Nucleotide Sequences Of PCR Products Research Articles

First Report of Alternaria tenuissima Causing Brown Spot Disease of Angelica dahurica in China.

Angelica dahurica (Fisch. ex Hoffm.) is an abundantly cultivated Chinese herbal medicine plant in China with about 4000 hectares grown, the annual production is up to 24,000 tons. The medicinal part of A. dahurica is its root, and mainly function for treat cold, headache, toothache, rhinitis, diabetes, etc. Besides, A. dahurica is also used as a spice in Asia. In September 2018, brown spot was observed on the leaves of A. dahurica in fields of Anguo City, Hebei Province, China. In the field investigated, the incidence of brown spot disease reached 15%. The infected leaves showed brown spots surrounded with pale yellow edge, resulting in withered of the whole leaf. It seriously endangers the growth of A. dahurica, reducing the yield and quality of medicinal materials, even leading to the death of plants. We isolated the pathogen from 10 leaves with same lesions, the small square leaf pieces of approximately 3 to 5 mm were obtained with the sterile scissors from the junction of infected and healthy tissues, sterilized with sodium hypochlorite (10%) for 1 min followed by washing in sterile water for 3 times, then incubated on potato dextrose agar (PDA) plates at 25°C for 4 days. The culture was transferred to new PDA plates and was cultivated in dark at 25°C for 10 days. A total of 3 species of fungi were isolated, and only one fungus species has been found to be able to cause the original pathological characteristics of A. dahurica leaves through the back-grafting experiment. The mycelium was black and began to sporulate after 8 days on PDA media by single spore separation. Multiple spores joined together to form spores chain. The spores were spindle-shaped, yellow to yellow brown, and size ranged from 45 to 55 × 15 to 20 µm (n=50), with zero to three longitudinal septa and one to five transverse septa. For pathogenicity tests, the spore suspension (3.5×105 spores/mL) were inoculated to healthy plants grown in experimental field, the test was repeated four times, and 10 leaves were inoculated in each repetition, and the sterile water was inoculated as the blank control. Inoculated leaves were covered with transparent plastic bags for 24 h to keep humidity. Nine days later, it was found that there were lesions on the leaves inoculated with the pathogen, and the traits were the same as those in the field, while the controls are healthy. The fungus was consistently isolated from the inoculated leaves. The similar isolates were re-isolated from the inoculated and infected leaves and identified as Alternaria tenuissima by DNA sequencing, fulfilling Koch's postulates. Fungal genomic DNA was extracted from 7-day-old culture. PCR amplifications were performed using primers ITS1 / ITS4 and TEFF / TEFR respectively (Takahashi et al. 2006, Du 2008). The nucleotide sequence of PCR products, which have been deposited in Genebank under the accession numbers MN153514 and MN735428, showed 99.8%-100% identity with the corresponding sequences of A. tenuissima (MW194297 and MK415954). In order to further identify the pathogen species, we constructed a phylogenetic tree by combining TEF sequence and ITS sequence to distinguish the relationship between the pathogen and other minor species in the genus Alternaria, the isolate was clustered in the Alternaria clade. Therefore, the pathogen was identified as A. tenuissima based on the morphological characteristics and molecular identification. To our knowledge, this is the first report of A. tenuissima causing leaf spot on A. dahurica in China.

Genetic diversity of tomato yellow leaf curl virus isolates and the effect of virus on the hormones content of tomato (Solanum lycopersicum) plants

Tomato yellow leaf curl virus (TYLCV) is one of the most economically important plant viruses globally and due to its continuing quick spread, now it afflicts many tomato growing countries in the Mediterranean basin, Africa, southern Asia, as well as South, Central and North America. Infection of tomato plants with TYLCV can reach 100% causing a severe reduction in tomato production, especially when plants are infected in the early stage of growth. Therefore, this study was conducted, during the growing season 2018-19 at the college of Agriculture, Kerbala University, Iraq, to isolate and diagnose three different isolates of TYLCV, infecting tomato, using the polymerase chain reaction technology (PCR) and determining the nucleotide sequences of PCR- amplified products and the effect of TYLCV infection on the plant contents of some hormones (gibberellin, cytokinin, and Indole acetic acid). Results of PCR amplification showed the possibility of amplifying a 789bp PCR product from each virus isolate (1, 2, 3, and 4). Analysis of the nucleotide sequences of PCR products using BLAST showed that all the diagnosed isolates belong to TYLCV. By comparing the sequences of nucleotide sequences, there was a genetic similarity of 100% among these isolates. However, all tomato genotypes tested in this study were found to be susceptible to TYLCV with different degrees of infection. TYLCV infection also had a significant effect in reducing the level of the hormone gibberellin in the infected genotypes. Increased levels of the cytokinin and indole acetic acid hormones were also observed in the TYLCV-infected genotypes.

