Detection of Polymyxa graminis in a barley crop in Australia.

Polymyxa graminis was detected in the roots of barley plants from a field near Wondai, Queensland, in 2009. P. graminis was identified by characteristic sporosori in roots stained with trypan blue. The presence of P. graminis f. sp. tepida (which is hosted by wheat and oats as well as barley) in the roots was confirmed by specific PCR tests based on nuclear ribosomal DNA. P. graminis is the vector of several damaging soil-borne virus diseases of cereals in the genera Furovirus, Bymovirus and Pecluvirus. No virus particles were detected in sap extracts from leaves of stunted barley plants with leaf chlorosis and increased tillering. Further work is required to determine the distribution of P. graminis in Australian grain crops and the potential for establishment and spread of the exotic soil-borne viruses that it vectors.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.

Panel of real-time PCRs for the multiplexed detection of two tomato-infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real-time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus-Israel (TYLCV-IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B.tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality-assurance purposes, two internal control assays were included in the assay panel for the co-amplification of solanaceous plant DNA or B.tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV-IL, 100 plasmid copies of ToLCV, 500fg B.tabaci MEAM1 and 300fg B.tabaci MED DNA. Evaluated methods for routine testing of field-collected whiteflies are presented, including protocols for processing B.tabaci captured on yellow sticky traps and for bulking of multiple B.tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality-assured diagnostic method for the identification and discrimination of tomato-infecting begomovirus and B.tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease-management programmes both in Australia and worldwide.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 ILL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles. (c) 2007 Elsevier B.V. All rights reserved.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Development of a probe-based blotting technique for the detection of Tobacco streak virus

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the Tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.