Separation of Gas Bubbles from Circulation Waters and Pulp Suspensions in Open Channel Flow

The objective of this thesis was to study the removal of gases from paper mill circulation waters experimentally and to provide data for CFD modeling. Flow and bubble size measurements were carried out in a laboratory scale open gas separation channel. Particle Image Velocimetry (PIV) technique was used to measure the gas and liquid flow fields, while bubble size measurements were conducted using digital imaging technique with back light illumination. Samples of paper machine waters as well as a model solution were used for the experiments. The PIV results show that the gas bubbles near the feed position have the tendency to escape from the circulation channel at a faster rate than those bubbles which are further away from the feed position. This was due to an increased rate of bubble coalescence as a result of the relatively larger bubbles near the feed position. Moreover, a close similarity between the measured slip velocities of the paper mill waters and that of literature values was obtained. It was found that due to dilution of paper mill waters, the observed average bubble size was considerably large as compared to the average bubble sizes in real industrial pulp suspension and circulation waters. Among the studied solutions, the model solution has the highest average drag coefficient value due to its relatively high viscosity. The results were compared to a 2D steady sate CFD simulation model. A standard Euler-Euler k-ε turbulence model was used in the simulations. The channel free surface was modeled as a degassing boundary. From the drag models used in the simulations, the Grace drag model gave velocity fields closest to the experimental values. In general, the results obtained from experiments and CFD simulations are in good qualitative agreement.

A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

Effect of extracts from citric biomass, rusted coffee leaves and coffee berry husks on Phoma costarricencis of coffee plants

Phoma leaf spot, caused by Phoma costarricensis poses a serious threat to coffee (Coffea arabica) production, especially in the highlands of the state of Minas Gerais, Brazil. Extracts of citric biomass, coffee berry husks and coffee leaves severely affected by rust caused by Hemileia vastatrix, were evaluated against P. costarricensis. In an in vitro assay, aqueous extracts of rusted leaves and berry husks plus the commercial extracts based on citric biomass named Ecolife® and Agromil® were tested at various dilutions on the mycelial growth inhibition of P. costarricensis. In vivo, coffee seedlings maintained in glasshouse, were sprayed with these extracts seven days before inoculation of P. costarricensis. Only extracts from citric biomass had inhibitory effects on the fungus. In vivo, Ecolife® (5 ml/l), Agromil® (5 g/l) and the aqueous extract of rusted coffee leaves (dilution 1:6) reduced Phoma leaf spot. Both, Ecolife® and the extract of rusted coffee leaves were significantly more effective in reducing the area under the lesion progress curve when applied at lower doses, indicating a possible effect on the induction of resistance.

Direct determination of calcium in milk by atomic absorption spectrometry using flow-injection analysis

A flow-injection system with sample and reagent addition by the synchronous merging zones approach for calcium determination in milk by flame AAS is proposed. Main parameters were optimized using a factorial design with central point. The optimum conditions were 2.5% (m/v) for La concentration, 8 mL min-1 for the carrier flow-rate, 20 cm for coiled reactor and 250 ìL for sample volume. Different sample preparation procedures were evaluated such as dilution in water or acid and microwave-assisted decomposition using concentrated or diluted acids. The optimized flow system was applied to determine Ca in eleven commercial milk samples and two standard reference materials diluted in water. Similar calcium levels were encountered comparing the results obtained by the proposed method (dilution in water) with those obtained using microwave-oven digestion. Results obtained in two standard reference materials were in agreement at 95% confidence level with those certified. Recoveries of spiked samples were in the 93% - 116% range. Relative standard deviation (n = 12) was < 5.4% and the sample throughput was 150 measurements per hour, corresponding to a consumption of 250 µL of sample and 6.25 mg La per determination.

Distribution and role of macrophytes in coastal lagoons : implications of critical shifts

