Browsing by Subject "qPCR"

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  • Malkamäki, S.; Näreaho, A.; Lavikainen, A.; Oksanen, A.; Sukura, A. (2019)
    Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10−5 ng/μl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables. © 2019 The Authors
  • Azinheiro, Sarah; Kant, Krishna; Shahbazi, Mohammad-Ali; Garrido-Maestu, Alejandro; Prado, Marta; Dieguez, Lorena (2020)
    Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ∼10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.
  • Rinta-Kanto, J. M.; Pehkonen, K.; Sinkko, H.; Tamminen, M. V.; Timonen, S. (2018)
    In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenar-chaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria.
  • Malkamäki, Sanna; Näreaho, Anu Susanna; Oksanen, Antti; Sukura, Antti Kalle Kalervo (2019)
    Potential role of wild forest berries as a transmission vehicle for taeniid eggs was examined using non-zoonotic Taenia laticollis eggs as a model. The berries studied were bilberries (Vaccinium myrtillus) (1 m(2) plot, n = 10) and lingonberries (Vaccinium vitis-idaea) (1 m(2) plot, n = 11). The plots in the managed forest were evenly sprayed with 30,000 or 60,000 T. laticollis eggs suspended in water, and berries were collected 24 h after spraying. The berries were rinsed with water, and the water was sieved through a 1-mm and a 63-mu m sieve to remove coarse material and through a 20-mu m sieve to collect possible eggs. A small proportion of the sieved material was examined by microscopy after treatment with fluorescent Calcofluor White stain, which binds to eggshell chitin. In the recovery tests in artificially spiked samples, the detection limit was 5 eggs in 100 g of commercial frozen bilberries and lingonberries. Taeniid eggs were detected in all of the 10 experimentally contaminated bilberry samples and in 10 of 11 lingonberry samples. The sieved debris was also analyzed for T. laticollis DNA using semiquantitative PCR. All samples were positive in quantitative SYBR Green real-time PCR using a T. laticollis-specific primer pair amplifying a short fragment of mitochondrial NADH dehydrogenase subunit 1 gene. This indicates that forest berries contaminated in shrubs contained T. laticollis eggs, and that berries can serve as a vehicle for taeniid eggs and may pose a possible risk to humans.
  • Pussila, Susanna (Helsingin yliopisto, 2014)
    The development of new antibiotics is very challenging work. However, it has become less attractive area of research for the pharmaceutical industry due to the rapid development of antibiotic resistance among bacteria. As a result, nowadays there are less new antimicrobial medicines appearing on the market. Increase in antibiotic resistance challenges the effective treatment of infectious diseases. Therefore, it is important to know about the existence and prevalence of antibiotic resistant bacteria and resistance genes. Waste water has been found to contain substantial amounts of antibiotics or their derivatives. Furthermore the bacterial density of the waste water is high. These factors may cause the selection pressure that assists the evolution, preservation and spread of antibiotic resistance in bacterial population. Wastewater treatment plants are believed to act as a reservoir of antibiotic resistant bacteria and antibiotic resistance genes. In this study, from samples taken from Viikinmäki wastewater treatment plant it was determined the quantity of resistance genes making bacteria resistance to a wide range of beta-lactam antibiotics. The samples were taken at the different stages of wastewater treatment process during three sampling days in June, September and December 2010. The samples were taken from the incoming wastewater and, from the final effluent and from the dried sludge. The DNA from wastewaters and sludge was isolated and the gene copy numbers of blaCTX-M-32 and blaOXA-58 antibiotic resistance genes were measured by quantitative PCR method. Antibiotic resistance gene copy numbers were normalized with 16S ribosomal RNA gene copy numbers. The antibiotic resistance genes investigated in this work make bacteria resistant to broad-spectrum penicillins, different groups of cephalosporins, monobactams, and carbapenems. It was found that blaOXA-58 and blaCTX-M-32 gene copies do exist in both incoming wastewater and dried sludge. From the incoming wastewater we found on average 3x10-3 blaOXA-58 gene copies/16S rRNA gene and 7x10-5 blaCTX-M-32 gene copies/16S rRNA gene. From dried sludge we found on average 3x10-3- blaOXA-58 gene copies/16S rRNA gene and 2x10-2 blaCTX-M-32 gene copies/16S rRNA. Both antibiotic resistance genes were also found in the final effluents. The amounts of antibiotic resistance genes /16S rRNA genes were found to decrease during of the wastewater treatment. In the final effluent the amount of blaOXA-58 genes was found to be two orders of magnitude less than in the incoming wastewater and for, blaCTX-M-32 gene copies it was found the decrease by one order of magnitude. Although the amount of antibiotic resistance genes decreased during of the wastewater treatment, the amount left in the final effluent is still notable and when it ends up in the water or soil it might have an effect to the antibiotic resistance in the nature.
