ANIMAL PATHOLOGY

Author: PARRA MUÑOZ M. DOLORES.
Year: 2003.
University: MURCIA [www.um.es].
Place of defense: CENTRO DE LECTURA (FACULTAD DE VETERINARIA).
Place of preparation: MURCIA.

Summary: In this paper we established several goals (methodological and clinical application) to try to resolve some of the constraints to the use of determinations of acute phase proteins in the canine species. As a first target is isolated and purified and C-reactive protein haptoglobin canine from serum acute phase. The CRP was purified by inmunopurificación using polyclonal anti-CRP canine antibodies and by affinity chromatography using a column Agarose coupled with fosforiletanolamina through liaison epoxy. This latter technique provided a protein purer than the first. The haptoglobin was purified by ammonium sulfate fractionation followed by gel filtration. The high purity of the CRP and Hp obtained allowed to use them as standards in fluoroinmunoensayos made in the present study for the determination of two proteins in the dog. The CRP and purified Hp could serve as immunogens in obtaining antibodies anti CRP and anti canine or as material control in other commercial methods for measuring these proteins. The second goal is focused on obtaining an immunoassay based fluorometría time delayed for the quantification of C-reactive protein canine. It won an essay or non-competitive type sandwich in three steps with antibodies biotinados as antibodies and antibody capture marked europio as antibody detection, such as using solid phase strips microtitre streptavidin-coated. The test was successfully validated in terms of analytical and clinical. Thus the method showed good precision and accuracy, with a very high sensitivity (detection limit of our method is the lowest of all existing at present) and the absence of interference by the use of samples hemolytic, lipémicas or bilirrubinémicas. As for the analysis of serum samples from healthy and sick animals, the values provided by the CRP test enabled differentiate clearly between the two groups of animals. Levels of protein detected in the various pathologies studied in dogs and healthy coincided with those of other authors. The third goal was implemented in samples of whole blood, saliva and efusiones the fluoroinmunoensayo developed for the measurement of serum CRP. The immunoassay was analytically validated for all three types of biological samples provide a good precision and accuracy. Additionally it was found that storage for a month of samples of whole blood and securing it with different anticoagulants did not affect the determination of the protein through fluorometría time delayed. The concentration of CRP in whole blood was slightly lower than that observed in plasma, possibly because of the volume occupied by red blood cells, however, it was found a good correlation between levels of CRP in whole blood and its related plasmas (y = 0.86 x - 1.03; R2 = 0.97). The salivary levels of CRP were significantly lower than in serum and did not show a high correlation (y = 0008x + 0034; R2 = 0.70). Perhaps some of the factors affecting the composition or salivary secretion may be responsible for this phenomenon. The highlight of this section was the clinical study, which showed that dogs with inflammation can be differentiated from healthy dogs by measuring their levels of CRP in blood samples 8 whole c e95 sa saliva, and that the determination of protein efusiones canine has a high sensitivity (100%, 88.2%) Yespecificidad (94.4%, 100%) to differentiate exudates of trasudados and exudates of trasudados modified. As the fourth goal was a test inmunofluorométrico time delayed for determining haptoglobin canine. One trial was conducted in two steps uncompetitive with antibodies biotinados and antibodies marked coneuropio on either side of the sandwich, and strips microtitre as streptavidin-coated solid phase. The test was validated in terms of showing adequate analytical precision and accuracy, a high detection limit (the lowest of all published so far) and the absence of interference by hemolysis, lipemia or bilirrubinemia. Most commercial trials for Hp canine affected by hemolysis, therefore compared with other methods, ours provides the great advantage of not being disturbed by it. As for the clinical validation, the results indicated that the test used to distinguish healthy from the sick dogs, providing distinct concentrations for both groups of animals that match those provided previously by other authors. The fifth and final goal was dedicated to the implementation of fluoroinmunoensayo for determining the HP canine in samples of whole blood, saliva and efusiones. That application was limited by problems of stability of haptoglobin purified, preventing a complete analytical and clinical validation on this kind of samples, although there were some interesting results. Concentrations of haptoglobin in whole blood were much lower than in serum, but the correlation between the two was good (y = O 15x - 0.01; R2 = 0.84). The salivary concentrations of HP were hundreds or thousands of times lower than serum and showed a weak correlation (R2 = 0.53) with the levels of serum Hp present. The low regression coefficient obtained could be explained by a possible delay in the passage of haptoglobin from the blood to the saliva or any of the factors affecting the saliva secretion. Nevertheless, it should be emphasized that the levels of Hp in whole blood and saliva of healthy dogs were significantly lower than those achieved in dogs sick. However, it would be necessary to conduct clinical studies with a larger number of animals, especially dogs sick, to determine the sensitivity and specificity of the test for this type of samples and evaluate the possible application of the determination of Hp in blood and salivary a future. Finally, although there are no methodological limitations for the quantification of haptoglobin in efusiones canine through fluorometría time delayed, the trial was not possible to distinguish between drips and trasudados, which limits the application of this protein in the differentiation efusiones.