Crossroads, crucibles and refuges are three words that may describe natural coastal lagoon environments. The words refer to the complex mix of marine and terrestrial influences, prolonged dilution due to the semi-enclosed nature and the function of a habitat for highly diverse plant and animal communities, some of which are endangered. To attain a realistic picture of the present situation, high vulnerability to anthropogenic impact should be added to the description. As the sea floor in coastal lagoons is usually entirely photic, macrophyte primary production is accentuated compared with open sea environments. There is, however, a lack of proper knowledge on the importance of vegetation for the general functioning of coastal lagoon ecosystems. The aim of this thesis is to assess the role of macrophyte diversity, cover and species identity over temporal and spatial scales for lagoon functions, and to determine which steering factors primarily restrict the qualitative and quantitative composition of vegetation in coastal lagoons. The results are linked to patterns of related trophic levels and the indicative potential of vegetation for assessment of general conditions in coastal lagoons is evaluated. This thesis includes five field studies conducted in flads and glo-flads in the brackish water northern Baltic Sea. Flads and glo-flads are defined as a Baltic variety of coastal lagoons, which due to an inlet threshold and post-glacial landuplift slowly will be isolated from the open sea. This process shrinks inlet size, increases exposure and water retention, and is called habitat isolation. The studied coastal lagoons are situated in the archipelago areas of the eastern coast of Sweden, the Åland Islands and the south-west mainland of Finland, where land-uplift amounts to ca. 5 mm/ per year. Out of 400 evaluated sites, a total of 70 lagoons varying in inlet size, archipelago position and anthropogenic influence to cover for essential environmental variation were chosen for further inventory. Vegetation composition, cover and richness were measured together with several hydrographic and morphometric variables in the lagoons both seasonally and inter-annually to cover for general regional, local and temporal patterns influencing lagoon and vegetation development. On smaller species-level scale, the effects of macrophyte species identity and richness for the fish habitat function were studied by examining the influence of plant interaction on juvenile fish diversity. Thus, the active election of plant monoand polycultures by fish and the diversity of fish in the respective culture were examined and related to plant height and water depth. The lagoons and vegetation composition were found to experience a regime shift initiated by increased habitat isolation along with land-uplift. Vegetation composition altered, richness decreased and cover increased forming a less isolated and more isolated regime, named the vascular plant regime and charophyte regime, respectively according to the dominant vegetation. As total phosphorus in the water, turbidity and the impact of regional influences decreased in parallel, the dominance of charophytes and increasing cover seemed to buffer and stabilize conditions in the charophyte regime and indicated an increased functional role of vegetation for the lagoon ecosystem. The regime pattern was unaffected by geographical differences, while strong anthropogenic impact seemed to distort the pattern due to loss of especially Chara tomentosa L. in the charophyte regime. The regimes were further found unperturbed by short-time temporal fluctuations. In fact the seasonal and inter-annual dynamics reinforced the functional difference between the regimes by the increasing role of vegetation along habitat isolation and the resemblance to lake environments for the charophyte regime. For instance, greater total phosphorus and chlorophyll a concentrations in the water in the beginning of the season in the charophyte regime compared with the vascular plant regime presented a steeper reduction to even lower values than in the vascular plant regime along the season. Despite a regional importance and positive relationship of macrophyte diversity in relation to trophic diversity, species identity was underlined in the results of this thesis, especially with decreasing spatial scale. This result was supported partly by the increased role of charophytes in the functioning of the charophyte regime, but even more explicitly by the species-specific preference of juvenile fish for tall macrophyte monocultures. On a smaller species-level scale, tall plant species in monoculture seemed to be able to increase their length, indicating that negative selection forms preferred habitat structures, which increase fish diversity. This negative relationship between plant and fish diversity suggest a shift in diversity patterns among trohic levels on smaller scale. Thus, as diversity patterns seem complex and diverge among spatial scales, it might be ambiguous to extend the understanding of diversity relationships from one trophic level to the other. All together, the regime shift described here presents similarities to the regime development in marine lagoon environments and shallow lakes subjected to nutrient enrichment. However, due to nutrient buffering by vegetation with increased isolation and water retention as a consequence of the inlet threshold, the development seems opposite to the course along an eutrophication gradient described in marine lagoons lacking an inlet threshold, where the role of vegetation decreases. Thus, the results imply devastating consequences of inlet dredging (decreasing isolation) in terms of vegetation loss and nutrient release, and call for increased conservational supervision. Especially the red listed charophytes would suffer negatively from such interference and the consequences are likely to also deteriorate juvenile fish production. The fact that a new species to Finland, Chara connivens Salzm. Ex. Braun 1835 was discovered during this study further indicates a potential of the lagoons serving as refuges for rare species.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli Campylobacters are a common cause of bacterial gastroenteritis worldwide, with Campylobacter jejuni and C. coli being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the C. jejuni and C. coli strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were in vitro highly effective towards Campylobacter species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected Campylobacter enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Vuosihuollon aikainen jäähdytteen boorihappopitoisuuden laimeneminen Loviisan ydinvoimalaitoksen riskianalyysissä

