Tesis sobre el tema "Wasting syndrome"

La síndrome d'aprimament post-deslletament (SAPD) és considerada com una malaltia porcina d'origen multifactorial en la qual el Circovirus porcí tipus 2 (CP2) és l'agent infecciós essencial. L'objectiu de la present tesi doctoral era expandir el coneixement en l'epidemiologia de la infecció per CP2 i el SAPD a través de la realització d'estudis de cas-control a nivell de camp. De manera general, es va investigar la potencial influència de la genètica del CP2, el moment d'infecció del CP2, els anticossos contra CP2 derivats de la immunitat maternal i la resposta humoral dels porcs contra el CP2, en la presentació de la SAPD.
En el primer estudi (estudi I) es van estudiar seqüències de PCV2 de porcs amb diferent condició clínica i patològica. Els resultats van confirmar l'existència de dos genogrups principals i es va proposar la definició de dos genotipus de CP2 (1 i 2). La metodologia suggerida per definir els genotipus està basada en la distribució de la relació p-distància/freqüència de les seqüències de CP2 conjuntament amb anàlisis filogenètics de CP2. Es va observar que el genotipus 1 era predominant en porcs de granges afectades per SAPD. Contràriament, totes les seqüències obtingudes de granges no afectades per la SAPD corresponien al genotipus 2. Així, es va suggerir que el genotipus 1 de CP2 podria ser potencialment més patogènic que el genotipus 2. Addicionalment, es va descriure la presència dels dos genotipus en el mateix moment en porcs individuals procedents de granges afectades per la SAPD.
La present tesi doctoral es va desenvolupar dins del marc del projecte de la Unió Europea (UE) titulat Control de les malalties associades al Circovirus porcí (MACP): Cap a la Millora en la Qualitat i Seguretat Alimentàries (www.pcvd.org), la qual era finançada pel 6è programa marc de la UE. El consorci de la UE d'aquest porjecte va publicar la carta presentada com a addendum de l'estudi I. En aquesta carta es va donar suport a la definició de genotipus proposada, a la vegada que es va proposar la nomenclatura de genotipus a de CP2 (CP2a) i genotipus b (CP2b), corresponent als genotipus 2 i 1, respectivament, per tal d'evitar confusions amb la ja existent diferenciació entre PCV2 i PCV1.
En el segon estudi (estudi II) es van comparar dues tècniques de PCR quantitativa (qPCR) de PCV2. Els resultats mostraven una associació lineal significativa entre les dues tècniques, i un biaix sistemàtic de 1.4 log10 copies of CP2 per mil·lilitre de mostra. Aquesta diferència indicava que la tècnica del laboratori danès generava un resultat sistemàticament més elevat que el generat a través de la tècnica del laboratori espanyol. A més, la tècnica del laboratori danès mostrava major sensibilitat que la tècnica del laboratori espanyol.
En els treballs III i IV es van realitzar estudis longitudinals de tipus cas-control en granges afectades per la SAPD de Dinamarca i Espanya tot utilitzant dissenys similars. Es van observar patrons similars en la dinàmica d'infecció per CP2 tant a Espanya com a Dinamarca, amb un retard en l'edat de presentació de la SAPD a Espanya en comparació a Dinamarca. El diagnòstic individual de la SAPD es va confirmar, mitjançant tests laboratorials, en només la meitat dels porcs en els quals hi havia la sospita clínica. Globalment, els resultats van mostrar que la quantitat de CP2 incrementava concomitantment a la caiguda dels nivells d'anticossos maternals, assolint valors màxims de càrrega vírica en el moment d'aparició dels símptomes clínics. De manera interessant, la reacció de fase aguda (RFA) en porcs afectats per la SAPD s'observava paral·lelament a l'evolució de la virèmia per CP2, suggerint que el CP2 és el principal responsable de l'estat d'inflamació sistèmica que pateixen els porcs afectats per aquesta malaltia. Col·lectivament, els porcs afectats tenien quantitats de CP2 i concentracions de porc-MFA i HPT superiors en sang, excretaven càrregues víriques superiors tant per via nasal com a través de les femtes, i tenien nivells inferiors d'anticossos maternals enfront al CP2 que els porcs que no estaven afectats per la SAPD. Addicionalment, es va observar una menor resposta humoral en els porcs afectats per la SAPD provinents d'Espanya a les 11 setmanes de vida (abans de l'aparició dels símptomes clínics) i en el moment de la necròpsia, suggerint que aquesta circumstància era podria associar-se com a causa més que no pas una conseqüència de la malaltia. D'altra banda, la falta de sensibilitat i/o especificitat observades de les tècniques de qPCR i/o de serologia suggereixen que aquestes tècniques no poden substituir la histopatologia més la detecció de CP2 en teixits per l'establiment del diagnòstic individual de la SAPD. Malgrat això, els resultats indicaven que la qPCR podria ser potencialment útil per diagnosticar la SAPD a nivell poblacional. Addicionalment, els resultats obtinguts donaven suport a la idea que, malgrat que les PFA són marcadors inespecífics d'inflamació, aquestes proteïnes podrien ser marcadors útils de salut, esdevenint una eina potencialment útil per monitoritzar el desenvolupament de la SAPD en estudis epidemiològics o per la valoració de l'eficàcia de vacunes de CP2 a nivell de camp.
