Restriction Enzyme Fragment Length Polymorphism Research Articles

Genotyping of Trichophyton tonsurans strains isolated between 2016 and 2020, and terbinafine susceptibility of the species in Japan.

Trichophyton tonsurans is the most prevalent fungus which causes dermatophytosis among contact sports players in Japan. We previously surveyed the epidemic of T.tonsurans in Japan from early 2000, and determined the genotypes of isolates by analysis of restriction enzyme fragment length polymorphisms in the non-transcription spacer (NTS) region of ribosomal RNA gene, which enabled discrimination of eight genotypes, namely NTS I to NTS VIII. In the present study, we performed genotyping of T. tonsurans isolated between 2016 and 2020, and investigated the trend of the epidemic and resistance of the pathogen to antibiotic terbinafine (TBF). Regardless of which contact sport they played, the genotype of all 123 strains of T. tonsurans isolated from athletes was NTS I. Genotypes NTS II and III, which were isolated in considerable numbers mainly from wrestlers between 2000 and 2015, were conspicuously absent. TBF susceptibility was screened in 237 T. tonsurans strains isolated between 2000 and 2020 with 28 of these further assessed for minimum inhibitory concentration of TBF and squalene epoxidase gene sequences. None of the strains showed TBF resistance. TBF may still be effective to control the epidemic of T. tonsurans.

Genetic characterization and phylogenetic of Anaplasma capra in Persian onagers (Equus hemionus onager)

Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclusions on RBC's were observed in two blood smears by light microscopy. Then, the blood specimens of both animals were analyzed by realtime-PCR for Anaplasma, Ehrlichia, and Theileria infections. A 1400 bp sequence of 16S rRNA belonging to Anaplasmataceae and 874 bp fragment for groEL gene for A. capra were amplified in Anaplasma positive samples and sequenced. Preliminary BLAST analysis of sequenced fragments showed high homology to A. capra strains in GenBank database. Finally, nested PCR and restriction enzyme fragment length polymorphism techniques confirmed the pathogen as A. capra. To the best of our knowledge, this study has reported the occurrence of A. capra in wild onagers for the first time and suggests that equines could be infected with this pathogen and act as reservoirs for A. capra.

A Functional Single Nucleotide Polymorphism in the Aγ Globin Gene Promoter Affects Globin Chain Synthesis

Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjunction with KLF1, play a part in activating the HbF switch upon birth. To date, the complete mechanism of how globin gene switching works is still elusive. A total of 355 DNA samples were tested. The majority of the DNA samples were obtained from an existing collection of normal to borderline HbA2 and HbF cases, while other samples were collected along the course of the study. Data from anonymous and randomized cord blood DNA samples were also used and obtained from the Laboratory of Molecular Genetics, University of Malta. These were tested for the rs368698783 polymorphism under study and were used for reference purposes. The SNP rs368698783 was amplified by PCR and checked by agarose gel electrophoresis using a 1.5% agarose gel. Restriction enzyme fragment length polymorphism analysis was then performed using Mnl1 restriction enzyme. Agarose gel electrophoresis was performed again in order to separate the Mnl1- incubated products according to size and thus determine the frequency of the mutation identified. Some samples were chosen at random and were genotyped by DNA sequencing. Amongst the total study population (~355 individuals), the frequency of the mutant allele was 0.14 and close to 0.25 for the random newborn cord blood DNA analyzed in a previous study. The total study population was divided into 125 healthy adults and 193 into borderline HbF/HbA2 The mean fetal hemoglobin and HbA2 for the normal sub-population were 0.39± 0.034 and 2.68± 0.02 respectively. The mean levels in the borderline population were slightly higher with a mean of 0.6± 0.04 for fetal hemoglobin and 3.7±0.04 for HbA2 The effects of rs368698783 A allele on HbF levels examined in the total population and furthermore in the two sub populations of (i) normal healthy adults and (ii) adults with borderline levels of HbF/HbA2 showed that the mutant A allele of HBG1-rs368698783 exhibited a significantly elevated level of HbF (p < 0.05) compared with the wildtype G allele in each of the cohorts from Malta. Intriguingly no association was determined between this point mutation and HbA2 levels. The data is suggestive that the variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in human adults. The SNP is located within the LYAR binding motif. The LYAR is a transcriptive factor which is said to silence the expression of fetal hemoglobin. thus, the G to A polymorphism reduces the DNA binding affinity increasing HbF levels invivo. Figure Disclosures No relevant conflicts of interest to declare.