This master's thesis examines scenarios, which can lead to a reactivity excursion due to boron dilution events in Loviisa nuclear power plant. This thesis also describes how the boron diluted slugs are modelled in the Probabilistic Risk Assessment (PRA) model. At the current model, the valuation of the reactivity risk due to boron dilution has been very conservative, and therefore the reactivity risk during an outage is as much as 9 % of Core Damage Frequency (CDF) and Large Release Frequency (LRF). The main objective of the thesis is to decrease the annual core damage and Large Release Frequency by reducing conservative assumptions in the probabilistic modelling of boron dilution events. The core behavior during boron dilution events was modelled and reported in 2011 by Fortum using three-dimensional core model of Apros. The results of these analyses were reported same year by Fortum. According to the reported results and the analyses made by StarNode visualization program, it seems that some changes could be made in the boron dilution fault trees of the PRA model. As a result, the reactivity risk was decreased to 0.7 % of the annual CDF. In the other words, the total annual CDF and LRF were decreased 8.5 % due to the changes made in the PRA model. However the analyses made by Apros include some uncertainty. The accuracy of the analyses should be validated before making these changes in the official PRA model.

Spin-Dynamics in Cold Gases of 87Rb and Atomic Hydrogen. The spins they are a-changin’

In this thesis the dynamics of cold gaseous atoms is studied. Two different atomic species and two different experimental techniques have been used. In the first part of the thesis experiments with Bose-Einstein condensates of Rb-87 are presented. In these experiments the methods of laser cooling and magnetic trapping of atoms were utilized. An atom chip was used as the experimental technique for implementation of magnetic trapping. The atom chip is a small integrated instrument allowing accurate and detailed manipulation of the atoms. The experiments with Rb-87 probed the behaviour of a falling beam of atoms outcoupled from the Bose-Einstein condensate by electromagnetic field induced spin flips. In the experiments a correspondence between the phases of the outcoupling radio frequency field and the falling beam of atoms was found. In the second part of the thesis experiments of spin dynamics in cold atomic hydrogen gas are discussed. The experiments with atomic hydrogen are conducted in a cryostat using a dilution refrigerator as the cooling method. These experiments concentrated on explaining and quantifying modulations in the electron spin resonance spectra of doubly polarized atomic hydrogen. The modifications to the previous experimental setup are described and the observation of electron spin waves is presented. The observed spin wave modes were caused by the identical spin rotation effect. These modes have a strong dependence on the spatial profile of the polarizing magnetic field. We also demonstrated confinement of these modes in regions of strong magnetic field and manipulated their spatial distribution by changing the position of the field maximum.