Characterization of PABPN1 expansion mutations in a large cohort of Mexican patients with oculopharyngeal muscular dystrophy (OPMD)

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal-dominant, adult-onset disorder defined by blepharoptosis, dysphagia, and proximal muscle weakness. OPMD arises from heterozygous expansions of a trinucleotide (GCN) tract situated at the...

Identification and characterization of two CD4 alleles in Microminipigs.

BackgroundWe previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.ResultsWe identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.ConclusionsWe identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0856-8) contains supplementary material, which is available to authorized users.

Diversity of MHC class I alleles in Spheniscus humboldti

The major histocompatibility complex locus (MHC) is a gene region related to immune response and exhibits a remarkably great diversity. We deduced that polymorphisms in MHC genes would help to solve several issues on penguins, including classification, phylogenetic relationship, and conservation. This study aimed to elucidate the structure and diversity of the so far unknown MHC class I gene in a penguin species. The structure of an MHC class I gene from the Humboldt penguin (Spheniscus humboldti) was determined by using an inverse PCR method. We designed PCR primers to directly determine nucleotide sequences of PCR products from the MHC class I gene and to obtain recombinant clones for investigating the diversity of the MHC class I gene in Humboldt penguins. A total of 24 MHC class I allele sequences were obtained from 40 individuals. Polymorphisms were mainly found in exons 2 and 3, as expected from the nature of MHC class I genes in vertebrate species including birds and mammals. Phylogenetic analyses of MHC class I alleles have revealed that the Humboldt penguin is closely related to the Red Knot (Calidris canutus) belonging to Charadriiformes.

Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand

BackgroundLeishmaniasis, caused by Leishmania siamensis, is an emerging disease in Thailand. Although reported cases have been increasing, epidemiological information of the disease including host and vector aspects is not clearly known. This study was a preliminary survey of the potential vector of L. siamensis in an affected area of leishmaniasis, Trang Province, southern Thailand.MethodsThe collection of sandflies was performed around the area where a case of leishmaniasis was reported using CDC light traps. Species of sandfly were identified based on morphological characteristics according to Lewis’s key. PCR amplification and sequencing of the heat shock protein 70 gene (hsp70) was used to identify L. siamensis DNA in sandflies.ResultsA total of 146 male and female sandflies were collected in the affected areas. Of 71 female sandflies, four species were identified, i.e., Sergentomyia (Neophlebotomus) gemmea, S. (Neophlebotomus) iyengari, S. (Parrotomyia) barraudi and Phlebotomus (Anaphlebotomus) stantoni. Among these species, S. (Neophlebotomus) gemmea was the most predominant species in all areas. DNA of L. siamensis was identified in S. (Neophlebotomus) gemmea. Nucleotide sequences of PCR products using DNA extracted from S. (Neophlebotomus) gemmea showed 99.8% identity to L. siamensis.ConclusionS. (Neophlebotomus) gemmea might be a potential vector of L. siamensis in an affected area, Trang Province, southern Thailand. However further studies are needed to prove whether these sandflies can be natural vectors of leishmaniasis.

Hepatitis G virus (HGV), its prevalence and genotypes in patients with hepatitis B & C in Ahvaz, Southwest Iran