  • Hyytiainen, Heidi K.; Jayaprakash, Balamuralikrishna; Kirjavainen, Pirkka V.; Saari, Sampo E.; Holopainen, Rauno; Keskinen, Jorma; Hämeri, Kaarle; Hyvarinen, Anne; Boor, Brandon E.; Taubel, Martin (2018)
    Background: Floor dust is commonly used for microbial determinations in epidemiological studies to estimate early-life indoor microbial exposures. Resuspension of floor dust and its impact on infant microbial exposure is, however, little explored. The aim of our study was to investigate how floor dust resuspension induced by an infant's crawling motion and an adult walking affects infant inhalation exposure to microbes. Results: We conducted controlled chamber experiments with a simplified mechanical crawling infant robot and an adult volunteer walking over carpeted flooring. We applied bacterial 16S rRNA gene sequencing and quantitative PCR to monitor the infant breathing zone microbial content and compared that to the adult breathing zone and the carpet dust as the source. During crawling, fungal and bacterial levels were, on average, 8- to 21-fold higher in the infant breathing zone compared to measurements from the adult breathing zone. During walking experiments, the increase in microbial levels in the infant breathing zone was far less pronounced. The correlation in rank orders of microbial levels in the carpet dust and the corresponding infant breathing zone sample varied between different microbial groups but was mostly moderate. The relative abundance of bacterial taxa was characteristically distinct in carpet dust and infant and adult breathing zones during the infant crawling experiments. Bacterial diversity in carpet dust and the infant breathing zone did not correlate significantly. Conclusions: The microbiota in the infant breathing zone differ in absolute quantitative and compositional terms from that of the adult breathing zone and of floor dust. Crawling induces resuspension of floor dust from carpeted flooring, creating a concentrated and localized cloud of microbial content around the infant. Thus, the microbial exposure of infants following dust resuspension is difficult to predict based on common house dust or bulk air measurements. Improved approaches for the assessment of infant microbial exposure, such as sampling at the infant breathing zone level, are needed.
  • Hyytiäinen, Heidi K; Jayaprakash, Balamuralikrishna; Kirjavainen, Pirkka V; Saari, Sampo E; Holopainen, Rauno; Keskinen, Jorma; Hämeri, Kaarle; Hyvärinen, Anne; Boor, Brandon E.; Täubel, Martin (BioMed Central, 2018)
    Abstract Background Floor dust is commonly used for microbial determinations in epidemiological studies to estimate early-life indoor microbial exposures. Resuspension of floor dust and its impact on infant microbial exposure is, however, little explored. The aim of our study was to investigate how floor dust resuspension induced by an infant’s crawling motion and an adult walking affects infant inhalation exposure to microbes. Results We conducted controlled chamber experiments with a simplified mechanical crawling infant robot and an adult volunteer walking over carpeted flooring. We applied bacterial 16S rRNA gene sequencing and quantitative PCR to monitor the infant breathing zone microbial content and compared that to the adult breathing zone and the carpet dust as the source. During crawling, fungal and bacterial levels were, on average, 8- to 21-fold higher in the infant breathing zone compared to measurements from the adult breathing zone. During walking experiments, the increase in microbial levels in the infant breathing zone was far less pronounced. The correlation in rank orders of microbial levels in the carpet dust and the corresponding infant breathing zone sample varied between different microbial groups but was mostly moderate. The relative abundance of bacterial taxa was characteristically distinct in carpet dust and infant and adult breathing zones during the infant crawling experiments. Bacterial diversity in carpet dust and the infant breathing zone did not correlate significantly. Conclusions The microbiota in the infant breathing zone differ in absolute quantitative and compositional terms from that of the adult breathing zone and of floor dust. Crawling induces resuspension of floor dust from carpeted flooring, creating a concentrated and localized cloud of microbial content around the infant. Thus, the microbial exposure of infants following dust resuspension is difficult to predict based on common house dust or bulk air measurements. Improved approaches for the assessment of infant microbial exposure, such as sampling at the infant breathing zone level, are needed.