Testate amoebae (thecamoebians) as indicators of aquatic mine impact

The environmental impacts of a single mine often remain local, but acidic and metal-rich acid mine drainage (AMD) from the waste materials may pose a serious threat to adjacent surface waters and their ecosystems. Testate amoebae (thecamoebian) analysis was used together with lake sediment geochemistry to study and evaluate the ecological effects of sulphidic metal mines on aquatic environments. Three different mines were included in the study: Luikonlahti Cu-mine in Kaavi, eastern Finland, Haveri Cu-Au mine in Ylöjärvi, southern Finland and Pyhäsalmi Zn-Cu-S mine in Pyhäjärvi, central Finland. Luikonlahti and Haveri are closed mines, but Pyhäsalmi is still operating. The sampling strategy was case specific, and planned to provide a representative sediment sample series to define natural background conditions, to detect spatial and temporal variations in mine impacts, to evaluate the possible recovery after the peak contamination, and to distinguish the effects of other environmental factors from the mining impacts. In the Haveri case, diatom analyses were performed alongside thecamoebian analysis to evaluate the similarities and differences between the two proxies. The results of the analyses were investigated with multivariate methods (direct and indirect ordinations, diversity and distance measure indices). Finally, the results of each case study were harmonized, pooled, and jointly analyzed to summarize the results for this dissertation. Geochemical results showed broadly similar temporal patterns in each case. Concentrations of ions in the pre-disturbance samples defined the natural baseline against which other results were compared. The beginning of the mining activities had only minor impacts on sediment geochemistry, mainly appearing as an increased clastic input into the lakes at Haveri and Pyhäsalmi. The active mining phase was followed by the metallic contamination and, subsequently, by the most recent change towards decreased but still elevated metal concentrations in the sediments. Because of the delay in the oxidation of waste material and formation of AMD, the most intense, but transient metal contamination phase occurred in the post-mining period at Luikonlahti and Haveri. At Pyhäsalmi, the highest metal contamination preceded effluent mitigation actions. Spatial gradients were observed besides the temporal evolution in both the pre-disturbance and mine-impacted samples from Luikonlahti and Pyhäsalmi. The geochemical gradients varied with distance from the main source of contaminants (dispersion and dilution) and with water depth (redox and pH). The spatial extent of the highest metal contamination associated with these mines remained rather limited. At Haveri, the metallic impact was widespread, with the upstream site in another lake basin found to be contaminated. Changes in thecamoebian assemblages corresponded well with the geochemical results. Despite some differences, the general features and ecological responses of the faunal assemblages were rather similar in each lake. Constantly abundant strains of Difflugia oblonga, Difflugia protaeiformis and centropyxids formed the core of these assemblages. Increasing proportions of Cucurbitella tricuspis towards the surface samples were found in all of the cases. The results affirmed the indicator value of some already known indicator forms, but such as C. tricuspis and higher nutrient levels, but also elicited possible new ones such as D. oblonga ‘spinosa’ and clayey substrate, high conductivity and/or alkalinity, D. protaeiformis ‘multicornis’ and pH, water hardness and the amount of clastic material and Centropyxis constricta ‘aerophila’ and high metal and S concentrations. In each case, eutrophication appeared to be the most important environmental factor, masking the effects of other variables. Faunal responses to high metal inputs in sediments remained minor, but were nevertheless detectable. Besides the trophic state of the lake, numerical methods suggested overall geochemical conditions (pH, redox) to be the most important factor at Luikonlahti, whereas the Haveri results showed the clearest connection between metals and amoebae. At Pyhäsalmi, the strongest relationships were found between Ca- and S-rich present loading, redox conditions and substrate composition. Sediment geochemistry and testate amoeba analysis proved to be a suitable combination of methods to detect and describe the aquatic mine impacts in each specific case, to evaluate recovery and to differentiate between the effects of different anthropogenic and natural environmental factors. It was also suggested that aquatic mine impacts can be significantly mitigated by careful design and after-care of the waste facilities, especially by reducing and preventing AMD. The case-specific approach is nevertheless necessary because of the unique characteristics of each mine and variations in the environmental background conditions.

Application of capillary electrophoretic methods for determining carbohydrates and aliphatic carboxylic acids formed during wood processing

Knowledge of the behaviour of cellulose, hemicelluloses, and lignin during wood and pulp processing is essential for understanding and controlling the processes. Determination of monosaccharide composition gives information about the structural polysaccharide composition of wood material and helps when determining the quality of fibrous products. In addition, monitoring of the acidic degradation products gives information of the extent of degradation of lignin and polysaccharides. This work describes two capillary electrophoretic methods developed for the analysis of monosaccharides and for the determination of aliphatic carboxylic acids from alkaline oxidation solutions of lignin and wood. Capillary electrophoresis (CE), in its many variants is an alternative separation technique to chromatographic methods. In capillary zone electrophoresis (CZE) the fused silica capillary is filled with an electrolyte solution. An applied voltage generates a field across the capillary. The movement of the ions under electric field is based on the charge and hydrodynamic radius of ions. Carbohydrates contain hydroxyl groups that are ionised only in strongly alkaline conditions. After ionisation, the structures are suitable for electrophoretic analysis and identification through either indirect UV detection or electrochemical detection. The current work presents a new capillary zone electrophoretic method, relying on in-capillary reaction and direct UV detection at the wavelength of 270 nm. The method has been used for the simultaneous separation of neutral carbohydrates, including mono- and disaccharides and sugar alcohols. The in-capillary reaction produces negatively charged and UV-absorbing compounds. The optimised method was applied to real samples. The methodology is fast since no other sample preparation, except dilution, is required. A new method for aliphatic carboxylic acids in highly alkaline process liquids was developed. The goal was to develop a method for the simultaneous analysis of the dicarboxylic acids, hydroxy acids and volatile acids that are oxidation and degradation products of lignin and wood polysaccharides. The CZE method was applied to three process cases. First, the fate of lignin under alkaline oxidation conditions was monitored by determining the level of carboxylic acids from process solutions. In the second application, the degradation of spruce wood using alkaline and catalysed alkaline oxidation were compared by determining carboxylic acids from the process solutions. In addition, the effectiveness of membrane filtration and preparative liquid chromatography in the enrichment of hydroxy acids from black liquor was evaluated, by analysing the effluents with capillary electrophoresis.