The aim of this study was to determine the prevalence and genotype distribution of hepatitis G virus (HGV, or GB virus C) in patients with hepatitis B and C in Ahvaz, southeastern Iran. Materials and methods: A total of 100 patients were selected; 50 were positive for hepatitis C virus (HCV) and 50 for hepatitis B virus (HBV). HGV status was examined among the HCV- and HBV-positive patients. An enzyme-linked immunosorbent assay (ELISA) kit was used to detect anti-E2 antibody. Nested reverse transcription polymerase chain reaction (RT-PCR) was used for RNA detection from serum samples of chronic hepatitis patients. Sequencing was done to determine genotypes Results: Using the ELISA method, co-infection of HGV with HCV and HBV was determined at 6% and 8%, respectively. RT-PCR showed that co-infection of HGV with HCV and HBV was 22% and 10%, respectively. Based on nucleotide sequencing of PCR products, the predominant genotype of HGV among the samples was 2a. Conclusion: Our study showed that the co-infection rate of HGV in patients with hepatitis B and C was somewhat high and was higher in HCV- than in HBV-infected patients. As our findings and other reports from Iran and neighboring countries indicate, genotype 2 of HGV may be the most common genotype of HGV in Middle Eastern countries.

Detection of Dengue Virus Genome in Urine by Real-Time Reverse Transcriptase PCR: a Laboratory Diagnostic Method Useful after Disappearance of the Genome in Serum

The reemergence of dengue virus (DENV) infection has created a requirement for improved laboratory diagnostic procedures. In this study, DENV genome detection in urine was evaluated as a diagnostic method. The DENV genome was detected by real-time reverse transcriptase PCR (RT-PCR) in urine and serum of dengue patients. The detection rate of DENV genome in urine was 25% (2/8) on disease days 0 to 3 and 32% (7/22) on days 4 to 5. The rate was 50% or higher on days 6 to 16, 52% (11/21) on days 6 to 7, 78% (7/9) on days 8 to 9, 80% (4/5) on days 10 to 11, 50% (2/4) on days 12 to 13, and 60% (3/5) on days 14 to 16. The last positive urine sample was on day 16. The detection rates in serum were highest on days 0 to 3 and were greater than 50% on days 0 to 7. Detection rates decreased thereafter, and the last positive detection was on day 11. These results indicate that the time frames for positive detection differ between urine and serum samples, whereby detection rates of 50% or higher are evident between days 6 to 16 for urine samples and days 0 to 7 for serum samples. Nucleotide sequences of PCR products were identical between urine and serum samples. The detection of DENV genome in urine samples by real-time RT-PCR is useful to confirm DENV infection, particularly after viremia disappears.

Histiocytic sarcoma arising in indolent small B-cell lymphoma: report of two cases with molecular/genetic evidence suggestive of a ‘transdifferentiation’ during the clonal evolution

The biologic relationship between small B-cell lymphoma and histiocytic sarcoma (HS) when occurring in the same patient remains unclear, though recent data suggest a possible ‘transdifferentiation’ from follicular lymphoma (FL) to HS. We investigated the clonal relationship in two cases of small B-cell lymphoma with subsequent HS. Case 1: A 62-year-old female with splenic marginal zone lymphoma (SMZL) developed HS in a groin lymph node 1 year after the primary diagnosis. PCR/sequence analysis of the IGH gene showed a monoclonal rearrangement carrying an identical nucleotide sequence of PCR products from the spleen with SMZL and the lymph node with HS. Case 2: A 61-year-old female with a remote history of FL developed supraclavicular lymphadenopathy, which was confirmed to be HS. PCR analysis of the HS detected a monoclonal rearrangement of the IGH gene and FISH analysis revealed IGH/BCL2 fusion, a genetic hallmark for FL. The transformed HSs showed partial retention of their prior B-cell lymphomas' signatures, including expression of OCT2 in both cases and expression of BCL6 and enhanced expression of BCL2 in case 2. Both HSs demonstrated hypermutated IGH variable regions, arguing against a common progenitor mechanism of the transformation process. The data suggest a common clonal origin of B-cell lymphoma and subsequent HS occurring in the same patient, indicating that ‘transdifferentiation’ occurs in other small B-cell lymphomas, in addition to the previously reported FL or B-cell lymphoma with IGH/BCL2.

Safety and efficacy of vaccination of seronegative bulls with modified-live, cytopathic bovine viral diarrhea viruses

The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) risk of prolonged testicular infection; and (c) protection against subsequent testicular infection due to viral challenge. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with a standard dose of vaccine ( n = 11) or were maintained as unvaccinated controls ( n = 11). Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry, and the identity of viral strains was determined by nucleotide sequencing of PCR products. Vaccination of peri-pubertal bulls with this vaccine caused a short-term, transient shed of only the type 1a strain of modified-live, cytopathic BVDV in semen for up to 10 d after vaccination. The vaccine did not cause prolonged testicular infection. Vaccination with this product prevented development of prolonged testicular infections after subsequent exposure to a field strain of BVDV.

