Dissertations / Theses on the topic 'Acute phase proteins'

Thesis (Doctoral)--Departments of Surgical Pathophysiology and Anaesthesiology, University of Lund, University Hospital MAS.
Added t.p. with thesis statement inserted. Summary in Swedish. Includes bibliographical references.

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

The principle aims of the work presented in this thesis were to further investigate the porcine acute phase proteins (APP) and determine the factors influencing the baseline concentrations of haptoglobin (Hp), C-reactive protein (CRP), Pig major acute phase protein (Pig-MAP) and transthyretin (TTR). The APP are used as markers of inflammation and sub-clinical disease and are considered potential biomarkers for pig health and welfare. They have also been identified as a possible means of breeding for disease resistance, however little is known about the genetics of the porcine APP. This study investigated associations between the APP genes and baseline concentrations and the heritability of those concentrations. An enzyme linked immunosorbent assay (ELISA) was developed for the measurement of porcine CRP, in-house methods were used for the determination of Hp and TTR and a commercial assay was used in the measurement of Pig-MAP. A population of pure line high health boars (n=~345) from 7 lines was used in the initial study each of which had an archived DNA sample and serum samples available for use. Baseline herd concentrations of the 4 APP were determined and correlations between Hp and CRP, Hp and Pig-Map and CRP and Pig-MAP were identified. Significant differences were found between the 7 breeding lines in CRP, Pig-MAP and TTR concentrations, indicating that selective breeding for performance traits may also have affected innate immune traits such as APP concentrations. Single nucleotide polymoiphisms (SNP) were identified in the 4 APP genes and 17 were genotyped across the boar population with line differences apparent in their allele frequencies for CRP, Pig-MAP and TTR. Statistical analysis showed that there were significant associations between 3 of the SNP located in the Hp gene and Hp baseline concentrations (p < 0.01); all 3 SNP were also in high linkage disequilibrium. The association indicates that Hp is under genetic control and would also be better suited to use as a biomarker due to the lack of line effects in the boars. A heritability study was earned out utilising a mixed sex population of 297 animals (120 male, 177 female) comprising 7 breeding lines located on 2 farms. Initial analysis identified significant differences between male and females in Hp concentration, between pig lines in Hp, CRP and TTR and between the 2 farm units for CRP concentrations. This study showed that the baseline concentrations of the 4 APP could be affected by a variety of factors such as sex, commercial line and individual farm units and this must be taken into account if they are to be used as biomarkers. Heritability was estimated at 0.15, 0.13, 0.12 and 0.07 for Hp, CRP, Pig-MAP and TTR, respectively. All 4 APP show low heritability in serum concentration, this may prove problematic if they are utilised as a breeding trait. Overall, the findings from this study indicate that baseline concentrations of porcine Hp, CRP, Pig-MAP and TTR are influenced by various factors including sex, breeding line, and farm unit and this must be taken into account before they are utilised as biomarkers. They are traits of low heritability but the evidence suggests there may be a genetic component to their regulation thus requiring further investigation.

Bovine mastitis continues to pose a major economic challenge to the dairy industry worldwide. Critical to the management and control of this condition, is the need for prompt and accurate diagnosis in field conditions, therefore a search for more sensitive and reliable biomarkers is required. In this thesis, studies focused on assessing milk samples from cows with various forms of mastitis were undertaken with a view to identifying new biomarkers for bovine mastitis. Three acute phase proteins (APP); haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in milk samples from composite milk samples of all lactating cows in a commercial dairy herd, mastitis cases, submitted to a diagnostic laboratory and following an experimental mastitis challenge of cows with Streptococcus uberis. A new enzyme linked immunosorbent assay (ELISA) was developed for measuring Hp, while commercial ELISA assay kits were used to assay M-SAA3 and CRP. Other mastitis related parameters evaluated in the samples included the somatic cell counts (SCC) and the presence of pathogens. A reliable and sensitive ELISA was developed and optimized for measuring milk Hp. A cut off value for Hp of 7.9 μg/ml was established for milk with SCC less than 200,000 cells/ml. Pathogen-specific variations were observed in the concentration of each APP in mastitic milk. It was observed that the environmental pathogens showed higher concentrations of APP compared to other pathogens, from the study of mastitis milk samples submitted to the diagnostic laboratory. Also, it was possible to distinguish between samples from subclinical and clinical mastitis and between samples from subclinical and healthy udders using each of the APP (P<0.05). Haptoglobin, M-SAA3 and CRP showed corresponding variation with stage of infection during the course of experimental mastitis, and specifically CRP was observed to rise earlier than other two APP. Furthermore, characterization of the profile of these APP in the immediate post-calving milk samples was carried out to determine how valuable they would be in recognizing new mastitis infections arising at the post-partum period. It was observed that there is a general moderately-high level of APP in milk immediately following parturition which drops a few days later in healthy milk. The immunohistochemical localization of Hp in the bovine mammary gland was also assessed. It could be concluded from that study that neutrophils and the mammary epithelial cells secrete Hp into milk during mastitis. Gel and non-gel based proteomics approaches were employed to study the protein profiles and variation in mastitic milk from normal samples. Several proteins were identified that confirmed previous findings and project new mastitis markers, for example, serotransferrin, serpins, alpha-macroglobulin and neutrophil gelatinase associated lipocalins. A capillary electrophoresis mass spectrometry system (CE-MS) was also employed to elucidate the changing peptidome in milk during the course of an experimental mastitis, which lead to the generation of a panel of 77 polypeptides, which were able to significantly differentiate critical stages of mastitis. Three of these polypeptides were found in mastitic milk samples from previous peptidomic analyses thereby indicating strong biomarker value. Finally, a liquid chromatography mass spectrometry based metabolomics approach was used to study the changing profile of small metabolites in milk during the course of an experimental infection. Several pathway-based changes that highlighted metabolites of potential significance in mastitis diagnosis were recognized including lactose synthesis, nitrogen containing compounds such as betaine, L-carnitine and lipid metabolites pathways namely sn-glycerophosphocholine and choline among others. Overall, this study has shown the value of APP, milk proteomics and metabolomics in bovine mastitis diagnosis; the changing proteins and metabolites or their patterns need to be further experimentally and clinically validated as specific and sensitive markers of mastitis. Ultimately, the applicability of APP, proteins, peptides and metabolites and/or their changing patterns as mastitis biomarkers would require their adaptation to rapid (on farm) and robust measurement formats.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs on immune function. The effect of the immune system on GC action, especially in the periphery, is not as well studied. The liver plays an important role in inflammation and stress in producing the acute phase proteins (APPs) required for the resolution of inflammation as well as in producing systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding effects of stress and inflammation and their interplay in the liver is thus not only useful to expand our understanding of these systems but could also have clinical applications in understanding the side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be developed as an anti-inflammatory drug that displays fewer side effects. The current study investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects at the protein level, using Western blotting and ELISA assays, the protein activity level, using enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes
AFRIKAANSE OPSOMMING: Kruiskommunikasie bestaan tussen die stres– en die immuunsisteem en hierdie kruiskommunikasie is van farmakologiese belang vir die gebruik van glukokortikoïede (GKe) as anti-inflammatoriese medikasie vir siektes soos asma en artritis. Tot dusver was die fokus van studies oor hierdie kruiskommunikasie hoofsaaklik op die effek van GKe op immuunfunksie. Die effek van die immuunsisteem op GK werking, veral in die periferie, is nie so goed bestudeer nie. Die lewer speel ʼn belangrike rol in inflammasie en stres deurdat dit die akute fase proteïene (AFPs) produseer wat benodig word vir die resolusie van inflammasie en omdat dit ook sistemiese glukose produseer, d.m.v. glukoneogenese, wat benodig word om die stres reaksie te dryf. ’n Beter insig in die effek van stres en inflammasie sowel as hul interaksie in die lewer is dus handig, nie net om ons begrip van hierdie sisteme te verbeter nie, maar ook omdat dit kliniese toepassing kan hê deurdat dit ons begrip van die newe-effekte wat gepaard gaan met die farmakologiese gebruik van GKe verbeter. Verbinding A (CpdA) is geïdentifiseer as ʼn selektiewe glukokortikoïed reseptor (GR) moderator (SERGM) omdat dit die vermoë het om gene te onderdruk maar nie te aktiveer d.m.v. die GR. Hierdie eienskap dui op die potensiaal van CpdA om ontwikkel te word as ʼn anti-inflammatoriese middel met minder newe-effekte. Die huidige studie het die effekte van dexamethasone, ʼn sterk GR agonis, en CpdA, beide in die teenwoordigheid en afwesigheid van interleukin 6 (IL6), op die GR, drie metaboliese ensiem gene wat betrokke is by glukoneogenese, sowel as drie APP gene, ondersoek en vergelyk. Die metaboliese ensiem gene wat ondersoek is, is tirosien aminotransferase (TAT), fosfoenolpirovaat karboksikinase (PEPCK), en gamma glutamieltransferase (GGT), terwyl die APP gene serum amiloïede A (SAA), C-reaktiewe proteïen (CRP), en kortikosteroïed bindings globien (CBG) was. Die studie het die effekte in ʼn muis hepatoma sellyn (BWTG3) op die proteïen vlak, deur van Western blotting en ELISA essays gebruik te maak, die proteïen aktiwiteits vlak, deur van ensiem aktiwiteits essays en vol-sel binding gebruik te maak, sowel as op die mRNA vlak, deur van kwantitatiewe polimerase ketting reaksie (qPCR) gebruik te maak, ondersoek. Die studie toon dat dexamethasone (Dex) en IL6 in die algemeen divergente effekte het op die GR en metaboliese ensieme deurdat Dex GR af-reguleer en die metaboliese ensieme op-reguleer, terwyl IL6 die GR op-reguleer en die metaboliese ensieme af-reguleer, en dat hulle funksies konvergerend is vir die APPs deurdat beide positiewe APPs opreguleer en negatiewe APPs afreguleer. In teenstelling met Dex het CpdA die GR op-gereguleer en die metaboliese ensieme af-gereguleer terwyl dit, soos Dex, die positiewe APPs op-gereguleer en die negatiewe APPs af-gereguleer het. Ons resultate vir Dex en IL6 word ondersteun deur vorige werk in die literatuur. Ons studie is wel uniek omdat dit die ondersoek van drie metaboliese ensieme kombineer met die ondersoek van drie APPs, sowel as GR vlakke in ʼn enkele sisteem onder dieselfde eksperimentele kondisies. Verder het ons resultate met CpdA verskeie nuwe aspekte, soos die af-regulering van metaboliese gene, opgelewer wat bydra tot die groeiende poel van kennis oor hierdie ongewone GR ligand en die moontlike farmakologiese gebruik daarvan.

Els pacients amb càncer presenten una taxa de supervivència superior si es diagnostiquen a estadis inicials, per la qual cosa és indispensable disposar de marcadors tumorals adequats. Glicoformes de proteïnes específiques es podrian utilizar com marcadors tumorals. S’han investigat les subformes i glicosilació de l’Antígen Prostàtic Específic (PSA) per millorar la seva capacitat de diagnosis de pacients amb càncer de pròstata vs aquells amb hiperplàsia benigna prostàtica. També s’han avaluat glicoproteïnes sèriques amb alteracions glucídiques en pacients de càncer de pàncrees, comparat amb pacients amb pancreatitis crònica i controls. S’ha observat una disminució de la fucosilació core i sialilació del PSA en càncer de pròstata i un augment de la fucosilació core i Sialyl-Lewis X en algunes Proteïnes de fase Aguda en càncer de pàncrees. Aquest canvis s’haurien d’avaluar en un cohort de pacients més gran per determinar el seu paper en el cribratge, diagnòstic o monitorització dels cancers estudiats.
The survival rate of cancer patients is increased when they are diagnosed at localized stage, for which the availability of adequate tumour markers is crucial. The determination of specific tumour‐associated glycoforms may either improve the specificity of known cancer biomarkers such Prostate-Specific Antigen (PSA) or allow the discovery of new tumour markers. This work has investigated PSA subforms and their glycosylation with the aim to improve the differentiation between prostate cancer and benign prostatic hyperplasia. In addition, serum glycoproteins with altered glycosylation have been evaluated in pancreatic cancer patients, compared to chronic pancreatitis patients and healthy controls. A decrease of PSA core fucosylation and sialylation in Prostate cancer and an increase in some Acute-Phase Proteins core fucosylation and Sialyl-Lewis X in pancreatic cancer were observed. These changes should be evaluated in a larger cohort of patients to determine their role as prostate and pancreatic cancer biomarkers, respectively.