RAD51 135G>C Single Nucleotide Polymorphism and Risk of Breast Cancer in Selected Filipino Cases

The RAD51 gene encodes the protein that plays a central role in the repair of DNA double-strand breaks (DSBs) through the homologous recombination pathway. Association of RAD51 single nucleotide genetic polymorphism (SNP) with the development of cancer has been observed to be tumor site- and race-specific. Thus, this study aimed to determine the potential association between RAD51 135G>C SNP and breast cancer among selected Filipinos. Patients with histologically confirmed breast cancer (n = 60) seen at the University of Santo Tomas Hospital (USTH Manila) were age- and sex-matched with clinically healthy controls (n = 60). Genomic DNA was extracted from the blood of participants and analyzed for RAD51 genotype by polymerase chain reaction – restriction enzyme fragment length polymorphism (PCR-RFLP). A significantly higher incidence of RAD51 C/C genotype was seen among the cases than the controls (p < 0.05). The more common G/C genotype was not associated with breast cancer development, while the recessive less common C/C genotype was observed to potentially increase the risk. However, passive smokers carrying the RAD51 G/C genotype had a significantly increased chance of developing breast cancer. RAD51 G/C genotype – even when combined with other established risk factors like alcohol use, active smoking, and family history – were not associated with breast cancer.

Impact of Apo Lipoprotein E Gene Polymorphism on Hepatitis C Virus Disease Progression and Response to Direct Acting Antivirals in the Egyptian Population

Background: Hepatitis C virus infection is a major health problem in Egypt. Apo lipoprotein E and lipid metabolism are closely associated with the life cycle of the virus and play an important role in facilitating this infection. Objectives: To identify the role of apolipoprotein E gene polymorphism on hepatitis C virus disease progression and response to direct-acting antivirals (DAAs). Methods: We included twenty patients with chronic hepatitis C virus infection (CHC) from those who failed to respond to DAAs treatment and 76 subjects from responders. Apo lipoprotein E genotypes were determined using PCR restriction enzyme fragment length polymorphism (RFLP). Results: Apolipoprotein genotype E3/E3 was prevalent in 75.9% of patients. The distribution for the rest of genotypes was 16.9%, 3.6%, 2.4% and 1.2 % for Apo E3/E4, E2/E3, E4/E4, and E2/E2 respectively. No statistically significant difference was found regarding any of the Apo E genotypes with liver cirrhosis or response to therapy. Conclusions: No significant impact for any Apo E genotypes on CHC disease severity or response to therapy with direct-acting antivirals was detected among Egyptian patients.

Anaplasma bovis infection in a horse: First clinical report and molecular analysis

A 23-year-old male Thoroughbred horse at the Korean Military Academy appeared thin with visible rib bones and presented clinical signs of fever, anorexia, lethargy, and severe dehydration. To determine the presence of various febrile disease-causing agents, the 23 cohabiting horses at the academy, including this horse, were subjected to hematology, blood chemistry, and molecular analysis using whole blood samples collected during regular medical check-ups. On the basis of clinical history, physical examination, hematology, blood chemistry, and fecal examination, differential diagnosis using molecular analyses was performed for various febrile disease-causing agents, including Lyme borreliae, Coxiella, piroplasms (Babesia and Theileria), Rickettsiales (Anaplasma, Ehrlichia, and Rickettsia), equine herpesvirus, equine infectious anemia virus, and equine arteritis virus. While other pathogens were not detected, PCR and phylogenetic analysis targeting the Anaplasma 16S rRNA gene revealed that the horse was infected with Anaplasma bovis. Although PCR targeting the groEL and gltA genes of A. bovis was not successful, the restriction enzyme fragment length polymorphism assay for differential diagnosis and determination of coinfectivity between Anaplasma phagocytophilum and A. bovis confirmed the pathogen as A. bovis. To the best of our knowledge, this is the first clinical report of A. bovis infection in a horse, suggesting a new reservoir host.

