Nitritation, the first stage of ammonia removal process is known to be limiting for total process performance. Ammonia oxidizing bacteria (AOB) which perform this process are obligatory activated sludge habitants, a mixture consisting of Bacteria, Protozoa and Metazoa used for biological wastewater treatment. Due to this fact they are an interesting bacterial group, from both the technological and ecological point of view. AOB changeability and biodiversity analyses both in wastewater treatment plants and lab-scale reactors are performed on the basis of 16S rRNA gene sequences using PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis) as a molecular biology tool. AOB researches are usually led with nested PCR. Because the application of nested PCR is laborious and time consuming, we have attempted to check the possibility of using only first PCR round to obtain DGGE fingerprinting of microbial communities. In this work we are comparing the nested and non-nested PCR-DGGE monitoring of an AOB community and presenting advantages and disadvantages of both methods used. The experiment revealed that PCR technique is a very sensitive tool for the amplification of even a minute amount of DNA sample. But in the case of nested-PCR, the sensitivity is higher and the template amount could be even smaller. The nested PCR-DGGE seems to be a better tool for AOB community monitoring and complexity research in activated sludge, despite shorter fragments of DNA amplification which seems to be a disadvantage in the case of bacteria identification. It is recommended that the sort of analysis approach should be chosen according to the aim of the study: nested-PCR-DGGE for community complexity analysis, while PCR-DGGE for identification of the dominant bacteria.
The use of lactoferrin (LF) and/or lactobacillus sp. (LB) to improve animal health and production has increased recently. However, information regarding the immune-modulatory role of LB supplementations either alone or in combination with LF in sheep remains unclear. Therefore, the present study was designed to evaluate the immune modulating properties and the antioxidant activity of supplementing commercially available LF and/or LB in healthy lambs. For this reason, twenty-four apparently healthy Ossimi lambs were used. After three weeks of acclimatization, the lambs were randomly allocated to four equal-sized groups and assigned to receive one of the following supplements: LB at a dose of ~ 1 g active ingredient/head (group 1), LF at a dose rate of 0.5 gm /head (group 2), a combination of both treatments using the same dosing regimens (group 3), and (group 4) received only 10 mL of isotonic saline and was considered as a control group. All supplements were given orally twice daily for 30 consecutive days. Blood samples were collected from each lamb before starting the experiment (T0) and two weeks (T15), and four weeks (T30) after giving supplements for hematological examinations, serum biochemical analyses, and RT-PCR assays. Our findings demonstrated that lambs receiving LB showed statistically significant (P<0.05) higher values of total leucocytes, lymphocytes and lysozyme activity than those receiving LF. In contrast, lambs that received LF had significantly (P< 0.05) higher values of serum catalase, nitric oxide and GSH with a significantly lower MDA level compared with those supplemented with LB. A combination of LF and LB supplementation elicited maximal up-regulation of Tollip, TLR4, IL-5, and IL-6 gene expression compared with other groups. The results suggest that bovine LF and or LB could be used as useful nutritional supplements to support the immune system in healthy lambs.
Faecal Enterococcus hirae from domestic ducks were studied for their bioactivity to select bioactive strain for more detailed study with its probable use in poultry and also to bring novelty in basic research. After defecation, faeces (n=23, faecal mixture of 40 ducks) were sampled from domestic ducks in eastern Slovakia; birds were aged from eight to 14 weeks. E. hirae strains were identified using Matrix-assisted laser desorption/ionization time-of flight mass spectrometry with a highly probable species identification score (2.300-3.000) or a secure genus identification/ /probable species identification score (2.000-2.299), confirmed by polymerase chain reaction and phenotypization in accordance with the properties for the type strain E. hirae ATCC 9790. Strains were hemolysis negative (γ-hemolysis), and did not have active enzyme stimulating disorders. Enterocin genes were detected in three strains out of seven. Three out of four Enterocin genes were detected in Kč1/b (Ent A, P, L50A); the most frequently detected was the Ent P gene. The strains inhibited indicator strains E. faecalis, listeriae, but also Escherichia coli and Buttiauxiella strains. Lactic-acid producing E. hirae were mostly susceptible to antibiotics. Based on parameter evaluation, E. hirae Kč1/b, Kč6 can be additionally studied to select the type of bioactive substance.