Hi ha un grup de proteïnes plasmàtiques, anomenades proteïnes de fase aguda (PFAs), la concentració de les quals canvia quan es produeixen lesions tissulars induïdes per traumes o estrès i per condicions infeccioses i inflamatòries. La funció principal de les PFAs és recuperar la homeòstasi i limitar el creixement microbià. La quantificació de la concentració de les PFAs pot ser utilitzada com a eina pel diagnòstic i pronòstic, així com pel seguiment de tractaments. A la bibliografia hi ha molts articles que descriuen els diferents patrons de les proteïnes enfront diferents malalties. No obstant, la utilitat de les APPs i la seva dinàmica en malalties pròpies de poblacions salvatges no ha estat determinada. Els mètodes de laboratori que s’utilitzen actualment per mesurar les APPs s’haurien de validar abans de proporcionar cap resultat. Això és especialment important en les espècies salvatges, on les tècniques dels animals domèstics s’extrapolen sense validació prèvia, i per tant la fiabilitat dels resultats es desconeguda. Els objectius principals d'aquest treball són validar els mètodes analítics disponibles per la determinació de proteïnes de fase aguda en ungulats domèstics per al seu ús en ungulats salvatges, així com proporcionar valors de referència per a les espècies estudiades i verificar la utilitat d’aquestes proteïnes de fase aguda. En l’ESTUDI I, realitzat en el senglar, es va determinar la concentració sèrica de sis APPs: l’haptoglobina (Hp), el sèrum amiloide A (SAA), la proteïna C-reactiva (CRP) i la proteïna de fase aguda major de porcí (Pig-MAP) utilitzant kits comercials. Els kits de les dues últimes proteïnes mencionades dissenyats específicament per la seva aplicació en mostres de porcí. També es van determinar la glicoproteïna àcida soluble (ASG) i la ceruloplasmina (Cp) mitjançant mètodes analítics descrits prèviament. Tots els mètodes utilitzats van demostrar una bona precisió (CV <15%), excepte per les proteïnes ASG i SAA que van presentar coeficients de variació (CVs) entre anàlisis més alts. Aquests dos CVs van ésser obtinguts pel pool de concentració baixa. L’estudi d’interferència va demostrar que l’hemòlisi produeix interferència en totes les proteïnes estudiades, sobretot el SAA. Totes les proteïnes estudiades van presentar diferències significatives entre les concentracions d’animals sans i malalts. Els valors de referència obtinguts pels senglars sans van ser similars als descrits prèviament en altres treballs realitzats en el porc domèstic. En l’ESTUDI II es van comparar dos mètodes diferents per la determinació de la CRP en el senglar. Un mètode immunoturbidimètric (TIA) específic de humana amb un mètode immunoenzimàtic (ELISA) específic de porcí. També es va validar el mètode immunoturbidimètric per al seu ús en els senglars. La regressió Passing-Bablok va demostrar que hi havia un error proporcional entre TIA i ELISA. Aquest error es va reduir quan es va utilitzar un calibrador de porcí fabricat en el laboratori. La validació del TIA amb el calibrador de porcí obtingut al laboratori va demostrar tenir una bona precisió i una bona exactitud. Es va demostrar també que l’hemòlisi produeix interferències importants en l’anàlisi de la CRP amb mostres de senglar amb el TIA. La validació dels mètodes de la Hp, SAA, ASG i Cp pel seu ús en l’isard es va realitzar en l’ESTUDI III. En general, tots els mètodes van presentar bona precisió i exactitud. Les mostres procedents d'animals sans van produir molts valors per sota del límit de detecció establert per les proteïnes Hp i SAA. La ASG es va veure afectada significativament per l'hemòlisi. Els valors obtinguts d'animals sans i malalts van ser significativament diferents entre sí. Els mètodes de determinació de la Hp i el SAA van ser avaluats pel seu ús en la cabra salvatge en l’ESTUDI IV. Aquests mètodes van demostrar tenir bona precisió intra-assaig i bona exactitud. En l’estudi inter-assaig es va observar una gran imprecisió per ambdues proteïnes. La inflamació induïda amb la injecció de trementina va produir un canvi significatiu en la concentració de Hp i SAA. En canvi, la infecció experimental amb llengua blava no va produir canvis significatius en cap de les proteïnes estudiades. Els mètodes validats per la determinació de les PFA en aquests quatre estudis van demostrar que els resultats que produeixen son fiables i que per tant es poden aplicar en les espècies estudiades, amb l’excepció d'alguns paràmetres que caldria tenir en compte quan s’utilitzin aquests mètodes. De la mateixa manera, hi va haver diferències significatives entre els animals sans i malalts en totes les proteïnes estudiades en el senglar i l’isard, i en l’haptoglobina i l’amiloide A sèric en la cabra salvatge. En aquesta última espècie les dues proteïnes estudiades van produir canvis de concentració suficients com per poder discriminar bé entre abans i després de l'establiment d’una resposta inflamatòria induïda per la injecció de trementina.
Hay un grupo de proteínas plasmáticas, llamadas proteínas de fase aguda (PFAs), la concentración de las cuales cambia cuando se producen lesiones tisulares inducidas por traumas o estrés y por condiciones infecciosas e inflamatorias. La función principal de las PFAs es restablecer la homeostasis y limitar el crecimiento microbiano. La cuantificación de la concentración de las PFAs puede ser utilizada como herramienta para el diagnóstico y pronóstico, así como para el seguimiento de tratamientos. En la bibliografía hay muchos artículos que describen los diferentes patrones de las proteínas frente distintas enfermedades. Sin embargo, la utilidad de las APPs y su dinámica en enfermedades propias de poblaciones salvajes no ha sido determinada. Los métodos de laboratorio que se utilizan actualmente para medir las APPs deberían ser validados antes de proporcionar ningún resultado. Esto es especialmente importante en las especies salvajes, donde las técnicas de los animales domésticos se extrapolan sin validación previa, y por tanto la fiabilidad de los resultados es desconocida. Los objetivos principales de este trabajo son validar los métodos analíticos disponibles para la determinación de proteínas de fase aguda en ungulados domésticos para su uso en ungulados salvajes, así como proporcionar valores de referencia para las especies estudiadas y verificar la utilidad de estas proteínas de fase aguda. En el ESTUDIO I, realizado en el jabalí, se determinó la concentración sérica de seis APPs: haptoglobina (Hp), el amiloide A sérico (SAA), la proteína C-reactiva (CRP) y la proteína de fase aguda mayor de porcino (Pig-MAP) utilizando kits comerciales. Los kits de las dos últimas proteínas mencionadas diseñados específicamente para su aplicación en muestras de porcino. También se determinaron la glicoproteína ácida soluble (ASG) y la ceruloplasmina (Cp) mediante métodos analíticos descritos previamente. Todos los métodos utilizados demostraron una buena precisión (CV <15%), excepto para las proteínas ASG y SAA que presentaron coeficientes de variación (CVs) entre análisis más altos. Estos dos CVs fueron obtenidos para el pool de concentración baja. El estudio de interferencia demostró que la hemólisis produce interferencia en todas las proteínas estudiadas, sobretodo el SAA. Todas las proteínas estudiadas presentaron diferencias significativas entre las concentraciones de animales sanos y enfermos. Los valores de referencia obtenidos por jabalíes sanos fueron similares a los descritos previamente en otros trabajos realizados en el cerdo doméstico. En el ESTUDIO II se compararon dos métodos diferentes para la determinación de la CRP en el jabalí. Un método immunoturbidimètrico (TIA) específico de humana con un método immunoenzimàtico (ELISA) específico de porcino. También se validó el método immunoturbidimètrico para su uso en los jabalíes. La regresión Passing-Bablok demostró que había un error proporcional entre TIA y ELISA. Este error se redujo cuando se utilizó un calibrador de porcino fabricado en el laboratorio. La validación del TIA con el calibrador de porcino obtenido en el laboratorio demostró tener una buena precisión y una buena exactitud. Se demostró también que la hemólisis produce interferencias importantes en el análisis de la CRP con muestras de jabalí con el TIA. La validación de los métodos de la Hp, SAA, ASG y Cp para su uso en el rebeco se realizó en el ESTUDIO III. En general, todos los métodos presentaron buena precisión y exactitud. Las muestras procedentes de animales sanos produjeron muchos valores por debajo del límite de detección establecido para las proteínas Hp y SAA. La ASG se vio afectada significativamente por la hemólisis. Los valores obtenidos de animales sanos y enfermos fueron significativamente diferentes entre sí. Los métodos de determinación de la Hp y el SAA fueron evaluados para su uso en la cabra montés en el ESTUDIO IV. Estos métodos demostraron tener buena precisión intra-ensayo y buena exactitud. En el estudio inter-ensayo se observó una gran imprecisión para ambas proteínas. La inflamación inducida con la inyección de trementina produjo un cambio significativo en la concentración de Hp y SAA. En cambio, la infección experimental con lengua azul no produjo cambios significativos en ninguna de las proteínas estudiadas. Los métodos validados para la determinación de las PFA en estos cuatro estudios demostraron que los resultados que producen son fiables y que por tanto se pueden aplicar en las especies estudiadas, con la excepción de algunos parámetros que habría que tener en cuenta cuando se utilicen estos métodos. Del mismo modo, hubo diferencias significativas entre los animales sanos y enfermos en todas las proteínas estudiadas en el jabalí y el rebeco, y en la haptoglobina y el amiloide A sérico en la cabra montesa. En esta última especie las dos proteínas estudiadas produjeron cambios de concentración suficientes como para poder discriminar bien entre antes y después del establecimiento de una respuesta inflamatoria inducida por la inyección de trementina.
Acute phase proteins (APPs) are a group of plasma proteins that change in concentration after any tissue injury induced by infection, inflammation, trauma or stress. Their main function is to restore homeostasis and limit microbial growth. The quantification of APPs can be used as a tool for diagnostic and prognostic as well as to monitor treatments. In the literature there are many articles describing the APPs patterns against different diseases in domestic animals, mainly livestock. However, the usefulness of APPs and their dynamics in diseases typical of wild populations has not been characterized. Analytical validation of the laboratory methods in use for the determination of APP should be assessed before the report of any value. This is especially important in wild animals, in which often the techniques in use in domestic animals are extrapolated without prior validation, so that the reliability of the results is unknown. The main goals of the present thesis are to validate analytical methods available for determination of APPs in domestic ungulates for its use in wild ungulates, to provide reference values and to verify the utility of several APPs for the species studied. In STUDY I six different APPs in wild boar were studied: serum haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP) and porcine major acute phase protein (Pig-MAP) concentrations were determined using commercial kits available, the last two were porcine-specific methods; Acid soluble glycoprotein (ASG) and ceruloplasmin (Cp) were analyzed using assay methods described previously in literature. All the methods demonstrated good precision (CVs<15%), except inter assay CVs for ASG and SAA with the low concentration pool. Hemolysis affected all the proteins studied, mostly the SAA. There were significant differences between healthy and diseased animals. Reference ranges obtained for healthy wild boars were similar to those reported previously in literature for domestic pigs. In STUDY II a human CRP turbidimetric immunoassay (TIA) was compared to the porcine specific enzyme linked immunosorbent assay (ELISA) CRP and validated for its use in wild boar. Passing bablok regression demonstrated that there was a proportional error between TIA and ELISA which was reduced using a porcine in-house calibrator. The validation of TIA CRP with the porcine in-house calibrator showed good precision and accuracy. Important interference was observed in the study of hemolysis. The method validation of Hp, SAA, ASG and Cp conducted in STUDY III in Pyrenean chamois demonstrated good precision and accuracy of all the proteins studied. Hp and SAA yielded many values below the limit of detection when samples from healthy animals were analyzed. ASG was significantly affected by hemolysis. The values obtained from healthy and diseased animals were significantly different from each other. Hp and SAA methods were evaluated for Spanish ibex in STUDY IV with good intra-assay precision and accuracy. An important inter-assay imprecision was observed for both proteins. Inflammation induced by turpentine injection produced a significant change in the concentration of Hp and SAA. No significant changes in any of the proteins studied were observed when a experimentally bluetongue virus infection was induced. APPs methods validated in these studies demonstrated to be reliable in the species studied, except for some parameters that should be kept in mind when implementing these methods. Likewise, significant differences between healthy and disease animals were observed for all the proteins studied in wild boar and Pyrenean chamois. In Spanih ibex, haptoglobin and serum amyloid A discriminated well before and after the establishment of an aseptic inflammation induced by turpentine injection.

The measurement of acute phase proteins is used by human clinicians to give valuable infonnation about a patient's inflammatory response, both when monitoring clinical disease and when assessing the effect of therapy. Levels of soluble receptors for the cytokine, tumour necrosis factor, also increase as a result of inflammatory stimuli and are useful prognostic markers over the asymptomatic phase of human immunodeficiency virus infection. The aim of the work presented in this thesis was to detennine whether these markers are of value when investigating feline disease. Reference ranges for two acute phase proteins, aI-acid glycoprotein (AGP) and haptoglobin were detennined by measuring their concentrations in serum samples from healthy cats. Analysis of samples from cats with feline infectious peritonitis (FIP) and from cats suffering from conditions with a similar clinical presentation revealed that measurement of AGP can be a useful adjunct to other laboratory tests when reaching a diagnosis. In contrast, measurement of haptoglobin was not found to be of value. Despite increases in the levels of pro-inflammatory cytokines in samples taken from cats during the asymptomatic phase of feline immunodeficiency virus (FIV) infection, no changes were detected in the levels of AGP and haptoglobin. It was concluded that these acute phase proteins are of no benefit as prognostic markers in FlY. The L929 bioassay was used to investigate anti-TNF-a activity in cell culture fluids from feline splenic cells. Cytotoxic activity was demonstrated in very few of the samples whilst anti-cytotoxic activity was detected in the majority of samples. This anti-cytotoxic activity was attributed to the presence of feline soluble TNF receptor type 1 (sTNFR-I) binding to and inhibiting the effects of TNF-a. This was not confinned because of the lack of specific neutralising antibody. Subsequent work was therefore directed towards the development of immune-based species-specific assays for feline soluble TNF receptors (sTNFRs). The polymerase chain reaction was used to amplify the sequences coding for feline sTNFRs. Most of the extracellular domain of feline TNFR-l and part of the intracellular domain of feline TNFR-2 were cloned and sequenced using this technique. The amplified regions demonstrated 85% and 77% homology at the nucleic acid level and 83% and 67% homology at the amino acid level to the corresponding regions of the human sequences for TNFR-l and 2 respectively. Feline sTNFR-l was expressed as a glutathione-S-transferase fusion protein. After purification, concentration and electrophoresis, the appropriate protein band was excised and used to inoculate a sheep. Antiserum taken from the sheep post-inoculation recognised the expressed protein by western blotting, but results were inconsistent and analysis of the antiserum was hampered by the very small amounts of expressed protein available. Two peptides were synthesised based on regions of antigenicity in feline sTNFR-l and were used to inoculate sheep. Antiserum to peptide A showed a strong reaction against peptide A in an ELISA and gave a positive result when used as the primary antibody to stain healthy feline liver tissue. In conclusion, both antiserum to expressed feline sTNFR-l and anti-peptide antibody based on a region of feline sTNFR-l have been raised in sheep and are available for the development of an assay for this protein. Further expression of feline TNFR-l will be required before these antisera can be analysed fully.

An initial assessment in stranded marine mammals, including evaluation of clinico-pathological variables, is a preliminary and critical step to define treatment and assessing the suitability of the animals for rehabilitation. Serum protein electrophoresis (SPE) is the most reliable method to determine the distribution of serum protein fractions and it is considered an essential step to evaluate the health status of animals, providing clinical useful information. The measurement of APPs in association to serum proteins’ fractions can supplement and extend the baseline information obtained from the complete blood cell count, fibrinogen, and standard serum chemistry panel. The thesis is divided in two main chapters, the first one focused on the serum protein electrophoresis (SPE) and the second one on the acute phase proteins evaluation In the first chapter, 38 under human care bottlenose dolphin serum samples were screened with agarose gel electrophoresis (AGE) to determine the reference intervals (RIs) of the serum proteins. Four main protein fractions were evident in all the animals tested: albumin, α-globulins, β-globulins, and γ-globulins. The RIs for the serum protein fractions were: albumin 45.0 ± 4.0 g/L, α-globulins 8.0 ± 1.0 g/L, β-globulins 5.0 ± 2.0 g/L, and γ-globulins 7.0 ± 2.0 g/L. Compared to previously published data in free ranging bottlenose dolphins, in our samples the concentration of total protein, α-globulins, and γ-globulins were slightly lower, while the concentration of albumin and the albumin/globulins ratio were slightly higher. The lower concentration of ‘’inflammatory’’ proteins associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio reported in our study could reflect a lower antigenic stimuli in the animals housed in aquaria compared to the free-ranging populations. Moreover, in 8 electropherograms, we noticed that the base of the albumin peak was wider compared to the electropherograms of the other animals. For this reason, the same serum samples used for AGE were evaluated also with capillary zone electrophoresis (CZE), a more sensitive electrophoretic method. With CZE 9 out of 38 samples showed a double albumin peak. However, all these samples except 2 had an albumin peak wider than that observed with AGE in dolphins classified as non bisalbuminemic by CZE; furthermore a wider albumin peak was also noted with AGE in one sample with normal CZE profile. We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration. To understand the genetic basis of bisalbuminemia, the albumin gene of 15 bottlenose dolphins belonging to two distinct families were reconstructed by direct comparison of its full length cDNAs with the provisional sequence of bottlenose dolphin albumin gene. Eighteen albumin gene variations were identified in the bottlenose dolphins studied (15 non-synonimous and 3 synonymous). In order to identify the non-synonimous variations able to cause bisalbuminemia, the genotype-phenotype correlations within the two families were studied. Two heterozygous non-synonymous variations that co-segregate with the ‘’bisalbuminemia’’ phenotype detected by SPE were identified: c.483C>G p.Phe146Leu in exon 4 and c.487T>C p.Tyr163His in exon 5. The genetic analysis of bottlenose dolphins’ albumin gene showed a significant polymorphism and two mutations associated with bisalbuminemia. Moreover, we were able to identify the autosomal codominant trait of this condition in dolphins, a similar pattern of inheritance to that in humans. The in silico analysis and the comparison between dolphin and human variations support the hypothesis that the variation p.Tyr163His could be more likely responsible for bisalbuminemia. In the second chapter double radial immunodiffusion (DRI), western blot (WB) analysis, and spectrophotometric measurement using immunologic or enzymatic assays were employed on serum samples of bottlenose dolphins and striped dolphins to validate, establish RIs, and evaluate the diagnostic accuracy of two positive acute phase proteins (C-reactive protein, CRP and serum amyloid-A, SAA) and one negative APP (serum paraoxonase-1, PON-1). With DRI none of the antibodies (Abs) against CRP and SAA cross-reacted with the serum samples of bottlenose dolphins and striped dolphins. Both the anti-SAA Abs tested were latex-conjugated, because produced for automatic immunoturbidimetric assays. The presence of latex associated to the Abs may have interfered to the migration of the Abs across the agarose gel. WB analysis for anti-CRP antibodies showed a weak positivity for striped dolphins and a pattern of positivity in the serum samples of bottlenose dolphins similar to those observed in dog, with multiple bands. However, we are not able to exclude the possibility that this pattern may represent an unspecific signal. The discouraging result obtained with the automated measurement of dolphins CRP (0.00 mg/L) seemed to confirm the hypothesis that the anti-human CRP Ab used does not recognize the cetaceans’ CRP, based also on the low homology of the amino acid sequence. On contrary, the SAA is highly conserved between different species. The automated measurement of SAA provided results with good precision; the SAA concentration in the whole set of bottlenose dolphins samples was 8.7 ± 11.8 mg/L. In addition, for the SAA concentration no differences were noted between different storage time, between the sex of the animals, and between pregnant and non-pregnant animals. The lack of differences in SAA concentration between males and females, and pregnant and non-pregnant animals allowed us to establish the SAA RIs using samples from the whole population instead of establish partitioned RIs .Moreover, a stability in SAA concentration in serum samples with long storage time was demonstrated. PON-1 activity was determined using 4 different substrates using enzymatic assays. The PON-1 activity using paraoxon as substrate provided results with good. The PON-1 activity in the whole set of bottlenose dolphins samples was 6.7 ± 4.6 U/L. As for the concentration of SAA, no differences in PON-1 activity were noted, based on sex, and between pregnant and non-pregnant animals. On contrary, the PON-1 activity for the long storage samples was significantly lower compared to the short storage samples. To evaluate the genetic influence of the single nucleotide polymorphisms (SNPs) in the PON-1 activity, we sequenced the two most studied SNPs of human PON-1 gene, the Q192R and the L55M. Based on the sequence analysis, all the dolphins were homozygous for methionine in L55M SNPs and for arginine in Q192R SNPs. Despite all the animals are homozygous for the phenotype associated to a higher paraoxonase activity in humans, the bottlenose dolphins’ PON-1 activity is low and it seems not useful to discriminate between healthy and diseased animals. The PON-1 activity using 4-nitrophenyl acetate (4-nPA) as substrate was higher compare to those obtained using paroxon, providing results with good precision and accuracy but no significant difference were noted between healthy dolphins and diseased dolphins. However, our results are based on a limited number of animals so we cannot exclude that, including a higher number of animals with different diseases, a more drastic change in PON activity will be evident.
An initial assessment in stranded marine mammals, including evaluation of clinico-pathological variables, is a preliminary and critical step to define treatment and assessing the suitability of the animals for rehabilitation. Serum protein electrophoresis (SPE) is the most reliable method to determine the distribution of serum protein fractions and it is considered an essential step to evaluate the health status of animals, providing clinical useful information. The measurement of APPs in association to serum proteins’ fractions can supplement and extend the baseline information obtained from the complete blood cell count, fibrinogen, and standard serum chemistry panel. The thesis is divided in two main chapters, the first one focused on the serum protein electrophoresis (SPE) and the second one on the acute phase proteins evaluation In the first chapter, 38 under human care bottlenose dolphin serum samples were screened with agarose gel electrophoresis (AGE) to determine the reference intervals (RIs) of the serum proteins. Four main protein fractions were evident in all the animals tested: albumin, α-globulins, β-globulins, and γ-globulins. The RIs for the serum protein fractions were: albumin 45.0 ± 4.0 g/L, α-globulins 8.0 ± 1.0 g/L, β-globulins 5.0 ± 2.0 g/L, and γ-globulins 7.0 ± 2.0 g/L. Compared to previously published data in free ranging bottlenose dolphins, in our samples the concentration of total protein, α-globulins, and γ-globulins were slightly lower, while the concentration of albumin and the albumin/globulins ratio were slightly higher. The lower concentration of ‘’inflammatory’’ proteins associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio reported in our study could reflect a lower antigenic stimuli in the animals housed in aquaria compared to the free-ranging populations. Moreover, in 8 electropherograms, we noticed that the base of the albumin peak was wider compared to the electropherograms of the other animals. For this reason, the same serum samples used for AGE were evaluated also with capillary zone electrophoresis (CZE), a more sensitive electrophoretic method. With CZE 9 out of 38 samples showed a double albumin peak. However, all these samples except 2 had an albumin peak wider than that observed with AGE in dolphins classified as non bisalbuminemic by CZE; furthermore a wider albumin peak was also noted with AGE in one sample with normal CZE profile. We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration. To understand the genetic basis of bisalbuminemia, the albumin gene of 15 bottlenose dolphins belonging to two distinct families were reconstructed by direct comparison of its full length cDNAs with the provisional sequence of bottlenose dolphin albumin gene. Eighteen albumin gene variations were identified in the bottlenose dolphins studied (15 non-synonimous and 3 synonymous). In order to identify the non-synonimous variations able to cause bisalbuminemia, the genotype-phenotype correlations within the two families were studied. Two heterozygous non-synonymous variations that co-segregate with the ‘’bisalbuminemia’’ phenotype detected by SPE were identified: c.483C>G p.Phe146Leu in exon 4 and c.487T>C p.Tyr163His in exon 5. The genetic analysis of bottlenose dolphins’ albumin gene showed a significant polymorphism and two mutations associated with bisalbuminemia. Moreover, we were able to identify the autosomal codominant trait of this condition in dolphins, a similar pattern of inheritance to that in humans. The in silico analysis and the comparison between dolphin and human variations support the hypothesis that the variation p.Tyr163His could be more likely responsible for bisalbuminemia. In the second chapter double radial immunodiffusion (DRI), western blot (WB) analysis, and spectrophotometric measurement using immunologic or enzymatic assays were employed on serum samples of bottlenose dolphins and striped dolphins to validate, establish RIs, and evaluate the diagnostic accuracy of two positive acute phase proteins (C-reactive protein, CRP and serum amyloid-A, SAA) and one negative APP (serum paraoxonase-1, PON-1). With DRI none of the antibodies (Abs) against CRP and SAA cross-reacted with the serum samples of bottlenose dolphins and striped dolphins. Both the anti-SAA Abs tested were latex-conjugated, because produced for automatic immunoturbidimetric assays. The presence of latex associated to the Abs may have interfered to the migration of the Abs across the agarose gel. WB analysis for anti-CRP antibodies showed a weak positivity for striped dolphins and a pattern of positivity in the serum samples of bottlenose dolphins similar to those observed in dog, with multiple bands. However, we are not able to exclude the possibility that this pattern may represent an unspecific signal. The discouraging result obtained with the automated measurement of dolphins CRP (0.00 mg/L) seemed to confirm the hypothesis that the anti-human CRP Ab used does not recognize the cetaceans’ CRP, based also on the low homology of the amino acid sequence. On contrary, the SAA is highly conserved between different species. The automated measurement of SAA provided results with good precision; the SAA concentration in the whole set of bottlenose dolphins samples was 8.7 ± 11.8 mg/L. In addition, for the SAA concentration no differences were noted between different storage time, between the sex of the animals, and between pregnant and non-pregnant animals. The lack of differences in SAA concentration between males and females, and pregnant and non-pregnant animals allowed us to establish the SAA RIs using samples from the whole population instead of establish partitioned RIs .Moreover, a stability in SAA concentration in serum samples with long storage time was demonstrated. PON-1 activity was determined using 4 different substrates using enzymatic assays. The PON-1 activity using paraoxon as substrate provided results with good. The PON-1 activity in the whole set of bottlenose dolphins samples was 6.7 ± 4.6 U/L. As for the concentration of SAA, no differences in PON-1 activity were noted, based on sex, and between pregnant and non-pregnant animals. On contrary, the PON-1 activity for the long storage samples was significantly lower compared to the short storage samples. To evaluate the genetic influence of the single nucleotide polymorphisms (SNPs) in the PON-1 activity, we sequenced the two most studied SNPs of human PON-1 gene, the Q192R and the L55M. Based on the sequence analysis, all the dolphins were homozygous for methionine in L55M SNPs and for arginine in Q192R SNPs. Despite all the animals are homozygous for the phenotype associated to a higher paraoxonase activity in humans, the bottlenose dolphins’ PON-1 activity is low and it seems not useful to discriminate between healthy and diseased animals. The PON-1 activity using 4-nitrophenyl acetate (4-nPA) as substrate was higher compare to those obtained using paroxon, providing results with good precision and accuracy but no significant difference were noted between healthy dolphins and diseased dolphins. However, our results are based on a limited number of animals so we cannot exclude that, including a higher number of animals with different diseases, a more drastic change in PON activity will be evident.