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.

The complete nucleotide sequence of a New World simian foamy virus

We determined the complete nucleotide sequence of the New World simian foamy virus (FV) from spider monkey (SFVspm). Starting from a conserved region in the integrase (IN) domain of the pol gene we cloned fragments of the genome up to the 5′ end of the long terminal repeat (LTR) into plasmid vectors and elucidated their nucleotide sequence. The 3′ end of the genome was determined by direct nucleotide sequencing of PCR products. Each nucleotide of the genome was determined at least two times from both strands. All protein motifs described to be conserved among primate FVs were found in SFVspm. At both the nucleotide and protein levels SFVspm is the most divergent primate FV described to date, reflecting the long-term phylogenetic separation between Old World and New World primate host species ( Catarrhini and Platyrrhini, respectively). The molecular probes developed for SFVspm will allow the investigation of trans-species transmissions of this New World foamy virus to humans by serological assays.

Nucleotide Sequence, Structural Investigation and Homology Modeling Studies of a Ca2+-independent α-amylase with Acidic pH-profile

The novel alpha-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of Ca(2+) and EDTA. Therefore, KRA acts as a Ca(2+)-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of Ca(2+) and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its T(m). The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis alpha-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the alpha-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

Small Anellovirus Infections in Korean Children

To the Editor: Recently, Jones et al. (1) identified circular DNA sequences, classified as Anellovirus genus, in plasma from patients with acute viral infection syndromes. These anelloviruses were then labeled as “small anellovirus (SAV)” because of their smaller genomes when compared with Torque Teno Virus (TTV) and Torque Teno Mini Virus (TTMV), which have small, circular, single-stranded DNA genomes. Although anelloviruses are not associated with any specific disease, TTV has been suggested to play a role in acute respiratory disease (ARD) and in asthma of children (2,3). Kawasaki disease and Henoch-Schonlein purpura are important vasculitis disorders in children, possibly triggered by unknown infectious agents. Recently, Gergely et al. (4) reported that molecular mimicry involving TTV and the generation of autoantibodies may have a role in the pathogenesis of systemic lupus erythematosus. The purpose of our study was to investigate the prevalence of SAV and its association with various clinical diseases in children. The study population comprised 81 serum samples from healthy children and 151 serum samples from children hospitalized with hepatitis (81 cases), ARD (40 cases), Kawasaki disease (12 cases), or Henoch-Schonlein purpura (18 cases) during the period January 2002–June 2006. Nasopharyngeal aspirates paired with serum samples were collected from 34 children with ARD, including upper respiratory tract infections, pneumonia, and acute bronchiolitis. Samples were collected after informed consent was obtained at admission from patients’ parents. PCRs for SAV were performed to amplify a 5′ noncoding region of SAV with specific primers, as described previously (5). PCR products were directly sequenced, and nucleotide sequences were registered in GenBank (accession nos. {type:entrez-nucleotide,attrs:{text:DQ978791,term_id:119656310,term_text:DQ978791}}DQ978791–DQ9788810). The χ2 test with Yates correction and Mann-Whitney U-test were used for statistical comparison by using MedCalc (MedCalc Software, Mariakerke, Belgium). A p value <0.05 was defined as statistically significant. In our study population, serum SAV DNA was detected in 28 (34.5%) of 81 children in the control group and in 66 (43.7%) of 151 children in the disease group. In the healthy control group, the SAV-positive rate was 7.4% (6/81) in children <12 months of age, 16.0% (13/81) in children 1–4 years of age, and 11.1% (9/81) in children 5–15 years of age. In the disease group, the SAV-positive rate was 35.8% (29/81) in patients with hepatitis, 67.5% (27/40) in ARD, 50% (6/12) in Kawasaki disease, and 22.2% (4/18) in Henoch-Schonlein purpura, respectively (Table). Among 34 nasopharyngeal aspirates collected from children with ARD, SAV DNA was detected in 19 (55.9%). Codetection of SAV and respiratory syncytial virus in nasopharyngeal aspirates was observed in 4 patients. Table Prevalence of SAV viremia in the study population, Republic of Korea, January 2002–June 2006* Percent similarity of nucleotide sequence of PCR products was 99% among SAV isolates. To our knowledge, this is the first report of SAV infections in children. The prevalence and role of SAV in clinical diseases have yet to be determined. Recently, Biagini et al. (5) reported that the prevalence of SAV infection was 20% (12/60) in French blood donors. In an Italian study (6), the positive rate of SAV DNA was 9.1% (5/55) in patients with hepatitis C compared with 8.6% (3/35) in healthy controls. Thus, the prevalence of SAV in Korean children is much higher than rates reported in adults from other countries (5,6). Further studies are needed to confirm this finding. In our study, the prevalence of SAV did not differ significantly between the hepatitis group and the healthy control group. Our results indicate that SAV presence does not appear to have a defining role in hepatitis, as do TTV or TTMV infection (7). In a previous study, several groups of viruses including TTV were ruled out as etiologic agents of Kawasaki disease (8), findings similar to those of our study. We found that the prevalence of SAV was significantly higher in ARD and that SAV-positive serum results were consistent with those of nasopharyngeal aspirates in 76% (26/34). These findings suggest that the respiratory tract may be a transmission route of SAV in children. In conclusion, we confirmed the presence of SAV in serum samples and nasopharyngeal aspirates from Korean children. A significantly higher detection of SAV DNA was observed in children with ARD compared with healthy children or children with other clinical diseases.

