DNA Restriction Fragment Length Polymorphism Research Articles

Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Cutaneous and Visceral Leishmaniasis Caused by the Same Zymodeme of Leishmania donovani in Kerala, India.

The tribal population in and around the Western Ghats region of India is affected by both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) with typical clinical symptoms. In this study, we recorded and analyzed seven CL and three VL cases from this emerging belt. All the cases were found as autochthonous transmission. Multiple genetic markers (minicircle kinetoplast DNA polymerase chain reaction and restriction fragment length polymorphism of 3'untranslated region heat shock protein (HSP) 70, a larger segment of HSP 70, and 6-phosphogluconate dehydrogenase [PGDH] gene sequences) were used to identify and characterize the parasite. It was found that both clinical manifestations are caused by zymodeme MON-37 of Leishmania donovani. We have investigated the detailed entomological and epidemiological aspects of disease transmission. An abundant population of the proven vector Phlebotomus argentipes was observed in the study villages.

Antimicrobial growth promoters approved in food-producing animals in South Africa induce shiga toxin-converting bacteriophages from Escherichia coli O157:H7.

In this study, four antimicrobial growth promoters, including virginiamycin, josamycin, flavophospholipol, poly 2-propenal 2-propenoic acid and ultraviolet light, were tested for their capacity to induce stx-bacteriophages in 47 Shiga toxin-producing E. coli O157:H7 isolates. Induced bacteriophages were characterized for shiga toxin subtypes and structural genes by PCR, DNA restriction fragment length polymorphisms (RFLP) and morphological features by electron microscopy. Bacteriophages were induced from 72.3% (34/47) of the STEC O157:H7 isolates tested. Bacteriophage induction rates per induction method were as follows: ultraviolet light, 53.2% (25/47); poly 2-propenal 2-propenoic acid, 42.6% (20/47); virginiamycin, 34.0% (16/47); josamycin, 34.0% (16/47); and flavophospholipol, 29.8% (14/47). A total of 98 bacteriophages were isolated, but only 59 were digestible by NdeI, revealing 40 RFLP profiles which could be subdivided in 12 phylogenetic subgroups. Among the 98 bacteriophages, stx2a, stx2c and stx2d were present in 85.7%, 94.9% and 36.7% of bacteriophages, respectively. The Q, P, CIII, N1, N2 and IS1203 genes were found in 96.9%, 82.7%, 69.4%, 40.8%, 60.2% and 73.5% of the samples, respectively. Electron microscopy revealed four main representative morphologies which included three bacteriophages which all had long tails but different head morphologies: long hexagonal head, oval/oblong head and oval/circular head, and one bacteriophage with an icosahedral/hexagonal head with a short thick contractile tail. This study demonstrated that virginiamycin, josamycin, flavophospholipol and poly 2-propenal 2-propenoic acid induce genetically and morphologically diverse free stx-converting bacteriophages from STEC O157:H7. The possibility that these antimicrobial growth promoters may induce bacteriophages in vivo in animals and human hosts is a public health concern. Policies aimed at minimizing or banning the use of antimicrobial growth promoters should be promoted and implemented in countries where these compounds are still in use in animal agriculture.

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

New splice site mutations in MYO7A causing Usher syndrome type 1: a study on a Chinese consanguineous family.

This study investigated the new splice site mutations of Myosin VIIA (MYO7A) in patients with Usher syndrome type 1 (USH1) from a three-generation Chinese consanguineous family. All subjects underwent comprehensive ophthalmic examinations and an audiometric test. Demographic data, family history, and peripheral blood leukocytes were collected. We performed whole exome sequencing (WES) to analyze the genomic DNA of the family. DNA sequence and restriction fragment length polymorphism (RFLP) analyses were also done. The identified genetic variants were validated by conducting polymerase chain reaction (PCR) in 100 healthy control subjects and comparing with the NCBI VARIANT database and the 1000 Genomes Project. The functional consequences were further analyzed. WES identified two new splice site mutations (c.5648G > A(rs111033215) and c.6238-1G > C) in MYO7A in two patients with USH1, i.e., the proband and her elder brother. DNA sequence and RFLP analyses showed that other members without USH1 carried only one of the two mutations. In the analysis of healthy controls, neither mutation existed. Both mutations were predicted to be damaging and were most likely associated with USH1. In the three-generation Chinese consanguineous family with USH1, c.5648G > A(rs111033215) and c.6238-1G > C mutations in MYO7A are most likely associated with the disease. Our findings expand the mutational spectrum of MYO7A, which will enhance the understanding of the genetic abnormalities in USH1 and provide more evidence for future investigations on therapeutic strategies such as precise gene replacement or gene editing.