The analysis of acute phase protein serum amyloid A (SAA) has recently been brought into clinical use in veterinary medicine. Some of the difficulties with incorporating the SAA method in clinical practice have been the expensive and rather large equipment required for the method. Due to these difficulties only larger clinics can afford to use the SAA analysis. The company Equinostic has recently developed a smaller instrument that costs one-tenth of a larger instrument. The instrument is named EVA1 and has so far only been used to analyze SAA in horses. The aim of this study was to investigate if the EVA1 instrument could be used to analyze SAA in cats. This study included 24 serum samples from cat, which were first analyzed twice on the EVA1 instrument and then sent to the Strömsholm Referral Animal Hospital in Sweden where they reanalyzed the samples using a validated reference method. Both instruments are based on an immunoturbidimetric assay. The correlation between the two instruments was good (r=0.97) but the EVA1 instrument showed constantly lower results than the reference method. The difference between the duplicates when analyzed on the EVA1 instrument was larger than expected. The conclusion is that EVA1 could be used to analyze SAA in cats. However, before it could be used clinically in veterinary practice an extended study is recommended.

A associação entre doenças periodontais e desfechos adversos durante a gestação tem ganho grande atenção nos últimos anos. Apesar da maior parte dos estudos mostrar uma relação entre doença periodontal e parto prematuro, estudos que avaliam o impacto do tratamento periodontal nem sempre tem encontrado reduções nas taxas de prematuridade. A explicação para este fato pode estar no impacto do tratamento periodontal sobre mediadores inflamatórios relacionados aos mecanismos dos desfechos adversos durante a gestação. Por este motivo, o estudo de marcadores inflamatórios, como a proteína C-reativa, é de suma importância para a melhor compreensão do impacto inflamatório sistêmico do tratamento periodontal em gestantes. O objetivo deste estudo foi comparar os níveis sistêmicos de PCR em gestantes que receberam ou não tratamento periodontal. Esta dissertação consiste na avaliação dos níveis de PCR de 89 gestantes que receberam tratamento periodontal durante (grupo teste, n=44) ou após a gestação (grupo controle, n=45). Foram realizados dois exames periodontais completos, o primeiro em um momento anterior à 20° semana de gestação e o segundo entre a 26a e a 28a semanas de gestação. As pacientes do grupo teste receberam tratamento periodontal que incluiu raspagem e alisamento supra e subgengivais e instrução para higiene bucal. Consultas de controle para deplacagem profissional e instrução de higiene bucal foram realizadas após o tratamento até o exame final, de acordo com necessidades individuais. Os níveis de PCR foram avaliados através de imunoturbidimetria. Nenhuma diferença estatisticamente significante foi encontrada entre os grupos nos níveis de PCR nos exames inicial e final (p=0.06 e p=0.19, respectivamente). A redução média encontrada nos níveis de PCR foi de 1.93mg/L (±9.69) e 0.44mg/L (±5.44) nos grupos teste e controle, respectivamente. Essas diferenças não foram estatisticamente significativas (p=0.38). O tratamento periodontal durante a gestação reduziu significativamente os parâmetros clínicos periodontais. Esta melhora clínica não apresentou um impacto significativo sobre os níveis sistêmicos de PCR.
The association between periodontal diseases and adverse pregnancy outcomes has been subject of great attention in recent years. Although most studies have shown an association between periodontal diseases and premature birth, studies assessing the impact of periodontal treatment have not found a reduction in these figures. The explanation for this may lie on the impact of periodontal treatment on inflammatory mediators associated with the mechanisms of adverse pregnancy outcomes. For this reason, the study of inflammatory markers such as C-reactive protein is of paramount importance for better comprehension of the impact of systemic inflammatory periodontal treatment in pregnant women. The aim of this study was to compare the systemic levels of CRP in pregnant women who received or not periodontal treatment. This paper was based in the assessment of CRP levels of 89 pregnant women who received periodontal treatment during (test group, n = 44) or after pregnancy (control group, n = 45). Two periodontal examinations were performed, before of 20 weeks of gestation and the second between the 26th and 28th weeks of gestation. Patients in the test group received treatment that included periodontal scaling and root planing and hygiene instructions. Professional cleaning and oral hygiene instruction were performed after treatment until the final exam, according to individual needs. CRP levels were evaluated by immunoturbidimetry. No statistically significant difference was found between the groups for the levels of CRP in the initial and final examinations (p=0.06 and p=0.19, respectively). The average reduction found in CRP was 1.93mg / L (± 9.69) and 0.44mg / L (± 5.44) in test and control groups, respectively. These differences were not statistically significant (p = 0.38). The periodontal treatment during pregnancy significantly reduced the clinical periodontal parameters, but this clinical improvement did not result in a significant reduction on the systemic levels of CRP.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O linfoma é o principal tumor hematopoiético no cão e é caracterizado pela proliferação de células originadas do tecido linfóide, histiócitos e seus precursores. Os animais com linfoma frequentemente apresentam alterações hematológicas e bioquímico-séricas, tais como anemia normocítica normocrômica não regenerativa, anemia hemolítica, hipercalcemia e gamopatia monoclonal. O objetivo desse estudo foi quantificar e qualificar as proteínas séricas totais e suas frações, em cães sadios e linfomatosos, sendo estes submetidos ao protocolo quimioterápico de Madison- Wisconsin. Após sinérese, centrifugação e obtenção das amostras de soro, de 10 cães sadios e 10 cães linfomatosos, as proteínas de fase aguda foram separadas por eletroforese em matriz de gel de poliacrilamida, e suas concentrações determinadas por densitometria computadorizada. Foram encontradas de 18 a 31 proteínas no fracionamento eletroforético, com pesos moleculares variando de 18 a 245 KD (kilodáltons). Os pesos moleculares obtidos foram: IgA, 170 KD; ceruloplasmina, 125 KD; transferrina, 85KD; albumina, 65 KD; 1-antitripsina, 60 KD; IgG (cadeia pesada), 50 KD; haptoglobina, 39 KD; 1-glicoproteína ácida, 37 KD; proteína nº 9, 33 KD; IgG (cadeia leve), 25 KD; e proteína nº 11, 23 KD. Os resultados mostraram que algumas proteínas de fase aguda se alteram no linfoma. A 1-antitripsina, 1-glicoproteína ácida, transferrina, IgG (cadeia pesada) e globulinas apresentaram diferenças significativas entre cães sadios e linfomatosos, no momento zero, antes da 1ª sessão de quimioterapia. Já nos cães linfomatosos, somente a concentração da proteína nº 9 (33KD) apresentou diferença significativa, durante o protocolo quimioterápico.
The lymphoma is the principal hematopoietic tumor in dogs and it is characterized by the proliferation of cells from lymphoid tissue, histiocytes and its precursors. Animals with lymphoma often showed changes in biochemical and hematological parameters of those animals such as non-regenerative normocromica normocytic anemia, hemolytic anemia, hypocalcemia and monoclonal gamopatia. The purpose of this study was quantify and qualify the serum total proteins and its fraction, in dogs healthy and with lymphoma, these under went the Madison-Wisconsin chemotherapy protocol. After centrifugation and fractioning of the serum samples, the acute phase proteins were separated by polyacrilamide gel electrophoresis, and their concentrations were determined by computer densitometry. Between eighteen and thirty proteins were separated by eletrophoresis, with molecular weights ranged from 18 to 245 KD (kilodaltons). The molecular weights of the proteins more found were: IgA, 170 KD; ceruloplasmin, 125 KD; transferrin, 85 KD; albumin, 65 KD; 1-antitripsin, 60 KD; IgG (weighty chain), 50 KD; haptoglobin, 39 KD; 1-acid glycoprotein, 37 KD; protein nº 9, 33 KD; IgG (light chain), 25 KD and protein nº 11, 23 KD. The results showed that some acute phase proteins changed in lymphoma. The 1-antitripsina, 1-acid glycoprotein, transferring, IgG (weighty chain) and globulins, showed significantly diferences between healthy and lymphoma dogs, in time zero, before 1ª chemotherapy session. In dogs with lymphoma, only protein nº 9 (33KD) showed significantly diference, during chemotherapy protocol.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Este trabalho objetivou avaliar a anatomopatologia, o hemograma e o perfil bioquímico sérico de aves de postura inoculadas por Salmonella Gallinarum (SG) contendo os genes cobS e cbiA inoperantes (SGcobScbiA) que mostrou ser avirulenta em trabalhos anteriores, comparando-a com cepas virulentas SGNalr e SGcobS, para mostrar se SGcobScbiA pode ser componente de vacina contra cepas selvagens de SG e S.Enteritidis. 280 pintainhas foram distribuídas em 4 grupos (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) e G4 (controle). Com exceção do G4, os grupos receberam 0,2 mL de suas respectivas cepas contendo aproximadamente 108 UFC/mL de inóculo, aos 5 dias de idade. A eutanásia foi realizada 24h antes (1DAI) e após a inoculação (1DPI), e 3 (3DPI), 5 (5DPI), 7 (7DPI), 10 (10DPI) e 15 (15DPI) dias após a administração do inóculo, sacrificando-se, em cada momento, dez aves de cada grupo. As aves foram sacrificadas, obtendo-se amostras de sangue utilizadas para os exames hematológicos e bioquímicos. Fragmentos de fígado, baço, timo, bursa de Fabricius, rins e coração foram destinados aos exames histológicos. As aves inoculadas com a cepa SGcobS tiveram comportamento semelhante às aves inoculadas por SGNalr, porém com algumas respostas diferentes nos exames hematológicos e bioquímicos. As aves inoculadas com a cepa SGcobScbiA tiveram comportamento semelhante ao grupo controle, entretanto foi verificado alterações brandas em alguns parâmetros, mostrando que estudos futuros devem ser feitos, verificando se as alterações constatadas não irão interferir no desempenho de aves vacinadas com a cepa SGcobScbiA.
The aim of the present study was to evaluate anatomopathology, hemogram and blood serum components of commercial layers experimentally inoculated with SGcobScbiA, which is a Salmonella Gallinarum (SG) strain it shows attenuation of the virulence in previous research and it was compared with high virulence SGNalr and SGcobS strains in order to show if SGcobScbiA has potential to be use as a vaccine against SG and S. Enteritidis wild strains. 280 commercial layers were divided into 4 groups (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) and G4 (control group). With exception of G4, all the other groups received 0,2 mL of their respective strain containing about 108 CFU/mL of the inoculum with five days of age. Birds were sacrificed 24 hours before (1DBI) and 24 hours after the inoculation (1DAI), and three (3DAI), five (5DAI), seven (7DAI) ten (10DAI), and fifteen (15DAI) days after the administration of the inoculum, slaughtering ten birds at a time in each group. Birds were submitted to euthanasia and blood samples were collected in order to make the hematological and blood serum components test. Samples of liver, spleen, thymus, bursa of Fabricius, kidneys and heart were collected for the histological test. The birds inoculated with SGcobS strain had similar behavior when compared with that ones who received SGNalr strain, however some different responses in the hematological and blood serum components were found. On the other hand, the birds inoculated with SGcobScbiA strain had similar behavior when compared with the control group, however, lower alterations in some parameters were found. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SGcobScbiA strain.

