Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.     

PCR-based restriction fragment length polymorphism (RFLP) analysis of Campylobacter jejuni isolates from humans, chickens and dogs in northern Taiwan

Two hundred and twenty strains of Campylobacter jejuni (70 human, 51 canine and 99 chicken strains) were isolated from September 2003 to September 2004 in northern Taiwan. These strains were subtyped by PCR-RFLP analysis of the flagellin (FlaA) gene. On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With the combination of these three enzymes, 47 distinct PCR-RFLP patterns were observed-25 each from human and chicken isolates, and 9 from canine isolates. In human strains, the most frequently occurring types were Cj-28 (14.3%), Cj-17 (10%), Cj-16 (8.6%), Cj-37 (7.1%) and Cj-46 (7.1%). In canine strains, the most prevalent types were Cj-1 (33.3%), Cj-26 (19.6%), Cj-3 (15.7%), Cj-2 (9.8%) and Cj-10 (9.8%). In chicken strains, the most frequently occurring types were Cj-46 (40.4%), Cj-29 (9.1%), Cj-45 (7.1%) and Cj-41 (5.1%). The results suggest that poultry is a source, but not the sole source, of C. jejuni infection in humans. Two RFLP types, Cj-17 and Cj-37, frequently occurring in human isolates in this study have also been found to be prevalent in human isolates in Japan, China and the Czech Republic, indicating a possible international clonal spread.     

Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis

Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.     

16S-23S rRNA基因间隔序列PCR及RFLP对奇异变形杆菌分离株的鉴别与系统发育分析

根据临床常见致病菌16S-23S rRNA基因间隔序列(ISR)两端的16S及23S rRNA保守序列设计PCR扩增的通用引物,对9株奇异变形杆菌和6株相近菌株应用通用引物PCR扩增16S-23S rRNA ISR序列.通过PCR长度多态性比较、RFLP分析以及部分序列测序比较,分析鉴别奇异变形杆菌.结果显示,PCR长度多态性可以将奇异变形杆菌同其余菌种进行区分;RFLP分析可以将所有试验菌种进行区分;部分序列测序可以对奇异变形杆菌进行分型.由此表明,16S 23S rRNA ISR序列PCR及RFLP分析可以简单、快速、准确的鉴定奇异变形杆菌.     

Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism analysis. The method was used to compare ORF 1 sequences of two divergent European-type PRRSV strains. Our results indicated that the structural and replicase parts of these two strains had evolved at overall similar rates.     

Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates.     

Identification of trichomonadid protozoa from the bovine preputial cavity by polymerase chain reaction and restriction fragment length polymorphism typing.

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.85 rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.85 rRNA gene and ITSR sequence resultsand PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.     

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.     

Somatic cell mapping and restriction fragment length polymorphism analysis of bovine insulin-like growth factor I.

The DNA isolated from cow-hamster hybrid somatic cells segregating bovine chromosomes was analyzed by Southern blotting and hybridization with a heterologous [32P]-labeled porcine cDNA probe encoding insulin-like growth factor I (IGF-I). Thirteen of 25 cow-hamster hybrid cell lines exhibited the bovine-specific IGF-I fragment. Analysis for the retention or loss of bovine IGF-I with markers previously screened against the same panel of hybrid cells revealed a 100% concordance with lactate dehydrogenase B of bovine syntenic group U3 located on bovine chromosome 5. Restriction fragment length analyses of genomic DNA from animals representing five breeds (Angus, Polled Hereford, Simmental, Gelbvieh, and Belgian Blue) and from seven half-sib Angus calves indicated that polymorphisms for the genomic composition of the bovine IGF-I gene may exist in cattle populations.     

Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus

OBJECTIVE: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs. SAMPLE POPULATION: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV. PROCEDURE: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined. RESULTS: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs. CONCLUSIONS AND CLINICAL RELEVANCE: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.     

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.     

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.     

Differentiation of twelve Actinobacillus pleuropneumoniae serotypes by outer membrane lipoprotein gene-based restriction fragment length polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950-base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR-based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR-based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.     

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.     

Differentiation by polymerase chain reaction - restriction fragment length polymorphism of some Oestridae larvae causing myiasis

The cytochrome oxidase I (COI) gene of the most wide-spread Italian species of Oestridae larvae causing myiasis (Gasterophilus spp., Hypoderma bovis, Hypoderma lineatum, Oestrus ovis and Przhevalskiana silenus) was amplified by polymerase chain reaction (PCR) using conserved primers. Restriction fragment length polymorphism (RFLP) of amplicons was also carried out and their restriction profiles compared. A clear genetic difference between the Oestridae larvae examined was demonstrated by using Taq I, Hinf I, Rsa I and Hpa II enzymes. No intra-specific variation in RFLPs was detected between the two species of Hypoderma. The results highlight the taxonomic and phylogenetic relationships among larvae belonging to the different subfamilies, and thus offer additional diagnostic and epidemiological instruments.     

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis

Infectious laryngotracheitis is a dramatic disease of the upper respiratory tract in poultry caused by a herpesvirus. In this study we investigated the characteristics of western European field isolates of infectious laryngotracheitis virus (ILTV) to gain more information on their diversity. The examined 104 isolates, collected from acute outbreaks during the last 35 years, originated from eight different countries: Switzerland (48), Germany (21), Sweden (14), the United Kingdom (9), Italy (5), Belgium (4), Austria (2), and Norway (1). Two vaccines, a chicken embryo origin product and a tissue culture origin product, were included in the survey. Polymerase chain reaction (PCR) was performed to amplify a 2.1-kb DNA fragment of ILTV using primers generated for the thymidine kinase (TK) gene. After digestion of the resulting PCR products by restriction endonuclease HaeIII, restriction fragment length polymorphism analysis was carried out. PCR amplicons of three field isolates and both vaccine strains were selected for sequencing. Here 98 field isolates showed the same cleavage pattern and were identical to both vaccine strains (clone 1). They differed from five Swiss isolates with identical cleavage pattern (clone 2) and one Swedish isolate (clone 3). The present study demonstrated that at least three clones of ILTV have been circulating in western Europe during the last 35 years. The 104 isolates analyzed showed a high genetic similarity regarding the TK gene, and a large majority of the field isolates (98/104) were genetically related to the vaccine strains.     

Genotyping of Mycobacterium bovis isolates from badgers in four areas of the Republic of Ireland by restriction fragment length polymorphism analysis

An analysis of the molecular epidemiology of Mycobacterium bovis in badgers was made in four selected areas of the Republic of Ireland in which an intensive badger removal programme was being carried out over a period of five years. Tissue samples from 2310 badgers were cultured. Restriction fragment length polymorphism (RFLP) analysis with IS6110, polymorphic GC-rich sequence (PGRS) and direct repeat sequence (DR) probes was applied to the isolates from 398 badgers, and 52 different rflp types were identified. Most of the isolates belonged to seven predominant types, and the other 45 types were represented by few isolates. An analysis suggests that some of these 45 types may have been introduced by the inward migration of badgers and others may have been the result of genetic changes to one of the prevalent types. The badgers were divided into groups on the basis of the sett at which they were captured, and RFLP typing was applied to isolates from two or more badgers from 85 groups. Multiple RFLP types were identified among isolates from 50 of these groups, suggesting that badgers probably moved frequently between group territories.