Characteristics and Analysis of Swine Streptococcosis Incidence in Binzhou Region in 2013

To fully understand swine stroptococcosis in Binzhou and surrounding areas, we studied its incidence rate and incidence characteristics in 2013, in order to put forward constructive suggestions for scientific prevention and control of swine streptococcosis. Isolation and purification, biochemical and molecular biolo- gy identification and drug susceptibility test of Streptococcus suis were carried out, and mixed infection of S. suis with classical swine fever virus, pseudorabics virus and porcine circovirus virus was statistically analyzed. The results showed that 46 strains of S. suis were screened, and the positive rate was approximately 24.9%, with high degree of resistance and multiple resistances. S. suis had different degrees of mixed infection with classical swine fever virus, pseudorabics virus and porcine cireovirus virus, and the total mixed infection rate was 58.7%.

Application Progresses of Glucose Oxidase(GOD)in Feed

Glucose oxidase （GOD） is a new pollution-free enzyme preparation which can improve morphological structure and micro ecological balance of intestine in livestock and poultry, perfect intestinal environment, and increase feed digestibility, growth performance and disease resistance. This paper summarizes the physical and chemical properties and biological function of GOD and its application status in feed.

Expression of Pseudorabies Virus gE Core Epitopes in Escherichia coli Strain BL21 and Utilization of Indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E （PRV gE） has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE core epitopes was expressed in E. coli BL21 expression system. SDS-PAGE and Western Blotting revealed that the expression product in culture supematant of E. coli BL21 was a recombinant protein, approximately 51.8 Kd. At 5 h post-induction, protein concentration assay showed that the expression product amounted to 1.65 mg/ml, accounting for 24. 17% of total proteins in the culture supematant. An indirect PRV gE-ELISA was established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. In addition, the assay was consistently reproducible. Comparison of detection results of 240 serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed that there was no significant difference between these two methods （P 〉 0.05 ）.

Establishment and Application of a SYBR Green I Real-time Quantitative PCR Assay for the Detection of LAT Gene of Pseudorabies Virus in Bama Miniature Pigs

[ Objective] This study aimed to establish a rapid, sensitive and specific SYBR Green I real-time quantitative PCR assay for the detection of latent pseudorabies virus （PRV） infection. [ Method ] SYBR Green I real-time quantitative PCR conditions and system for early detection of latent pseudorabies virus in- fection were optimized and compared with conventional PCR to investigate the sensitivity and specificity. Subsequently, the established assay was applied to detect different clinical samples. [ Result] The sensitivity of SYBR Green I real-time quantitative PCR assay （52 copies/μl） was 1 000 times higher than that of conven- tional PCR （5.2×1^04 copies/μl） and the detection time was shortened by 1/2. The established assay could be used to detect PRV but could not be used to detect PCV2, PPV, CSFV or PRRSV. Various tissues were collected from Bama miniature pigs with latent PRV infection under sterile conditions for real-time PCR detection. Results showed that viral copy number in the brain, nasal swab, inguinal lymph node, liver, lung and spleen was above 20, while PRV was not detected in the kidney and heart tissues. [ Conclusion] The established SYBR Green I real-time quantitative PCR assay for PRV/.AT detection was specific, sensitive and rapid, which could be used for pathogen monitoring, epidemiological investigation and quantitative study of PRV.

Prokaryotic Expression of Gene Encoding Glutamate Dehydrogenase of Streptococcus suis Serotype 2 and Preparation of Polyclonal Antibodies against Its Expressed Products

[ Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [ Method] The complete ORF of glutamate dehy- drogenase （GDH） gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by poly- merase chain reaction （PCR）. The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coil BL21 （DE3）. The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [ Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot as- say, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could re- act specifically with serum samples collected from five pigs experimentally infected by strain SC22. [ Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.

Detection of Patulin in Dairy Products by ELISA

The content of patulin in dairy products was detected by rapid ELISA assay. The pretreatment of samples was simple, with wide range of application. The standard product showed good linear relationship with the range of 0-5.4 μg/kg, and the linear relationship reached 0.999 3, with high sensitivity. It took shorter time, with a total time-consumption of 1 h. Moreover, it had good repeatability for analytical requirements.