Molecular Detection and Phylogenetic Analysis of Anaplasma phagocytophilum in Horses in Korea.

The identification and characterization of pathogenic and zoonotic tick-borne diseases like granulocytic anaplasmosis are essential for developing effective control programs. The differential diagnosis of pathogenic Anaplasma phagocytophilum and non-pathogenic A. phagocytophilum-like Anaplasma spp. is important for implementing effective treatment from control programs. The objective of the present study was to investigate the prevalence of Anaplasma spp. in horses in Korea by nucleotide sequencing and restriction enzyme fragment length polymorphism assay. Of the 627 horses included in the study, only 1 (0.2%) was infected with A. phagocytophilum. Co-infection with A. phagocytophilumlike Anaplasma spp. was not detected in the study. The 16S rRNA sequence of A. phagocytophilum was similar (99.5–100%) to A. phagocytophilum 16S rRNA isolated from horses in other countries. PCR adapted to amplify A. phagocytophilum groEL and msp2 genes failed to generate amplicons, suggesting genetic diversity in these genes. This study is the first molecular detection of A. phagocytophilum in horses in Korea. Human granulocytic anaplasmosis and animal infection of A. phagocytophilum have been reported in Korea recently. Because of vector tick distribution, global warming, and the increase of the horse industry, horses should be considered as a potential reservoir for A. phagocytophilum, and cross infectivity should be evaluated even though a low prevalence of infection was detected in this study. Furthermore, continuous surveillance and effective control measures for A. phagocytophilum should be established to prevent disease distribution and possible transmission to humans.

CYP1A1 gene (6235T

This case-control study was carried out to examine the association between CYP1A1 gene (6235T<C) polymorphism among Egyptian women with polycystic ovarian syndrome (PCOS). One hundred and twenty women with PCOS, as well as 120 apparently healthy women as controls, were included in this study. Blood samples were collected on the second or third day of the menstrual cycle for laboratory work-up. CYP1A1 gene (6235T<C) polymorphism was determined using restriction enzyme fragment length polymorphism – polymerase chain reaction (RFLP-PCR) technique. Women who carried the TC and CC genotypes showed insignificant increase in risk (1.4 and 2.7, respectively) for developing PCOS (p = 0.331 and p = 0.124, respectively). The risk showed a significant association among PCOS women with ovarian sonographic stigmata of polycystic ovaries (PCO) who carried the TC genotype (Odds Ratio (OR) = 1.92, p = 0.032), as well as among carriers of the CC genotype (OR = 4.2, p = 0.048). Both TC and CC genotypes were significantly associated with the ovarian volume (p < 0.001) and abdominal obesity (p = 0.025), while no associations were shown with the hormonal profile. In conclusion, both TC and CC genotypes of the CYP1A1 gene showed increased susceptibility to PCOS. CYP1A1 gene polymorphism may affect the folliculogenesis and the hormonal status along with other risk factors leading to the development of the full picture of PCOS.