Survey of the association between polymorphisms of CTLA-4 exon 1 49 A/G genes with rheumatoid arthritis in Iran

ABSTRACT Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which suppresses T cell proliferation, is a promising candidate for the susceptibility genes to rheumatic arthritis diseases (RA). This study aims to examine the association between the polymorphisms of the CTLA-4 exon 1(+ 49) genes with RA in the Qazvin city of Iran population. The polymerase chain reaction of genomic DNA-restriction fragment length polymorphism (PCR-RFLP) was applied to genotype the CTLA-4 exon 1(+ 49) polymorphisms in 105 RA patients and 90 control subjects. Laboratory diagnostic tests were also measured for RA and control groups. Our results did not demonstrate a significant difference in allele and genotype frequencies of the CTLA-4 exon 1(+ 49) between RA patients and the control group (p < .0001). There was no significant difference in age at onset, CRP, RF value in patients with RA according to the CTLA-4 polymorphisms; just anti-CCP showed a significant difference. Our data declared that polymorphisms of CTLA-4 exon 1(+ 49) genes are not correlated with RA susceptibility and its clinical and paraclinical manifestations.

TCF7L2 (rs7903146) GENE POLYMORPHISM OF SELECTED HUMAN POPULATION RESIDENT WITHIN QUARRIES IN AKAMKPA LGA, CROSS RIVER STATE, NIGERIA

This work investigated the DNA concentration and TCF7L2 (rs7903146) gene polymorphism of selected human population resident within quarries in Akamkpa LGA, Cross River State, Nigeria. Forty (40) blood samples from volunteers aged 40 years and above were obtained from Old Netim, while 10 blood samples (control) were obtained from Oban Okoroba (20km from Old Netim) DNA extraction was done using Zymo DNA extraction kit, DNA concentration was determined using NanoDrop 1000 Spectrophotometer, and DNA amplification and Restriction Fragment Length Polymorphism (RFLP) was performed by Polymerase Chain Reaction (PCR) system. In the study group, males had a DNA concentration between 15.79 ng/µL and 39.99 ng/µL while females had a DNA concentration between 35.34 ng/ul and 43.15 ng/µL. DNA concentration was highest in 31 – 40 age group (43.15 ng/µL) and lowest in 51 – 60 age group (15.79 ng/µL). Residents had a rs7903146 TCF7L2 gene genotype of CC, signifying zero polymorphism and implying quarry activities had no deleterious impact on rs7903146 TCF7L2 gene. Thus, suggesting residents had low diabetic risk associated with SNP rs7903146 TCF7L2 gene.

Association of NFkB1 Gene Polymorphism with Inflammatory Markers in Patients of Type 2 Diabetes Mellitus with or without Renal Involvement in Eastern India

Aims: To evaluate the association of Nuclear factor kappa B1(NFkB1) gene polymorphism with inflammatory markers Urinary Monocyte Chemoattractant Protein 1 (UMCP1) and Tumor Necrosis Factor alfa (TNF alfa) in Patients of diabetes mellitus with or without renal involvement in Eastern India. Material and Methods: Consecutive Patients of Type 2 Diabetes Mellitus (DM) with or without microalbuminuria attending SCB MEDICAL COLLEGE and HOSPITAL Medical OPDs in between September 2018 to September 2019 were recruited in this study. Patients were subjected to blood and urine investigations. DNA extraction and Restriction fragment Length Polymorphism (RFLP) was done in Department of Biochemistry. Controls were unrelated healthy attendants with no history of Diabetes Mellitus, HTN, Chronic Kidney Disease (CKD). Results: Mean Systolic BP, Fasting Blood Glucose, Post Prandial Blood Glucose, HBA1c, Total Cholesterol were significantly higher in diabetes mellitus and diabetic nephropathy groups than control group. Estimated Glomerular Filtration Rate was significantly lower in diabetic nephropathy (p value < 0.001). UMCP1, Urinary Albumin Creatinine Ratio, TNF alfa were higher in diabetes mellitus and nephropathy with p value (<0.001, 0.006 < 0.001) respectively. In between DM and Diabetic Nephropathy groups nfkb1 gene expression, umcp1 and tnf alfa levels were significantly increased in Diabetic nephropathy with p value 0.019, <0.01, 0.001 respectively. Insertion/insertion NFkB1 gene polymorphisms were more in diabetic nephropathy group and were positively correlated with inflammatory markers UMCP1 (r = 0.517, p < 0.01) and TNF alfa (r = 0.172, p = 0.19). Conclusion: insertion/insertion NFkB1 gene polymorphism increases the risk of nephropathy by 2.52 times (OR = 2.52, 95% CI: 0.04 - 0.63, p value = 0.019) in diabetes patients in eastern India.