Postweaning multisystemic wasting syndrome (PMWS) is considered a multifactorial pig disease in which Porcine circovirus type 2 (PCV2) is the essential infectious agent. The present thesis aimed to expand the epidemiological knowledge on PCV2 infection and PMWS through the realization of case-control field studies. Mainly, the potential influence of PCV2 genetics, the timing of PCV2 infection, the PCV2 maternal derived humoral immunity and the pig humoral response against PCV2 infection in PMWS presentation were investigated.
In the first study consisted in the sudy of PCV2 sequences obtained from pigs with different clinical and pathological conditions. Results further confirmed the existence of two main genogroups and the definition of two PCV2 genotypes (1 and 2) was proposed. The suggested methodology to define PCV2 genotypes is based on the p-distance/frequency distribution of PCV2 sequences together with PCV2 phylogenetic analyses. Genotype 1 was shown to be predominant within pigs coming from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Consequently, it was suggested that PCV2 genotype 1 might potentially be more pathogenic than PCV2 genotype 2. In addition, infection of single pigs from PMWS affected farms harbouring both genotypes at the same time was described.
The present thesis was developed within the European Union (EU) project entitled Control of Porcine Circovirus Diseases (PCVD): Towards Improved Food Quality and Safety (www.pcvd.org), which was funded by the EU Sixth Framework Programme. The EU consortium on PCVD published the letter here presented as an addendum of study I. In this letter it was supported the genotype definition, but it was also proposed the nomenclature of PCV2 genotype a (PCV2a) and genotype b (PCV2b), corresponding to genotypes 2 and 1, respectively, in order to avoid potential confusions with the already existent PCV2 and PCV1.
In the second study (Study II) two different real-time quantitative PCR (qPCR) assays were compared. Results showed a significant linear association between the assays, and a systematic difference of 1.4 log10 copies of PCV2 per millilitre of sample. This difference indicated that the assay from the Danish laboratory yielded a higher output than the assay from the Spanish laboratory. Moreover, the Danish assay had higher sensitivity than the Spanish one.
In studies III and IV, longitudinal case-control studies were performed in PMWS affected farms from Denmark and Spain using similar designs. Similar PCV2 infection dynamic patterns were observed in Spain and Denmark, with a delay in PMWS age-presentation in Spain compared to the one in Denmark. Results showed that PCV2 load increased concomitantly to maternal antibody level waning, reaching the maximum viral load concurrently with the development of clinical signs. Interestingly, the acute phase response (APR) in PMWS affected pigs occurred in parallel to PCV2 viremia, suggesting that PCV2 is the main responsible for the systemic inflammatory status suffered by diseased pigs. As a collective, PMWS affected pigs harboured higher PCV2 loads and higher Pig-MAP and HPT concentrations in sera, shed higher viral loads through both nasal secretions and faeces, and had lower level of maternal antibodies against PCV2 than non-PMWS affected pigs. Furthermore, an impaired humoral response was observed in PMWS affected pigs from Spain at 11 weeks of age (prior to the appearence of clinical signs) and at the moment of necropsy, suggesting that this circumstance might be more a cause rather than a consequence of the disease. On the other hand, the lack of sensitivity and/or specificity observed from qPCR and/or serological techniques suggests that those techniques are not able to substitute histopathology plus detection of PCV2 in tissues for the individual PMWS diagnosis. However, results indicated that qPCR might potentially be a reliable technique to diagnose PMWS on a population basis. Additionally, obtained results supported the idea that although APPs are unspecific markers of inflammation, they might be useful indicators of health, becoming a potentially interesting tool to monitor PMWS development in epidemiological studies or in the assessment of the efficacy of PCV2 vaccines in the field.