Survey of Anaplasma phagocytophilum and Anaplasma sp. \u2018Omatjenne\u2019 infection in cattle in Africa with special reference to Ethiopia

BackgroundAs evidence of the infection of domestic animals by Anaplasma phagocytophilum and Anaplasma sp. ‘Omatjenne’ is presently becoming available, understanding the epidemiological and ecological significance of infection is important to quantify the clinical and socio-economic impact of the diseases they cause.MethodsThe first aim of this study was to analyse the occurrence of A. phagocytophilum and Anaplasma sp. ‘Omatjenne’ in cattle samples collected from selected African countries using a polymerase chain reaction and restriction enzyme fragment length polymorphism. Secondly, this study was aimed at the molecular identification of Ehrlichia spp. and Anaplasma spp. infection in ruminants raised under different production systems in selected sites in central Ethiopia.ResultsIn total, 695 samples from cattle in six African countries were analysed. Overall, 45 positive results were obtained for Anaplasma sp. ‘Omatjenne’ (6.47%) and 19 for A. phagocytophilum (2.73%). Anaplasma sp. ‘Omatjenne’ was detected in all countries except Tanzania while A. phagocytophilum was detected only in samples from Ethiopia. The proportion of samples tested positive for Anaplasma sp. ‘Omatjenne’ ranged from 1.2% in Morocco to 16% in Rwanda. The occurrence of both agents is now confirmed in African cattle. For the survey in Ethiopia a semi-nested 16S rDNA polymerase chain reaction followed by restriction fragment length polymorphism was used for the identification of Ehrlichia spp. and Anaplasma spp. in blood samples. Randomly selected samples were also analysed by pCS20 polymerase chain reaction for the detection of E. ruminantium. Positive results were obtained for E. ruminantium and five species of Anaplasma including a zoonotic species. To our knowledge, this is the first report of infection of domestic ruminants with A. phagocytophilum, A. ovis and Anaplasma sp. ‘Omatjenne’ in Ethiopia.ConclusionThe present study showed widespread occurrence of Anaplasma sp. 'Omatijenne' in African cattle and five Anaplasma species in Ethiopia.

IL-6 promoter polymorphism increased risks of recurrent stroke in the young patients with moderate internal carotid artery stenosis.

Interleukin-6 (IL-6) is a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory properties. Increased serum IL-6 is associated with higher risk of cerebral vascular diseases, however, the circulating levels of IL-6 vary greatly between individuals for bioenvironmental or genetic interferes by IL-6 transcription, therefore, this study was to investigate if IL-6 promoter polymorphisms predict the recurrence of young stroke. A total of 94 patients with moderate internal carotid artery stenosis and 94 healthy subjects were selected. Color Doppler was used to screen internal carotid artery stenosis, PCR restriction enzyme fragment length polymorphism for genotypes. Stroke recurrence rates in patients with different genotypes of IL-6 promoter were evaluated by 1-year follow-up. The frequencies of 634C/G genotype of IL-6 promoter were CC 38.29%, CG 46.81%, and GG 14.90%, while CC 59.51%, CG 35.11%, GG 6.38% in the control group (P < 0.01). The frequency of G allele was significantly higher than that in the control group (38.30% vs 21.28%, P < 0.01). The relative risk was 2.2838, and 95% confidence interval was 1.0026 to 5.2026, adjusted age, sex, blood pressure, BMI, blood lipid levels and IL-6 (P < 0.05). The recurrent rate was 25.53% at 1-year follow-up; the recurrent rate among carriers with wild-type G allele (CG + GG genotype) was 32.76%, but CC 13.89% (P < 0.01). IL-6 G allele promoter increased stroke recurrent risk, therefore, it would be a predictor for recurrence of stroke in the young with moderate internal carotid artery stenosis.