Author: CARBONERO MARTINEZ ALFONSO.
Year: 2004.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: There has been an observational epidemiological study to find out factors act on the HIV status of the major viral agents of Bovine Respiratory Syndrome: Bovine Herpesvirus type 1, the Virus Diarrhea Vírica Bovina, Virus Respiratory Sincitial Bovinoy Virus the Parainfluenza3 . People in the study were undergoing breast-feeding calves from dairy farms in the provinces of Cordoba and Santa Fe. In total, took blood samples from 852 calves from 55 farms, between the years 2000 and 2002. The samples were subjected to serological testing kits ELlSA commercial use. The result of these tests was taken as dependent variable for the epidemiological study, while 101 of the 155 variables initially included in the questionnaire epidemiological served as independent variables. The statistical study was descriptive analysis followed by a bivariate and, finally, the multivariate analysis, conducted by logistic regression, which is extracted predictive models parala seropositividadde each of the agents. The seroprevalence in front of each of the viruses studied ranged from 38.4% for the virus Parainfluenza 3 and the a 66.3% to Bovine Herpesvirus type 1, while the Respiratory Sincitial Bovine Virus, with a 46.6 % and the Virus Diarrhea Vírica Bovina, with a 59.9% mostraronvalores intermediate. Significant associations were found between Respiratory Sincitial Bovine Virus and the rest of agents surveyed, and between Virus the DiarreaVírica Bovinay Virus the PArainfluenza3. By multivariate analysis were obtained logistic regression models that allow us to know the variables that influence seropositive viral agents studied. In the case of Bovine Herpesvirus type 1 model included eight variables, seven for the Virus Diarrhea Vírica Bovina, seven for Respiratory Sincitial Bovine Virus, and finally five for the virus the Parainfluenza3.