Applications and Benefits of Adaptive Pulsed GMAW

In ship and offshore terminal construction, welded cross sections are thick and the number of welds very high. Consequently, there are two aspects of great importance; cost and heat input. Reduction in the welding operation time decreases the costs of the work force and avoids excessive heat, preventing distortion and other weld defects. The need to increase productivity while using a single wire in the GMAW process has led to the use of a high current and voltage to improve the melting rate. Unfortunately, this also increases the heat input. Innovative GMAW processes, mostly implemented for sheet plate sections, have shown significant reduction in heat input (Q), low distortion and increase in welding speed. The aim of this study is to investigate adaptive pulsed GMAW processes and assess relevant applications in the high power range, considering possible benefits when welding thicker sections and high yield strength steel. The study experimentally tests the usability of adaptive welding processes and evaluates their effects on weld properties, penetration and shapes of the weld bead.The study first briefly reviews adaptive GMAW to evaluate different approaches and their applications and to identify benefits in adaptive pulsed. Experiments are then performed using Synergic Pulsed GMAW, WiseFusionTM and Synergic GMAW processes to weld a T-joint in a horizontal position (PB). The air gap between the parts ranges from 0 to 2.5 mm. The base materials are structural steel grade S355MC and filler material G3Si1. The experiment investigates heat input, mechanical properties and microstructure of the welded joint. Analysis of the literature reveals that different approaches have been suggested using advanced digital power sources with accurate waveform, current, voltage, and feedback control. In addition, studies have clearly indicated the efficiency of lower energy welding processes. Interest in the high power range is growing and a number of different approaches have been suggested. The welding experiments in this study reveal a significant reduction of heat input and a weld microstructure with the presence of acicular ferrite (AF) beneficial for resistance to crack propagation. The WiseFusion bead had higher dilution, due to the weld bead shape, and low defects. Adaptive pulse GMAW processes can be a favoured choice when welding structures with many welded joints. The total heat reduction mitigates residual stresses and the bead shape allows a higher amperage limit. The stability of the arc during the process is virtually spatter free and allows an increase in welding speed.

Vuosihuollon aikainen käyttöturvallisuus

Tässä työssä on tarkasteltu Suomessa käytössä olevien ydinvoimalaitosten vuosihuoltojen aikaista käyttöturvallisuutta yleisesti sekä arvioitu voimayhtiöiden vuosihuoltojen aikaisten häiriö- ja hätätilanteiden varalta laatimien ohjeiden kattavuutta. Kattavuuden arviointi suoritettiin tarkastelemalla seisokkitiloja käsitteleviä todennäköisyysperusteista riskianalyysia (PRA), lopullista turvallisuusselostetta (FSAR) ja turvallisuusteknisiä käyttöehtoja (TTKE). PRA:n mukaan Olkiluodon 1 ja 2 laitosyksiköiden sydänvauriotaajuudesta noin 25 % liittyy vuosihuollon aikaisiin alkutapahtumiin. Loviisan laitosyksiköillä vastaava osuus on noin 61 %. Merkittävimmät vuosihuoltojen aikaiset alkutapahtumat sydänvaurioriskin kannalta olivat Olkiluodossa tulipalot, jäähdytteen menetykset ja jälkilämmön poiston menetykset sekä Loviisassa raskaan taakan pudotukset, booripitoisuuden laimeneminen ja öljyonnettomuudet. Saatujen tulosten perusteella voitiin todeta, että voimayhtiöiden laatimat häiriö- ja hätätilanneohjeet olivat pääosiltaan asianmukaiset ja ne kattoivat hyvin erilaiset seisokin aikaiset alkutapahtumat. Tarkastelun perusteella tehtiin ohjeistoon muutamia parannusehdotuksia. Seisokkitiloja koskevat TTKE ja FSAR havaittiin asianmukaisiksi molemmilla tarkastelluilla laitoksilla.

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10% at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65% of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p<0.05), when compared to the other CYS concentrations. The BTS (10.20±0.39) treated group showed a lower rate of estrus return when compared to other (BTSCYS; 86.05±039), and it showed also the highest total number of piglets borne per treatment (12.71±3.38 vs. 9.00±3.38, respectively). In conclusion, the addition of CYS in the BTS semen extender did not maintain spermatic viability of boar cooled spermatozoa and it results in a higher percentage of return to estrus and lower number of piglets borne.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.