Potato white mold caused by Sclerotinia sclerotiorum is an important plant disease occurring in many potato-producing areas throughout the world. In this study, a specific diagnostic method was used to detect and quantify S. sclerotiorum ascospores, and its forecasting ability was assessed in potato fields during flowering periods of 2011 to 2014 in Bahar County, Hamedan Province. Using GenEMBL database, a primer pair, HZSCREV and HZSCFOR, was designed and optimized for the pathogen. After testing the sensitivity of primers, DNA was extracted from samples of outdoor Burkard traps from potato fields. A linear association was observed between pathogen DNA and the number of ascospores using the quantitative PCR (qPCR) technique in the presence of SYBR dye. The qPCR could successfully detect DNA amounts representing two S. sclerotiorum ascospores and was not sensitive to a variety of tested fungi such as Botrytis cinerea, Alternaria brassicae, Fusarium solani. In contrast to the amount of rainfall, a direct relationship was found between ascospore numbers and the incidence of potato white mold from 2011 to 2014.
The present study was conducted to characterize the infectious bursal disease virus (IBDV) circulating in clinically diseased broiler chicken flocks with previous vaccination history during 2015-2016 in Egypt. IBDVs were isolated from 48 out of 63 of the investigated bursae from 10 flocks onto embryonated chicken eggs (ECEs) and verified by reverse transcriptase-poly- merase chain reaction (RT-PCR). Histopathologically, bursae lesions revealed some lymphocytes depletion as well as the presence of vesicles in the lining epithelium. The hyper variable region (HVR) of VP2 and VP1 genes of the 10 isolates (1 isolate/flock) were partially sequenced and subjected to comparative alignment and phyologenetic analysis. Phylogenetically, IBDV isolates were clustered into two distinct genetic lineages: variants of classical virulent (cv) and very viru- lent (vv) IBDV strains based on VP1 and VP2 amino acid (aa) sequences. Alignment analysis of HVR-VP2 aa sequences has demonstrated that the vvIBDV isolates have the conserved residues of the vvIBDV pathotype (A222, I242, and I256), while, the cvIBDV isolates have the same aa sequences of the classical attenuated vaccine strain (D78). Expected single point mutation occurred at position 253 (H253N). All previously characterized isolates were re-subjected to molecular analysis with VP1 protein due to its correlation with virulence and pathogenicity of IBDVs. vvIBDV isolates have the conserved tripeptide (TDN), while, the cvIBDV isolates have aa substitutions at conserved tripeptide including NEG at 145-147 amino acid. The present study has demonstrated that variants of classical virulent and very virulent IBDV circulated among vaccinated flocks in Egypt during 2015-2016.
Water samples were collected from irrigation ditches and drainage canals surrounding fields in southern Greater Poland. Initially, the samples were subjected to low and highspeed centrifugation and obtained pellets were used to perform biological assays. Viral identification involved biological, electron microscopic as well as molecular methods. The occurrence of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) was demonstrated in 12 of the 17 examined water sources. The molecular analysis results showed TMV and ToMV co-infections in the analysed water samples. To our knowledge, this is the first report of tobamoviruses being found in environmental water in Poland.
The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.
Fusarium crown rot (FCR), caused by Fusarium culmorum (Wm.G.Sm) Sacc., is an important disease of wheat both in Iraq and other regions of wheat production worldwide. Changes in environmental conditions and cultural practices such as crop rotation generate stress on pathogen populations leading to the evolution of new strains that can tolerate more stressful environments. This study aimed to investigate the genetic diversity among isolates of F. culmorum in Iraq. Twenty-nine samples were collected from different regions of wheat cultivation in Iraq to investigate the pathogenicity and genetic diversity of F. culmorum using the repetitive extragenic palindromic (REP-PCR) technique. Among the 29 isolates of F. culmorum examined for pathogenicity, 96% were pathogenic to wheat at the seedling stage. The most aggressive isolate, from Baghdad, was IF 0021 at 0.890 on the FCR severity index. Three primer sets were used to assess the genotypic diversity via REP, ERIC and BOX elements. The amplicon sizes ranged from 200–800 bp for BOX-ERIC2, 110–1100 bp for ERIC-ERIC2 and 200–1300 bp for REP. A total of 410 markers were polymorphic, including 106 for BOX, 175 for ERIC and 129 for the REP. Genetic similarity was calculated by comparing markers according to minimum variance (Squared Euclidean). Clustering analysis generated two major groups, group 1 with two subgroups 1a and 1b with 5 and 12 isolates, respectively, and group 2 with two subgroups 2a and 2b with 3 and 9 isolates, respectively. This is the first study in this field that has been reported in Iraq.