Associated Bacteriostasis Research of Coptidis Rhizoma-Folium Isatidis and Coptidis Rhizoma-Flos Poprli against Escherichia coli O2

[Objective] The paper was to explore the combined inhibitory effect of rhizoma coptidis-folium isatidis and rhizoma coptidis-flos poprli against Escherichia coli O2.[Method] Contrast test of single and associated bacteriostasis against known serotype E. coli O2 was conducted using microcheckerboard method.[Result] The MIC of rhizoma coptidis, folium isatidis and flos poprli were 1/8 extracting liquid, 1/8 extracting liquid and 1/2 extracting liquid, respectively. When combined with folium isatidis or flos poprli, the MIC of rhizoma coptidis was 1/8 extracting liq-uid or 1/16 extracting liquid compared with single use. When combined with rhizoma coptidis, the MIC of folium isatidis and flos poprli were 1/8 extracting liquid and 1/16 extracting liquid.[Conclusion] When rhizoma coptidis was combined with folium isatidis or flos poprli, the FIC values were 2 and 0.625, performing independent action and additive effect, respectively.

Isolation and Identification of a High-pathogenicity Strepto- coccus suis Serotype 7 Strain

In order to diagnose the diseased pigs in a certain large pig farm in Binzhou City, Shandong Province, the dead piglets with joint swelling were subjected to necroscopy, and the pathogenic bacterium was isolated and identified. One Gram-positive Streptococcus was isolated. The strain was subjected to characteristic culture, microscopic examination and molecular biological identification, and resistance detection, animal regression experiment and mouse pathogenicity test were carried out. The results showed that the isolate was identified to be Streptococcus suis serotype 7, which was resistant to multiple drugs; and the pathogenicity test showed that the strain had high pathogenicity to pigs, resulting in neurosis on partial pigs, and the strain had no pathogenicity to Kunming and BALB/c mice but certain pathogenicity to CD1 mice.

In Vitro Antibacterial Test of Alkaline Hydrogen Water on Four Kinds of Common Pathogenic Microorganisms

[Objective] The paper was to investigate in vitro antibacterial effect of alkaline hydrogen water on Escherichia coli,Salmonella,Pseudomonas aeruginosa and Staphylococcus aureus.[Method] With Cortex cinnamomi extract（1 g/m L） and pure water as the control,the minimal inhibitory concentrations（MIC） against four kinds of common pathogenic microorganisms were tested through microdilution method.[Result] When the alkaline hydrogen water was diluted to 1/8 times of the original concentration,it had significant antibacterial effects on four kinds of common mi-croorganisms with the concentration of 1.5 ×10^5 CFU/m L,which had equivalent effect with C.cinnamomi extract group.[Conclusion]The alkaline hydrogen water has remarkable antibacterial effects on the four kinds of common microorganisms,which may provide a new important way for pre-venting disease occurrence and reducing the harms of pathogenic microorganisms.

Research Progress on a Novel Duck Flavivirus Disease

A severe egg-drop disease caused by the infection of a novel duck flavivirus outbroke successively in many provinces in southeastern China since 2010.It was identified as a duck Tembusu virus（DTMUV）and named by Chinese Association of Animal Science and Veterinary Medicine in the first symposium on waterfowl disease control,which was presumed to be a mosquito-borne flavivirus of the Ntaya virus subgroup in the genus Flavivirus,family Flaviviridae.Currently,a large number of studies have been conducted on the epidemiology,clinical symptoms and pathological changes,etiology,and rapid diagnoses of the virus.The disease remains a constant threat to the duck industry.In order to provide reference for subsequent in-depth study,in this paper,research progress on the disease was summarized based on previous studies.Furthermore,the potential infection or asymptomatic infection in humans should be evaluated as soon as possible.

Diagnosis and Treatment Report of the First Case of Mycoplasma bovis in Shandong Province

[Objective] The paper was to introduce the first case of Mycoplasma bovis in Shandong Province.[Method] Samples were collected from sick cattle,and identified through M.bovis isolation,biochemical identification,PCR,16S RNA sequence analysis and clinical treatment and other research work.[Result] A strain of M.bovis was successfully isolated,and typical colonies in fried egg shape were grown.It was further confirmed as M.bovis via molecular biological detection.The use of vaccine and sensitive drugs in clinical could quickly control the epidemic situation of cat-tle herds.[Conclusion] This is the first reported case of M.bovis in Shandong Province,and its diagnosis and treatment process is conducive to further prevention and control of the disease.

