The role of Mycoplasma canis in the pathogenesis of infertility and urogenital diseases in dogs

Namen doktorske naloge je bil razjasniti pomembnost okužbe z M. canis pri psih v patogenezi obolenj sečil in spolovil in z njimi povezanih plodnostnih motenj. V klinično študijo smo vključili 52 psov, od tega 7 psov z znaki obolenj sečil in spolovil, 14 psov s kliničnimi znaki plodnostnih motenj in 31 psov brez kliničnih znakov okužbe. Povzročitelja smo dokazovali s pomočjo PCR (angl. polymerase chain reaction) metode v vaginalnih in prepucialnih brisih psov. Analizo sestave celotne bakterijske raznovrstnosti v vzorcih brisov smo nadgradili z metagenomskim pristopom. Prisotnost specifičnih protiteles smo ugotavljali z metodo DIBA (angl. dot immunobinding assay) in rezultate potrdili z metodo po Westernu. Pri pacientih smo poleg kliničnih znakov bolezni ugotavljali odstopanja v hematoloških in izbranih biokemijskih parametrih v krvi, odstopanja v analizi urina ter specifičnih parametrih analize plodnostnih motenj in skupine medsebojno primerjali. Z metodo PCR smo M. canis dokazali pri 3/7 (43 %) pacientov z obolenji sečil in spolovil, pri 6/14 (43 %) pacientih s plodnostnimi motnjami in pri značilno nižjem deležu 6/31 (19 %) zdravih psov iz kontrolne skupine. Z metagenomsko analizo bakterijske sestave med skupinami nismo ugotovili značilnih razlik. Prisotnost specifičnih protiteles na vsaj 1 od 5 izbranih sevov smo z metodo DIBA ugotovili pri 54/55 (98 %) vseh vzorcev. Pri vseh pacientih, razen pri 2 iz kontrolne skupine, smo zabeležili reakcijo na sev M. canis Larissa. Specifičnost reakcije smo potrdili z metodo po Westernu. Rezultati doktorske naloge so nam v pomoč pri izboru ustreznih diagnostičnih metod, planu zdravljenja in spremljanju odgovora nanj pri pacientih s ponavljajočimi se vnetnimi obolenji sečil in spolovil ali plodnostnimi motnjami. Serološki presejalni testi so smiselni, vsaj pri pacientih s plodnostnimi motnjami. Za dokaz okužbe pa je najprimernejša uporaba specifčne metode PCR za dokazovanje M. canis.The aim of this dissertation was to investigate the role of M. canis infection in dogs with urogenital diseases and with fertility problems. The clinical study included 52 dogs7 dogs with urogenital disease symptoms, 14 dogs with fertility problems, and 31 healthy dogs. The pathogen was detected by PCR (polymerase chain reaction) method on vaginal and preputial swabs. Swab samples were analysed to determine the full bacterial spectrum, and these analyses were extended by metagenomic analysis. The presence of antibodies was tested by the Dot-immunobinding assay (DIBA) method, while the results were confirmed by the Western Blot method. In addition to clinical signs, we looked for abnormalities in haematological and biochemical parameters in blood, urinalysis, and specific parameters of fertility disorders, and compared the groups with each other. M. canis was detected by PCR method in 3/7 (43 %) patients with urogenital disorders, in 6/14 (43 %) patients with fertility disorders, and in a statistically significantly lower proportion of healthy dogs in the control group (6/31, 19 %). Metagenomic analysis of bacterial composition showed no significant differences between the groups. The DIBA method showed the presence of specific antibodies against at least one of the five selected strains in 54/55 samples (98 %). With all patients, except two from the control group, we detected a reaction to the M. canis Larissa strain. The results of the DIBA test were confirmed by the Western Blot method. The results of the thesis will help us to select appropriate diagnostic methods and treatment plan and to monitor the response to treatment in canine patients with chronic urinary tract diseases or fertility disorders. The results show that serological screening tests are useful at least for patients with fertility disorders. The specific PCR method is the most suitable for the detection of M. canis infections