Author: MONLEÓN MOSCARDÓ EVA.
Year: 2004.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The diagnosis of bovine spongiform encephalopathy and scrapie under the surveillance and control of transmissible spongiform encephalopathies is done using the techniques established by the International Organization of Epizootics: study characteristics of the lesions in the central nervous system by examining histopathology and the detection of PrPsc, the only known molecular marker for these diseases, by applying the techniques of immunohistochemistry, Western Blotting, and the observation of the fibrils associated with scrapie by electron microscopy. Additionally, starting in the year 2001 introduced the so-called rapid tests for diagnosis, positive or equivocal results should be confirmed by the techniques established by the International Organization of Epizootics. The main objective of this thesis, developed in four different studies, is to contribute to improved diagnostic techniques of transmissible spongiform encephalopathies, mainly from the immunohistochemistry and implemented at the national program for monitoring and controlling these enfermedades.En the first paper presented (study) described the first cases of BSE detected in Spain. These cases were diagnosed using histological techniques and Immunohistochemical, with a wide distribution of PrPsc in the samples tested, even in areas where the vacuolisation characteristic of these diseases was not present. On the other hand, both the lesion profile as the pattern of accumulation of PrPsc observed in these cases were similar between them, as well as those described in other European countries, which suggests, in agreement with other authors, that the majority of BSE cases are caused by a single strain of the agent causal.Durante the course of the monitoring program TSE is often a significant proportion of samples submitted for diagnosis submit an advanced state of autolisis in these samples lack Integrity tissue prevents applying techniques for histological diagnosis. In the second of the studies in this thesis (study B) shows that the technique is suitable immunocytochemistry for the diagnosis of bovine spongiform encephalopathy and scrapie samples in an advanced state of autolisis, even in a state líquido.Por another However, the sensitivity of the detection of PrPsc by immunohistochemical technique depends largely on the methodology and the antibodies used. In this regard, over the past two works included in this thesis (studios C and D) is studying the effect of different visualization systems and antibodies on the sensitivity of the technique applied in immunohistochemistry samples lymphoreticular system of sheep affected by the disease scrapie. The results of both studies indicate that the sensitivity of the technique studied for the detection of PrPsc varies significantly depending on the display system and the antibody used in the studies performed the best results have been obtained with the imaging system CSA and the antibody L42.Finalmente, in the last work (study B) provides an additional study done in order to compare the detection capability of the PrPsc two rapid tests (validated for the diagnosis of bovine spongiform encephalopathy) regarding the immunohistochemical technique, used on samples of the central nervous system and the lymphoreticular system of sheep affected by the disease scrapie. The rapid tests examined (Prionics Western immunoblotting and LIA) detected the PrPsc, both in tissue samples as nervous tissue linfoid 8 and, but 3c9 with a sensitivity lower than immunohistochemical technique. The main conclusion of this study was that only analyze samples from the two tissues, and lymphoreticular nervous, we could detect all positive cases, irrespective of the technique used (rapid tests or immunohistochemistry).

Author: OLIVEIRA PETRUCCI CARLOS GUILHERME DE.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: MURCIA.

Summary: In this work has been validated in terms of analytical and clinical canine species, a commercial kit for determining haptoglobin (Hp) (Izasa SA, Barcelona, Spain), the kit is based on a reaction inmunoturbidimétrica employing antibodies against haptoglobin human. In a first tested for cross-reactivity between the antibody anti-haptoglobina provided by the kit and haptoglobin of the canine species. To this were used techniques immunodifusión the agarose gel and an ELISA. Both methods showed that the antibody against human haptoglobin recognizes haptoglobin dog, and that the union between the two is proportional to the concentration of HP. Therefore kit object of our study can be used in dogs. It also studied the effect produced in the commercial kit using a standard dog, which led to an improvement in the accuracy and specificity, and an increase in the measurement range, thus avoiding the need to dilute previously samples dog, as is the case with current commercial kits. Once optimized method was performed to validate analytical. Initially, we calculated the precision, accuracy and limit of detection; yielding results in all cases highly satisfactory, especially highlighted the increased accuracy with regard to manual methods, and low detection limit shows that the ability of the method to detect small quantities of HP, as well as absence of interference between components of the kit. It was subsequently studied the effects of substances capable of causing interference as hemolysis, lipemia and bilirrubinemia; very significant declines that produces hemolysis in the concentrations of haptoglobin, it is not recommended to use this method in serum or plasma samples with different as anticoagulants heparin, EDTA or sodium citrate. Lastly was conducted clinical validation, demonstrating that the method can detect high levels of haptoglobin various problems associated with inflammatory (such as pyometra or leishmaniasis), neoplasms or treatment with glucocorticoids. Therefore, the validated method in this paper can be used to measure samples of dog haptoglobin, being advised to use own standards of the canine and not using samples hemolytic. While it would be advisable to validate the method every time you get a new batch of antiserum.

Author: PARRA MUÑOZ M. DOLORES.
Year: 2005.
University: MURCIA [www.um.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: MURCIA.