Analysis of a polymorphism within a TATA box of horse myostatin gene promoter in different breeds

Myostatin or growth and differentiation factor 8 (MSTN or GDF8, respectively) is a member of the transforming growth factor-β superfamily that acts as a negative regulator of skeletal muscle development and growth. The myostatin encoding gene (MSTN) and the 5’ regulative region in different species has been investigated. The MSTN gene consists of three exons and two intronic regions. In cattle, the gene has well-characterized mutations determining phenotypes with increased muscle mass. In a previous study, by alignment of nucleotide sequences of PCR products of horse MSTN gene, we identified some SNPs of which two T=C transitions in the promoter of the gene. The aim of this work is to study in different horse breeds the frequency of the polymorphism located 516 nt upstream of the ATG start codon. This SNP is within a TATA box sequence (NATAAAA, where N= T or C) and the comparative sequence analysis of mammals myostatin gene promoters reveals that this TATA-box motive is conserved in position and sequence among some species including cattle, sheep and goat. The point mutation is not recognised by restriction enzymes, then the genotyping was done by restriction site insertion-PCR. The primer pair was designed on the basis of the obtained nucleotide sequences. The reverse primer was modified and designed with a single nucleotide mismatched with respect to target sequence to introduces an SspI artificial restriction site when the T nucleotide occurred. The PCR products of 203 bp were digested and the resulting fragments were resolved on 3.5% agarose gel. Two hundred and six samples of DNA belonging to 18 horse breeds (Bardigiano, 20; Breton, 5; Criollo, 10; Esperia Pony, 4; Haflinger, 9; Italian Heavy Draught Horse, 24; Italian Saddle, 21; Italian Trotter, 16; Lipizzan, 11; Maremmano, 13; Murgese, 12; Norico, 10; Purebred Spanish Horse, 10; Salernitano, 12; San Fratellano, 3; Tolfetano, 7; Thoroughbred, 11 and Ventasso Horse, 8) were genotyped. The T allele occurs in all the breeds. The C allele, which eliminates TATA box motif, was detected in 11 breeds and its value frequency ranged from 0.006 (Thoroughbred) to 0.400 (Norico). The homozygous C/C genotype was detected in Haflinger, Bardigiano, Norico and Tolfetano breeds. The presence of the polymorphism within a conserved region of promoter of horse myostatin gene could affect expression gene. This marker could be used for association studies with performance traits in horses.

