Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).     

Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains.

2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain.

3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity.

4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains.

5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.     

Detection of infectious laryngotracheitis virus in chickens using a non-radioactive DNA probe.

A DNA hybridization assay using a non-radioactive probe has been developed for the detection of infectious laryngotracheitis virus (ILTV) DNA. A 1.4-kilobase pair BamHI fragment of ILTV genomic DNA was cloned and then labeled by one of two methods; nick translation using 32P-dATP or non-radioactive labeling using a commercially available DNA labeling and detection kit. The non-radioactive DNA labeling method proved to be as sensitive as the radioactive method. Using the non-radioactive probe, ILTV DNA was readily detected in tracheal samples from acutely infected chickens and also from convalescent chickens at a time when viral antigen could no longer be detected by the enzyme-linked immunosorbent assay or the virus could no longer be reisolated. This technique provides a safe and effective means of identifying field outbreaks of ILTV and also may detect latent ILTV infections relatively quickly and inexpensively.     

Comparison of a vaccine strain and field isolates of avian reovirus by T1-oligonucleotide mapping.

Total RNA of eight avian reovirus isolates and the S1133 strain were compared by RNase T1-oligonucleotide mapping. The viruses were propagated in Vero cell cultures, and viral genomes were extracted from purified virions for comparison. Pairwise comparisons of the oligonucleotide maps showed genetic variation among reovirus isolates ranging from 78% to 99%. The T1 fingerprints of the RNA of isolates 1103, 724, 615, and 684 differed slightly from the standard S1133 strain, suggesting that the vaccine strain might have changed and became part of the circulating reoviruses. In contrast, when compared with the vaccine strain, isolates 902, 644, and 6207 showed greater differences in the fingerprint pattern. This genomic diversity may be due to the differences in immunological status of the affected avian population and/or due to simultaneous coinfection with different reovirus strains.     

DNA fingerprinting for differentiation of field isolates from reference vaccine strains of Pasteurella multocida in turkeys

The genomes from field isolates of Pasteurella multocida in turkeys and those of P multocida reference CU and M9 vaccine strains were analyzed and compared after cleavage with restriction endonucleases. The electrophoretic profiles obtained with DNA fragments from field isolates and vaccine strains of the same serotype were characteristic and reproducible. These features indicated the existence of differences among the isolates of the same serotype that cannot currently be detected, using available serotyping methods. However, several field isolates had electrophoretic profiles similar to those of either CU or M9 vaccine strain. It was concluded that restriction endonuclease analysis of DNA genomes from P multocida isolated from turkeys provides the information for differentiation of field isolates from vaccine strains of the same serotype.     

Differentiation between vaccine strain and field isolates of classical swine fever virus using polymerase chain reaction and restriction test.

The CS vaccine strain of Classical Swine Fever Virus is a derivative from the LK parental strain that has been used in Russia for more than 30 years. A 10697 nucleotide fragment of the CS strain's genome has been sequenced. Sixteen unique restriction markers have been found in the CS genome comparing to the following strains: Alfort187, Alfort Tubingen, Brescia, CAP, Glentorf, ALD, GPE-, Chinese, C-strain, Riems, P97. Fourteen of these sites (AflII, AvaI, CfoI, Eco47II, HaeII, KpnI, MunI, NspI, PstI, ScaI, SmaI, SpeI, StyI, VspI) are only present in the CS strain genome. The 2 sites (BgII, NdeI) are present in all other genomes except for the genome of vaccine strain. A PCR/restriction test has been developed based on these findings in order to distinguish the vaccine strain from field isolates. Two pairs of nested primers and a criteria of analysis have been designed for each restriction marker site. The tests have been conducted first on the reference strains resulting in predicted restriction patterns. Finally, the tests have been applied to a number of field isolates obtained at different locations in Russia in different years. These results give further evidence that PCR/restriction tests can identify the LK and CS vaccines helping to avoid confusion with field strains.     

Tissue-print hybridization using a non-radioactive probe for the detection of infectious bursal disease virus.