Summary: In this paper we established several goals (methodological and clinical application) to try to resolve some of the constraints for employment delas determinations of acute phase proteins in the canine species. As a first target is isolated and purified and C-reactive protein haptoglobin canine from serum acute phase. The CRP was purified by inmunopurificación using polyclonal anti-CRP canine antibodies and cormatografía affinity column using an agarose coupled with fosforiletanolammina through liaison epoxy. This latter technique provided a protein purer than the first. The haptoglobin was purified by ammonium sulfate fractionation followed by gel filtration. The high purity of the CRP and Hp obtained allowed to use them as standards in fluorinmunoensayos made in the present study for the determination of two proteins in the dog. The CRP and purified Hp could serve as immunogens in obtaining antibodies anti-CRP and anti canine or as material control in other commercial methods for measuring these proteins. The second goal is focused on obtaining an immunoassay based flurometría time delayed for the quantification of C-reactive protein canine. It won an essay or non-competitive type sandwich in three steps with antibodies biotinados as antibodies and antibody capture marked europio as antibody detection, such as using solid phase strips microtitre streptavidin-coated. The test was successfully validated in terms of analytical and clinical. Thus the method showed good precision and accuracy, with a very high sensitivity (detection limit of our method is the lowest of all existing at present) and the absence deinterferencias by using samples hemolytic, lipémicas or bilirrubinémicas. As for the analysis and serum samples from healthy and sick animals, the values provided by the CRP test enabled differentiate clearly between the two groups of animals. Levels of protein detected in the various pathologies studied in dogs and healthy coincided with those of other authors. The third objective was implemented in samples of whole blood, saliva and efusiones the fluoroinmunoensayo developed for the quantification d serum CRP. The immunoassay was analytically validated for all three types of biological samples provide a good precision and accuracy. Additionally it was found that storage for a month of samples of whole blood and obtaining this with different anticoagulants did not affect the determination of the protein through flurometría time delayed. The concentration and CRP in whole blood was slightly lower than that observed in plasma, possibly because of the volume occupied by red blood cells, however, it was found a good correlation between levels of CRP in the blood between woe in their plasmas (y = 0.86 x -1.03; R2 = 0.97). The salivary levels of CRP were significantly lower than in serum and did not show a high correlation (y = 0008x +0034; R2 = 0.70). Perhaps some of the factors affecting the composition or salivary secretion may be responsible for this phenomenon. The highlight of this section was the clinical study, which showed that dogs with inflammation can be differentiated from healthy dogs by measuring their levels of CRP in blood samples of saliva as a whole, and that determines 8 of e66 the protein efusiones canine has a high sensitivity (100%, 88.2%) and specificity (94.4, 100%) to differentiate exudates of trasudados and exudates of trasudados modified. As the fourth goal was a test inmunofluoromético time delayed for determining haptoglobin canine. One trial was conducted in two steps uncompetitive with antibodies biotinados and antibodies marked europio on either side of the sandwich, and strips microtitre as streptavidin-coated solid phase. The test was validated in terms of showing adequate analytical precision and accuracy, a high detection limit (the lowest of all published so far) and the absence of interference by hemolysis, lipemia or bilirrubinemia. Most commercial trials for Hp canine affected by the hemólis therefore compared with other methods, ours provides the great advantage of not being disturbed by it. As for the clinical validation, the results indicated that the test used to distinguish healthy from the sick dogs, providing distinct concentrations for both groups of animals that match those provided previously by other authors. The fifth and final goal was dedicated to the implementation of fluoroinmunoensayo for determining the HP canine in samples of whole blood, saliva and efusiones. That application was limited by problems of stability of haptoglobin purified, preventing a complete analytical and clinical validation on this kind of show, even though some interesting results were obtained. The haptoglobin concentrations in whole blood were lower than many serum concentrations, but the correlation between the two was good (y = 0.15 x -0.01; R2 = 0.84). The salivary concentrations of HP were hundreds or thousands of times lower than serum and showed a weak correlation (R2 = 0.53) with the levels of serum Hp present. The low regression coefficient obtained could be explained by a possible delay in the passage of haptoglobin from the blood to the saliva or any of the factors that affect the salivary secretion. Nevertheless, it should be emphasized that the levels of Hp in whole blood and saliva d healthy dogs were significantly lower than those achieved in dogs sick. However, it would be necessary to conduct clinical studies with a larger number of animals, especially dogs sick, to determine the sensitivity and specificity of the test for this type of samples and evaluate the possible application of the determination of Hp in blood and salivary a future. Finally, although there are no methodological limitations for the quantification of a haptoglobin in efusiones canine through fluorometría time delayed, the trial was not possible to distinguish between drips and trasudados, which limits the application of this protein in the differentiation efusiones.