Pseudomonas syringae pv. syringae (Pss) constitutes a diverse group of bacterial strains that cause canker of stone fruits, blight of cereals and red streak of sugarcane. The purpose of this study was to determine how diverse Iranian strains of Pss are when they come from different hosts. We compared a total of 32 Pss strains isolated from stone fruits, barley, wheat and sugarcane from different geographical regions of Iran based on their phenotypic and molecular properties. Strains showed some variation regarding carbon and nitrogen utilization. Pss strains were similar in their protein banding patterns. Additional bands were found in sugarcane strains. Most strains showed one indigenous plasmid DNA and a few had two and some none. The genes of syrB and syrD encoding syringomycin synthesis and secretion, respectively, were amplified using specific primers in polymerase chain reaction. Syringomycin, producing strains amplified two DNA fragments of 752 and 446 bp representing syrB and syrD genes, respectively. Primer specificity was shown for Pss using various genera. Based on the results of this study, it is suggested that Pss strains from different hosts and geographical regions show diversity in phenotypic and molecular characters. It is thought that phenotypic variation is due to adaptation to specific hosts and niches for survival and pathogenicity.
Field and laboratory protocols that originally led to the success of published studies have previously been only briefly laid out in the methods sections of scientific publications. For the sake of repeatability, we regard the details of the methodology that allowed broad−range DNA studies on deep−sea isopods too valuable to be neglected. Here, a com− prehensive summary of protocols for the retrieval of the samples, fixation on board research vessels, PCR amplification and cycle sequencing of altogether six loci (three mitochondrial and three nuclear) is provided. These were adapted from previous protocols and developed especially for asellote Isopoda from deep−sea samples but have been successfully used in some other peracarids as well. In total, about 2300 specimens of isopods, 100 amphipods and 300 tanaids were sequenced mainly for COI and 16S and partly for the other markers. Although we did not set up an experimental design, we were able to analyze amplification and sequencing success of different methods on 16S and compare success rates for COI and 16S. The primer pair 16S SF/SR was generally reliable and led to better results than universal primers in all studied Janiroidea, except Munnopsidae and Dendrotionidae. The widely applied universal primers for the barcoding region of COI are problematic to use in deep−sea isopods with a success rate of 45–79% varying with family. To improve this, we recommend the development of taxon−specific primers.
The full-length cDNA of LeTIR1 gene was isolated from tomato with EST-based in silico cloning followed by RACE amplification. LeTIR1 contained an open reading frame (ORF) 1872 bp long, encoding 624 amino acid residues. The predicted protein LeTIR1 had one F-box motif and eleven leucine-rich repeats (LRRs), all of which are highly conserved in TIR1 proteins of other plant species. Phylogenetic analysis showed that the LeTIR1 protein shared high similarity with other known TIR1 proteins. Both sequence and phylogenetic analysis suggested that LeTIR1 is a TIR1 homologue and encodes an F-box protein in tomato. Semi-quantitative RT-PCR indicated that LeTIR1 was expressed constitutively in all organs tested, with higher expression in stem than root, leaf, flower and fruit. Its expression level was positively correlated with the auxin distribution in stem or axillary shoot, and was induced by spraying exogenous IAA.