Weißbüschelaffen (WBA) sind wissenschaftlich häufig genutzte Modelltiere für diverse Humanerkrankungen. Zur Gesunderhaltung dieser Primaten sind grundlegende diagnostische Blutparameter unverzichtbar. Bisher erhobene Daten zeichneten sich jedoch durch große Divergenz aus. Ob Veränderungen in Haltungsbedingungen einen Einfluss auf diese Blutparameter nehmen, ist bis heute unklar. Somit war ein Ziel dieser Arbeit die Erhebung aktueller hämatologischer und klinisch-chemischer Blutparameter von WBA. Zudem wurde der Einfluss der routinemäßigen Umsetzung in eine neue Behausung auf die erhobenen Parameter sowie den Kortisolspiegel im Kot untersucht. Des Weiteren leiden WBA in menschlicher Obhut rezidivierend an gastrointestinalen Erkrankungen, die mittels klinischer Standardparameter allein nicht diagnostizierbar sind. Dabei spielt vor allem die Sensitivität gegenüber Futtermittelinhaltsstoffen (z. B. Gluten) eine Rolle, welche ursächlich im Zusammenhang mit dem Wasting Marmoset Syndrome (WMS) diskutiert wird. Im zweiten Teil der vorliegenden Arbeit sollten deshalb die gastrointestinalen Erkrankungen von in menschlicher Obhut lebenden WBA ätiologisch beleuchtet werden, vor allem hinsichtlich einer möglichen Sensitivität gegenüber Gluten. Im ersten Teil dieser Studie wurden von 54 WBA hämatologische und klinischchemische Richtwerte erhoben. Die ermittelten hämatologischen Blutrichtwerte ähneln denen aus den achtziger Jahren, die Daten der klinischen Chemie nur bedingt: Die Richtwertbereiche von Laktatdehydrogenase, Alaninaminotransferase, Lipase sowie Alkalische Phosphatase und Gesamtbilirubin weichen von den ehemals erhobenen Daten ab. Zudem wurden in der vorliegenden Arbeit geschlechtsabhängige Unterschiede ermittelt: Weibliche Tiere wiesen signifikant höheres mittleres Erythrozytenvolumen und mittleren Hämoglobingehalt des Einzelerythrozyten auf als männliche Tiere, wohingegen bei diesen ein signifikant höheres Gesamt- und Low density lipoprotein- Cholesterol im Vergleich zu weiblichen Affen messbar war. Des Weiteren wurden 16 Tiere über einen vierwöchigen Zeitraum in eine neue Umgebung verbracht, bevor sie in ihre Heimatbehausung zurückkehrten. Durch diese Umsetzung war bei den untersuchten Tieren die Leuko- und Lymphozytenzahl auch vier Wochen nach der Umsetzung erniedrigt. Zeitgleich lag ein erhöhter Kortisolspiegel vor, der im Kot bestimmt wurde. Im zweiten Teil der Studie wurden anhand humandiagnostischer Standards IgAAntikörper (AK) gegen Gliadin (AGA), Gewebstransglutaminase (tTG), deamidiertes Gliadin (ADGA) sowie Glykoprotein 2 (AGP2A) im Plasma von 24 WBA mittels eines ELISAs während glutenhaltiger (Diät 1) und glutenfreier Ernährung (Diät 2) bestimmt. Dabei wurden die klinische Symptomatik von WMS und das Körpergewicht der Tiere ebenfalls untersucht. Zudem erfolgte die Analyse von Kotproben antikörperpositiver Tiere hinsichtlich Qualität und Trockenmassegehalt während Diät 2 und einer darauf folgenden glutenhaltigen Provokationsdiät. Die serologische Diagnostik ergab einen signifikanten Rückgang von AGA, AK gegen tTG und AGP2A während Diät 2 bei Tieren, die nach Diät 1 erhöhte Werte aufwiesen. Diät 2 führte zu einem Rückgang der klinischen Symptome und einer signifikanten Gewichtszunahme bei antikörperpositiven WBA. Die glutenhaltige Provokationsdiät ergab eine verminderte Kotqualität mit einem niedrigeren Trockenmassegehalt. Es wurden im Rahmen dieser Arbeit aktuelle, hämatologische und klinisch-chemische Blutrichtwerte des WBA erhoben. Der durch Umsetzung in eine neue Behausung bedingte Stress ist bei WBA bis vier Wochen lang nachweisbar. Es ist sinnvoll, dies in der zeitlichen Planung wissenschaftlicher Studien zu berücksichtigen, um das Wohlbefinden der Tiere vor Versuchsbeginn sicherzustellen und den Einfluss von Stress auf experimentelle Ergebnisse zu minimieren. Der Nachweis grundlegender, an der Pathogenese der Zöliakie beteiligter Antikörper, in Kombination mit den klinischen Symptomen, deutet auf Glutensensitivität mit ätiologischer Beteiligung an WMS bei WBA hin. Die glutenfreie Ernährung von WBA in menschlicher Obhut ist daher sinnvoll und empfehlenswert