Author: Romero Brey Inés.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: Insituto de Acuicultura.
Place of preparation: Instituto de Acuicultura.

Summary: This study reflects the findings on virological analysis of 7 species of fish living in the Flemish Cap (Newfoundland). A preliminary analysis by transmission electron microscopy revealed that these fish are natural hosts (because they do not cause disease apparent) mainly birnavirus, which appeared almost always in the company of tubular structures associated. In addition, it was observed in minimum quantities of viral particles type picomavirus / nodavirus / calcivirus, flavivirus, coronavirus, arenavirus / paramyxoviruses and orotmixovirus / paramyxoviruses. Secondly there was a characterization of genetic birnavirus observed, as well as an exhaustive study of tubular structures, trying to ascertain the reason for its existence and training, and its possible role. Finally, the analysis of the genomic sequences of 2 segments of some strains of birnavirus revealed both coinfection and recombination between genomic segments of these birnavirus.

Author: VIDAL BARBA ENRIC.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE VETERINARIA.
Place of preparation: LABORATORI PRIOCAT, CReSA.

Author: LACASTA LOZANO DELIA MARÍA.
Year: 2005.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: Knowing the current conditions of the sheep sector in Aragon and the low profitability that is offered to livestock at the moment, we decided to further investigate one of the major diseases that cause economic losses; respiratory processes. To that end, we chose four farms in the southern part of Huesca, with different management systems and facilities. For three years conducted a comprehensive follow-up on them, both their health parameters as productive. We necropsy of all lambs that they died in this period and determine the cause of death. In parallel, climatological data were collected from the meteorological station in the area then, complying with the targets set, as parameters were analyzed thoroughly, and caneas mortality rates, age at which the death occurred, the type of lung injury and microorganisms isolated from the respiratory process. Finally, it became statistically study the possible influence of environmental factors on the onset of pneumonia. The results demonstrate the enormous importance of respiratory processes in our farms and the grave economic losses they cause. The processes respiratory and digestive alternated their importance as the year and exploitation in the study, but all of them agree on a dramatic increase in pneumonia during the year 2003, which introduced significant climatic differences regarding the other two, being one years rain and warmer than is usual in the study area. The predominant lung injury in sheep was bronchopneumonia fibrinosa acute, but during the year 2003, the year warm and rainy, processes septicémicos from respiratory tract, increased their relative importance. Mannheimia haemolytica was the main microorganism appearance in respiratory processes, usually associated with bronchopneumonia fibrinosas. Pasteurella multocida associated greater processes septicémicos increased a very important occurrence in years warm and rainy (2003). Mycoplasma ovipneamoniae was isolated in a 33% of the samples, which were obtained some form of isolation. Despite the fact that we know that we need the presence of a microorganism to develop a respiratory process, there are many studies that demonstrate the importance of risk factors, primarily from climatology In our work we try to establish relationships between the influence of weather factors and the onset of pneumonia. Winds had a clear influence on the development of respiratory processes in our farms. In the nave C also noted an increase in respiratory processes days and warmer with higher thermal oscillations. It was on this farm where older Pasteurella multocida isolates occurred, microorganism associated in the literature with warmer climates. The ship D, the worst ventilated, it was the weather that influenced an increase in humidity within the vessel, which contributed to the development of respiratory processes. The year also was decisive, when developed respiratory pathology The 2003 was a year particularly damaging to the emergence of these processes. It was a year with no wind and fewer days in which, in addition, the presence of Southeast wind was increased significantly. We could partly explain why 2003 was a year in which there was a dramatic increase in the occurrence of respiratory processes. In developing the present work we have been able to corroborate impression that there are many factors that influence the development of respiratory processes, being the clear influence of climatology at the onset of pneumonia. However, further studies are necessary to 8 s for i 32d nvestigate the subject more deeply and see the influence of the weather in other parts of Spain and the world, with different management systems and facilities.