Surveying Mutation of FLT3 Genes in Children with Acute Leukemia

Background and purpose: Mutation of FMS like tyrosine kinase (flt3) gene causes uncontrolled proliferation of leukemic cells and a bad prognosis. The present study is aimed at implementing molecular tests to diagnose and screen the mutations in acute leukemia patients. Methodology: Totally, 91 children with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) were examined as to flt3 mutation, internal tandem duplication (ITD) mutation, and point mutation in exon 17 (e17). ITD mutation in flt3 receptor was carried out in exon 12&amp;12 and intron 11. As to point mutation in e17, PCR products of the subjects after PCR on genomic DNA of them were examined using restriction enzyme (ECORV) and restriction fragment length polymorphism (RFLP). As to ITD positive, sequencing method was used. Findings: ITD mutation was observed in seven (7.7%) of acute leukemia patients and two (2.2%) patients were diagnosed with point mutation D835. Distribution in different subgroups of FAB was not identical. Conclusion: FLT3 mutation was highly prevalent in children with acute leukemia. Therefore, molecular diagnosis of these mutation, regardless of FAB categorization and before initiation of intervention, can be used to make better decision about therapeutic protocol.

Anaplasma platys-like strains in ruminants from Tunisia

Molecular diagnosis of Anaplasma platys and related strains (A. platys–like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.

Role of Hyperhomocysteinemia and Methylene Tetrahydrofolate Reductase C677T Polymorphism in Idiopathic Portal Vein Thrombosis.

Purpose:Portal vein thrombosis (PVT) is a rare and life-threatening vascular disorder characterized by obstruction or narrowing of the portal vein. Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been studied in PVT patients with conflicting results. In the present study the association of hyperhomocysteinemia and MTHFR C677T polymorphism with PVT risk was investigated in Iranians.Materials and Methods:Our study population consisted of 10 idiopathic PVT patients and 80 healthy control subjects matched for age and sex. MTHFR C677T polymorphism was genotyped by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) technique and plasma total homocysteine (tHcy) levels were determined by enzyme immunoassay method.Results:Mean plasma tHcy levels were significantly higher in PVT patients (20.2±6.8) than control subjects (10.9±4.7) (P=0.001). Moreover, plasma tHcy levels were significantly higher in 677T allele carriers relative to 677C allele carriers in both PVT patients (P=0.01) and control subjects (P=0.03). Neither homozygote nor heterozygote genotypes of MTHFR C677T polymorphism correlated significantly with PVT risk (P>0.05). Moreover, MTHFR C677T polymorphism didn’t increase the risk of PVT under dominant (CT+TT vs. CC) or recessive (TT vs. CC+CT) genetic models analyzed (P>0.05). The difference in frequency of minor 677T allele between PVT patients and control subjects was not statistically significant (P>0.05).Conclusion:Based on the current study, we suggest that hyperhomocysteinemia constitutes a significant and common risk factor for PVT. Also, MTHFR C677T polymorphism is not a risk factor for PVT but is a contributing factor for elevated plasma tHcy levels.

Association of Methylenetetrahydrofolate Reductase C677T Polymorphism with Hyperhomocysteinemia and Deep Vein Thrombosis in the Iranian Population.

Purpose:Deep venous thrombosis (DVT) is a common but elusive condition characterized by a high morbidity and mortality rate. The aim of the present study was to investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with plasma total homocysteine (tHcy) levels and DVT risk in an Iranian population.Materials and Methods:Our study population consisted of 67 patients with a diagnosis of DVT and 67 healthy subjects as controls. Genotyping of MTHFR C677T polymorphism was performed by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) and measurement of tHcy levels was done by enzyme immunoassay method.Results:Plasma tHcy levels were significantly higher in DVT patients than controls (18.09±7.6 vs. 10.5±4.3, P=0.001). Also, plasma tHcy levels were significantly higher in MTHFR 677TT genotypes compared to 677CC genotypes in both DVT patients (P=0.016) and controls (P=0.03). Neither heterozygote nor homozygote genotypes of MTHFR C677T polymorphism was significantly correlated with DVT (P>0.05). The distribution of MTHFR C677T genotypes was similar between men and women in both DVT patients and controls (P>0.05). Moreover, the frequency of mutant 677T allele did not differ significantly between the two groups (28.3% vs. 21.6%, P=0.15).Conclusion:Based on this study, we propose that hyperhomocysteinemia but not homozygosity for MTHFR C677T polymorphism is a significant risk factor for DVT in the Iranian population. Also, MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels.