  • Oikkonen, Hanna (Helsingin yliopisto, 2022)
    The use of recycled fibers in paper production has increased during recent years. Recycled fibers are a more sustainable alternative compared to virgin fibers made from wood. However, paper mills utilizing recycled fibers have more microbiological problems compared to mills using only virgin fibers. Especially, anaerobic bacteria are harmful for papermaking processes utilizing recycled fibers. Bacteria of the class Clostridia comprise a very diverse group and have many different metabolic properties. Bacteria of class Clostridia can ferment different substrates, for example cellulose and starch, crucial in paper mills utilizing recycled fibers. Fermentation does not only decrease material efficiency, but also the acids produced during fermentation deteriorate papermaking processes. Volatile fatty acids are odorous compounds causing bad odors in the mills and in the final products. Clostridia can also produce, for example, hydrogen which is an explosive gas endangering the safety of the mill employees. Quantitative PCR is a feasible detection method for microbes. Here, a qPCR method was developed for the detection of most abundant bacteria in the class Clostridia in the recycled fiber mills. The designed primers targeted the most harmful bacteria from the genera Clostridium, Ethanoligenens, Fonticella and Ruminococcus identified in the recycled fiber mills. Three primer sets were designed for the target bacterial group. Positive controls of each target bacterial genus was included and close relatives from class Bacilli were used as negative controls. The designed primer sets were compared in efficiency, specificity and performance with process samples collected from paper mills using recycled fibers. One of the primer sets was found the most potential for the qPCR detection method for the diverse target bacterial group. All positive controls were amplified with the designed qPCR assay, whereas the designed primers discriminated well each negative control in vitro. The applicability of the designed qPCR assay was yet confirmed with process samples collected from mills utilizing recycled pulp. Even though the efficiency of the designed primer set was not optimal, the designed assay was determined feasible for the detection of the target group in the recycled fiber mills usually high in bacterial density.
  • Mielonen, Outi I.; Pratas, Diogo; Hedman, Klaus; Sajantila, Antti; Perdomo , Maria (2022)
    Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (+/- paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63-159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.
  • Virtanen, Jenni; Aaltonen, Kirsi; Vapalahti, Olli; Sironen, Tarja (2020)
    Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), causes significant welfare problems to mink, and financial losses to the farmers. As there is no vaccine or treatment available, reliable diagnostics is important for disease control. Here, we set up a probe-based real-time PCR (NS1-probe-PCR) to detect all strains of AMDV. PCR was validated and compared to two other real-time PCR methods (pan-AMDV- and pan-AMDO-PCR) currently used for AMDV diagnostics in Finland. The NS1-probe-PCR had a similar detection limit of 20 copies/reaction based on plasmid dilution series, and similar or better diagnostic sensitivity, when evaluated using spleen samples from mink, and stool samples from mink and foxes. None of the three PCR tests cross-reacted with other parvoviruses. The NS1-probe-PCR also showed a significantly higher specificity than the pan-AMDO-PCR with spleen samples and the best specificity with stool samples. Furthermore, it produced the results more rapidly than the other two PCRs making it a promising tool for both diagnostic and research purposes.
  • Pulkkinen, Katja; Pekkala, Nina; Ashrafi, Roghaieh; Hamalainen, Dorrit M.; Nkembeng, Aloysius N.; Lipponen, Anssi; Hiltunen, Teppo; Valkonen, Janne K.; Taskinen, Jouni (2018)
    Understanding ecological and epidemiological factors driving pathogen evolution in contemporary time scales is a major challenge in modern health management. Pathogens that replicate outside the hosts are subject to selection imposed by ambient environmental conditions. Increased nutrient levels could increase pathogen virulence by pre-adapting for efficient use of resources upon contact to a nutrient rich host or by favouring transmission of fast-growing virulent strains. We measured changes in virulence and competition in Flavobacterium columnare, a bacterial pathogen of freshwater fish, under high and low nutrient levels. To test competition between strains in genotype mixtures, we developed a quantitative real-time PCR assay. We found that a virulent strain maintained its virulence and outcompeted less virulent strains independent of the nutrient level and resource renewal rate while a less virulent strain further lost virulence in chemostats under low nutrient level and over long-term serial culture under high nutrient level. Our results suggest that increased outside-host nutrient levels might maintain virulence in less virulent strains and increase their contribution to epidemics in aquaculture. The results highlight a need to further explore the role of resource in the outside-host environment in maintaining strain diversity and driving evolution of virulence among environmentally growing pathogens.