A manheimiose pneumônica bovina (MPB) gera significativa lesividade quando predomina no Complexo Doença Respiratória Bovina. Visto isso, considera-se importante o estudo do diagnóstico precoce para melhor estadiamento e tratamento da doença. As concentrações plasmáticas e séricas das proteínas de fase aguda (PFA) têm sido adotadas na medicina veterinária como marcadores precoces de doenças inflamatórias e algumas vezes, relacionadas ao bem-estar animal. O presente estudo objetivou revisar o painel inflamatório da MPB; investigar a contribuição que o emprego da flunixina meglumina no tratamento de bovinos manifestando MPB; e investigar marcadores inflamatórios para o diagnóstico precoce da doença e evolução do tratamento, empregando referências de bem estar animal. Para isto foram utilizados 12 bezerros da raça holandesa com seis meses de idade, hígidos e divididos em dois grupos experimentais, o primeiro tratado com antimicrobiano e o segundo com antimicrobiano e anti-inflamatório. Os animais foram submetidos à inoculação intratraqueal de M. haemolytica e tiveram suas amostras de lavado broncoalveolar e de sangue colhidas em quatro momentos, onde foram realizados exame clínico, coleta de sangue e lavado broncoalveolar para realização de hemograma, determinação das proteínas de fase aguda, análise de cortisol e cortisona. A análise estatística foi realizada utilizando programa SAS 9.3 para Windows. Os dados foram testados quanto à normalidade pelo teste de Shapiko Wilk, considerando significância de 5%. Foi efetuada a análise de variância simples com comparação entre médias por meio do teste de LSD, sendo considerado significante p<0.05. O resultados mostraram que a MPB é uma doença de grande importância no cenário mundial; não foram encontradas diferenças estatísticas entre os marcadores precoces entre os grupos com ou sem anti-inflamatório, porém o uso concomitante com o antimicrobiano promoveu incremento do bem estar.
The bovine pneumonic mannheimiosis (MPB) generates significant lesivity when it predominates in the Complex Bovine Respiratory Disease. Thus being important the study of the previous diagnosis for better understanding and treatment of the disease. As plasma and serum concentrations of acute phase proteins (PFA) have been adopted in veterinary medicine as early markers of inflammatory diseases and sometimes related to animal welfare. The aim of the study was review the inflammatory panel of MPB; to investigate a contribution that the use of flunixin meglumina without treatment of bovines manifesting MPB; and to investigate inflammatory markers for the previous diagnosis of the disease and treatment evolution, using references of animal welfare. Twelve six-month-old, healthy and divided into two experimental groups, the first treated with antimicrobial and the second with antimicrobial and anti-inflammatory were used in this study. The animals were submitted to intratracheal inoculation of M. haemolytica and had their bronchoalveolar lavage and blood samples collected at four moments, where clinical examination, blood collection and bronchoalveolar lavage were carried out to perform hemogram, determination of the acute phase proteins, analysis of cortisol and cortisone. Statistical analysis was performed using SAS 9.3 software for Windows. The data were tested for normality by the Shapiko Wilk test, considering a significance of 5%. The analysis of simple variance was performed with a comparison of means by means of the LSD test, being considered significant p <0.05. The results showed that MPB is a disease of great importance in the world scenario; no statistical differences were found between the early markers between the groups with or without anti-inflammatory, but the concomitant use with the antimicrobial promoted an increase in animal welfare.

Os objetivos deste estudo foram: determinar a dinâmica de concentrações séricas de proteínas de fase aguda em cadelas gestantes e aspectos ultra-sonográficos da gestação, além de estabelecer uma possível correlação entre níveis séricos das mesmas proteínas, idade gestacional e aspectos ultra-sonográficos da gestação. Examinou-se 09 cadelas gestantes da referida raça com um aparelho portátil da marca GE®, modelo Logic α 100 MP, equipado com um transdutor convexo 5,0 MHz e outro linear de 7,5 MHz, de 2 a 3 vezes por semana a partir do 18º dia de gestação até o parto com a finalidade de mensurar as seguintes estruturas no concepto: DBP, DC e EP. Nos mesmos dias, coletou-se soro para dosagem de Hp, GAα1 e CRP por meio de kits comerciais (Phase Haptoglobin Assay ®, ,Canine α1 Acid Glycoprotein measurement kit ®; Phase Canine C - Reactive Protein Assay ®, Tridelta Development Limited, respectivamente). As cadelas do grupo NG tiveram o soro coletado a partir do proestro ao fim do diestro. Foi possível dividir o período gestacional em 9 semanas, cada uma delas, da terceira à nona semana de gestação, foi associada a um determinado conjunto de características ultra-sonográficas no concepto. As mensurações ultra-sonográficas foram altamente correlacionadas à idade gestacional e resultou em um modelo ; Y = 22,886 + 1,26 DBP + 0,107 DC, onde Y = idade gestacional (p < 0001 e R2 = 0,99). As concentrações séricas de Hp, GAα1 e CRP, no grupo G, foram significativamente maiores que no grupo NG a partir da 4ª semana de gestação. Essas variáveis não foram tão bem correlacionadas com a idade gestacional, quanto às mensurações ultra-sonográficas no concepto, contudo, pode-se afirmar que as concentrações séricas máximas de Hp, GAα1 e CRP (11,11 ± 1,7 mg/ml; 449,44 ± 146,91 µg/ml e 58,30 ± 14,69 µg/ml) foram atingidas na 5ª semana de gestação. Foi possível ilustrar a dinâmica das proteínas e fase aguda (Hp, GAα1 e CRP) e das mensurações ultra-sonográficas (DBP, DC e EP), possibilitando a visualização do comportamento dessas variáveis, uma em relação às outras, durante a gestação de cadelas da raça boxer.
The aims of this study were: to set up the dynamics of acute phase proteins serum concentrations in pregnant bitches and ultrasonographis features of conceptus and make a correlation among acute phase proteins serum levels, ultrasonographic features of conceptus and gestational age. Serial ultrasonographic examinations were performed on 09 boxer bitches, 2 or 3 times per week from 18º day of gestation until parturition. The used device is a portable GE®, Logic α 100 MP, equipped with a sectorial transducer 5,0 MHz and another linear one of 7,5 MHz. Embryonic and fetal parameters had been measured: DBP, DC e EP. Serum samples were collected in the same day and acute phase proteins (Hp, GAα1 e CRP) were measured by commercial kits (Phase Haptoglobin Assay ®, Canine α1 Acid Glycoprotein measurement kit ®; Phase Canine C - Reactive Protein Assay ®, Tridelta Development Limited). Serum samples of non pregnant bitches were collected from the beginnig of proestrous to the end of diestrous. The gestational period was divided into 9 weeks, each one of them (from the 3rd to the 9th week of gestation) was associated to a range of ultrasonographic features of the conceptus. Ultrasonographic mensurations (DBP, DC e EP) were linearly correlated to gestational age. The R2 values were > 0,9 in all cases, whitch indicates that more than 90% of the variability of gestational age was explained by any one of these measurements. Those mensurations were put in a multivariated model and resulted in a equation: Y = 22,886 + 1,26 DBP + 0,107 DC (p < 0,0001 e R2 = 0,99) that is able to estimate the gestational age. Hp, GAα1 e CRP seric concentrations were statisticaly more elevated in pregnant than in non pregnant bitches, from the 4th week of gestation until parturition. The acute phase proteins didn´t presented goog correlation to the gestational age like the ultrasonographic mensurations did, however it is possible to affirm that their concentrations reached maximum values (Hp = 11,11 ± 1,7 mg/ml, GAα1 = 449,44 ± 146,91 µg/ml e CRP = 58,30 ± 14,69 µg/ml) on the 5th week. The dynamic of the acute phase proteins (Hp, GAα1 e CRP) and ultrasonographic mensurations (DBP, DC e EP) was performed in order to visualise the profile of all mean measurements, one regarding to the other, during gestation in boxer bitches.

Orientador: Aureo Evangelista Santana
Banca: Alessandra Kataoka
Banca: Maria Angélica Dias
Resumo: O linfoma é o principal tumor hematopoiético no cão e é caracterizado pela proliferação de células originadas do tecido linfóide, histiócitos e seus precursores. Os animais com linfoma frequentemente apresentam alterações hematológicas e bioquímico-séricas, tais como anemia normocítica normocrômica não regenerativa, anemia hemolítica, hipercalcemia e gamopatia monoclonal. O objetivo desse estudo foi quantificar e qualificar as proteínas séricas totais e suas frações, em cães sadios e linfomatosos, sendo estes submetidos ao protocolo quimioterápico de Madison- Wisconsin. Após sinérese, centrifugação e obtenção das amostras de soro, de 10 cães sadios e 10 cães linfomatosos, as proteínas de fase aguda foram separadas por eletroforese em matriz de gel de poliacrilamida, e suas concentrações determinadas por densitometria computadorizada. Foram encontradas de 18 a 31 proteínas no fracionamento eletroforético, com pesos moleculares variando de 18 a 245 KD (kilodáltons). Os pesos moleculares obtidos foram: IgA, 170 KD; ceruloplasmina, 125 KD; transferrina, 85KD; albumina, 65 KD; 1-antitripsina, 60 KD; IgG (cadeia pesada), 50 KD; haptoglobina, 39 KD; 1-glicoproteína ácida, 37 KD; proteína nº 9, 33 KD; IgG (cadeia leve), 25 KD; e proteína nº 11, 23 KD. Os resultados mostraram que algumas proteínas de fase aguda se alteram no linfoma. A 1-antitripsina, 1-glicoproteína ácida, transferrina, IgG (cadeia pesada) e globulinas apresentaram diferenças significativas entre cães sadios e linfomatosos, no momento zero, antes da 1ª sessão de quimioterapia. Já nos cães linfomatosos, somente a concentração da proteína nº 9 (33KD) apresentou diferença significativa, durante o protocolo quimioterápico.
Abstract: The lymphoma is the principal hematopoietic tumor in dogs and it is characterized by the proliferation of cells from lymphoid tissue, histiocytes and its precursors. Animals with lymphoma often showed changes in biochemical and hematological parameters of those animals such as non-regenerative normocromica normocytic anemia, hemolytic anemia, hypocalcemia and monoclonal gamopatia. The purpose of this study was quantify and qualify the serum total proteins and its fraction, in dogs healthy and with lymphoma, these under went the Madison-Wisconsin chemotherapy protocol. After centrifugation and fractioning of the serum samples, the acute phase proteins were separated by polyacrilamide gel electrophoresis, and their concentrations were determined by computer densitometry. Between eighteen and thirty proteins were separated by eletrophoresis, with molecular weights ranged from 18 to 245 KD (kilodaltons). The molecular weights of the proteins more found were: IgA, 170 KD; ceruloplasmin, 125 KD; transferrin, 85 KD; albumin, 65 KD; 1-antitripsin, 60 KD; IgG (weighty chain), 50 KD; haptoglobin, 39 KD; 1-acid glycoprotein, 37 KD; protein nº 9, 33 KD; IgG (light chain), 25 KD and protein nº 11, 23 KD. The results showed that some acute phase proteins changed in lymphoma. The 1-antitripsina, 1-acid glycoprotein, transferring, IgG (weighty chain) and globulins, showed significantly diferences between healthy and lymphoma dogs, in time zero, before 1ª chemotherapy session. In dogs with lymphoma, only protein nº 9 (33KD) showed significantly diference, during chemotherapy protocol.
Mestre

A variety of inflammatory markers, coupled with changes in a number of haematological and biochemical parameters have classically been used to diagnose, monitor, and prognosticate disease in horses. Unfortunately these traditional markers respond fairly slowly to the presence of disease and inflammation and have wide normal ranges (Allen and Cold 1988; Pepys et al., 1989). Serum amyloid A (SAA), and haptoglobin are acute phase proteins common to humans, cattle, sheep, mice, and several other species, including the horse. In several of these species, plasma concentration of SAA has been shown to increase 1000-fold following tissue injury, cellular necrosis, inflammation, and infection and decline rapidly in the recovery phase. In the studies presented here the concentration of serum amyloid A (SAA), haptoglobin and fibrinogen and other indices of health were measured in a number of different groups of horses, including; normal horses, those subjected to operative surgery, horses with surgical colic, racehorses in training, some of which had evidence of gastric ulceration, and foals with respiratory disease. Acute phase protein concentration was modeled with the outcome and with other commonly measured indices of health relevant to the disease states of interest. The study indicates that there is an association between acute and chronic inflammation and between the present of disease both overt and latent and suggests that the concentration of a number of acute phase proteins could be used to aid decision making when planning diagnostic or treatment interventions in horses.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
No hospedeiro bovino o nível de resistência ao carrapato Rhipicephalus (Boophilus) microplus varia de acordo com a raça sendo os fenótipos contrastantes herdáveis. O presente trabalho explora esses fenótipos a fim de estabelecer os perfis das respostas imunes, humoral e inflamatória, correlacionados com resistência em raça zebuína (Nelore) e susceptibilidade em raça taurina (HPB). Os animais foram expostos a infestação natural pelo R.(B.) microplus e amostras de soros foram coletadas em pontos estratégicos da cinética das infestações. Os níveis de imunoglobulinas totais (IgG1 e IgG2), bem como os de anticorpos IgG1, IgG2 e IgE anti-extrato de ovo, anti-extrato de larva não alimentada e anti-saliva foram determinados. Elementos da resposta inflamatória, como as principais proteínas de fase aguda e óxido nítrico, também foram dosados. Os resultados obtidos mostram que as infestações muito intensas, em hospedeiros suscetíveis, são capazes de modular os níveis séricos de IgG1 e IgG2 total, diminuindo-os significativamente em relação aos níveis observados durantes infestações menores. Também modulam negativamente a produção de todos os anticorpos específicos IgG1 e IgG2 avaliados a nível sistêmico. Bovinos susceptíveis ao carrapato produzem níveis mais altos de anticorpos IgE para todos os antígenos. O fenótipo susceptivel de infestação se diferencia pela maior freqüência do alótipo de IgG?2a, herdado por herança Mendeliana co-dominante. Animais suscetíveis, quando infestados, produzem níveis mais altos da proteína de fase aguda a1- glicoproteína ácida, de padrão anti-inflamatório, enquanto que animais resistentes produziram relativamente mais proteínas de fase aguda pró-inflamatórias, haptoglobina e amilóide sérica A. Em ambas as raças não houve diferença nos níveis de transferrina e óxido nítrico sistêmico, porém a produção de ambos é influenciada pelos níveis de infestação.
In bovine hosts resistance to the cattle tick, Rhipicephalus (Boophilus) microplus, varies according to the breed, being the phenotypes of infestations inherited. The present work exploits these contrasting phenotypes in order to determine the profile of the humoral, inflammatory and acute phase responses that are correlated with resistance seen in a zebuine breed (Nelore) and susceptibility seen in a taurine breed (Holstein). Bovines were exposed to natural infestations with R.(B.) microplus and they presented, as expected, different levels of infestation that also varied in intensity according to the season of the year. Samples of sera were collected at strategic points during the kinetics of different cycles of infestations. The levels of total serum IgG1 and IgG2 immunoglobulins, as well as those of IgG1, IgG2 and IgE anti-egg extracts, anti-unfed larvae extracts and anti-saliva antibodies were measured. Components of the inflammatory response, nitric oxide, as well as acute phase proteins, were also measured. The results show that very intense infestations in ticksusceptible bovines modulate the serum levels of IgG1 and IgG2, which are significantly diminished relative to those observed during less intense infestations. Intense infestations also modulate the production of all specific IgG1 and IgG2 antibodies. Susceptible animals produced more specific IgE, suggesting that this isotype does not participate in resistance against ticks. The susceptible cattle also have a higher frequency of IgG?2a , which are encoded by Mendelian co-dominant alleles. When infested susceptible animals produced higher levels of the anti-inflammatory acute phase protein, a1-acid glycoprotein, whereas resistant animals produced relatively higher levels of the pro-inflammatory proteins, haptoglobin and serum amyloide A.