Nanodiagonastic methods in plant pathology are used for enhancing detection and identification of different plant pathogens and toxigenic fungi. Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by using some nanoparticles is emerging as a new area of research. In the current research, silver, zinc, and gold nanoparticles were used to increase the yield of DNA for two plant pathogenic fungi including soil-borne fungus Rhizoctonia solani and toxigenic fungus Alternaria alternata. Gold nanoparticles combined with zinc and silver nanoparticles enhanced both DNA yield and PCR products compared to DNA extraction methods with ALB buffer, sodium dodecyl sulfate, ALBfree from protinase K, ZnNPs and AgNPs. Also, by using ZnNPs and AgNPs the DNA yield was enhanced and the sensitivity of random amplified polymorphic DNA (RAPD) PCR products was increased. Application of nanomaterials in the PCR reaction could increase or decrease the PCR product according to the type of applied nanometal and the type of DNA template. Additions of AuNPs to PCR mix increased both sensitivity and specificity for PCR products of the tested fungi. Thus, the use of these highly stable, commercially available and inexpensive inorganic nano reagents open new opportunities for improving the specificity and sensitivity of PCR amplicon, which is the most important standard method in molecular plant pathology and mycotoxicology.
Wheat dwarf virus (WDV) has been one of the most common viruses on cereal crops in Poland in the last years. This single stranded DNA virus is transmitted by the leafhopper spec, Psammotettix alienus (Dahlb.) in a persistent manner. It induces yellowing and streaking of leaves, dwarfing or even death of infected plants. The presence of barley- and wheat-specific forms of WDV (WDV-B and WDV-W) and their vector were previously reported in the country, however the literature data did not include any information on the infectivity of the vector in Poland. A duplex polymerase chain reaction (PCR) procedure was developed and optimized for simultaneous detection and differentiation of both forms in the vector. Two sets of primers amplify 734 bp and 483 bp specific fragments for WDV-W and WDV-B, respectively. The results were verified by a sequencing method. The studies were carried out on insect samples collected in autumn from four different locations in Greater Poland. The results confirmed the presence of WDV-W in the tested samples. They also suggested the concomitant of both forms of the virus in the vector. Additional studies to determine virus-vector relationships should be undertaken.
Perinatal calf mortality in dairy herds has been reported worldwide. The etiology of stillbirth is multifactorial, and can be caused by various species of bacteria and environmental factors. Among them some potential pathogens from the Mollicutes class such as Mycoplasma (M.) spp. and Ureaplasma (U.) diversum can be isolated from the bovine genital tract and other organs of the suspected cattle. The aim of this study was to evaluate if the bacteria belonging to the Molli- cutes class i.e. M. bovis, M. bovigenitalium, M. canadense, M. canis, M. arginini, M. bovirhinis, M. dispar, M. alkalescens and U. diversum could have an impact on perinatal calf mortality in selected Polish dairy farms. The material was: 121 stillborn calves (SB), 21 live born calves (C) and 131 cows (dams) from 30 Polish Holstein-Friesian herds. Samples were examined from all the SB calves’ and six control euthanized calves’ abomasal contents and lung samples collected during necropsy, and from the dams’ serum and placenta. In dams the serological ELISA, and in calves and placenta samples molecular PCR/denaturing gradient gel electrophoresis, methods were used. Screening of dams’ sera for antibodies to M. bovis (ELISA) showed seven dams positive for M. bovis, whereas none of the nine examined Mollicutes microorganisms were detected in the placenta and calves.
Cucumber mosaic virus (CMV; family Bromoviridae, genus Cucumovirus) is the most cosmopolitan plant virus occurring worldwide. In the present study, leaf samples showing deformations, mosaics, and chlorotic spots symptoms were collected from naturally infected Basella alba, Telfairia occidentalis and Talinum fruticosum in a home yard garden in Ibadan, Nigeria. Total nucleic acid was extracted from leaves and used as template for cDNA synthesis. RT-PCR was carried out using CMV-specific primers targeting RNA-1 segment. Samples were also tested by RT-PCR using Potyvirus and Begomovirus genusspecific primers. DNA fragments with the expected sizes of ~500 bp were amplified by using CMV-specific primers; however, the expected amplicons were not produced using specific primers used for the detection of potyviruses and begomoviruses. The nucleotide and deduced amino acid sequences obtained for the isolates studied contained 503–511 nt and 144 aa, respectively. The isolates shared 81.9–85.3% nucleotide and 74.3–77.8% amino acid sequence identities with each other. The results of BLASTN analyses showed the highest identities of the isolates (80–93%) with CMV strains from Japan, USA and South Korea. Alignment of deduced partial protein revealed multiple amino acid substitutions within the three isolates and high identities with CMV subgroup I. Phylogenetic analyses putatively categorized the isolates in close association with subgroup IB isolates. The three isolates clustered together into a separate subclade, indicating possible new CMV strains. The results provide the first molecular evidence for CMV infections of T. fruticosum and B. alba in Nigeria and seem to show the possible presence of new strain(s). These findings also add three new hosts to the list of natural host range of the virus in Nigeria.