  • Tiwari, Ananda; Kauppinen, Ari; Räsänen, Pia; Salonen, Jenniina; Wessels, Laura; Juntunen, Janne; Miettinen, Ilkka T.; Pitkänen, Tarja (Elsevier BV, 2023)
    Science of The Total Environment
    Highlights •Coliphages decayed 22–27 times, and E. coli 4.8 times slower in 4 °C than 22 °C. •E. coli, enterococci, and som. coliphages were more sensitive to light than temperature. •Bacterial DNA/RNA, and Legionella were more sensitive to temperature than light. •Norovirus GII and F-specific coliphages were also more sensitive to temperature. •Indication power of fecal indicators can vary between seasons of the year. Abstract Knowledge of the decay characteristics of health-related microbes in surface waters is important for modeling the transportation of waterborne pathogens and for assessing their public health risks. Although water temperature and light exposure are major factors determining the decay characteristics of enteric microbes in surface waters, such effects have not been well studied in subarctic surface waters. This study comprehensively evaluated the effect of temperature and light on the decay characteristics of health-related microbes [Escherichia coli, enterococci, microbial source tracking markers (GenBac3 & HF183 assays), coliphages (F-specific and somatic), noroviruses GII and Legionella spp.] under simulated subarctic river water conditions. The experiments were conducted in four different laboratory settings (4 °C/dark, 15 °C/dark, 15 °C/light, and 22 °C/light). The T90 values (time required for a 90 % reduction in the population of a target) of all targets were higher under cold and dark (2.6–51.3 days depending upon targets) than under warm and light conditions (0.6–3.5 days). Under 4 °C/dark (simulated winter) water conditions, F-specific coliphages had 27.2 times higher, and coliform bacteria had 3.3 times higher T90 value than under 22 °C/light (simulated summer) water conditions. Bacterial molecular markers also displayed high variation in T90 values, with the greatest difference between 4 °C/dark and 22 °C/light recorded for HF183 DNA (20.6 times) and the lowest difference for EC23S857 RNA (6.6 times). E. coli, intestinal enterococci, and somatic coliphages were relatively more sensitive to light than water temperature, but F-specific coliphages, norovirus, and all bacterial rDNA and rRNA markers were relatively more sensitive to temperature than light exposure. Due to the slow microbial decay in winter under subarctic conditions, the microbial quality of river water might remain low for a long time after a sewage spill. This increased risk associated with fecal pollution during winter may deserve more attention, especially when river waters are used for drinking water production.
  • Jokinen, Maija (Helsingin yliopisto, 2019)
    Parvoviruses are among the smallest known viruses. The parvovirus genome is a single stranded DNA, approximately 5 kb in size. The virion has a small (20 to 30 nm), rugged, non-enveloped icosahedral capsid. Parvoviruses can cause a number of diseases. Possibly the most recognized human parvovirus is parvovirus B19 (B19V), which can cause the so-called fifth disease, anemias and fetal death. Another relatively well characterised parvovirus is human bocavirus 1 (HBoV1), which causes respiratory tract infections in young children. Bufavirus (BuV) tusavirus (TuV) and cutavirus (CuV) are emerging parvoviruses, discovered during the years 2012-2016 using next generation sequencing methods. All three viruses were originally discovered in feces of patients suffering from diarrhea. BuV was originally found in Burkina Faso and has since been detected in fecal samples with polymerase chain reaction (PCR)-based methods from Europe, Asia and Africa. The seroprevalence of BuV differs between countries. TuV was found in a single stool sample from Tunisia, but no further reports of it have since emerged. CuV was found in 2016 and it has been linked to cutaneous T-cell lymphoma, but it is not known if the virus is the cause of the cancer or if the virus simply prefers quickly dividing cancer cells for its replication. BuV, TuV and CuV belong to the Protoparvovirus genus, but it is still unclear whether TuV is a human pathogen. More research is needed to study the epidemiology of these viruses and their role in illnesses. There were two