Uncovering Human History: Origin through Preserved Names of Places!!!

When we are trying to uncover history and evolution of humanity we rely on Stonehenge evidence, paintings on rocks, writing on rocks, etc. With the advent of modern molecular biological techniques such as Human Leucocyte Antigen (HLA) typing, Polymerase Chain Reaction, Restriction enzyme Fragment Length Polymorphism (RFLP) analysis of DNA, whole genome sequencing we try to map out the origin of human evolution with reference to genetics and the associated cultural practices, migration patterns, beliefs and so on. With pros and cons of each methods one technique may not be superior/comprehensive over other. Instead, all the data and evidences can complement each other and come up with a comprehensive interpretation and conclusions.

Diversity of acidophilic prokaryotes at two acid mine drainage sites in Turkey.

The biodiversity of acidophilic prokaryotes in two acidic (pH 2.8-3.05) mine drainage (AMD) sites (Balya and Çan) in Turkey was examined using a combined cultivation-based and cultivation-independent approach. The latter included analyzing microbial diversity using fluorescent in situ hybridization (FISH), terminal restriction enzyme fragment length polymorphism (`T-RFLP), and quantitative PCR (qPCR). Numbers of cultivatable heterotrophic acidophilic bacteria were over an order of magnitude greater than those of chemolithotrophic acidophiles in both AMD ponds examined. Isolates identified as strains of Acidithiobacillus ferrivorans, Acidiphilium organovorum, and Ferrimicrobium acidiphilum were isolated from the Balya AMD pond, and others identified as strains of Leptospirillum ferriphilum, Acidicapsa ligni, and Acidiphilium rubrum from Çan AMD. Other isolates were too distantly related (from analysis of their 16S rRNA genes) to be identified at the species level. Archaeal diversity in the two ponds appeared to be far more limited. T-RFLP and qPCR confirmed the presence of Ferroplasma-like prokaryotes, but no archaea were isolated from the two sites. qPCR generated semiquantitative data for genera of some of the iron-oxidizing acidophiles isolated and/or detected, suggesting the order of abundance was Leptospirillum > Ferroplasma > Acidithiobacillus (Balya AMD) and Ferroplasma > Leptospirillum > Acidithiobacillus (Çan AMD).

Rickettsiae and Ehrlichiae Within a City Park: Is the Urban Dweller at Risk?

Tick-borne diseases, such as spotted fever rickettsioses and ehrlichioses, are potentially severe and life-threatening infections. The incidences of these infections increase during warm weather months as ticks become active. Clinicians often consider outdoor activities in rural areas to be a risk factor for exposure to ticks and the pathogens they carry, but are those who live, work, and play within an urban environment excluded from this risk? In this study, we collected ticks from two urban parks in Little Rock, AR, to assess the presence of rickettsiae and ehrlichiae within an urban setting. A total of 273 ticks were collected during July, 2011. Amblyomma americanum was the predominant tick species, with 255 (93%) of those collected. The remaining 18 (7%) were Dermacentor variabilis. Ticks were separated and pooled into groups for further testing. Forty-two of the 43 (98%) A. americanum pools demonstrated molecular evidence for the presence of rickettsiae. None of the D. variabilis contained rickettsiae. Restriction enzyme fragment length polymorphism analysis and DNA sequencing revealed Rickettsia amblyommii to be the species present. One A. americanum pool from park A demonstrated the presence of Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis. These results indicate that tick-borne pathogens are not limited to rural or suburban areas.

Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria

PCR targeting the 16S–23S rRNA gene internally transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Mycobacterium species in human clinical specimens. Because of variation in ITS sequences amongst Mycobacterium species, a single PCR amplification can be used to differentiate slowly growing and rapidly growing species within this genus. In the present study, analysis by ITS-PCR and ITS-restriction fragment length polymorphism (RFLP) was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from 13 reference strains and 59 fish isolated mycobacteria using a set of published PCR primers. After PCR, the banding patterns generated allowed slowly growing mycobacteria to be differentiated from all other rapidly growing species, with the exception of Mycobacterium conceptionense. HaeIII was selected as one of two restriction enzymes that, together with the knowledge about amplicon sizes, would produce an acceptable level of discrimination in the resulting RLFP patterns, especially in the rapidly growing group of mycobacteria. After digestion with Sau96I, the amplified products of most isolates of Mycobacterium fortuitum, including subtypes II and V and those 2 isolates with new patterns (220, 100bp), presented identical or very similar patterns as obtained by HaeIII digestion. All isolates of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium gordonae, whose PCR products were not digested with HaeIII, produced two well-defined fragments with the Sau96I restriction enzyme.

Detection of avian reoviruses causing tenosynovitis in breeder flocks in Iran by reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme fragment length polymorphism (RFLP)

BACKGROUND:Avian reoviruses (ARVs) are members of the Orthoreovirus genus; one of the 12 genera of the Reoviridae family. The ARVs are the cause of some important diseases in poultry such as reovirus-induced arthritis, tenosynovitis, chronic respiratory disease, and mal-absorption syndrome. OBJECTIVES: In this study, the presence of ARVs in the Iranian breeder flocks was investigated through reverse transcription- polymerase chain reaction (RT-PCR) and restriction enzyme fragment length polymorphism (RFLP). METHODS: A total of 800 fecal swab samples were initially collected from breeder flocks (older than 45 weeks of age). They were then sent to the laboratory in containers with PBS, and after that they were pooled and finally to 120 samples were obtained. The total RNA extracted from the pooled fecal samples were used to amplify the selected parts of the S1 (1023 bp) and S4 (437 bp) genes from the ARV field isolates using RT-PCR. The positive RT-PCR amplified products were further analyzed by RFLP using five restriction enzymes. RESULTS: Based on the findings, 5 samples were positive with the S1 primer and 6 samples were with the S4 one. The patterns observed after the digestion of PCR products revealed that the isolates of this study were identical to both the S1133 vaccine and standard strains. CONCLUSIONS: The findings suggested that the RT-PCR/RFLP analysis might be considered as a simple and rapid approach for the differentiation of ARV isolates. This study was the first molecular detection of the ARVs presence in the Iranian breeder flocks using the RT- PCR amplification of the S1 and S4 genes and RFLP analysis.

Evolution of microbial "streamer" growths in an acidic, metal-contaminated stream draining an abandoned underground copper mine.

A nine year study was carried out on the evolution of macroscopic “acid streamer” growths in acidic, metal-rich mine water from the point of construction of a new channel to drain an abandoned underground copper mine. The new channel became rapidly colonized by acidophilic bacteria: two species of autotrophic iron-oxidizers (Acidithiobacillus ferrivorans and “Ferrovum myxofaciens”) and a heterotrophic iron-oxidizer (a novel genus/species with the proposed name “Acidithrix ferrooxidans”). The same bacteria dominated the acid streamer communities for the entire nine year period, with the autotrophic species accounting for ~80% of the micro-organisms in the streamer growths (as determined by terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis). Biodiversity of the acid streamers became somewhat greater in time, and included species of heterotrophic acidophiles that reduce ferric iron (Acidiphilium, Acidobacterium, Acidocella and gammaproteobacterium WJ2) and other autotrophic iron-oxidizers (Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans). The diversity of archaea in the acid streamers was far more limited; relatively few clones were obtained, all of which were very distantly related to known species of euryarchaeotes. Some differences were apparent between the acid streamer community and planktonic-phase bacteria. This study has provided unique insights into the evolution of an extremophilic microbial community, and identified several novel species of acidophilic prokaryotes.