Antibiotics are a group of substances potentially harmful to the environment. They can play a role in bacterial resistance transfer among pathogenic and non-pathogenic bacteria. In this experiment three representatives of medically important chemotherapeutics, confirmed to be present in high concentrations in wastewater treatment plants with HPLC analysis were used: erythromycin, sulfamethoxazole and trimethoprim. Erythromycin concentration in activated sludge was not higher than 20 ng L−1. N-acetylo-sulfamethoxazole concentration was 3349 ± 719 in winter and 2933 ± 429 ng L−1 in summer. Trimethoprim was present in wastewater at concentrations 400 ± 22 and 364 ± 60 ng L−1, respectively in winter and summer. Due to a wide variety of PCR-detectable resistance mechanisms towards these substances, the most common found in literature was chosen. For erythromycin: erm and mef genes, for sulfamethoxazole: sul1, sul2, sul3 genes, in the case of trimethoprim resistance dhfrA1 and dhfr14 were used in this study. The presence of resistance genes were analyzed in pure strains isolated from activated sludge and in the activated sludge sample itself. The research revealed that the value of minimal inhibitory concentration (MIC) did not correspond with the expected presence of more than one resistance mechanisms. Most of the isolates possessed only one of the genes responsible for a particular chemotherapeutic resistance. It was confirmed that it is possible to monitor the presence of resistance genes directly in activated sludge using PCR. Due to the limited isolates number used in the experiment these results should be regarded as preliminary.
The present study investigated the expression of androgen receptor (AR) in neurons of the anterior pelvic ganglion (APG) and celiac-superior mesenteric ganglion (CSMG; ganglion not involved in the innervation of reproductive organs) in the male pig with quantitative real-time PCR (qPCR) and immunohistochemistry. qPCR investigations revealed that the level of AR gene expression in the APG tissue was approximately 2.5 times higher in the adult (180-day-old) than in the juvenile (7-day-old) boars. Furthermore, in both the adult and juvenile animals it was sig- nificantly higher in the APG than in CSMG tissue (42 and 85 times higher, respectively). Immu- nofluorescence results fully confirmed those obtained with qPCR. In the adult boars, nearly all adrenergic (DβH-positive) and the majority of non-adrenergic neurons in APG stained for AR. In the juvenile animals, about half of the adrenergic and non-adrenergic neurons were AR-posi- tive. In both the adult and juvenile animals, only solitary CSMG neurons stained for AR. The present results suggest that in the male pig, pelvic neurons should be considered as an element of highly testosterone-dependent autonomic circuits involved in the regulation of urogenital func- tion, and that their sensitization to androgens is a dynamic process, increasing during the prepu- bertal period.
Ammonia-oxidizing bacteria communities were evaluated in a completely mixed, laboratory scale membrane reactor (MBR) working under anoxic conditions for 5 months. The microorganisms in activated sludge were fed a synthetic medium containing 66-150 mg NH4 +-N/l. The age of the activated sludge in MBR was 50 days and the hydraulic retention time (HRT) was 3.3 days. The estimation of the diversity and complexity of the AOB community together with the identification of the dominant bacteria in the activated sludge under anoxic conditions were performed using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. Molecular analysis of the microbial community carried out with two microbial molecular markers, 16S rRNA gene and amoA gene, suggested that nitrification was led by a Nitrosomonas-like species. In the biocenosis of the investigated bioreactor, oxygen was the crucial selective parameter. The results obtained in this work showed that amoA gene research is more suitable to study the stability and effectiveness of ammonia oxidation. This information emphasizes the necessity of the usage of molecular markers based on functional genes instead of ribosomal ones in order to present the actual state of the process performed in bioreactors. It was also stated that Nitrosomonas -like bacteria are able to perform nitritation even in anoxic environment, that is probably the reason why these bacteria are the most common AOB in different bioreactors.