main aims in this thesis: to set up an IgM µ-capture enzyme immunoassay (EIA) for human protoparvoviruses using BuV1 as an example and to screen three stool sample cohorts for BuV, TuV and CuV using an in-house multiplex quantitative PCR (qPCR). The IgM EIAs developed for B19V and HBoV1 was used as the base for developing human protoparvovirus IgM EIA, using Virus-like particles (VLP) as antigens. Setting up the EIA required a great amount of optimization and finally troubleshooting, since the assay did not work as expected. The troubleshooting revealed that the ambiguous results in the IgM µ-capture EIA were possibly due to degraded VLPs or that the sensitive µ-capture format requires extremely carefully purified VLPs. More optimizing is needed for this assay, however, the work done in this thesis offers a good base for further development of protoparvovirus IgM EIA. All three viruses were found in the stool samples during multiplex qPCR screening. Based on the qPCR and sequencing results one sample was positive for BuV DNA, one sample for TuV DNA and a total of 12 samples for CuV DNA. This is the first time TuV DNA has been found since its discovery. In addition to that, CuV DNA was identified in fecal samples for the first time since the discovery, previously CuV DNA had been found mostly in skin biopsies. As for TuV, based on the parvovirus phylogenetic analyses, its sequence is more closely related to rodent parvoviruses than CuV or BuV. More research is needed, possibly with animal and human samples, to establish the role of TuV as a human virus.
  • Puntila-Dodd, R.; Bekkevold, D.; Behrens, J. W. (Springer, 2021)
    Hydrobiologia 848: 2
    Species invasions often occur on coasts and estuaries where abiotic conditions vary, e.g. salinity, temperature, runoff etc. Successful establishment and dispersal of non-indigenous species in many such systems are poorly understood, partially since the species tend to show genetic and ecological plasticity at population level towards many abiotic conditions, including salinity tolerance. Plasticity may be driven by shifting expression of heat shock proteins such as Hsp70, which is widely recognized as indicator of physical stress. In this study, we developed a qPCR assay for expression of the hsp70 gene in the invasive round goby (Neogobius melanostomus) and tested the expression response of fish collected from a brackish environment in the western Baltic Sea to three different salinities, 0, 10 and 30. hsp70 expression was highest in fresh water, indicating higher stress, and lower at brackish (ambient condition for the sampled population) and oceanic salinities, suggestive of low stress response to salinities above the population’s current distribution. The highest stress in fresh water was surprising since populations in fresh water exist, e.g. large European rivers and Laurentian Great Lakes. The results have implications to predictions for the species’ plasticity potential and possible range expansion of the species into other salinity regimes.
  • Verhagen, Irene; Laine, Veronika N.; Mateman, A. Christa; Pijl, Agata; de Wit, Ruben; van Lith, Bart; Kamphuis, Willem; Viitaniemi, Heidi M.; Williams, Tony D.; Caro, Samuel P.; Meddle, Simone L.; Gienapp, Phillip; van Oers, Kees; Visser, Marcel E. (2019)
    The timing of breeding is under selection in wild populations as a result of climate change, and understanding the underlying physiological processes mediating this timing provides insight into the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in the timing of breeding in females are reflected in differences in candidate gene expression and, if so, whether these differences occur in the upstream (hypothalamus) or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg laying, which were housed in climate-controlled aviaries and went through two breeding cycles within 1 year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on the timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with the timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues, with the most pronounced differences in the hypothalamus. Differences between time points, and early- and late-laying females, were found exclusively in the ovary and liver. Altogether, we show that fine-tuning of the seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.
