidelity, application scope, operation and conditions of the constructed fusion fragments were compared. The results showed that construction with DNA ligase was more mature technology as manifested by more stable conditions and more extensive application. The fusion PCR method had high fidelity and simple operation, and the transformation rate was 9.5 times as high as that of ligation with DNA ligase. For this reason, this method was more suitable for construction of multi-fragment targeting genes. The study lays a foundation for establishing an efficient operating system of targeting gene of Haemophilus parasuis in the future.

Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

Isolation and Identification of Haemophilus parasuis SD02 strain

A bacterial strain was isolated from the sick pigs suspiciously infected by polyserositis and arthritis in a pig farm in Shandong Province, and identified through morphological observation, culture traits, bioehemical characteristics and PCR amplifieation. Additionally, primers were de- signed according to the 16S rRNA sequence of Haemophihzs parasuis, and the bacterial strain was amplified by PCR. The amplified fragments of approximately 1 400 bp was sequenced, and aligned with the sequence in GenBank. The results showed that it shared the homology of 97%-99% with the 16S rRNA sequence of foreign H.parasuis, and confirmed as H.parasuis （HPS）. The strain was determined as serotype 4 through serotype identification. The strain was named SD02.

副猪嗜血杆菌sodA基因的表达及其理化性质

为探究副猪嗜血杆菌(Haemophilus parasuis)sodA基因编码的超氧化物歧化酶(Superoxide Dismutase,SOD)的活性及理化性质,设计特异性引物从HPS中扩增得到目的基因sodA,利用原核表达系统对HPS SOD蛋白进行表达,通过硫酸铵分级沉淀、Sephacryl S-300凝胶过滤层析以及DEAE Sepharose Fast Flow阴离子交换层析等方法对表达蛋白进行了分离纯化,利用邻苯三酚自氧化法对纯化产物进行SOD酶活性检测,通过HPSEC法、火焰原子吸收分光光度法对其理化性质进行了初步探究。结果表明,表达的重组HPS SOD蛋白质为可溶性蛋白质,单亚基分子质量为26 ku,纯化后目的蛋白质纯度为95%,纯化后的重组HPS SOD蛋白质比活性为215.9 U/mg,经HPSEC法检测分析,重组HPS SOD呈多聚体,通过火焰原子吸收分光光度法检测纯化的重组HPS SOD中锰元素含量为0.71 mg/L。

Epidemiological Investigation on the Etiology of Glasser＇s Disease in China

Haemophilus parasuis（HPS） is one of the most important bacterial infectious diseases harming China＇s pig industry for the last few years.To understand the prevalence status of glasser＇s disease in China, the disease samples submitted by Shandong, Henan, Heilongjiang, Jiangxi, Liaon-ing, Guangdong, Shaanxi and Sichuan provinces were isolated and identified. The synovial fluid, lung, brain tissue, heart blood and nasal cavity ofsick pigs in 194 pig farms collected from 2006 to 2009 were detected, and 47 strains of HPS were eventually isolated, with the separation rate of24.2%. Serotypes of the isolates were identified through KRG （Kieletein-Rapp-Gabriedson） agar diffusion method. There were 13 strains of serotype4 （27.7%）, 10 strains of serotype 5 （21.3%）, 8 strains of serotype 12 （17%） and7 strains of serotype 13 （14.9%）, indicating the major serotypes ofHPS in China were serotype 4, serotype 5, serotype 12 and serotype 13.