Characteristics and limitations of web design

V diplomskem delu so opisani dejavniki, ki vplivajo na načrtovanje, oblikovanje in razvoj spletnih vsebin. Pasovna širina, zaslonska ločljivost in vrsta brskalnika so le nekatere izmed njih. Predstavljeni sta glavni tehniki zasnove spletnega mesta, še posebej izpostavljen pa je problem uporabe poljubne tipografije na spletu. Besedilo analizira podporo transparence, izpostavlja tehnologije za povečevanje interaktivnosti kot so Flash in Ajax, preučuje značilnosti in omejitve oblikovanja spletnih pasic in promocijskih e-sporočil, razkriva značilnosti Web 2.0, 3.0 in 4.0, za konec pa se poda v odkrivanje prihajajočih trendov spletnega oblikovanja. Diplomsko delo poleg pregleda dejavnikov, ki služijo kot izhodišče za uspešno spletno oblikovanje, izpostavlja nekatere aktualne probleme spleta in za njih ponuja možne rešitve.The final thesis describes factors (such as bandwidth, screen resolution, type of a browser, etc...), that influence the planning, designing and developing process of a web site. We compare two main web layout techniques and highlight the problem of using an optional font on the web. The work analyzes transparency support, points out the technologies used to increase interactivity, such as Flash and Ajax, and studies characteristics and limitations of banner and newsletter design. It exposes characteristics of Web 2.0, 3.0 and 4.0 and finishes with uncovering the future web design trends. Not only is the thesis an overview of the factors, that serve as a starting-point for successful web design, it is also highlighting some of the current web design problems for which it offers possible solutions

Straw management and slurry application affect the soil microbial community composition and its activity

In low-input farming systems, where plant nutrition strongly depends on either organic matter inputs or nitrogen fixation by legumes, it is crucial to understand the effects of different crop residue management strategies on microbially mediated processes that influence nutrient dynamics. To evaluate different straw management options, we conducted a pot experiment with the following treatments: (1) control with no straw added (CON), (2) applying straw to the soil surface (SRF), simulating no-till, and (3) straw incorporating straw to the top 10 cm (INC), simulating minimum tillage. Another factor was slurry fertilization (with 3.2 t d.m. ha$^{-1}$ or without). Throughout the experiment, CO$_2$ efflux ([mu]mol/m$^2$/s) was measured regularly over a period of four months. Soil sampling was performed to monitor the abundance of the total bacterial, crenarchaeal and fungal communities, as well as nitrogen cycle microbial guilds and the dynamics of labile N and C fractions in the soils at different depths. Among straw treatments, significantly higher soil respiration was observed in INC and SRF compared to CON. Slurry fertilization increased soil respiration in all straw treatments. In the top 10 cm soil layer, the fungal abundance increased in INC compared to SRF and CON, whereas bacteria and crenarchaea did not respond. This effect was even more pronounced with slurry addition. The slurry also stimulated the abundance of certain N-cycle guilds, with the greatest increase in bacterial amoA-bearing nitrifiers and nirS, nosZI and nozII-bearing denitrifiers, particularly in the uppermost 0–1 cm soil layer. Straw treatment effects on N-cycle microbial guilds were less pronounced, while the coupled effect of straw incorporation and slurry was beneficial mostly for nosZ, again mainly in the top 1 cm soil layer. Compared to straw management, slurry fertilization had more potent effects on the abundance and performance of the investigated microbial communities

Plants play a crucial role in the development of soil fungal communities in the remediated substrate after EDTA washing of metal-contaminated soils

In this study, we investigated the importance of plant cover for secondary succession and soil fungal community development in remediated substrates after EDTA washing of metal-contaminated soils. The abundance of the total fungal community, determined by ITS fungal marker genes (Internal Transcribed Spacer region), and root colonisation by arbuscular mycorrhizal (AM) fungi were monitored in two types of soil material (calcareous and acidic) sown with perennial ryegrass (Lolium perenne L.) and without plant cover (bulk soil). Four months after the start of the experiment, the abundance of ITS genes in the soil clearly showed that the presence of plants was the main factor affecting the total fungal community, which increased in the rhizosphere soil in most treatments, while it remained at a low level in the bulk soil (without plants). Interestingly, the addition of environmental inoculum, i.e., rhizosphere soil from a semi-natural meadow, did not have a positive effect on the abundance of the total fungal community. While fungal ITS genes were detected in soils at the end of the first growing season, arbuscular mycorrhizal (AM) structures were scarce in Lolium roots in all treatments throughout the first season. However, in the second season, more than a year after the start of the experiment, AM fungal colonisation was detected in Lolium roots in virtually all treatments, with the frequency of colonised root length ranging from 30% to >75% in some treatments, the latter also in remediated soil. This study demonstrates the importance of plants and rhizosphere in the development and secondary succession of fungal communities in soil, which has important implications for the revitalisation of remediated soils and regenerative agriculture

Effects of transitioning from conventional to organic farming on soil organic carbon and microbial community