Tissue-print hybridization was evaluated as a simplified means for detection of infectious bursal disease virus (IBDV) in the bursa of Fabricius from infected chickens. The assay employed a biotin-labeled synthetic oligonucleotide as a probe. The bound probe was detected using a color assay consisting of streptavidin conjugated to alkaline phosphatase. Bursae were imprinted onto nitrocellulose and then hybridized with the biotinylated probe. Bursal prints from IBDV-infected chickens were readily distinguished from control prints by color development and differences in signal intensity.     

Evaluation of the non-temperature-sensitive field clonal isolates of the Mycoplasma synoviae vaccine strain MS-H

The live attenuated temperature-sensitive (ts+) Mycoplasma synovia (MS) strain, MS-H, is used as a vaccine in a number of countries to control virulent MS infection in commercial chicken flocks. Nine out of 50 isolates made from flocks vaccinated with MS-H were found to have lost the ts+ phenotype of the original vaccine strain. In order to examine the influence of the ts- phenotype on virulence of the isolates, four of the ts- isolates, the MS-H vaccine, and the vaccine parent strain 86079/7NS were administered by aerosol in conjunction with infectious bronchitis virus to 3-wk-old specific-pathogen-free chickens. The four ts- clones induced only minimal air sac lesions that were not different in severity from those caused by MS-H vaccine; however, the vaccine parent strain 86079/7NS caused air sac lesions that were significantly greater than those of MS-H and all ts- clones. The vaccine parent strain 86079/7NS and two of the ts- clones were recovered from the air sacs of the respectively infected chickens whereas the MS-H vaccine and two other ts- clones were not. Three of the ts- isolates caused increased tracheal mucosal thicknesses that were significantly greater than those from birds inoculated with MS-H, and one caused increased tracheal mucosal thicknesses that were significantly less than those from birds inoculated with 86079/7NS. In conclusion, unlike the MS-H vaccine, the MS-H ts- clones were associated with minor changes in tracheal mucosa; however, unlike the vaccine parent strain, they did not induce lesions in the air sacs. These results suggest that factors other than ts+ phenotype are involved in the attenuation of the MS-H vaccine.     

Differentiation of field isolates of Pasteurella multocida serotype 3,4 from live vaccine strain by genotypic characterization

Fifty-five serotype 3,4 isolates of Pasteurella multocida, isolated from turkeys dead from fowl cholera, were characterized (fingerprinted) genotypically for comparison with the serotype 3,4 live fowl cholera vaccine principally used in turkeys in California. Twenty-three isolates were obtained from turkeys vaccinated with the M9 live vaccine, and 32 additional isolates were from turkeys not vaccinated for fowl cholera. Methods of characterization included restriction endonuclease analysis of chromosomal DNA and ribotyping, a technique for highlighting restriction site heterogeneity of highly conserved ribosomal RNA genes and associated sequences using a radiolabeled rRNA probe. Eight different genotypes or ribotypes were detected in these isolates by the above methods. Of 23 isolates from M9-vaccinated turkeys flocks, 19 were the same ribotype as M9. Thirty of 32 isolates recovered from unvaccinated turkeys were different ribotypes from M9. The remaining two isolates resembled M9 and were recovered from two different flocks placed in succession on a turkey farm where a flock placed previously had been vaccinated with M9, suggesting interflock transmission. Ribotyping and restriction endonuclease analysis appear to be useful tools to aid in the determination of the role that the live vaccine plays in fowl cholera epidemiology.     

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele’s clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.     

Comparison of genome segments 2, 7 and 10 of bluetongue viruses serotype 2 for differentiation between field isolates and the vaccine strain

Bluetongue (BT) virus serotype 2 (BTV 2) was first confirmed in Tunisia in February 2000 and has since spread northward and westward, infecting several other countries and islands, including Corsica, where clinical disease was reported in October 2000. BT was again reported on the Island in July 2001, some six months after a vaccination campaign against BTV 2. The molecular relationship between isolates of the BTV 2 Corsican wild-type viruses from 2000 and 2001, and the attenuated BTV 2 vaccine were determined by comparing corresponding sequences of genome segments 2, 7 and 10 with each other and with already published sequences available in the genome database. Complete genetic stability was observed between the isolates of the Corsican BTV 2. There was some divergence between the nucleotide sequences of segment 10 obtained from the wild-type and vaccine virus strains. Based on these differences, primers were selected that could be used in RT-PCR to differentiate between the wild-type and the vaccine viruses.     

Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.     

Identification of Trypanosoma evansi by DNA hybridisation using a non-radioactive probe generated by arbitrary primer PCR: short communication

A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer-polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes.     

Differentiation of Erysipelothrix rhusiopathiae strains by nucleotide sequence analysis of a hypervariable region in the spaA gene: discrimination of a live vaccine strain from field isolates.

Erysipelothrix rhusiopathiae causes erysipelas in swine and is considered a reemerging disease contributing substantially to economic losses in the swine industry. Since an attenuated live vaccine was commercialized in 1974 in Japan, outbreaks of acute septicemia or subacute urticaria of erysipelas have decreased dramatically. In contrast, a chronic form of erysipelas found during meat inspections in slaughterhouses has been increasing. In this study, a new strain-typing method was developed based on nucleotide sequencing of a hypervariable region in the surface protective antigen (spaA) gene for discrimination of the live vaccine strain from field isolates. Sixteen strains isolated from arthritic lesions found in slaughtered pigs were segregated into 4 major patterns: 1) identical nucleotide sequence with the vaccine strain: 3 isolates; 2) 1 nucleotide substitution (C to A) at position 555: 5 isolates; 3) 1 nucleotide substitution at various positions: 5 isolates; and 4) 2 nucleotide substitutions: 3 isolates. Isolates with the same nucleotide sequence as the vaccine strain were further characterized by other properties, including the mouse pathogenicity test. One strain isolated from pigs on a farm where the live vaccine had been used was found to be closely related to the vaccine strain. The phylogenetic tree constructed based on the spaA sequence suggests that the evolutionary distance of the isolates is related to the pathogenicity in mice. The new strain-typing system based on nucleotide sequencing of the spaA region is useful to discriminate the vaccine strain from field isolates.     

猪流行性腹泻病毒野毒株及弱毒疫苗株RT-PCR鉴别检测方法的建立及应用

为建立猪流行性腹泻病毒(PEDV)野毒株及弱毒疫苗株快速鉴别检测方法,根据Gen Bank中公布的PEDV野毒株及弱毒疫苗株的ORF3基因序列,在缺失区的两端设计合成1对特异性扩增引物,用以特异性的扩增PEDV ORF3基因片段,根据目的片段大小判断PEDV的毒株类型;通过退火温度等反应条件优化,建立了区分PEDV野毒株和弱毒疫苗株的RT-PCR鉴别检测方法。结果显示:所建立的RT-PCR鉴别检测方法能特异性区分PEDV野毒株和弱毒疫苗株;PEDV野毒株扩增出234 bp目的片段,PEDV弱毒疫苗株扩增出185 bp目的片段;与猪传染性胃肠炎病毒(TGEV)、A群猪轮状病毒(PRo VA)、猪嵴病毒(PKV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪乙型脑炎病毒(JEV)及猪细小病毒(PPV)均无交叉反应;敏感性试验显示,该方法能检测到病毒滴度为1.3×10~3TCID_(50)/mL。利用该方法对采集自广西部分地区93份临床腹泻样品进行检测,临床腹泻样品中PEDV野毒株阳性率为61.29%(57/93),弱毒株阳性率为5.38%(5/93)。结果表明:该RT-PCR鉴别检测方法特异性强、灵敏度高、操作简便,能快速、准确地区分PEDV自然感染野毒株和弱毒疫苗毒株,为猪流行性腹泻的快速诊断及病原分子鉴别检测研究提供了可供借鉴的技术手段。     

Antigenic heterogeneity among the field isolates of Newcastle disease virus (NDV) in relation to the vaccine strain: 1. Studies on viruses isolated from wild birds in Israel

In order to reveal the viruses strongly differing from the VH NDV strain used in Israel for poultry vaccination, 54 NDV strains isolated during the last 15 years in Israel from feral birds were studied by means of the panels of 39 monoclonal antibodies. Six isolates were found to have considerable antigenic differences in envelope proteins as compared to the vaccine strain. In four cases, the differences were related mostly to the hemagglutinin-neuraminidase glycoprotein, in one case to the fusion glycoprotein, and in one case to the matrix protein.