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Este estudo teve como objetivo geral comparar os efeitos da laparotomia exploratória e do uso da fenilbutazona em bovinos e bubalinos. Para isso foram avaliados o perfil bioquímico, o proteinograma e o hemograma de 28 animais dos quais 14 eram búfalas e 14 vacas, todas fêmeas não lactantes, distribuídas em quatro grupos - VT: vacas tratadas com fenilbutazona; VC: vacas controle; BT: búfalas tratadas com fenilbutazona e BC: búfalas controle. Os animais dos grupos VT e BT receberam dose única de 10 mg/kg de fenilbutazona, por via intravenosa, uma hora antes da cirurgia. Foram coletadas amostras de sangue antes da laparotomia (0h) e 1(1h), 6(6h), 12(12h), 24(24h), 48(48h), 72(72h), 96(96h) e 120 horas (120h) e 15(15d) e 30(30d) dias após a intervenção. Os valores de creatinina foram mais elevados nas búfalas, e não sofram alteração devido à cirurgia, ou ao tratamento. O tratamento com fenilbutazona aumentou a concentração sérica de HDL em vacas e búfalas. Dentre as proteínas de fase aguda, a concentração de ceruloplasmina se elevou em todos os grupos de 48 horas a 120 horas após a cirurgia. A concentração sérica de haptoglobina se elevou mais nos grupos das búfalas do que nas vacas. A hemopexina foi verificada apenas na espécie bubalina e apenas até o 5º dia. O tratamento com fenilbutazona não alterou a contagem de hemácias nem o volume globular. Todas as vacas e búfalas apresentaram neutrofilia 12 horas após a laparotomia. O teor de fósforo aumentou no grupo controle das búfalas (BC) e no grupo tratado (BT), às 72h e 96h, respectivamente. O magnésio se apresentou, no início do experimento (0h) e ao final (30d), significativamente maior nos grupos das búfalas (BT e BC). A laparotomia e ou o tratamento com fenilbutazona não interferiram nas concentrações de sódio, potássio e cálcio ionizado
This study aims to compare the effect of exploratory laparotomy and treated with phenylbutazone in bovine and buffaloes. To pursue this purpose, biochemical profile, proteinogram and blood count cell tests were performed in 28 animals, of which 14 were buffaloes and 14 cows, all of then non-nursing, distributed in four groups - VT: cows treated with phenylbutazone; VC: control cows; BT: buffaloes treated with phenylbutazone and BC: control buffaloes. Animals from VT and BT received a single administration of 10 mg/kg of phenylbutazone, intravenously one hour before surgery. Blood samples were taken before laparotomy (0h) and 1(1h), 6(6h), 12(12h), 24(24h), 48(48h), 72(72h), 96(96h) e 120 horas (120h) e 15(15d) e 30(30d) days after surgery. Creatinine values were higher in buffaloes, and there were no changes, despite surgery or treatment. HDL showed different values in both species, due to surgical procedure. The use of phenylbutazone increased serum HDL in cows and buffaloes. Among acute phase proteins, serum haptoglobin showed higher values in buffaloes than in cows. Hemopexina was foundetected only in buffaloes and until the 5th day. Treatment with phenylbutazone did not change RBCs or globular volume values. Cows and buffaloes showed neutrophilia 12 hours after laparotomy. Phosphorus content increased in control buffaloes (BC) and in the treated buffaloes (BT) at 72h and 96h, respectively. Magnesium presented higher itself at the beginning of the experiment (0h) and end (30d) in buffaloes groups (BT and BC). Laparotomy or treatment with phenylbutazone did not changed sodium, potassium and calcium ionized values

Resumo: Este trabalho objetivou avaliar a anatomopatologia, o hemograma e o perfil bioquímico sérico de aves de postura inoculadas por Salmonella Gallinarum (SG) contendo os genes cobS e cbiA inoperantes (SGcobScbiA) que mostrou ser avirulenta em trabalhos anteriores, comparando-a com cepas virulentas SGNalr e SGcobS, para mostrar se SGcobScbiA pode ser componente de vacina contra cepas selvagens de SG e S.Enteritidis. 280 pintainhas foram distribuídas em 4 grupos (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) e G4 (controle). Com exceção do G4, os grupos receberam 0,2 mL de suas respectivas cepas contendo aproximadamente 108 UFC/mL de inóculo, aos 5 dias de idade. A eutanásia foi realizada 24h antes (1DAI) e após a inoculação (1DPI), e 3 (3DPI), 5 (5DPI), 7 (7DPI), 10 (10DPI) e 15 (15DPI) dias após a administração do inóculo, sacrificando-se, em cada momento, dez aves de cada grupo. As aves foram sacrificadas, obtendo-se amostras de sangue utilizadas para os exames hematológicos e bioquímicos. Fragmentos de fígado, baço, timo, bursa de Fabricius, rins e coração foram destinados aos exames histológicos. As aves inoculadas com a cepa SGcobS tiveram comportamento semelhante às aves inoculadas por SGNalr, porém com algumas respostas diferentes nos exames hematológicos e bioquímicos. As aves inoculadas com a cepa SGcobScbiA tiveram comportamento semelhante ao grupo controle, entretanto foi verificado alterações brandas em alguns parâmetros, mostrando que estudos futuros devem ser feitos, verificando se as alterações constatadas não irão interferir no desempenho de aves vacinadas com a cepa SGcobScbiA.
Abstract: The aim of the present study was to evaluate anatomopathology, hemogram and blood serum components of commercial layers experimentally inoculated with SGcobScbiA, which is a Salmonella Gallinarum (SG) strain it shows attenuation of the virulence in previous research and it was compared with high virulence SGNalr and SGcobS strains in order to show if SGcobScbiA has potential to be use as a vaccine against SG and S. Enteritidis wild strains. 280 commercial layers were divided into 4 groups (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) and G4 (control group). With exception of G4, all the other groups received 0,2 mL of their respective strain containing about 108 CFU/mL of the inoculum with five days of age. Birds were sacrificed 24 hours before (1DBI) and 24 hours after the inoculation (1DAI), and three (3DAI), five (5DAI), seven (7DAI) ten (10DAI), and fifteen (15DAI) days after the administration of the inoculum, slaughtering ten birds at a time in each group. Birds were submitted to euthanasia and blood samples were collected in order to make the hematological and blood serum components test. Samples of liver, spleen, thymus, bursa of Fabricius, kidneys and heart were collected for the histological test. The birds inoculated with SGcobS strain had similar behavior when compared with that ones who received SGNalr strain, however some different responses in the hematological and blood serum components were found. On the other hand, the birds inoculated with SGcobScbiA strain had similar behavior when compared with the control group, however, lower alterations in some parameters were found. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SGcobScbiA strain.
Orientador: Ângelo Berchieri Júnior
Coorientador: José Jurandir Fagliari
Banca: Antonio Carlos Alessi
Banca: Raimundo Souza Lopes
Mestre

Orientador: Helio Langoni
Resumo: A mastite bovina é responsável por grandes perdas econômicas na bovinocultura de leite, além de aspectos de saúde pública que devem também ser considerados. Animais com mastite clínica ou subclínica apresentam diversas alterações reprodutivas. No primeiro artigo da tese, os resultados obtidos demonstraram que a mastite causada por patógenos maiores (20,1%) ou por gram-negativos (15,4%) levaram a queda na taxa de prenhez na primeira inseminação comparado com o grupo controle (32,6%). Foi observado maior taxa de perda gestacional para os grupos de patógenos maiores (22,2%) e gram-negativos (30,1%) comparados com o controle (12,8%). Foi observado diferença entre todos os grupos para o número de dias em aberto. O grupo controle apresentou o menor intervalo (126,5 dias) seguido pelo grupo patógenos menores (162,0 dias) e pelo grupo de patógenos maiores (175,1 dias). Comportamento similar observado para patógenos gram-positivo (172,7 dias) e patógenos gram-negativos (191,1 dias). Portanto, é importante controlar a mastite em rebanhos leiteiros e, para tanto, o diagnóstico é uma etapa importante. Diferentes proteínas de fase aguda foram descritas como bons biomarcadores para o diagnóstico da mastite. No segundo artigo da tese, foi observado que diferentes patógenos podem alterar a liberação de proteínas de fase aguda no leite. Bactérias que causam inflamações mais graves (E. coli e Klebsiella pneumoniae) levaram maior concentração das APP. Patógenos que causam infecções crônica (Str... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Bovine mastitis is responsible for major economic losses in dairy cattle farming, as well as public health aspects that should also be considered. Animals with clinical or subclinical mastitis present several reproductive alterations. In the first article of this thesis, the results showed that mastitis caused by major pathogens (20.1%) or gram-negative (15.4%) led to a decrease in pregnancy rate in the first insemination compared to the control group (32.6%). Higher gestational loss rates were observed for the major (22.2%) and gram-negative (30.1%) pathogen groups compared with the control (12.8%). The difference was observed between all groups for the number of days open. The control group had the shortest interval (126.5 days) followed by the minor pathogens group (162.0 days) and the major pathogens group (175.1 days). Similar behavior observed for gram-positive pathogens (172.7 days) and gram-negative pathogens (191.1 days). Therefore, it is important to control mastitis in dairy herds and, so, diagnosis is a critical step. Different acute phase proteins have been described as good biomarkers for the diagnosis of mastitis. In the second article of this thesis, it was observed that different pathogens may alter the release of acute phase proteins in milk. Bacteria that cause more severe inflammation (E. coli and K. pneumoniae) led to a higher concentration of APP. Pathogens causing chronic infections (Environmental Streptococcus, S. aureus, Mycoplasma spp.) led to interm... (Complete abstract click electronic access below)
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Acute phase proteins are glycoproteins produced by hepatocytes and released into the bloodstream in response to tissue damage resulting from acute inflammatory processes. Currently, they are considered biomarkers of inflammation or infection, compared with other variables such as hyperthermia, leukocytosis and neutrophilia, but in donkeys have not yet been analyzed. This study aimed to evaluate acute inflammatory response in donkeys submitted to ovariectomy by two different approaches. We randomly selected 18 adult females animals weighing on average 100 kg, which were divided into two groups: Group I, 9 animals were ovariectomized by laparotomy and group II, 9 animals by laparoscopy. After the surgeries were evaluated the following variables in whole blood: blood count, white blood cell count differential cell count, fibrinogen, haptoglobin, albumin, antitrypsin, alpha-1-acid glycoprotein, ceruloplasmin, transferrin, immunoglobulins A and G (acute phase proteins) . In the same time, the peritoneal fluid were measured: number of erythrocytes and leukocytes, cytological analysis differential (leukocytes and mesothelial cells), total protein, pH and acute phase proteins. All variables were analyzed before surgery, 12, 24, 48, 72 hours, 8 and 16 days after surgery. There was an increase (p <0.05) in the number of leukocytes with neutrophilia after surgery, regardless of the surgical serum and peritoneal procedure. Acute phase proteins had different behaviors in blood and peritoneal fluid. For the first time, we identified a P23.000kD protein in serum and peritoneal samples in donkeys. The interest on the role of acute phase proteins in the veterinary medicine, particularly in horses is growing, mainly aiming to use them as early markers of inflammation
Proteínas de fase aguda são glicoproteínas produzidas pelos hepatócitos e liberadas na corrente sanguínea em resposta a dano tecidual, decorrente de processos inflamatórios agudos. Atualmente, são consideradas biomarcadores da resposta inflamatória e infecciosa, quando comparada com outras variáveis como hipertermia, leucocitose e neutrofilia, porém em asininos ainda não foram bem analisadas. Objetivou-se avaliar resposta inflamatória aguda em asininos submetidos à ovariectomia por duas diferentes abordagens. Foram utilizados 18 animais, fêmeas adultas, pesando em média 100 kg, as quais foram divididas em dois grupos, de 09 animais cada, onde grupo I, foram ovariectomizadas por laparotomia e o grupo II por laparoscopia. Após as cirurgias foram avaliadas as seguintes variáveis no sangue total: hemograma, fibrinogênio, haptoglobina, albumina, antitripsina, alfa-1 glicoproteína ácida, ceruloplasmina, transferrina, imunoglobulinas A e G. Nos mesmos momentos, no líquido peritoneal, foram mensuradas: número de hemácias e leucócitos, análise citológica diferencial (leucócitos e células mesoteliais), proteínas totais, pH e proteínas de fase aguda. Todas as variáveis foram analisadas antes das cirurgias, 12, 24, 48, 72 horas, 8 e 16 dias após os procedimentos cirúrgicos. Houve um aumento (p<0,05) do número de leucócitos com neutrofilia após as cirurgias, independente do procedimento cirúrgico, no soro e líquido peritoneal. As proteínas de fase aguda tiveram comportamentos diferentes no sangue e líquido peritoneal. Neste trabalho identificamos, pela primeira vez, a proteína P23.000kD no soro e amostras peritoneais de asininos. O interesse sobre o papel das proteínas de fase aguda na medicina veterinária, em especial em equídeos vem crescendo, principalmente, visando utilizá-las como marcadores precoces do processo inflamatório
2017-04-26