Inducible Expression on Multi-epitope of Porcine Circovirus Type 2 and Its Immunological Competence

In order to obtain induction expressed porcine circovirus type 2（PCV2）multi-epitope with good immunogenicity in vitro.Major epitopes of PCV2 were screened in the test,epitopes were composed sequentially,the c DNA sequences were artificially synthesized.Bam HⅠand SalⅠwere directionally cloned into prokaryotic expression vector PEGX-4T-1 multiple cloning site,then BL21 competent cells were transformed,positive clones were screened,IPTG inducible expression was conducted.Expression on target gene was analyzed by SDS-PAGE,fusion protein polypeptide was extracted and purified,immunocompetence of the expressed multi-epitope protein was identified by Westernblot.BALB/c mouse was immuned by fusion protein polypeptide,the antibody was determined by ELISA,immunogenicity was evaluated.Results showed that expression recombinant plasmid pEGX-4T-1-ep contained with seven PCV2 antigen epitopes had been constructed successfully.SDS-PAGE analysis showed that fusion protein polypeptide was expressed effectively in Escherichia coli（E.coli）,and the molecular weight was about 35ku,which existed in the form of solubility.Results of Westernblot showed that the extraction and purification of fusion protein polypeptide and PCV2 positive serum had good reactogenicity.Results of ELISA showed that the purified fusion protein polypeptide could stimulate the body to produce PCV2 specific antibody which had good immunogenicity.Results indicated that the constructed PCV2 multi-epitope had good expression characteristics in vitro,and the expression protein had good immunogenicity.The study provided a basic for the study on PCV2 epitope screening,functional identification and multi-epitope vaccine.

Establishment on Genotype Identification PCR Detection of Porcine Circovirus Type 2

ORF2 gene fragment was amplified from Porcine circovirus type 2（ PCV2） Shandong isolates by PCR,homology analysis and genotype identification were conducted on PCV2 ORF2 gene combining with the sequences listed in Gen Bank. The dominating popular genotype of PCV2 in Shandong Province was developed and the gene genetic variation characteristics of PCV2 was discussed. Specific PCR primers were designed and synthesized according to the differences among PCV2 genetypes,PCV2 gene identification PCR detection was established,and feasibility of the method was evaluated from the respects of sensitivity,specificity and repeatability,etc A total of 28 PCV2 ORF2 complete genes were obtained including 4 PCV2 a isolates,21 PCV2 b isolates and 3 PCV2 d isolates,indicating that the dominating popular genotype of PCV2 in Shandong Province was PCV2 b. Analysis on gene homology showed that homology of isolates in different regions from2000 to 2012 was 90. 5%- 99. 8%,amino acid sites sequences existed in different genotypes of PCV2 were different. Fragments amplified by the established PCR method were 341 bp and 177 bp,the lowest content of DNA could be detected was 5 ng / μL. Specific test results showed that the DNA amplifications on PCV2 a and PCV2 b were both positive,while amplifications on Porcine circovirus virus 1（ PCV1）,Porcine reproductive and respiratory syndrome virus（ PRRSV）,Pseudorabies virus（ PRV）,Porcine parvovirus（ PPV）,Classical swine fever virus（ CSFV）,Japanese encephalitis virus（ JEV） and Escherichia coli（ E. coli） were all negative,all the tests were conducted for 10 times,and the results were consistent. Test results indicated that the established PCR method had good specificity and repeatability,which could be applied to the identification of PCV2 genotypes.

Diagnosis and Treatment of Rex Rabbit Infectious Coryza and Isolation of the Pathogen

Rex rabbit Bordetella bronchiseptica was isolated from a large rabbit field in Shandong Province. And which could be diagnosed to be Rex rabbit infectious coryza combining with the incidence,clinical symptoms,pathological changes and laboratory examination. Kanamycin soluble powder was used to treat the disease according to the antibiotics usage and course,and the treatment was proved to be effective.

Prokaryotic Expression and Immunogenicity of Dominant Epitope Region of Goose Parvovirus Structural Protein VP3

[Objective] The paper was to develop a subunit vaccine candidate for prevention and control of goose parvovirus infection. [Method]Based on the prokaryotic expression system, the antigenic epitopes and locations of the structural protein VP3 were predicted by software analysis,and the region displaying a large portion of antigenic epitopes was amplified by PCR. The target VP3 DNA fragment was inserted into pET-30 a-VP3 vector, was transformed into Escherichia coli BL21 competent cells for protein expression and animal test. The SPF chickens were immunized with the recombinant protein and the antisera were collected for neutralization test by using a goose embryo fibroblast. [Result] The recombinant plasmid was constructed, and the target region of VP3 protein was expressed efficiently in a soluble form. The neutralizing titers of antisera could reach up to-2.608. [Conclusion] The target region displaying a large portion of antigenic epitopes of the structural protein VP3 could be expressed efficiently in soluble form, and the expressed protein could induce neutralizing antibodies in SFP chicken.