Noncytopathic bovine viral diarrhea virus can persist in testicular tissue after vaccination of peri-pubertal bulls but prevents subsequent infection

The objectives of this research were to evaluate the risk of prolonged testicular infection as a consequence of vaccination of peri-pubertal bulls with a modified-live, noncytopathic strain of BVDV and to assess vaccine efficacy in preventing prolonged testicular infections after a subsequent acute infection. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with an approximate minimum immunizing dose or a 10× standard dose of modified-live, noncytopathic BVDV or were maintained as unvaccinated controls. Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry and the identity of viral strains was determined by nucleotide sequencing of PCR products. The vaccine strain of BVDV was detected in testicular tissue of vaccinated bulls as long as 134 days after immunization. Prolonged testicular infections with the challenge strain were detected only in unvaccinated bulls as long as 85 days after challenge. Whereas vaccination caused prolonged testicular infection in some bulls, it did prevent subsequent infection of testicular tissue with the challenge strain. This research demonstrates that subcutaneous vaccination of naïve, peri-pubertal bulls with a noncytopathic, modified-live strain of BVDV can result in prolonged viral replication within testicular tissue. The risk for these prolonged testicular infections to cause venereal transmission of BVDV or subfertility is likely to be low but requires further investigation.

Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification

A total of 124 blood samples were collected from 92 sheep and 32 goats from 21 randomly selected herds located in two regions of Greece. Data on the characteristics of the animals (species, gender, age, tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. Nineteen animals (15%) produced the DNA fragment specific for Babesia of which 16 were sheep and three were goats. Nucleotide sequence of PCR products revealed 100% homology with Babesia ovis 18S rRNA gene. Nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%. The relative risk of the presence of ticks in sheep and goats ( p < 0.01) and farm dogs ( p < 0.01) for PCR-positive results for B. ovis in sheep and goats was found 6.63 and 4.14, respectively.

CAPS markers improved by cluster-specific amplification for identification of octoploid strawberry (Fragaria × ananassa Duch.) cultivars, and their disomic inheritance

Cleavage amplified polymorphic sequence (CAPS) markers of strawberry (Fragaria x ananassa Duch.) can be useful for identifying mislabeled or patent-infringing cultivars in the marketplace. However, CAPS markers in octoploid strawberry tend to give unclear bands because multiple homologous sites are simultaneously amplified by the non-selective PCR. To overcome this problem, we used "cluster-specific amplification" based on the nucleotide sequences of PCR products and were able to improve the band clarity of 18 CAPS markers. By analyzing the marker segregation ratio, we demonstrated that 13 clarified markers were derived from single diploid loci that were transmitted to progeny in a manner consistent with Mendelian inheritance. We discuss the genomic structure of octoploid strawberry from the viewpoint of cluster and segregation analysis and suggest that it comprises independent genomes. We tested the utility of all of the markers we developed for cultivar identification and confirmed their ability to distinguish among 64 strawberry cultivars.

Enterovirus related metabolic myopathy: a postviral fatigue syndrome

Objective: To detect and characterise enterovirus RNA in skeletal muscle from patients with chronic fatigue syndrome (CFS) and to compare efficiency of muscle energy metabolism in enterovirus positive and negative...

Change in Activity of Soybean Trypsin Inhibitor by Removal of C-terminal Amino Acid Residues during Seed Germination

Kunitz-type soybean trypsin inhibitor (KSTI) is a major factor lowering the nutritional quality of soybean proteins. KSTI might be degraded during protein processing. In the present work, we determined the effect of removal of C-terminal amino acid residues on the inhibitory activity of KSTI in the cotyledons during germination in Japanese soybean cultivars, Toyokomachi and Wasekogane. The type of KSTI in both cultivars was determined by analysis of the complete nucleotide sequences of PCR products amplified from genomic DNA and partial amino acid sequences of both cultivars. These KSTI were identical to Tib in Toyokomachi and Tia in Wasekogane. KSTI from cotyledons at 4 days after germination in both cultivars was separated clearly into two bands Tl and T2 in Toyokomachi and Wl and W2 in Wasekogane upon native-PAGE. Based on the C-terminal amino acid sequences, KSTIs with higher mobility (Tl, Wl) were found to lack 13 amino acid residues at the C-terminus. The KSTI with lower mobility (T2, W2) lacked 14 or 15 amino acid residues at the C-terminus. Further, KSTI with lower mobility showed high inhibitory activity compared with that of KSTI with higher mobility. Thus, the changes in the form of KSTI from Tl or Wl to T2 or W2 by removal of C-terminal amino acid residues during seed germination may change the structure at the active site and consequently increase the inhibitory activity of KSTI.