Exploration of the Microbial Biodiversity Associated with North Apulian Sourdoughs and the Effect of the Increasing Number of Inoculated Lactic Acid Bacteria Strains on the Biocontrol against Fungal Spoilage

In this study, we explored the diversity of yeasts and lactic acid bacteria (LAB) associated with six spontaneous sourdough fermentations from the northern part of the Apulian region (Italy). Bacterial and yeast isolates from sourdough were investigated by amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism (RFLP) analysis, respectively. The identification of the isolates was confirmed by sequencing bacterial 16S gene and yeast ITS1–5.8S–ITS2 rRNA gene amplicons. Microbiological analysis of all sourdough samples revealed that LAB and yeast counts ranged between 1.7 × 105 and 6.5 × 108 cfu/g, and 7.7 × 105 and 2.5 × 107 cfu/g, respectively. The molecular identification at species level revealed the occurrence of Lactobacillus plantarum as the dominant LAB and Saccharomyces cerevisiae as the dominant yeast species in all different sourdough samples. Then, the ability of all isolated strains to inhibit and/or reduce the growth of several selected fungi was valued through the overlay method. In light of their antifungal performances, ten LAB strains were inoculated, singularly and in combination, in subsequent bread-making trials. Overall, we confirmed the potential of LAB to extend the shelf life of bread through spoilage inhibition and, for the first time, we observed a synergistic effect due to the combination of several isolated LAB on the inhibition behavior against selected fungal spoilage strains. Our findings suggest the exploration of a LAB-based approach in order to extend the shelf life of bread, reducing, at the same time, the use of chemical agents for food preservation.

Diversity of Saccharomyces cerevisiae strains isolated of the spontaneous fermentation of cachaça from northeastern Brazil

Cachaca is a beverage obtained by distilling fermented sugar cane juice. The state of Bahia in northeastern Brazil is the second-largest producer of traditional cachaca, and this region has the potential to improve the quality and quantity of its beverage production. The aim of this study was to analyze the genetic diversity of Saccharomyces cerevisiae populations isolated from must in six distilleries in Bahia using mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP). Among the three hundred and thirty S. cerevisiae strains isolated, mtDNA-RFLP analysis identified a total of 30 molecular patterns. Analysis of molecular variance (AMOVA) revealed that the greatest genetic variation was found among, rather than within, the populations. Population structure analyses showed the presence of three distinct gene pools, thereby corroborating the AMOVA results. This study represents an important contribution to better understanding the molecular characterization and genetic variability of S. cerevisiae strains during the fermentation of cachaca. The dominant molecular patterns identified here may be used to select S. cerevisiae strains that could improve the quality and volume of traditional cachaca production in Bahia.

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No native trout on Cyprus

Toxoplasma gondii infection in stranded St. Lawrence Estuary beluga Delphinapterus leucas in Quebec, Canada.

The St. Lawrence Estuary (SLE) beluga Delphinapterus leucas in Quebec, Canada, is endangered due to intensive hunting in the 19th and 20th centuries and subsequent anthropogenic contamination and human activities in the region. Infectious disease is a primary cause of death in this population. The protozoan parasite Toxoplasma gondii is reported in numerous marine mammal species, including beluga. In the present study, 55 tissue samples (heart and brain) collected from 34 stranded SLE beluga were analysed by PCR followed by DNA sequencing and restriction fragment length polymorphism analysis (RFLP) to determine the PCR prevalence and genotypes of T. gondii in these beluga. Of 34 beluga tested, 44% were positive for T. gondii by PCR, with males having a higher prevalence of infection than females and with more infected neonates and juveniles than adults. Molecular analyses indicated that all T. gondii infecting stranded SLE beluga grouped into genotype II, which predominates in humans. While our results indicate that a high prevalence of stranded beluga are PCR-positive for T. gondii infection, very few deaths are attributed to toxoplasmosis based on published necropsy results. Toxoplasma gondii can cause a range of diseases, including neurological deficits, and more data are needed to investigate this parasite's effect on population recovery.