  • Rosato, Giuliana; Ruiz Subira, Andres; Al-Saadi, Mohammed; Michalopoulou, Eleni; Verin, Ranieri; Dettwiler, Martina; Nordgren, Heli; Chiers, Koen; Grossmann, Ernst; Köhler, Kernt; Suntz, Michael; Stewart, James P.; Kipar, Anja (2021)
    The genus Macavirus, subfamily Gammaherpesvirinae, comprises ungulate viruses that infect domestic and wild ruminants and swine. They cause asymptomatic latent infections in reservoir hosts and malignant catarrhal fever in susceptible species. Lung, spleen, bronchial lymph node, and tongue were collected from 448 cattle (348 necropsied, 100 slaughtered) in Switzerland, United Kingdom, Finland, Belgium, and Germany to determine their infection with bovine herpesvirus-6 (BoHV-6) and gammaherpesviruses of other ruminants, i.e., ovine herpesvirus-1 and -2, caprine herpesvirus-2, and bison lymphotropic herpesvirus, using quantitative PCR. Only BoHV-6 was detected, with an overall frequency of 32%, ranging between 22% and 42% in the different countries. Infection was detected across all ages, from one day after birth, and was positively correlated with age. There was no evidence of an association with specific disease processes. In positive animals, BoHV-6 was detected in all organs with high frequency, consistently in the lungs or spleen. Viral loads varied substantially. In BoHV-6-positive gravid cows, organs of fetuses tested negative for infection, indicating that the virus is not vertically transmitted. Our results confirm previous data indicating that BoHV-6 is a commensal of domestic cattle not associated with disease processes and confirm that infections with other macaviruses are rare and sporadic.
  • Pyöriä, Lari; Jokinen, Maija; Toppinen, Mari; Salminen, Henri; Vuorinen, Tytti; Hukkanen, Veijo; Schmotz, Constanze; Elbasani, Endrit; Ojala, Päivi M.; Hedman, Klaus; Välimaa, Hannamari; Perdomo, Maria F. (2020)
    Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95 of similar to 10 to similar to 17 copies/reaction), with a dynamic range of 10' to 10 6 copies/p.I. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs. IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.
  • Lääveri, Tinja; Pakkanen, Sari H.; Antikainen, Jenni; Riutta, Jukka; Mero, Sointu; Kirveskari, Juha; Kantele, Anu (2014)
  • Gawriyski, Lisa (Helsingin yliopisto, 2018)
    Life historyresearch seeks to explain how natural selection and ecological challenges shape organisms to optimize their fitness. A strong immune defense is energetically demanding to upkeep and there may be trade-offs among other life history traits. Investing a lot of energy to upkeep a strong immune defense in conditions where there are less pathogens and parasites might have negative fitness effects. Heliconius eratois a neotropical species of butterfly found widely in South America. The immune defense, ecologicalfactors affecting its immune defense, and possible life history trade-offs of the butterfly are currently not well known. Environmental moisture conditions have been shown to affect the diversity, quality and amount of microorganisms and parasites. The aim of this thesis was to use real-time quantitative PCR (RT-qPCR) to quantify immune gene expression of individuals of the butterfly species Heliconius eratocollected from different environmental moisture conditions. Additionally, individual variation in encapsulation rates, a physiological measure of immunity, was compared across the moisture gradient. Results indicate reduced expression of the gene encoding the antimicrobial peptide attacin in dry conditions, but no difference in encapsulation rates across the moisture gradient. Additionally, differential expression of the prophenoloxidase encoding gene was found between male and female butterflies. These results indicate a possibility of differential immune threats in different environmental moisture conditions in H. erato, but requires further study.
  • König, Walter; Konig, Emilia; Weiss, Kirsten; Tuomivirta, Tero T.; Fritze, Hannu; Elo, Kari; Vanhatalo, Aila; Jaakkola, Seija (2019)
    BACKGROUND Nitrite and hexamine are used as silage additives because of their adverse effects on Clostridia and Clostridia spores. The effect of sodium nitrite and sodium nitrite/hexamine mixtures on silage quality was investigated. A white lupin-wheat mixture was treated with sodium nitrite (NaHe0) (900 g t(-1) forage), or mixtures of sodium nitrite (900 g t(-1)) and hexamine. The application rate of hexamine was 300 g t(-1) (NaHe300) or 600 g t(-1) (NaHe600). Additional treatments were the untreated control (Con), and formic acid (FA) applied at a rate of 4 L t(-1) (1000 g kg(-1)). RESULTS Additives improved silage quality noticeably only by reducing silage ammonia content compared with the control. The addition of hexamine to a sodium nitrite solution did not improve silage quality compared with the solution containing sodium nitrite alone. The increasing addition of hexamine resulted in linearly rising pH values (P <0.001) and decreasing amounts of lactic acid (P <0.01). Sodium nitrite based additives were more effective than formic acid in preventing butyric acid formation. Additives did not restrict the growth of Saccharomyces cerevisiae compared to the control. CONCLUSION The addition of hexamine did not improve silage quality compared with a solution of sodium nitrite. (c) 2018 Society of Chemical Industry