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.     

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.     

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.     

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.     

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 infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)

Infectious laryngotracheitis (ILT) is a highly contagious, acute respiratory disease of chickens, of worldwide distribution, that affects growth and egg production and leads to significant economic losses during periodic outbreaks of the disease. Live attenuated vaccines (chicken embryo origin [CEO] and tissue-culture origin [TCO]) have been widely used to control the disease in the United States. It is believed that most of the outbreaks in the United States are caused by vaccine-related isolates that persist in the field and spill over into na?ve poultry populations. The objective of this study was to utilize the previously developed polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis to genotype recent ILT virus (ILTV) isolates from commercial poultry. Forty-six samples were collected during January 2006 to April 2007 from five poultry production regions of the United States and were characterized within PCR-RFLP groups III-VI. Sixty-three percent of the samples analyzed were categorized as closely related to the vaccine strains (groups III-V), whereas 33% were categorized as group VI viruses that differed in six and nine PCR-RFLP patterns from the CEO and TCO vaccines; a mixture of group IV and V viruses was detected in two samples (4%). In general, groups V and VI were the most prevalent viruses, found in 52% and 33% of the samples tested respectively. Both types of viruses were detected in vaccinated and nonvaccinated flocks. Although genetically different, both viruses produced severe disease in the field.     

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cvarepsilon gene

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Cvarepsilon sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.     

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

Efficacy of live virus vaccines against infectious laryngotracheitis assessed by polymerase chain reaction-restriction fragment length polymorphism

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.     

A restriction fragment length polymorphism-based polymerase chain reaction as an alternative to serotyping for identifying Salmonella serotypes

The phase 1 (fliC) and phase 2 (fljB) Salmonella flagella genes were analyzed by restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR) to aid in the identification of different Salmonella serotypes. Twenty-four phase 1 flagellin and eight phase 2 flagellin genes could be differentiated among each other with restriction endonucleases Sau3A and HhaI in RFLP-PCR analysis. These flagellin genes comprise the major antigenic formulas for 52 serotypes of Salmonella sp., which include the common serotypes found in poultry and other important food animal species. With the knowledge of the O antigen composition determined from conventional O serotyping, 90% of the Salmonella serotypes could be identified by this double restriction enzyme RFLP analysis of fliC and fljB genes. This RFLP-PCR flagellar typing scheme was successfully applied to the identification of serotype for 112 Salmonella isolates obtained from poultry environment. There was a significant correlation between RFLP-PCR and conventional serotyping (chi-square, P < 0.001). Overall, PCR-RFLP proved to be a fast, accurate, and economical alternative approach to serotyping Salmonella sp.     

Molecular characterization of Streptococcus suis strains by 16S–23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.     

Bovine leukemia virus genotype surveillance in cattle at a slaughterhouse in Aichi Prefecture,Japan, in 2019 using polymerase chain reaction combined with restriction fragment length polymorphism

Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) is commonly used for genotyping bovine leukemia virus (BLV) in slaughterhouses. However, unclassified BLV genotypes have been sporadically reported. To assess the current status of BLV genetic characterization in cattle, PCR-RFLP was performed on blood samples of 170 cattle (84 Japanese Black, 60 Japanese Black x Holstein, and 26 Holstein) from 17 farms (5 prefectures) at a slaughterhouse in Aichi Prefecture in 2019. A total of 65 samples (38.2%) were BLV positive, and genotype 1 was the most predominant (56/65 samples), followed by genotypes 3 (6 samples) and 5 (1 sample), and two unclassified samples. No relationship between the genotypes and breeds was observed. Sequence and phylogenetic analyses demonstrated that unclassified BLV genotypes clustered with genotype 1 sequences were, therefore, not new genotypes.     

Polymerase chain reaction-based restriction fragment length polymorphism pattern of porcine reproductive and respiratory syndrome virus directly from lung tissues without virus isolation in Korea.

Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis was developed for directly typing porcine reproductive and respiratory syndrome virus (PRRSV) from lung specimens without virus isolation. Twenty nine lung specimens collected from postweaning pigs were isolated for PRRSV. When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig. The results suggest that the PCR-based RFLP analysis method may be useful to distinguish PRRSV isolates directly from lung specimens without virus isolation.     

Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan

The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.     

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.