Prevalence of Theileria sergenti infection in Korean native cattle by polymerase chain reaction

This study was performed to investigate the prevalence of theileriosis and to compare the prevalence of this disease in Korean native cattle reared under different environmental conditions, namely, in a grazing area and a non-grazing area by polymerase chain reaction. Three hundred and one Korean native cattle (276 cows and 25 bulls) that had not received prior treatment or been vaccinated to prevent theileriosis were examined by PCR for Theileria sergenti infection from 2001 to 2002. In our study, the parasitemia range in T. sergenti-positive cattle by microscopy were from 0.1 to 3% (mean 0.8%). In terms of mean prevalence, 204 of the 301 Korean native cattle (67.8%) were positive reaction by PCR. Our results also revealed that the infection rate among cows (70.3%) was significantly higher than that among bulls (40.0%) (p < 0.01). T. sergenti infection among the over 3 year-old-group (75%) had a significant higher prevalence than that among the less than 3 year-old-group (61.8%) (p < 0.05). Our data also showed that grazing areas (76.1%) had the significant higher prevalence than non-grazing areas (51%) (p < 0.001). In conclusion, this study demonstrates that the prevalence of T. sergenti infection is high and that its prevalence in grazing cattle is higher than that in non-grazing cattle. Therefore, life-long treatment and the development of an optimal vaccine are needed to reduce the numbers of bovine theileriosis in both grazing and non-grazing areas.     

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3' end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.     

Molecular cloning and characterisation of 23-kDa piroplasm surface proteins of Theileria sergenti and Theileria buffeli.

The cDNA encoding a 23-kDa piroplasm membrane protein (p23) of Theileria sergenti Chitose (C)-type was isolated and its nucleotide sequence was determined. The gene encodes a polypeptide of 223 aa with a 28 residue N-terminal signal sequence and a hydrophobic, valine-rich, C-terminal transmembrane domain, as deduced from its nucleotide sequence. Southern blot hybridisation analysis proved that p23 gene was a single copy gene and had allelic forms of the gene in the parasite population. By PCR, the open reading frames of T. sergenti Ikeda (I)-type and Theileria buffeli (B)-type p23 were amplified from genomic DNA and their nucleotide sequences were also determined. Comparison of C-type sequence with that of I-type and B-type revealed 90.5% and 93.5% sequence similarity, respectively, at the aa level. These results suggest that a conserved molecule in these benign Theileria spp. could be a candidate antigen for the development of an anti-piroplasm vaccine.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide probes

A rapid procedure for determining apolipoprotein E genotype from genomic DNA has been developed. In this procedure, DNA is amplified by the polymerase chain reaction, and allele-specific oligonucleotide probes are used to detect the cysteine-arginine interchanges at residues 112 and 158 that distinguish the three common isoforms of apolipoprotein E. The method was tested with 68 subjects, representing the six common phenotypes, and yielded results consistent with the known phenotype.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.     

Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.     

Detection of the Bcl I polymorphism of the glucocorticoid receptor gene by single-tube allele-specific polymerase chain reaction

The Bcl I polymorphism of the glucocorticoid receptor gene, recently identified as an intronic C to G change 646 nucleotides downstream of exon 2, has been associated with increased sensitivity to glucocorticoids and its potential relevance in metabolic disturbances and in various disorders has been extensively investigated. In the present study, we designed a single-tube allele-specific polymerase chain reaction for genotyping this polymorphism in peripheral blood DNA samples. When the Bcl I polymorphism was detected with this novel method in a cohort of 247 healthy subjects, the observed genotype distribution matched the Hardy–Weinberg equilibrium (100 subjects homozygous for the wild-type, 124 heterozygous and 23 homozygous for the mutant allele). In 50 randomly selected subjects the Bcl I polymorphism was also determined using a traditional restriction fragment length polymorphism technique and DNA sequencing, and the results showed 100% coincidence with those obtained by our novel method. The method proved to be more rapid and less labour-intensive compared to currently used techniques, and it avoided the use of extensive instrumentals. We assume that this novel method may have a broad utility in clinical and molecular epidemiological studies aimed to elucidate the impact of the Bcl I polymorphism of the glucocorticoid receptor gene either on metabolic disturbances, or various disorders, including cancer treatment and hormone substitution therapies.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.     

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.     

Sex determination using the polymerase chain reaction

A practical class experiment on the PCR is described which has been used over several years as part of an undergraduate biochemistry and molecular biology course for science students. A major aim is to provide experience in the use of the polymerase chain reaction (PCR) and its interpretation. Students are given small coded DNA samples and use the PCR reaction to determine whether the sample is from a male or a female.     

Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene

AIMS: This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus. METHODS AND RESULTS: A primer set (CbsA2F-CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI-Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1-A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F-CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI-Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq. CONCLUSIONS: A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.     

Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene

The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.     

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction

Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.