Common marmosets are often used as animal models for human diseases. For their health maintenance, diagnostic blood values are absolutely essential. Previously obtained reference values are characterized by great value-specific differences. Moreover, the influence of routine measures on these blood parameters, e. g. changes in housing conditions, has not been examined yet. Therefore, the first aim of the present study was to update haematological and clinical chemical blood parameters of common marmosets. Further, the influence of stress, caused by relocation to a new housing, on these parameters and the cortisol level in feces was examined. In addition to that, common marmosets under human management are often affected by gastrointestinal diseases, which are difficult to diagnose with basic standard blood values. In this context, sensitivity to nutritional elements, e. g. gluten, plays an important role and is discussed as a potential cause of wasting marmoset syndrome (WMS). In the second part of this study, the recurrent gastrointestinal diseases of common marmosets under human management were aetiologically investigated, with special regard to possible gluten sensitivity. In the first part of this study, blood samples were obtained from 54 female and male common marmosets to evaluate standard values of haematology and clinical chemistry. The determined haematological parameters are similar to the already obtained data, the clinical chemistry values differ somewhat: The enzyme activities of lactate dehydrogenase, alanine aminotransferase and lipase in addition to the ranges of alkaline phosphatase and total bilirubin diverge from the data ascertained in this study. Moreover, female animals presented significantly higher mean corpuscular volume and mean corpuscular haemoglobin than males, whereas male common marmosets showed significantly higher total- and low density lipoprotein-cholesterol, compared to females. Further, 16 animals were relocated to a new environment for a time period of four weeks, before they returned to their home cages. The change of housing caused a decreased leuko- and lymphocyte count in all examined animals that was still measurable four weeks after the relocation. At the same time, an increased fecal cortisol level was determined. The aim of the second study was to investigate the modification of plasma antibodies to gliadin (AGA), tissue transglutaminase (tTG), deamidated gliadin (ADGA) and glycoprotein 2 (AGP2A) during two successive diets in 24 animals: A gluten-containing diet (diet 1) and a gluten-free diet (diet 2). Further, clinical symptoms of WMS and the animals’ body weight were also examined. An analysis of the feces of antibody-positive animals regarding changes in quality and dry matter content was carried out with samples collected during diet 2 and a successive gluten challenge diet of two months duration. The serological diagnostics resulted in a significant decline of AGA, antibodies to tTG and AGP2A during diet 2 in animals that had shown increased antibody concentrations during diet 1. Diet 2 also caused an amelioration of clinical symptoms and an increased body weight in antibody-positive animals. The gluten challenge resulted in a decreased feces quality and a lower fecal dry matter, compared to fecal samples of diet 2. In the context of this dissertation, parameters of haematology and clinical chemistry of the common marmoset were updated. Stress caused by relocation to a new housing was still measurable for a period of four weeks. It is therefore essential to consider this time span in the design of scientific studies to secure animal welfare prior to the study and to reduce the influence of stress on experimental results. In combination with the clinical symptoms, the detection of antibodies that are part of the pathogenesis of coeliac disease in humans strongly suggests gluten sensitivity with an aetiological connection to WMS in common marmosets. Therefore, gluten-free nutrition of common marmosets under human management is highly recommendable

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated disease (PCVAD), an economically important swine disease that causes wasting in pigs 5-18 weeks of age. There exist two different types of porcine circoviruses: porcine circovirus type 1 (PCV1) was discovered as a contaminant of porcine kidney (PK-15) cells and was determined to be nonpathogenic in swine; whereas porcine circovirus type 2 (PCV2) is pathogenic. A recently released vaccine for PCVAD was generated by inserting the gene encoding the immunogenic capsid protein of PCV2 into the genetic backbone of the non-pathogenic PCV1. This chimeric PCV vaccine, called PCV1-2, was shown to induce protective immunity against PCV2 infection in pigs. The vaccine is currently on the market in a killed form. In order to develop a live version of the vaccine, the genetic stability of the chimeric PCV1-2 vaccine virus was investigated by in vitro and in vivo passaging of the vaccine virus. In vitro passaging of the PCV1-2 vaccine virus was done in a porcine kidney PK-15 cell line. Cells were infected with the PCV1-2 vaccine virus and then serially passaged 11 times. The passaged vaccine viruses recovered from passages 5 and 11 were sequenced, and the sequences were compared to that of the original PCV1-2 vaccine virus. The in vitro serial passage