Transition period is defined as 3 weeks prepartum until 3 weeks postpartum and it is a period marked by changes in endocrine status to accommodate parturition and lactogenesis. Most infectious diseases and metabolic disorders occur during this time. Probably nutrition of transition cows is the key for the success of a dairy farm, since cows during the transition period are very sensible and a well fed herd will probably have more opportunity to be in good health status; but it is important at the same time to consider the environment and the management of cows groups within the herd in order to prevent stress which could influence feeding behavior. Forestomach motility of ruminants, especially cattle, is of major concern to the veterinarian, especially during the transition period. Among disturbs of rumen fermentation we can include Subacute Ruminal Acidosis (SARA) that is primarily a variation in rumen fermentation, tipical at the beginning of lactation…but not only. The disease seems to represent one of the most important fermentative/metabolic disorders in intensive dairy farms that affects rumen fermentations, animal welfare, productivity and farm profitability. According to the literature SARA may be caused by formulation of rations that contain excessive amounts of rapidly fermentable carbohydrates but also by a deficency of fiber or errors in delivery of the rations. In fact we can find SARA also in herds with correctly formulated diets in a chemical point of view and in this case the problem is probably related to management and physical treatment of the ration. This is probably the reason why we have SARA not only in early lactation but also in mid lactation: in the lattest situation SARA is not caused by inadequate adaptation of rumen papillae to the lactation diet because SARA appears long time after calving. In mid-lactation the development of SARA is linked to managerial factors like feeding frequency, processing of feed, e.g. pelleting, and housing and similar influences. Many authors studied the acute phase response during SARA. It has been suggested that low rumen pH could result in death and lysis of gram-negative bacteria that are in the rumen and hence increase free endotoxins in the rumen. It has been suggested that the acidic rumen environment, changes in osmotic pressure, and ruminal LPS may render the rumen epithelium susceptible to injury resulting in translocation of rumen endotoxin (which is a strong inducer of acute phase response) into the bloodstream. The role of free ruminal LPS in SARA remains difficult to ascertain because free LPS is detoxified in the liver and hence is not detectable in peripheral blood circulation. Acute phase proteins are the best indicators of an acute phase response but other indicators of inflammation such as fibrinogen and white blood cells can also be used as markers. The use of other blood parameters like base excess 2 (BE) or blood pH has been cited as diagnostic tool of non-acute ruminal acidosis. Because of an absorption of SCFA by the ruminal wall, the BE may be reduced. The objective was to investigate, analyze and study changes in blood, urine and faeces parameters in dairy cows affected by SARA in field conditions. The project was a collaboration which involved the Department of Veterinary Clinical Science of the University of Padova, the Istituto Zooprofilattico Sperimentale delle Venezie and the animal feed company Cortal Extrasoy s.p.a. that follow 10 of these farms for the nutrition plan. Twelve farms were investigated; farms where selected with similar characteristics about production, management and structures in the north-east of Italy (Veneto region). Many samples were collected (rumen liquid, urine, faeces and blood) from 132 lactating cows. Rumen liquid, urine and faeces pH was measured in field; ematochemical, ematological and blood gas analysis were then perfomed on blood and urine samples to the IZS delle Venezie and acute phase proteins were measured to the Faculty of Veterinary Medicine of Glasgow. Results showed the absence of strong correlation between SARA and APR leading to think that SARA as described in the literature is not really SARA that we find in field. According to our data we can not say that there is a direct relationship between SARA and metabolism and between SARA and blood gases; some intermediate products like homocysteine or other toxic products like valerate could affect general metabolism but this subject must be investigated more. We found alteration in blood parameters (leukocytosis with stress-like leukogram that included altered neutrophils/lymphocytes ratio, albumin alteration, some alteration in APP that could be related to stress as well): these data suggest that SARA could be related to a general unhealthy status and welfare decrease. It would be interesting to investigate more on causes and effects of SARA to understand if this fermentative disturb is the consequence of stressful situations: neuroendocrine conditions, management and environment could be related to SARA since several neuropeptides control voluntary feed intake and their effect may result in sorting out feeds from the diet. We hypothesize also that at the base of SARA onset in our region there could be a gastro-intestinal motility problem: low rumen pH values detectable during SARA could lead to weaker rumen motility inhibited by certain mechanisms arising during low pH phases within the reticulo-ruminal environment. Our data confirmed that SARA must be considered as a herd problem and even if the transition period is the most delicate in the cow's carrier SARA must be considered in every lactation stage and related not only to feeding management but to the equilibrium between the animals and the environment (general herd management, structures and welfare of cows).
Il periodo di transizione viene definite come il periodo che va da 3 settimane prima del parto a 3 settimane dopo il parto ed è un periodo caratterizzato da profondi cambiamenti a livello endocrine che hanno lo scopo di sopperire alle necessità del parto e della lattogenesi. La maggior parte delle patologie infettive e metaboliche si verificano in questo periodo. Probabilmente la nutrizione delle bovine è la chiave di successo di una azienda da latte, dal momento che le vacche nel periodo di transizione sono molto sensibili e che una mandria ben nutrita probabilmente avrà maggiore opportunità di godere di buona salute. È però importante allo stesso tempo considerare l'ambiente circostante e la gestione dei gruppi all'interno dell'intera mandria con lo scopo di prevenire qualunque elemento di stress che potrebbe a sua volta influenzare il comportamento alimentare degli animali. La motilità dell'apparato gastroenterico dei ruminanti, specialmente per i bovini, è una delle maggiori preoccupazioni per il veterinario, specialmente durante il periodo di transizione. Tra i disturbi fermentativi che colpiscono la vacca da latte possiamo includere l'acidosi ruminale subacuta (SARA) che è primariamente una variazione delle fermentazioni ruminali, e si verifica all'inizio della lattazione…ma non solo. Questa patologia sembra rappresentare uno dei maggiori problemi metabolico-fermentativi delle aziende da latte intensive che interessa le fermentazioni ruminali ma anche il benessere animale, la produzione ed il profitto. Secondo quanto riportato in letteratura SARA potrebbe essere causata dalla formulazione di razioni che contengono un eccesso di carboidrati rapidamente fermentescibili ma anche da una carenza di fibra o errori nella preparazione e distribuzione della razione. Infatti possiamo trovare SARA anche in mandrie con diete correttamente formulate dal punto di vista della composizione chimica e in questo caso probabilmente il problema è collegato al management e ai trattamenti fisici dell'alimento. Questo spiegherebbe perché troviamo SARA non solo a inizio lattazione ma anche a lattazione avanzata: in quest'ultima situazione SARA non può essere causata da un inadeguato adattamento delle papille ruminali alla dieta da lattazione perché compare molto tempo dopo il parto. Durante la lattazione avanzata lo sviluppo di SARA è legato a fattori manageriali come la frequenza di alimentazione, processi di trattamento dell'alimento come ad esempio l'uso del pellet, e i ricoveri o fattori simili. Molti autori hanno studiato la risposta di fase acuta in condizioni di SARA. È stato suggerito che il pH basso del rumine possa risultare nella morte e lisi dei batteri gram-negativi contenuti nel rumine stesso e quindi nell'aumento di endotossine libere (LPS). Si suppone che l'ambiente acido a livello ruminale, i cambi di pressione osmotica e i lipopolisaccaridi (LPS) liberi possano rendere l'epitelio 4 ruminale suscettibile a insulti che permetterebbero il passaggio delle endotossine (che sono forti induttori della risposta di fase acuta) nel circolo sanguigno. Il ruolo dei LPS in corso di SARA resta difficile da definire perché i LPS liberi vengono detossificati nel fegato e quindi non sono misurabili nel sangue periferico. Le proteine di fase acuta sono i migliori indicatori di una risposta di fase acuta ma altri indicatori di infiammazione come il fibrinogeno e i leucociti possono essere usati come markers. L'uso di altri parametri come il base excess (BE) o il pH sanguigno è stato citato come strumento diagnostico di acidosi ruminale non acuta: a causa dell'assorbimento di acidi grassi volatili a catena corta attraverso la parete ruminale, il BE dovrebbe ridursi. L'obiettivo di questo lavoro era di approfondire, analizzare e studiare i cambiamenti di parametri a livello ematico, urinario e fecale in vacche da latte con SARA in condizioni di campo. Il progetto è stato realizzato grazie ad una collaborazione tra il Dipartimento di Scienze Cliniche Veterinarie dell'Università di Padova, l'Istituto Zooprofilattico Sperimentale delle Venezie e la ditta mangimistica Cortal Extrasoy s.p.a. che segue 10 delle aziende dal punto di vista nutrizionale. Dodici aziende sono state incluse; sono state scelte nel nord-est dell'Italia (in regione Veneto) con caratteristiche simili relativamente alla produzione, la gestione e le strutture. Diversi campioni sono stati raccolti (liquido ruminale, urine, feci e sangue) da 132 vacche in lattazione. Il pH del liquido ruminale, delle urine e delle feci è stato misurato in campo; sui campioni di sangue e di urine sono stati fatti profili biochimici, oltre all'esame emocromocitometrico, l'emogasanalisi (presso IZS delle Venezie) e la misurazione delle proteine di fase acuta (presso la Facoltà di Medicina Veterinaria di Glasgow). I risultati hanno mostrato l'assenza di correlazioni forti tra SARA e risposta di fase acuta portando a pensare che SARA così come è descritta in letteratura non è realmente la SARA che troviamo in campo. Secondo i nostri risultati non esiste una relazione diretta tra SARA e metabolismo e tra SARA e parametri dell'emogas; alcuni prodotti intermedi come l'omocisteina o altri prodotti tossici come l'acido valerianico potrebbero influire sul metabolismo generale ma questo argomento meriterebbe di essere approfondito ulteriormente. Abbiamo trovato alterazioni in parametri sanguigni (leucocitosi con leucogramma assimilabile a situazione di stress, compreso un alterato rapporto neutrofili/linfociti, alterazione dell'albumina, alterazioni delle APP che potrebbero far pensare anch'esse a stress): questo suggerisce che SARA potrebbe essere legata ad uno stato di cattiva salute generale e mancanza di benessere. Sarebbe interessante approfondire le cause e gli effetti di SARA per capire se questo disturbo fermentativo è la conseguenza di una situazione stressante: condizioni neuroendocrine, management e ambiente potrebbero essere correlati a SARA dal momento che neuro peptidi 5 controllano l'assunzione volontaria di alimento e il loro effetto potrebbe risultare in alterati comportamenti alimentari. Abbiamo ipotizzato anche che alla base dell'insorgenza di SARA nella nostra regione ci possa essere un problema di motilità gastrointestinale: un basso pH registrabile durante SARA potrebbe portare a diminuita motilità inibita da meccanismi scatenati durante le fasi di abbassamento del pH nell'ambiente reticolo-ruminale. I nostri dati confermano che l?acidosi ruminale subacuta deve essere considerata come problema di mandria e, anche se il periodo di transizione è quello più delicato nella carriera di una vacca, SARA deve essere considerata in ogni stadio di lattazione e correlata non solo all'alimentazione ma anche all'equilibrio tra l'animale e l'ambiente (management, strutture e benessere).

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Trypanosomiasis is a disease of worldwide distribution which in Brazil is called "mal das cadeiras", owing the clinical signs shown by horses, which are the major species affected. The disease is caused by the protozoan Trypanosoma evansi, characterized by clinical signs such as weight loss, pale mucous membranes, swelling of the eyelid and vulva. Rabbits have been reported as a resistant species to T. evansi, however, is not yet known how immune aspects, such as innate immune and cholinergic system, behave in this species, not even as this inflammations pathways influence in rabbits's trypanotolerance. Thus, the aim of this study was to investigate the inflammatory response and cholinergic anti-inflammatory pathway and its relation to trypanotolerance in rabbits infected with T. evansi. For this, twelve female adult New Zealand rabbits, weighing 3.6 4.5 kg were used in this study. The animals were divided into two groups, a control group and an infected group, both with six animals. The rabbits belonging to the infected group received, intraperitoneally, 0.5 ml of rat blood containing 108 parasites per animal, while the control group received physiological solution by the same route. The experimental period reached 118 days. Blood was collected on days 0, 2, 7, 12, 27, 42, 57, 87, 102, and 118, for to determine the cholinesterases, and days 0, 5, 20, 35, 65, 95 and 118, for to evaluate blood count, total protein, serum proteinogram, immunoglobulins and acute phase proteins. There was an increase in the activity of butyrylcholinesterase (BChE), at 7th day PI, and in the activity of acetylcholinesterase (AChE), in 27th day PI. Furthermore, the infected group showed an increase in total protein and the fractions alpha, beta and gamma globulins along of experimental period. The reduction in albumin and hematocrit were observed in precise periods of experimental infection, as well as the increase in immunoglobulin G. Infection with T. evansi stimulated the production of acute phase proteins, such as C-reactive protein, haptoglobin, α-2 macroglobulin, being observed increase in immunoglobulin M (IgM) throughout the experimental period (118 days post-infection). From these results, it is concluded that the cholinergic pathway had influence the inflammatory response, through the action of AChE and BChE in the regulation of concentrations of acetylcholine, resulting in increased concentrations of cytokines and, consequently, in the production phase protein acute. The increase in IgM, associated with increased C-reactive protein and haptoglobin, suggests the involvement of these proteins in host defense against flagellated, with possible participation in trypanotolerance of rabbits infected with T. evansi.
A tripanossomose é uma enfermidade de distribuição mundial que, no Brasil, é denominada mal das cadeiras , em função dos sinais clínicos apresentados pelos equinos, principal espécie afetada. A doença é causada pelo protozoário Trypanosoma evansi, sendo caracterizada por sinais clínicos como perda de peso, mucosas pálidas, edema de pálpebra e vulva. Coelhos têm sido relatados como uma espécie resistente ao T. evansi, contudo, ainda não se sabe de que modo aspectos imunitários, como a imunidade inata e o sistema colinérgico, comportam-se nessa espécie, nem mesmo como essas vias da inflamação influenciam a tripanotolerância de coelhos. Assim, o objetivo deste trabalho foi investigar a resposta inflamatória e a via anti-inflamatória colinérgica e sua relação na tripanotolerância em coelhos infectados experimentalmente pelo T. evansi. Para tanto, foram utilizados 12 coelhos adultos, fêmeas, da raça Nova Zelândia, com peso corporal entre 3,6 a 4,5 Kg, divididos em dois grupos, um grupo controle e um grupo infectado, ambos com seis animais. Os coelhos pertencentes ao grupo infectado receberam, pela via intraperitoneal, 0,5 mL de sangue de rato contendo 108 tripanossomas por animal, enquanto que o grupo controle recebeu, pela mesma via, solução fisiológica. O período experimental foi de 118 dias, sendo o sangue coletado nos dias 0, 2, 7, 12, 27, 42, 57, 87, 102, e 118, para determinar as colinesterases, e nos dias 0, 5, 20, 35, 65, 95 e 118, para avaliar hemograma, proteínas totais, proteinograma sérico, imunoglobulinas e proteínas de fase aguda. Foi observado aumento na atividade da butirilcolinesterase (BChE) no 7° dia PI e da atividade da acetilcolinesterase (AChE) no 27° dia PI. Além disso, o grupo infectado apresentou aumento nas proteínas totais, bem como nas frações alfa, beta e gama globulinas ao longo do período experimental. A redução na albumina e no hematócrito foram observados em períodos pontuais da infecção experimental, bem como o aumento na imunoglobulina G. A infecção pelo T. evansi estimulou a produção de proteínas de fase aguda como a proteína C-reativa, haptoglobina, α-2 macroglobulina, sendo observado um aumento na imunoglobulina M (IgM) em todo o período experimental (118 dias pós-infecção). A partir desses resultados, conclui-se que a via colinérgica pode ter influência na resposta inflamatória, através da ação da BChE e AChE na regulação das concentrações de acetilcolina, acarretando em aumento nas concentrações de citocinas e, consequentemente, na produção de proteínas de fase aguda. A elevação na IgM, relacionada ao aumento da proteína C-reativa e haptoglobina, sugere o envolvimento dessas proteínas na defesa do hospedeiro contra o flagelado, e, possivelmente, na tripanotolerância de coelhos infectados com T. evansi.

Orientador: Gervásio Henrique Bechara
Banca: Hélio José Montassier
Banca: Osvaldo Augusto Brasil Esteves Sant'Anna
Resumo: No hospedeiro bovino o nível de resistência ao carrapato Rhipicephalus (Boophilus) microplus varia de acordo com a raça sendo os fenótipos contrastantes herdáveis. O presente trabalho explora esses fenótipos a fim de estabelecer os perfis das respostas imunes, humoral e inflamatória, correlacionados com resistência em raça zebuína (Nelore) e susceptibilidade em raça taurina (HPB). Os animais foram expostos a infestação natural pelo R.(B.) microplus e amostras de soros foram coletadas em pontos estratégicos da cinética das infestações. Os níveis de imunoglobulinas totais (IgG1 e IgG2), bem como os de anticorpos IgG1, IgG2 e IgE anti-extrato de ovo, anti-extrato de larva não alimentada e anti-saliva foram determinados. Elementos da resposta inflamatória, como as principais proteínas de fase aguda e óxido nítrico, também foram dosados. Os resultados obtidos mostram que as infestações muito intensas, em hospedeiros suscetíveis, são capazes de modular os níveis séricos de IgG1 e IgG2 total, diminuindo-os significativamente em relação aos níveis observados durantes infestações menores. Também modulam negativamente a produção de todos os anticorpos específicos IgG1 e IgG2 avaliados a nível sistêmico. Bovinos susceptíveis ao carrapato produzem níveis mais altos de anticorpos IgE para todos os antígenos. O fenótipo susceptivel de infestação se diferencia pela maior freqüência do alótipo de IgG?2a, herdado por herança Mendeliana co-dominante. Animais suscetíveis, quando infestados, produzem níveis mais altos da proteína de fase aguda a1- glicoproteína ácida, de padrão anti-inflamatório, enquanto que animais resistentes produziram relativamente mais proteínas de fase aguda pró-inflamatórias, haptoglobina e amilóide sérica A. Em ambas as raças não houve diferença nos níveis de transferrina e óxido nítrico sistêmico, porém a produção de ambos é influenciada pelos níveis de infestação.
Abstract: In bovine hosts resistance to the cattle tick, Rhipicephalus (Boophilus) microplus, varies according to the breed, being the phenotypes of infestations inherited. The present work exploits these contrasting phenotypes in order to determine the profile of the humoral, inflammatory and acute phase responses that are correlated with resistance seen in a zebuine breed (Nelore) and susceptibility seen in a taurine breed (Holstein). Bovines were exposed to natural infestations with R.(B.) microplus and they presented, as expected, different levels of infestation that also varied in intensity according to the season of the year. Samples of sera were collected at strategic points during the kinetics of different cycles of infestations. The levels of total serum IgG1 and IgG2 immunoglobulins, as well as those of IgG1, IgG2 and IgE anti-egg extracts, anti-unfed larvae extracts and anti-saliva antibodies were measured. Components of the inflammatory response, nitric oxide, as well as acute phase proteins, were also measured. The results show that very intense infestations in ticksusceptible bovines modulate the serum levels of IgG1 and IgG2, which are significantly diminished relative to those observed during less intense infestations. Intense infestations also modulate the production of all specific IgG1 and IgG2 antibodies. Susceptible animals produced more specific IgE, suggesting that this isotype does not participate in resistance against ticks. The susceptible cattle also have a higher frequency of IgG?2a , which are encoded by Mendelian co-dominant alleles. When infested susceptible animals produced higher levels of the anti-inflammatory acute phase protein, a1-acid glycoprotein, whereas resistant animals produced relatively higher levels of the pro-inflammatory proteins, haptoglobin and serum amyloide A.
Mestre

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A modulação da resposta imune inata seria uma ferramenta capaz de elhorar a capacidade do sistema imune de combater a infecção por patógenos e outros estímulos externos. A utilização de vias sinalizadoras com a participação de alguns receptores específicos, como o peroxisome proliferator-activated receptor alpha (PPARα) surgiria como importante hipótese para modular a produção de proteínas de fase aguda e do complemento em hepatócitos bovinos, órgão principal envolvido nessa síntese. Nesse sentido, o presente trabalho se utilizou de técnicas de cultivo primário de hepatócitos bovinos, provenientes de 5 animais diferentes, que foram isolados, cultivados e estimulados com um ligante desse receptor nuclear, o fenofibrato. Após 48 horas de cultivo as células tiveram seu ácido ribonucleico (RNA) extraído e quantificado, seguindo-se à síntese de ácido desoxirribonucleico complementar (cDNA) e execução das reações em cadeia da polimerase quantitativo (qPCR), a fim de se verificar a expressão e produção de proteínas de fase aguda e do sistema complemento por meio de de reações de qPCR. Os sobrenadantes de cultura também foram avaliados após esse período de tempo, onde foram dosadas as mesmas proteínas, por meio da técnica de Enzyme-linked immunosorbent assay (ELISA). Os resultados demonstraram que o cultivo se provou ineficiente no que diz respeito à viabilidade celular e à quantificação de RNA extraído, indicando que mais estudos devem ser feitos para padronização dessa técnica. Além disso, os resultados demonstraram que o PPARα, ativado frente ao seu ligante específico, o fenofibrato, não foi capaz de modular a expressão e tampouco a produção dos fatores de complemento e das proteínas de fase aguda analisadas, podendo indicar que em hepatócitos bovinos a modulação por esse receptor não ocorre da mesma maneira como em humanos e em murinos. No entanto, devido à grande variabilidade racial dos animais cujos fígados foram coletados e ao pequeno número amostral utilizado, análises mais refinadas são necessárias. Dessa maneira, estudos devem ser realizados visando novas moléculas capazes de modular proteínas relacionadas ao sistema imune em bovinos.
The innate immune modulation is a tool capable of improve the immune system capacity to fight infections by pathogens and other external stimuli. The use of signaling paths with participation of some specific receptors, such as peroxisome proliferator-activated receptor alpha (PPARα) arises as an important hypothesis to modulate the acute phase proteins (APP) and complement proteins in bovine liver cells, where most of these molecules are produced in a systemic level. In this way, this work aimed to establish a protocol of primary culture of bovine hepatocytes suitable to analyze intracellular signaling paths and genetic expression and synthesis of proteins related to innate immune system. For that, bovine hepatocytes from five different animals were isolated, cultivated and activated by a receptor ligand - fenofibrate, both alone and associated with proinflammatory stimuli, in different concentrations. After 48 hours of culture, ribonucleic acid (RNA) was extracted and quantified, complementary deoxyribonucleic acid was synthetized and real-time polymerase chain reactions were setup to verify the acute phase and complement proteins expression and synthesis. The culture supernatant was used to analyze the protein dosage by Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the hepatocytes isolation and culture were not very effective, since the extracted RNA showed low concentration and bad integrity, indicating that more studies must be performed to standardize this technique. Considering the low quantification and bad quality of RNA, it was not possible to access the role of the receptor PPARα in complement factors and acute phase proteins genetic expression and production, when activated by its specific ligand (fenofibrate). Thus, the development of isolation and culture techniques for bovine hepatocytes must continue to be developed in order to better evaluate the intracellular signaling pathways and the search for new molecules capable to modulate proteins related to the immune system in bovines.