Detection of Cytochrome P450 Polymorphisms in Breast Cancer Patients May Impact on Tamoxifen Therapy

Background:Breast cancer is the most common cancer among women worldwide. Tamoxifen (TAM), a selective estrogen receptor modulator, is widely used in its treatment. TAM is metabolized by cytochrome P450 (CYP450) enzymes, including CYP2D6, CYP3A5 and CYP2C19, whose genetic variations may have clinicopathological importance. However, reports on the association of various P450 polymorphisms with certain cancers are contradictory.Methods:We here investigated whether the prevalence of the four most common polymorphism in the CYP2D6*4 (G1934A), CYP2D6*10 (C188T), CYP3A5*3 and CYP2C19*2 alleles has any link with breast cancer using genomic DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis.Results:Prevalences of CYP2D6*4, CYP2D6*10 and CYP2C19*2 genotypes were differed significantly (P = 0.01 and P = 0.004) between breast cancer patients and controls. The CYP3A5*3 genotype did not demonstrate statistically significant variation.Conclusion:Polymorphisms in CYP2 appear to be associated with breast cancer risk. Our data taken together with other reports indicates that drug resistance gene polymorphisms might be indicators of response to tamoxifen therapy in breast cancer cases.

Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China

BackgroundMembers of the genus Acanthamoeba are widely distributed throughout the world, and some of them are considered pathogenic, as they are capable of causing corneal and central nervous system diseases. In this study, we isolated Acanthamoeba strains from soil and tap water in Yanji, China.MethodsWe identified four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3, and CJY/W1) using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis. Nuclear 18S rDNA sequences were used for phylogenetic analysis and species identification.ResultsGenotypic characterization of the isolates showed that they belonged to genotypes T4 (CJY/S1 and CJY/S2), T5 (CJY/S3), and T16 (CJY/W1). Sequence differences between CJY/S1 and Acanthamoeba castellanii Neff, CJY/S2 and Acanthamoeba KA/E7, and CJY/S3 and Acanthamoeba lenticulata 68–2 were 0.31, 0.2, and 0.26%, respectively. 18S ribosomal deoxyribonucleic acid (rDNA) of CJY/W1 had 99% sequence identity to that of Acanthamoeba sp. U/H-C1. Strains CJY/S1 and CJY/S2, isolated from soil, had similar mtDNA RFLP patterns, whereas strain CJY/W1, isolated from tap water, displayed a different pattern.ConclusionsTo the best of our knowledge, this is the first report on the identification of genotypes T4, T5, and T16 from environmental sources in Yanji, China.

Differential diagnosis for pythiosis using thermophilic helicase DNA amplification and restriction fragment length polymorphism (tHDA-RFLP).

Pythiosis is caused by Pythium insidiosum, a fungus-like microbe belonging to the kingdom Stramenopila. Its diagnosis is challenging due to clinical and histopathological similarities with the fungal microbes that cause mucormycosis and entomophthoramycosis. In addition, the proper identification of P. insidiosum in the clinical laboratory is difficult. We have developed a rapid and accurate, species-specific identification method using a thermophilic helicase DNA amplification (tHDA) technique, to differentiate this pathogen from closely related pathogenic fungi. Sixty-seven fungal isolates, including 39 of P. insidiosum, were evaluated. A 91 base-pair (bp) DNA fragment was consistently amplified using a COX2 primer. The limiting concentrations of the one- and two-step tHDA protocols were 100 picograms (1.74 × 102 copies) and 100 femtograms (1.74 × 10-1 copies), respectively. The CviKI-1 enzyme in restriction fragment length polymorphism (RFLP) with the 91bp amplicons accurately separated P. insidiosum from other fungal species. The data suggest that this tHDA-RFLP assay is a rapid and accurate test for the identification of P. insidiosum. The potential use of the assay directly in clinical samples is also discussed.

Omentin Val109Asp polymorphism and risk of coronary artery disease.

Background Omentin is an adipocytokine with antiinflammatory properties. It has been reported to be involved in atherosclerosis and coronary artery disease. We aimed to investigate the association of omentin Val109Asp polymorphism with coronary artery disease in an Iranian population. Methods For a case-control study, 400 individuals were recruited: 200 with coronary artery disease and 200 healthy controls. Patients with coronary artery disease were diagnosed by angiography as having at least one main coronary artery with more than 50% stenosis. Genotyping of Val109Asp was carried out using a polymerase chain reaction DNA-restriction fragment length polymorphism technique. Results There was no association between Val109Asp polymorphism and the risk of coronary artery disease in our study population ( p = 0.20). However, when subgroup analysis was performed according to sex, there was a significant difference in the distribution of alleles between groups for men ( p = 0.031, odds ratio = 0.57, 95% confidence interval: 0.35-0.95) but not for women ( p = 0.88, odds ratio = 1.03, 95% confidence interval: 0.66-1.61). Conclusions Our results indicate that the Asp allele of Val109Asp (T allele of rs2274907) is more frequent among men with coronary artery disease than healthy men, so it is possibly a risk factor for coronary artery disease in men only. The difference in association between men and women may be due to the different distribution and metabolism of adipose tissue in men and women. More studies with larger sample sizes and in different populations are required to validate our study results.