Author: VELA MARTINEZ ANTONIO.
Year: 2006.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE VETERINARIA DE LUGO.
Place of preparation: FACULTAD DE VETERINARIA DE LUGO.

Summary: Tapping into the possession of a veterinary clinic open and running, an epidemiological study and clinical canine babesiosis Lugo. In the first, which lasted 15 years (1989-2003) were handled 624 cases of sick animals, while the second, covering 9 years (1989-1997) studied 376 cases of clinical disease confirmed. At the same time, a recording of total visits to search during the same years, noting an incidence rate of 17.07 â ° over the total. There was a bimodal distribution over the year, with a peak in fall and another significant increase in the spring, to coincide with periods of activity vector most recognized ticks Dermacentor reticulatus. Of the total patients, there was a 64.5% of males and 35.5% of females, which are often repeated in the literature. The average age of sick animals was 4.01 years, determining that the universe studied accounted for a majority of young population (63% in 4 years or younger). Regarding fitness, it was found that about half of the troops were pets, with upward trend. About 40% were hunting dogs with downward trend and the remaining 10% went to dog care in proportion stable. The average age of hunting dogs (3.55 years) was nearly a year less than that of dogs Company (4.41 years). Regarding the environment, it was found that 4 out of 5 patients (80%) live in rural areas or had frequent contact with him. The average age of presentation of babesiosis in the urban environment (3.74 years) was a year less than those of urban environment (4.77 years). A study of the acute mortality, which was carried out for 10 years (1990-1999), showed a mortality rate of 11.9% of those affected, with a neat and interesting difference between males (7.7%) and females (19.7%). Moreover, these rates were maintained with very few changes over the years of study. Mortality was directly proportional to age. Moreover, during those years were collected 381 ticks with the aim of identifying them up to level of genus. It was found that almost half were Rhipicephalus, a 38.1% were Ixodes and 13.1% were identified as Dermacentor. In clinical signs, the most frequent were anorexia and weight loss (85% incidence), then fell apathy and depression (74.5%) and in third place was found general weakness in the strength (65, 7%). One of the most visible symptoms of the disease such as the coluria, was observed only in one out of every 3 patients (34.3%). The fever appeared in 7 out of 10 patients but had a mortality rate of 7%, while in 4% of patients who suffered hypothermia, the mortality was 25%. It was noted pale mucous in more than 80% of all patients and jaundice in only one out of 7. The assessment of recovery after treatment, it was found that at 72 am the same, 87.4% of patients showed clear improvement in the general state of health. A 69.8% of patients suffered from anemia, which have the greatest impact on youth (under 6 years). Those who also showed leukopenia had lower mortality than those of leukocytosis. A 91.8% of all patients showed thrombocytopenia. In biochemistry blood hypoproteinaemia was observed in more than half of patients. All other parameters showed widely varying results, can intuit sometimes renal or hepatic of unfavorable prognosis. The study showed clotting light extensions of time studied. Still, in those patients in whom were seen 3 or more alterations simultaneous coagulation parameters 8, not 3b8 caused any casualties. The study of the experimental infection, there was an incubation period of 4.5 days, while the recovery time after treatment was 48 hours. The study of clotting in these dogs endorsed the results obtained in the natural infection.