The combination of conservation tillage (non-inversion and no-till) with organic farming is rare due to weed problems. However, both practices have the potential to improve soil quality and increase soil organic C (SOC). This study investigated the changes in SOC, microbial biomass, and microbial composition during the transition from conventional to organic farming (from 2014 to 2020) in a long-term tillage trial established in 1999. Non-inversion minimum tillage to a depth of 10 cm (MT) resulted in SOC stratification, whilst conventional soil tillage with 25-cm-deep mouldboard ploughing (CT) maintained an even SOC distribution in the plough layer. After 12 years of contrasting tillage in 2011, the uppermost soil layer under MT had a 10% higher SOC content (1.6% w/w) than CT (1.45% w/w). This difference became even more pronounced after introducing organic farming in 2014. By the fall of 2020, the SOC content under MT increased to 1.94%, whilst it decreased slightly to 1.36% under CT, resulting in a 43% difference between the two systems. Conversion to organic farming increased microbial biomass under both tillage systems, whilst SOC remained unchanged in CT. Abundances of total bacterial and Crenarchaeal 16S rRNA and fungal ITS genes indicated shifts in the microbial community in response to tillage and depth. Fungal communities under MT were more responsive to organic farming than bacterial communities. The improved soil quality observed under MT supports its adoption in both organic and conventional systems, but potentially large yield losses due to increased weed cover discourage farmers from combining MT and organic farming

Association of Mycoplasma canis with fertility disorders in dogs

The role of Mycoplasma canis in canine fertility disorders is still poorly understood. As infection is often asymptomatic, there is an increasing need for appropriate diagnostic methods and treatment plans that would allow the reliable detection of M. canis infection and rapid alleviation of infection symptoms in affected dogs. In this study, we included 14 dogs with fertility problems and 16 dogs without fertility disorder signs. We compared clinical examination data and selected laboratory parameters (hematology and biochemistry) between the groups. We performed PCR-based detection of M. canis and 16S rRNA gene-based microbiota profiling of DNA isolated from vaginal and preputial swabs. Dog sera were tested for the presence of M. canis-specific antibodies. Hematological and selected biochemical parameters showed no differences between groups. PCR-based detection of M. canis in the samples was consistent with the results of 16S microbiota profiling. Several other bacterial taxa were also identified that could potentially be involved in different fertility disorders. Serological methods were not accurate enough since high cross-reactivity rates were observed. In the future, more accurate and efficient methods will be needed to determine the role of M. canis and its true role in the pathogenesis of specific fertility disorders in dogs

Trade-offs and synergies of soil carbon sequestration

Soil organic carbon (SOC) sequestration in agricultural soils is an importanttool for climate change mitigation within the EU soil strategy for 2030 and canbe achieved via the adoption of soil management strategies (SMS). These strategies may induce synergistic effects by simultaneously reducing greenhousegas (GHG) emissions and/or nitrogen (N) leaching. In contrast, other SMS maystimulate emissions of GHG such as nitrous oxide (N$_2$O) or methane (CH$_4$),offsetting the climate change mitigation gained via SOC sequestration. Despitethe importance of understanding trade-offs and synergies for selecting sustain-able SMS for European agriculture, knowledge on these effects remains lim-ited. This review synthesizes existing knowledge, identifies knowledge gapsand provides research recommendations on trade-offs and synergies betweenSOC sequestration or SOC accrual, non-CO$_2$ GHG emissions and N leachingrelated to selected SMS. We investigated 87 peer-reviewed articles that addressSMS and categorized them under tillage management, cropping systems, watermanagement and fertilization and organic matter (OM) inputs. SMS, such as conservation tillage, adapted crop rotations, adapted water management, OMinputs by cover crops (CC), organic amendments (OA) and biochar, contributeto increase SOC stocks and reduce N leaching. Adoption of leguminous CC orspecific cropping systems and adapted water management tend to create trade-offs by stimulating N$_2$O emissions, while specific cropping systems or application of biochar can mitigate N$_2$O emissions. The effect of crop residues on N$_2$O emissions depends strongly on their C/N ratio. Organic agriculture and agro-forestry clearly mitigate CH$_4$ emissions but the impact of other SMS requiresadditional study. More experimental research is needed to study the impact ofboth the pedoclimatic conditions and the long-term dynamics of trade-offs andsynergies. Researchers should simultaneously assess the impact of (multiple)agricultural SMS on SOC stocks, GHG emissions and N leaching. This reviewprovides guidance to policymakers as well as a framework to design fieldexperiments and model simulations, which can address knowledge gaps andnon-intentional effects of applying agricultural SMS meant to increase SOC sequestration