Neste trabalho foram estudados os perfis renal, hepático, energético, hormonal e de proteínas de fase aguda (proteinograma) para avaliar possíveis interações com o desempenho reprodutivo em bovinos. Para isso, foram delineados três experimentos. No experimento 1 o objetivo foi verificar se a inseminação artificial (IA) causa alterações nos perfis renal, hepático, energético, hormonal e de proteínas de fase aguda e estudar as relações entre esses perfis e a hemodinâmica uterina. Foram utilizadas amostras de sangue de vacas Nelore que foram inseminadas (GIA, n=9) ou não (GC, n=9). As amostras foram coletadas 30 horas antes da IA, 4, 24, 48 e 168 horas após a IA. No experimento 2 o objetivo foi estudar os efeitos da qualidade do sêmen sobre o perfil hepático e proteinograma, e estudar a relação dos perfis renal, hepático, energético, hormonal e proteinograma sobre a vascularização uterina. Foram utilizadas amostras sanguíneas de 362 vacas, que foram divididas em três grupos experimentais de acordo com a qualidade do sêmen: Boa (n=121), Média (n=121) e Regular (n=120). As amostras foram coletadas 30 horas antes da IA, 4 e 24 horas após a IA. Por fim, o experimento 3 é um estudo retrospectivo, realizado com o objetivo de comparar os perfis renal, hepático, energético, hormonal, e proteínas de fase aguda entre animais prenhes e vazios após a IA, e verificar se há relação entre a hemodinâmica uterina e a fertilidade. Neste experimento, os animais foram divididos em dois grupos experimentais de acordo com o resultado da IA (prenhe, n=76 X vazia, n=45). Em todos os experimentos, nos mesmos momentos da coleta de sangue, foram realizadas avaliações ultrassonográficas do útero no modo color Doppler e espectral. As amostras dos experimentos 1, 2 e 3 foram submetidas à quantificação das proteínas de fase aguda e dos componentes metabólicos utilizando analisador bioquímico automático (RX Daytona) e à dosagem hormonal, pela técnica de radioimunoensaio. Os dados foram analisados pelo PROC MIXED (SAS, versão 9.2, 2010). Foram consideradas diferenças estatísticas quando P<0,05. No experimento 1, os grupos não diferiram quanto aos perfis renal, hepático, energético, hormonal e proteinograma, no entanto, o RI apresentou correlações positivas com AST e BHB e correlação negativa com estradiol. O estradiol também foi correlacionado com EV, entretanto essa correlação foi positiva. No experimento 2, os animais inseminados com sêmen B, M ou R apresentaram concentrações semelhantes das variáveis do perfil hepático e proteínograma. O RI foi correlacionado positivamente com colesterol, HDL, LDL, e progesterona, e negativamente com glicose, estradiol, albumina e proteína total. Já o EV apresentou correlações negativas com ureia, GGT e cortisol. No experimento 3, os grupos Vazio e Prenhe foram semelhantes quanto aos perfis renal, hepático, energético, hormonal, proteiograma e hemodinâmica uterina. Sendo assim, conclui-se que o processo da IA e a qualidade do sêmen utilizado não causam alterações sistêmicas, bem como a fertilidade não pode ser explicada por estas alterações. Adicionalmente, a hemodinâmica uterina é correlacionada com diversos parâmetros, no entanto, o padrão vascular do útero não mostrou relação com a fertilidade
In this study, were evaluated kidney, liver, energy, hormonal and acute phase proteins profiles to evaluate the possible interactions with the reproductive performance in cattle. For this, three experiments were designed. In experiment 1 the objective was to verify if artificial insemination (AI) causes changes in renal, liver, energetic, hormonal and acute phase proteins profiles and to study the relationship between these profiles and uterine hemodynamics. Blood samples from inseminated (GIA, n = 9) or non inseminated Nellore cows (CG, n = 9) were used. Samples were collected 30 hours before AI, 4, 24, 48 and 168 hours after AI. In experiment 2 the objective was to study the effects of semen quality on liver and protein profiles and study the relationship of renal, liver, energetic, hormonal and protein profiles on uterine vascularization. Blood samples of 362 cows were used, which were divided into three groups according to semen quality: Good (n = 121), medium (n = 121) and Regular (n = 1200. Samples were collected 30 hours before AI, 4 and 24 hours after AI. Finally, experiment 3 is a retrospective study, carried out in order to compare the renal, liver, energetic, hormonal, and acute phase proteins profiles between pregnant and non pregnant animals after AI, and check for relationship between uterine hemodynamics and fertility. In this experiment, animals were divided into two groups according to the result of AI (pregnant, n = 76 and non pregnant, n = 45). In all experiments, at the same time of blood sampling were performed sonographic evaluations of the uterus in color Doppler and spectral mode. The samples of experiments 1, 2 and 3 were subjected to quantification of acute phase proteins and metabolic components using automatic biochemical analyzer (RX Daytona) and to hormone dosage, by radioimmunoassay. Data were analyzed using PROC MIXED (SAS, version 9.2, 2010). Statistics differences were considered when P<0,05. In experiment 1, the groups did not differ about kidney, liver, energetic, hormonal and protein profiles, however, the RI showed positive correlations with AST and BHB and negative correlation with estradiol. Estradiol was also correlated with EV, however this correlation was positive. In experiment 2, the animals inseminated with semen B, M or R showed similar concentrations of the variables of liver and proteinogram profiles. The RI was positively correlated with cholesterol, HDL, LDL, and progesterone, and negatively with glucose, estradiol, albumin and total protein. EV showed negative correlations with urea, GGT and cortisol. In Experiment 3, the non pregnant and pregnant groups were similar about kidney, liver, energetic, hormonal, proteiogram profiles and uterine hemodynamics. Thus, in this study were not observed systemic changes caused by AI process and by quality of semen, and systemic differences did not notice is between non pregnant and pregnant animals. Additionally, uterine hemodynamic is correlated with various parameters, however, the vascular pattern of the uterus was not correlated with fertility

A modulação da resposta inflamatória uterina equina recebe atenção, devido a um distúrbio imunológico, que parece ocorrer em uma população de éguas, classificadas como suscetíveis à endometrite. O objetivo deste estudo foi verificar o efeito da corticoterapia aplicada na presença e na ausência de inflamação uterina sobre a proteômica e a concentração de óxido nítrico no líquido endometrial de éguas suscetíveis à endometrite. Éguas foram sincronizadas com 5 mg de prostaglandina F2α e após a verificação dos sinais de estro foram submetidas a quatro tratamentos. O primeiro foi o Controle, que não recebeu nenhum tipo de tratamento. O segundo foi o GC, onde as éguas receberam aplicação de um glicocorticóide, a cada 12 horas, por três dias consecutivos. O terceiro foi o Infectado, onde as éguas receberam uma infusão intrauterina de Streptococcus zoopepidemicus (1x109/mL) e o quarto foi o GC + Infectado, onde as éguas receberam a aplicação do glicocorticóide e a infusão intrauterina descrita acima. Doze horas após o final de cada tratamento, amostras de líquido endometrial puro e de lavados uterinos foram coletadas para análise proteômica e determinação de óxido nítrico, respectivamente. O primeiro artigo relata os dados preliminares da análise proteômica, com a contagem das bandas protéicas e a observação de 33 bandas protéicas no Controle, 54 no GC, 51 no Infectado e 72 no GC + Infectado. A corticoterapia pode induzir o aparecimento de um número maior de bandas protéicas, pois os géis com as maiores contagens foram nos tratamentos onde ela foi aplicada. No segundo artigo, foi realizada a identificação das bandas protéicas significativas, em relação à densidade óptica relativa e à frequência. A corticoterapia provocou uma alteração na proteômica do líquido endometrial, caracterizada pelo aumento e diminuição na densidade óptica relativa e na frequência de proteínas da fase aguda da inflamação, com as maiores alterações observadas quando a corticoterapia foi aplicada na presença do processo infeccioso. A infusão de Streptococcus zooepidemicus provocou alterações na proteômica do líquido endometrial, caracterizadas pelo aumento e diminuição na densidade óptica relativa e na frequência de proteínas da fase aguda da inflamação. Os resultados do estudo indicam que a corticoterapia provoca alterações imunológicas no endométrio equino, não apenas como depressiva, mas estimuladora da defesa local, através de uma ação imunomoduladora. No terceiro artigo, foi realizada a determinação da concentração de óxido nítrico, não sendo observada diferença significativa nos quatro tratamentos. Portanto, a corticoterapia provoca alterações proteômicas no líquido endometrial de éguas suscetíveis em estro, onde a presença de um estímulo inflamatório causado pela infusão intrauterina bacteriana induz a uma maior alteração, do que a ausência. A infecção do lúmen uterino provoca alterações proteômicas no líquido endometrial e a corticoterapia não influencia a concentração de óxido nítrico de éguas em estro.
The modulation of the equine uterine inflammatory response recieves much attention, due to an immunological disorder, which appears to happen in a population of mares, classified as suscetipble to endometritis. The aim of this study was to verify the effect of corticotherapy applied in the presence and in the absence of uterine inflammation on the proteomics and nitric oxide concentration of the endometrial fluid of mares susceptible to endometritis. Mares were synchronized with 5 mg prostaglandin F2α and after the observation of the signs of estrus were submitted to four treatments. The first was the Control, which did not recieve any kind of treatment. The second was the GC, where mares recieved the administration of a glucocorticoid, each 12 hours, for three consecutive days. The third was the Infected, where mares received an intrauterine infusion of Streptococcus zoopepidemicus (1x109/mL) and the fourth was the GC + Infected, where mares received the administration of glucocorticoid and intrauterine infusion as described above. Twelve hours after the end of each treatment, pure endometrial fluid and uterine flushings were collected for proteomic analysis and nitric oxide determination, respectively. The first article reports the preliminary data of the proteomic analysis, where protein band counts were done, being observed 33 protein bands in Control, 54 in GC, 51 in Infected and 72 in GC + Infected. Corticotherapy can induce the appearance of a higher number of protein bands, because the gels with the highest counts were in the treatments where it was applied. In the second article, the identification of the significative protein bands was done, regarding the relative optic density and frequency. Corticotherapy provoked an alteration in the endometrial proteomics, characterized by an increase and a decrease on the relative optic density and frequency of inflammatory acute phase proteins, with the major alterations observed when corticotherapy was applied in the presence of an infectious process. Streptococcus zooepidemicus infusion provoked alterations in the endometrial fluid proteomics, characterized by an increase and a decrease on the relative optic density and frequency of inflammatory acute phase proteins. Results from this study indicate that corticotherapy provokes immunological alterations in the equine endometrium, not only as depressor, but enhancer of local defense, through an immunomodulatory action. In the third article, the nitric oxide concentration was determined, with no significative diference observed in the four treatments. So, corticotherapy provokes alterations in the proteomics of the endometrial fluid of susceptible mares in estrus, where the presence of an inflammatory stimulus caused by intrauterine bacterial infusion induces a major alteration, than the absence. Uterine lumen infection provokes alterations in the proteomics of the endometrial fluid and corticotherapy does not influence nitric oxide concentration in mares in estrus.

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Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS
A pesquisa por marcadores precoces de inflamação tem sido o foco na medicina humana e veterinária durante as últimas décadas. Acredita-se que tanto o exercício físico quanto a obesidade sejam capazes de gerar a reação de fase aguda. O primeiro estudo teve como objetivo caracterizar a reação de fase aguda e sua relação com desempenho de cavalos submetidos a uma competição de longa distância. Foram avaliados 23 equinos os quais foram divididos em dois grupos sendo o grupo 1 composto pelos 10 primeiros colocados na competição e o grupo 2 composto pelos 13 animais que concluíram a competição em colocações inferiores. Foram efetuadas coletas sanguíneas em repouso (dia 0) e no ultimo dia de competição e foram realizados o teste de eletroforese em gel de poliacrilamida (SDS-PAGE). Os resultados demonstraram que o exercício físico imposto influenciou de forma significativa (p<0,0001) as concentrações séricas das proteínas inflamatórias. A haptoglobina foi superior nos animais com baixo desempenho. Conclui-se que o exercício prolongado é capaz de gerar reação de fase aguda e que o monitoramento da concentração de haptoglobina pode ser um sinalizador de processo inflamatório e baixo desempenho. O segundo estudo teve como objetivo associar a adiposidade corporal e a forma de criação de potros com o perfil energético sanguíneo, as concentrações sanguíneas de proteínas inflamatórias e lesões osteoarticulares no tarso comparando animais criados em regimes intensivo ou extensivo. Foram avaliados 40 potros com 18 meses de idade da raça crioula, sendo 20 animais criados em sistema extensivo e 20 animais em sistema intensivo. Foram efetuadas avaliação bioquímica e eletroforese proteica. Foram efetuadas a mensuração de acumulo de gordura através de ultrassonografia e estudo radiológico da região por tarso. Foi observado maiores níveis de colesterol total e LDL, glicemia, amiloide A sérica, transferina, haptoglobina, glicoproteina acida e proteína de 23Kda não identificada no animais do grupo intensivo com relação aos do grupo extensivo. Em 100% dos animais do grupo intensivo foram observados lesões articulares enquanto 23% dos animais do grupo extensivo apresentaram tais alterações. No teste de Fisher foi observado que os animais do grupo intensivo apresentaram 105% mais chance de desenvolver osteoartrite que os animais do grupo extensivo. Ainda, no teste de Pearson foi observada correlação positiva entre a gordura na crista do pescoço com o grau de comprometimento articular. Conclui-se que tanto o exercício quanto o sobre peso foram capazes de gerar uma reação de fase aguda, sendo que durante o exercício prolongado a haptoglobina apresentou relação com o desempenho dos animais. Já os potros em criação intensiva, além da reação de fase aguda, foi observada elevada incidência de osteoartrite juvenil e alterações no metabolismo energético
The search for early markers of inflammation has been the focus in human and veterinary medicine during the last decades. To determine the value of the measurement of proteins in the situations described, two studies were conducted. It is believed that both the physical exercise as obesity can generate an acute phase reaction. The first study aimed to characterize the acute phase reaction and its relation to performance of horses subjected to long distance competition. Was developed during the March of Resistance of Crioulo horses, consisting of a route of 750 km traveled. Were evaluated 23 horses, of wich were divided into two groups, with the first group consisting of the top 10 in the competition and the second group comprises the 13 animals that completed the competition in lower positions. Blood samples were collected at rest (day 0) and on the last day of competition and testing polyacrylamide electrophoresis (SDS-PAGE) gel were performed. The results showed that imposed physical exercise influenced significantly (p<0.0001) in serum concentrations of haptoglobin, ceruloplasmin, immunoglobulin A, acid glycoprotein and the protein with molecular weight of 23 kDa. Haptoglobin was higher in animals with low performance. The second study aimed to associate adiposity and the shape of creation of foals with blood energy profile, blood concentrations of inflammatory proteins and osteoarticular lesions in the tarsus comparing animals raised in intensive or extensive regimes. Were evaluated 40 Crioulo foals at 18 months old, 20 animals kept in extensive systems and 20 animals in intensive system. Protein electrophoresis and biochemical evaluation were performed. Measurement of fat accumulation were made by ultrasound and radiological study of the tarsus region. Higher levels of total cholesterol and LDL, blood glucose, serum amyloid A, transferrin, haptoglobin, acid glycoprotein and unidentified protein 23Kda of animals in the intensive group compared to the extensive group was observed. The intensive group also showed greater deposit of fat in the crest of the neck, retroperitoneal area and base of the tail region. In 100% of the animals in the intensive group were observed lesions compatible with juvenile osteoarthritis while 23% of the animals in extensive group showed such changes. In Fisher's exact test was observed that the animals in the intensive group had 105% more likely to develop osteoarthritis than animals from extensive group. Still, the Pearson test positive correlation between fat on the crest of the neck with the degree of joint involvement of foals was observed. We conclude that both the exercise and the overweight were able to generate an acute phase reaction, and during prolonged exercise haptoglobin were related to animal performance. Already foals in intensive farming, besides the acute phase reaction, high incidence of juvenile osteoarthritis and changes in energy metabolism was observed.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Rangelia vitalii is the etiologic agent of rangeliosis, a hemorrhagic disease that was described in dogs from South and Southeast Brazil, Uruguay and Argentina. The immunological aspects involved in this disease have not been fully elucidated. This study aimed to investigate the immunological parameters in rangeliosis by the determination of serum cytokines, nitric oxide, electrophoretic profile, acute phase proteins and immunoglobulins of dogs experimentally infected with R. vitalii. Twelve dogs were divided into two groups: Group A (control) composed by uninfected animals (n=5) and Group B consisting of animals inoculated with R. vitalii (n=7). After inoculation, animals were monitored by blood smear. Blood samples were collected through the jugular vein on days 0, 10 and 20 post-inoculation (PI) to determine the serum levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), oxidation products of nitric oxide (NOx), electrophoretic profile, acute phase proteins (C-reactive protein and alpha 1 acid glycoprotein) and immunoglobulins (IgG, IgM, IgA and IgE). The parasite was found within erythrocytes, neutrophils and monocytes on day 5 PI in infected group. This study showed an increase (P<0.01) of cytokines levels in the infected group when compared to control group. Serum levels of NOx increased on days 10 (P<0.01) and 20 PI (P<0.05) in infected animals. Total protein decrease on days 10 (P<0.01) and 20 (P<0.05) PI. There was a reduction (P<0.01) in albumin in infected group when compared with control group. Alpha-1 globulin and gamma globulin fractions increased (P<0.01) on day 20 PI in infected animals. Alpha-2 globulin (days 10 and 20 PI) and beta-2 globulin (day 10 PI) were decreased (P<0.05) in infected animals when compared to control group. Beta-1 globulin fraction did not differ statistically. Serum C-reactive and alpha 1 acid glycoprotein concentrations increased (P<0.05) on days 10 and 20 PI in infected animals. IgG was increased on days 10 (P<0.05) and 20 PI (P<0.01) in infected animals. It was observed an increase (P<0.01) in the levels of IgM, IgA, and IgE in infected animals compared to control group. From these results, we can conclude that rangeliosis causes alterations in serum cytokines, nitric oxide metabolites, serum proteins and stimulates the synthesis of acute phase proteins and immunoglobulins. These alterations may be associated with the host immune protection against the piroplasms, similar to what occurs in other hemoparasite infections.
Rangelia vitalii é o agente etiológico da rangeliose, uma doença de curso hemorrágico que foi descrita em cães no Sul e Sudeste do Brasil, Uruguai e Argentina. Os aspectos imunológicos envolvidos nessa doença não foram totalmente elucidados. Nesse estudo objetivou-se investigar os parâmetros imunológicos na rangeliose, através da determinação sérica de citocinas, óxido nítrico, perfil eletroforético, proteínas de fase aguda e imunoglobulinas de cães experimentalmente infectados por Rangelia vitalii. Foram utilizados 12 cães divididos em dois grupos: Grupo A (controle) composto por animais não infectados (n=5) e Grupo B composto por animais inoculados com R. vitalii (n=7). Após inoculação, os animais foram monitorados por esfregaço sanguíneo. Foi coletado sangue da veia jugular nos dias 0, 10 e 20 pós-inoculação (PI) para a dosagem sérica das citocinas interferon gama (IFN-γ), fator de necrose tumoral alfa (TNF-α), interleucina 1 (IL-1) e interleucina 6 (IL-6), dosagem de produtos de oxidação do óxido nítrico (NOx), perfil eletroforético, dosagem de proteínas de fase aguda (proteína C-reativa e alfa 1 glicoproteína ácida) e imunoglobulinas (IgG, IgM, IgA e IgE). No grupo infectado o parasita foi encontrado no interior de eritrócitos, neutrófilos e monócitos no dia 5 PI. Esse estudo demonstrou aumento (P<0,01) dos níveis de citocinas séricas no grupo infectado em relação ao grupo controle. Os níveis séricos de NOx aumentaram nos dias 10 (P<0,01) e 20 PI (P<0,05) nos animais infectados. A proteína total reduziu nos dias 10 (P<0,01) e 20 PI (P<0,05). Ocorreu redução (P<0,01) de albumina no grupo infectado em relação ao grupo controle. As frações alfa-1 globulina e gama globulina aumentaram (P<0,01) nos animais infectados no dia 20 PI. Alfa-2 globulina (dias 10 e 20 PI) e beta-2 globulina (dia 10 PI) decresceram (P<0,05) nos animais infectados em comparação ao grupo controle. A fração beta-1 globulina não diferiu estatisticamente. As concentrações séricas de proteína C- reativa e alfa-1 glicoproteína ácida aumentaram (P<0,05) nos dias 10 e 20 PI nos animais infectados. Ocorreu aumento de IgG nos dias 10 PI (P<0,05) e 20 PI (P<0,01) no soro dos animais infectados. Foi observado aumento (P<0,01) de IgM, IgA e IgE nos animais infectados em relação ao grupo controle. A partir dos resultados pode-se concluir que a rangeliose causa alteração na concentração de citocinas séricas, nos metabólitos do óxido nítrico, nas proteínas séricas e estimula a síntese de proteínas de fase aguda e imunoglobulinas. Estas alterações podem ser associadas à resposta imune do hospedeiro contra o piroplasma.