Effect of GH and DGAT1 gene polymorphism on feeding qualities of bull calves

The aim of this paper is to study effects of GH and DGAT1 gene polymorphisms on feeding qualities of Hereford and Limousin bull calves bred in conditions of the Cis-Ural steppe zone. SNPs of genes GH and DGAT1 are investigated by polymerase chain reaction (PCR) and DNA restriction fragment length polymorphism (RFLP). The studied population of animals was assessed by defining the allele frequency and animal genotype occurrence for the studied gene SNPs, indicators of the actual and expected heterozygosity, and Pearson’s test. A study of polymorphism C214G of gene GH revealed that genotype LL prevails in Hereford and Limousin animals, 47.37 and 57.7%, respectively, while frequency of allele L is higher in Limousin bull calves (0.731). A study of polymorphism K232A of gene DGAT1 gene in both the populations showed absence of genotypes AA, which can be related to the low number of the studied animals. Expected heterozygosity indicators of gene GH are higher than the observed ones, and the observed heterozygosity is higher for DGAT1. The number of efficient alleles for the studied genes is higher for Hereford bull calves. In general, according to Pearson’s test, both the studied populations are in equilibrium. There is a significant effect of GH gene polymorphism on live weight gain rates at the end of sagination and total and average daily weight gains during animal raising.

Cigarette smoking has a differential effect on the plasma level of clozapine in Taiwanese schizophrenic patients associated with the CYP1A2 gene -163A/C single nucleotide polymorphism.

The efficacy of clozapine clearance has been shown to be associated with smoking and genetic polymorphism of CYP1A2. This study aims to investigate the effect of smoking on the plasma level of clozapine in Taiwanese schizophrenic patients and its relevance to the CYP1A2 gene -163A/C single nucleotide polymorphism. A total of 143 hospitalized schizophrenic patients who had received clozapine therapy for at least 14 days were enrolled in this study. The trough plasma concentration of clozapine was measured with LC/MS/MS. The -163A/C variant in the CYP1A2 gene was identified by DNA sequencing and restriction fragment length polymorphism analysis. The effect of smoking on the clozapine level was examined by multiple linear regression analysis and its relation to the -163A/C variant of the CYP1A2 gene was analyzed using a general linear model with Bonferroni correction. Patients with smoking habits showed a significantly lower plasma level of clozapine than those without smoking habits (P=0.022) and the difference in clozapine levels between smokers and nonsmokers appeared to be significant in the individuals carrying the homozygous -163A allele (P=0.02). It was also found that nonsmokers carrying the -163A allele tended to have higher plasma levels of clozapine. This tendency was not found in the individuals with smoking habits. Cigarette smoking has a significant impact on the plasma level of clozapine in Taiwanese schizophrenic patients carrying the homozygous -163A allele in the CYP1A2 gene. Cigarette smoking may increase the clearance of clozapine in these patients.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Differentiation of polyvalent bacteriophages specific to uropathogenic Proteus mirabilis strains based on the host range pattern and RFLP.

Urinary tract infections (UTIs) caused by P. mirabilis are difficult to cure because of the increasing antimicrobial resistance of these bacteria. Phage therapy is proposed as an alternative infection treatment. The aim of this study was to isolate and differentiate uropathogenic P. mirabilis strain specific polyvalent bacteriophages producing polysaccharide depolymerases (PDs). 51 specific phages were obtained. The plaques of 29 bacteriophages were surrounded by halos, which indicated that they produced PDs. The host range analysis showed that, except phages 58B and 58C, the phage host range profiles differed from each other. Phages 35 and 45 infected all P. mirabilis strains tested. Another 10 phages lysed more than 90% of isolates. Among these phages, 65A, 70, 66 and 66A caused a complete lysis of the bacterial lawn formed by 62% to 78% of strains. Additionally, phages 39A and 70 probably produced PDs. The phages' DNA restriction fragment length polymorphism (RFLP) analysis demonstrated that genomes of 51 isolated phages represented 34 different restriction profiles. DNA of phage 58A seemed to be resistant to selected EcoRV endonuclease. The 33 RFLP-EcoRV profiles showed a Dice similarity index of 38.8%. 22 RFLP patterns were obtained from single phage isolates. The remaining 12 restriction profiles consisted of 2 to 4 viruses. The results obtained from phage characterization based on the pattern of phage host range in combination with the RFLP method enabled effective differentiation of the studied phages and selection of PD producing polyvalent phages for further study.