MOLECULAR DETECTION AND SEROPREVALENCE OF MYCOPLASMAS IN CLINICALY HEALTHY WORKING DOGS

In this study seroprevalence and prevalence of mycoplasmas in clinically healthy dogs were studied. Thirty-four working dogs of various breeds, gender and age were included in this research. Among them, 27 were working dogs from Slovene armed forces and 7 were working sheepdogs. We used dot-immunobinding assay (DIBA) as a serological test for the detection of specific antibodies to Mycoplasma cynos, Mycoplasma canis and Mycoplasma molare and consensus PCR for detection of genes for 16S rRNA or 16S/23S IGS region of mycoplasmas. Specific antibodies against at least one of the canine mycoplasmas were detected in 94.1% dogs. Of them 23.5% samples showed positive reaction only to M. cynos, 20.6% were positive only to M. canis and none of the samples were positive only to M. molare. Altogether 47.0% of samples were positive to M. cynos and M. canis whereas only one dog (2.9%) had specific antibodies to all three mycoplasmas tested. The presence of mycoplasmas detected by PCR was 57.14% in younger dogs (≤ 1 year) and 18.52% to 35.29% in older dogs, depending on year of the sampling. Genital swabs were positive in more cases (60%) in comparison with oral swabs (46.67%). M. canis was detected in 40% of positive cases, in the same percent of samples mixed not determined mycoplasma infections were confirmed. Mycoplasma species such as:  M. cynos, M. edwardii, M. maculosum, M. spumans were determined each in single cases and in one case mixed ureaplasma infection was confirmed.   MOLEKULARNA DETEKCIJA IN SEROPREVALENCA MIKOPLAZEM PRI KLINIČNO ZDRAVIH DELOVNIH PSIH Namen raziskave je bil določiti seroprevalenco in prevalenco mikoplazem pri klinično zdravih delovnih psih. V raziskavo je bilo vključenih 34 delovnih psov različnih pasem in starosti, od tega 27 psov iz Slovenske vojske in 7 ovčarskih psov. Za dokazovanje specifičnih protiteles proti bakterijam Mycoplasma cynos, Mycoplasma canis in Mycoplasma molare smo uporabili metodo točkastega imunskega odtisa (ang. Dot Immuno Binding Assay- DIBA) in konvencionalni PCR, ki temelji na pomnoževanju odseka gena za ribosomalno RNK 16s ali intergenskega odseka genoma med genoma ribosomalnih RNK 16s in 23s. Specifična protitelesa proti vsaj eni od izbranih vrst mikoplazem so bila ugotovljena pri 94.1 % psov. Med njimi je 23.5 % vzorcev reagiralo pozitivno samo na M. canis, 20.6 % samo na M. canis in noben od vzorcev ni reagiral pozitivno samo na M. molare. Skupno je 47.0 % vzorcev reagiralo pozitivno na M. canis in M. canis hkrati, en pes (2.9 %) je imel specifična protitelesa proti vsem trem testiranim mikoplazmam. Z metodo PCR smo mikoplazme dokazali v vzorcih 57.1 % psov mlajših od enega leta, in pri 18.5 % do 35.3 % starejših od enega leta, odvisno od leta vzorčenja. Genitalni brisi so bili pozitivni v 60 % primerov v primerjavi z oralnimi kjer je bil delež 46.7 %. M. canis je bila ugotovljena v 40 % pozitivnih primerov, v enakem deležu so bile ugotovljene tudi mešane nedeterminirane mikoplazemske okužbe. Mikoplazme, kot so M. cynos, M. edwardii, M. maculosumin, M. spumans so bile ugotovljene posamično. V enem primeru je bila ugotovljena mešana okužba z ureaplazmami. Ključne besede: delovni psi; pasje mikoplazme; Mycoplasma canis; Mycoplasma cynos; DIBA; PC

Structure and physical properties of the tectosphere in the metastable Europe

The Earth's structure and the geod) nam ic processes that originate it are strictly interconnected: an accurate knowledge of the Earth·s structure and its physical properties is fundamental for the understanding of the dynamics of our planet and critical for realistic simulations of seismic ground motion. On the other side the separation and the definition of the different dynamic processes acting in a certain area help to better understand the structure and the physical properties of the investigated region. In recent years much emphasis has been given to the inversion or observed phase and waveform data for the best fitting set or model parameters at a variety of scale-lengths