Serum amyloid A protein (SAA) is an acute phase protein that has recently gained increasing interest as a potential marker for disease and treatment monitoring. We investigated SAA and CRP levels in (a) patients with various common infectious diseases (n=98), (b) patients with pyelonephritis (n=37) versus patients with cystitis (n=32), (c) healthy individuals of varying ages (n=231), (d) very immature newborn infants with or without nosocomial infections (NIs) (n=72) and (e) patients with bacterial infections treated with cefuroxime (n=81).

SAA significantly correlated with CRP in viral as well as in bacterial infections (for the total group: r²=0.757, p<0.0001) and showed a systemic inflammatory response in 90% of the patients with cystitis as compared with 23% for CRP. Equally high efficiencies (0.96 and 0.94 for SAA and CRP, respectively) were observed in discriminating between pyelonephritis and cystitis. SAA and high sensitive (hs) CRP were lower in umbilical cords (p<0.0001) and higher in elderly adults (p<0.0001-0.03) than in the other age groups; higher in immature newborn infants than in term infants; and higher in the NI group than in the non-NI group. Interindividual variabilities of the time course of the biomarkers SAA and CRP were considerable. Because of the smoothed distribution of SAA and CRP (i.e. elevations were both essentially unchanged during the first 3 days of cefuroxime treatment), these markers were not useful when deciding parenteral-oral switch of therapy, which occurred within this time period in most cases.

SAA is a sensitive systemic marker in cystitis. SAA and hsCRP in umbilical cord blood are close to the detection limit and increase with age. They increase in relation to NI in very immature newborn infants and might therefore be used in diagnosis and monitoring. Finally, SAA and CRP in adults with bacterial infections could not predict an early parenteral-oral switch of antimicrobial therapy.

O cachorro-do-mato (Cerdocyon thous - Linnaeus, 1766) é um canídeo de médio porte com distribuição ampla na América do Sul e que ocorre em quase todo o Brasil. Dentre as principais ameaças à sua conservação estão os atropelamentos causados principalmente pela perda de habitat. A escassez de dados laboratoriais de cachorro-do-mato prejudica o atendimento médico veterinário dificultando a aplicação de terapias adequadas. Este trabalho teve como objetivo avaliar os níveis de Proteína C Reativa, Albumina, Pré-albumina, Ceruloplasmina, Haptoglobina e Alfa 1 Glicoproteína Ácida bem como o Indice Prognóstico Inflamatório Nutricional (IPIN) nesta espécie, obtendo portanto uma primeira descrição destes marcadores prognósticos. Foram coletados 1,5 ml de sangue por acesso jugular de oito exemplares de Cachorro-do-mato (Cerdocyon thous) provenientes do acervo do Laboratório de Ensino e Pesquisa em Animais Selvagens (LAPAS) da Faculdade de Medicina Veterinária da Universidade Federal de Uberlândia para exames de rotina. As amostras foram coletadas através da veia jugular após contenção física dos animais e tricotomia da região. Após análise estatística, os valores encontrados foram: albumina: entre 2,7 e 3,0 g/dl, alfa 1 glicoproteína ácida: entre 0,19 e 0,21 g/l, proteína C reativa: entre 1,7 e 2,2, pré-albumina: entre 30 e 35 mg/l e haptoglobina: entre 0,078 e 0,156 e IPIN ≤ 0,006 sendo considerado normal e valores ≥ 0,006 considerados altos. Esta prima descrição servirá como base para estudos utilizando animais com doenças específicas e, após as análises, comparadas com os valores encontrados neste trabalho verificando se o comportamento segue a semelhança de cães domésticos.
The dog-eating fox (Cerdocyon thous - Linnaeus, 1766) is a medium sized canid widely distributed in South America and occurs in almost all of Brazil. Among the main threats to their conservation are the roadkill mainly caused by habitat loss. The shortage of laboratory bush dogs data affect the veterinary medical care hindering the application of appropriate therapies. This study aimed to evaluate the levels of C-reactive protein, albumin, pre-albumin, ceruloplasmin, haptoglobin and Afla 1 acid glycoprotein and the Prognostic Index Inflammatory Nutritional (IPIN) in this species, thus obtaining a first description of these prognostic markers. They collected 1.5 ml of blood by jugular access 8 of Mato Dogs copies (thous thous) from the Laboratory of collection of Teaching and Research in Wildlife (limpets), Faculty of Veterinary Medicine, Federal University of Uberlândia for exams routine. The samples were collected via the jugular vein after physical restraint of animals and trichotomy of the region. After statistical analysis, the values were: albumin: between 2.7 and 3.0 g / dl, alpha 1-acid glycoprotein: between 0.19 and 0.21 g / l, C-reactive protein: between 1.7 and 2 2, prealbumin between 30 and 35 mg / l haptoglobin: between 0.078 and 0.156 and IPIN ≤ 0.006 being considered normal and values ≥ 0.006 considered high. This press description will serve as a basis for studies where animals may be used with specific diseases and, after analysis, compared with the values found in this study and verified the behavior follows the likeness of domestic dogs.
Tese (Doutorado)

Malnutrition is associated with an increased incidence of morbidity and mortality after surgical operations. The acute phase plasma protein response is believed to be an important mechanism in the repair and resolution of inflammation following operation. This study attempted to determine if the acute phase response is attenuated by malnutrition and thus a possible contributor to the observed increase in the incidence of complications after surgery. Four groups of patients were studied. Normally nourished patients with benign disease of the upper gastrointestinal tract, normally nourished patients with malignant disease of the oesophagus or stomach, malnourished patients with malignant disease of the oesophagus or stomach and malnourished patients with malignant disease of the oesophagus or stomach who had received intravenous nutrition for 7 days prior to operation. Firstly, nutritional indices of the patients were assessed. Then, in the in vivo study, the acute phase proteins C-reactive protein, alpha₁-antitrypsin, and alpha₁-acid glycoprotein were measured sequentially following radical surgery for upper gastrointestinal carcinoma in groups 2, 3 and 4. Finally liver biopsies from all 4 groups were incubated in vitro in the presence of interleukin-1 an acute phase stimulant and the production of C-reactive protein measured using an ELISA assay. The magnitude of the acute phase plasma protein response in vivo and in vitro was compared amongst the groups and correlations were sought between the magnitude of the acute phase plasma protein response in vivo and in vitro and the various nutritional indices. No influence of nutritional factors could be demonstrated in either arm of the study. This could be a genuine finding or the result of inadeguacy in the design of the study or the power of the study in relation to the number of patients studied. Further studies using methods to improve the preservation of the liver slices by shorter incubation and improved oxygenation are suggested.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
O cálcio é um mineral essencial para uma variedade de processos fisiológicos do organismo da vaca leiteira, sua deficiência pode causar vários distúrbios metabólicos, que levam a redução do desempenho produtivo e reprodutivo, com consequentes perdas econômicas. Atualmente há no mercado veterinário algumas estratégias para a prevenção da hipocalcemia, como: dieta aniônica, dieta deficiente de cálcio, administração de vitamina D3 no pré-parto e tratamento com cálcio oral no pós-parto recente, mas todas essas requerem ajustes a serem melhorados. Com isso, o objetivo desta Tese, é desenvolver uma estratégia farmacêutica eficiente no pré-parto, para prevenir a hipocalcemia subclínica (HSC) no pós-parto, para tal foi realizado dois experimentos in vivo, aprovados pelo Comitê de Ética de Experimentação animal da Universidade Federal de Pelotas. O primeiro foi relacionar a queda do cálcio no periparto com a síntese de proteínas de fase aguda, observamos que vacas com concentrações de cálcio  8,5 mg/dL alteram a síntese de proteínas de fase aguda já no pré-parto, causando diminuição no escore de condição corporal, no peso e na produção de leite. O segundo experimento foi analisar o efeito da suplementação de diferentes dietas cátion-anionica no pré-parto e relacionar com a hemogasometria e concentrações de cálcio sanguíneo. Observamos que a suplementação com dieta aniônica, proporciona um aumento na concentração de cálcio ionizado disponível no plasma e redução nas globulinas, demonstrando que o pH levemente ácido provavelmente altera a síntese de glubulinas.
Calcium is an essential mineral for a variety of physiological processes in the body of the dairy cow, its deficiency can cause various metabolic disorders, leading to reduced production and reproductive performance, with consequent economic losses. There are currently in the veterinary market some strategies for the prevention of hypocalcemia, such as anionic diet, deficient in dietary calcium, vitamin D3 administration in pre-partum and treatment with oral calcium in the recent post-partum, but all of these require adjustments to be improved. Thus, the aim of this thesis is to develop an effective strategy in the pre-partum to prevent subclinical hypocalcemia (HSC) postpartum, for this was done two in vivo experiments, approved by the Animal Experimentation Ethics Committee of the University Federal of the Pelotas. The first was to correlate the drop of calcium in the peripartum with the synthesis of acute phase proteins, we observed that cows in concentrations of calcium  8.5 mg / dL alter the synthesis of acute phase proteins, causing decrease in body condition score, in weight and milk production. The second experiment was to evaluate the effect of supplementation of different cation-anion diets in prepartum and relate to the blood gas analysis and blood calcium levels. We observed that supplementation with dietary anion, provides an increase in concentration of available calcium in ionized plasma and reduction in globulins, demonstrating that pH may change glubulinas synthesis.

Die Bestimmung der Plasmaviskositaet und des Haptoglobin beim Rind erweisen sich als sinnvolle Ergaenzungen in der Labordiagnostik. Ihre Bestimmung als Suchtest zur Fruehdiagnostik von praxisrelevanten Rinderkrankheiten ist zu empfehlen.

Type 2 diabetes is a metabolic disorder with globally increasing prevalence. Therefore, the identification of etiological factors is of ascending relevance for the understanding, treatment, and prevention of the disease. Levels of the acute-phase serum amyloid A (A-SAA) protein have been found to be elevated in type 2 diabetic subjects, but little is known about their causal implication in the development of type 2 diabetes so far. This doctoral thesis presents an epidemiological perspective on the association between circulating levels of A-SAA and risk of type 2 diabetes and assesses a possible causality in this association using a genetic approach. Three studies were conducted. In a prospective cohort study, A-SAA levels were measured in 836 initially non-diabetic, elderly, Western European subjects without clinically overt inflammation who participated in a seven-year follow-up examination. Results of this study provided first evidence that levels of A-SAA are elevated years before the manifestation of type 2 diabetes independent of other type 2 diabetes risk factors. However, adjustment for parameters related to glucose metabolism, particularly levels of 2h-glucose, attenuated the association suggesting a potential link via post-challenge hyperglycemia in the association between elevated levels of A-SAA and type 2 diabetes or, alternatively, a possible reverse causality between levels of A-SAA and 2h-glucose. In a meta-analysis of genome-wide association studies (GWAS) on levels of A-SAA conducted in three population-based studies and one prospective case-cohort study including a total of 4,212 participants of European descent two biologically highly plausible genetic susceptibility loci for A-SAA proteins at chromosome 11p15.5-p13 and chromosome 1p31 were identified. One of these loci represented a suitable candidate for a Mendelian Randomization study. In Mendelian Randomization studies, genetic variants are used as proxies for a biomarker. These studies benefit from the fact that genotypes are randomly assorted at meiosis and are largely independent of non-genetic confounding and disease processes. Thus, they constitute a genetic approach to assess whether the association between a biomarker and a disease is causal. The associations between genetic variants of the candidate locus and type 2 diabetes were extracted from the results of a meta-analysis of eight GWAS (8,130 cases, 38,987 controls) published by DIAGRAM, a large diabetes and genetic consortium. In spite of sufficient power, the above mentioned associations were not significant suggesting that there are genetic mechanisms that raise plasma levels of A-SAA without translating into an increase in type 2 diabetes risk. In conclusion, results of this doctoral thesis indicated that levels of A-SAA are elevated years before the manifestation of type 2 diabetes but could not provide evidence that the association is truly causal using a genetic approach. Rather it seems likely that the association between levels of A-SAA and risk of type 2 diabetes is substantially influenced by post-challenge hyperglycemia. Time-series studies are warranted to elucidate the role of post-challenge hyperglycemia in this association.