result showed that no mutation occurred during the 11 in vitro passages. The in vivo passaging was done using specific-pathogen-free (SPF) pigs. In in vivo â passage 1â , nine piglets were divided into 3 groups of 3 each: group 1 each inoculated with 200ug of PCV1-2 plasmid, group 2 each with 1Ã 103 TCID50 live PCV1-2 vaccine virus, and group 3 each with 3ml phosphate buffered saline (PBS) buffer as a control. One pig from each group was necropsied at 14, 21, and 28 days post-inoculation (DPI), respectively. A panel of tissue samples including lymph nodes and thymus were collected from each pig. Tissue homogenates from DPI 28 that were positive by PCR for PCV1-2 DNA were used to inoculate new piglets in the in vivo passage 2 experiment. Viruses recovered from passage 2 pigs were subsequently used for inoculation in the in vivo passage 3 experiment. The PCV1-2 vaccine virus DNA from pigs in each passage was amplified and sequenced. The results of the in vivo serial passage experiment showed that, after 3 passages of the PCV1-2 vaccine virus in pigs, there were no new mutations in the viruses recovered from pigs. The PCV1-2 vaccine contained an introduced marker mutation at amino acid position number 79, which is in the capsid region. During the in vivo passaging of the vaccine virus in pigs, this marker mutation quickly reverted back to its original nucleotide. This marker back mutation occurred between DPI 21 and DPI 28 of passage 1 in the PCV1-2 live vaccine virus group, and between DPI 28 of passage 1 and DPI 14 of passage 2 in the PCV1-2 vaccine plasmid group, and remained stable throughout the reminder of the in vivo study. Based upon the results from this study, we conclude that the PCV1-2 chimeric vaccine virus is genetically stable in vitro and in pigs, and thus should serve as a good candidate for a live vaccine against PCV2.
Master of Science

The non-pathogenic porcine circovirus type 1 (PCV1) was originally isolated as a persistent contaminant of the porcine kidney cell line PK-15. Whereas, porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) in pigs, which is devastating to the swine industry. My objectives were to determine the effect of maternally derived antibodies on PCV2 infection, assess the role of 2 amino acid substitutions in the PCV2 capsid protein in PCV2 attenuation, evaluate the effect of Rep gene exchange between PCV1 and PCV2 on growth characteristics of a chimeric PCV2, and evaluate the role of open reading frame (ORF) 3 of PCV2 in virus replication and pathogenesis in pigs. Under field conditions, PCV2 infection is widespread and most breeding pigs are seropositive. Assessment of the role of PCV2 maternal antibodies in preventing PCV2 infection in piglets provided evidence that higher levels of maternal antibody provide more protection to piglets. Two amino acid substitutions in the PCV2 capsid protein that enhanced virus replication in vitro and attenuated the virus in vivo were evaluated for their pathogenicity in pigs. The results indicated that P110A and R191S are collectively responsible for virus attenuation. PCV1 replicates better in PK-15 cells and grows at least 1-log titer higher than PCV2. A chimeric PCV with the rep gene of PCV1 replacing that of PCV2 in the genomic backbone of PCV2 replicated more rapidly than PCV1 and PCV2, and more efficiently than PCV2, although to a titer similar to PCV1. The ORF3 of PCV2 is believed to encode a protein involved in apoptosis. The ORF3 start codon was mutated from ATG to GTG and the resulting mutant muPCV2 was infectious in vitro and in pigs; therefore ORF3 is dispensable for virus replication. The pathogenicity of muPCV2 was compared with PCV2 in vivo. Delayed viremia and seroconversion, decreased viral loads, lower level of IgG antibodies, and lower amounts of PCV2 antigen in mesenteric lymph nodes suggested attenuation of muPCV2. However, there was no significant difference in histological or gross lesions in tissues between PCV2- and muPCV2-inoculated groups. The role of ORF3 in attenuation needs to be further elucidated.
Ph. D.

A síndrome de emagrecimento progressivo (SEP) dos calitriquídeos representa importante causa de morbidade e mortalidade de sagüis mantidas em cativeiro. A etiologia dessa síndrome não está estabelecida e suas principais características são emagrecimento progressivo, diarréia, colite, anemia, paralisia dos membros posteriores e alopecia. Com esse estudo pretende-se responder se a síndrome é um processo de má-absorção ou de desnutrição protéico-calórica primária, caracterizar o quadro histológico intestinal de base e a resposta imunológica tecidual local. Foram estudados três grupos de sagüis: 1) 40 doentes com SEP pertencentes ao criadouro Mucky, 2) 9 controles vivos sadios, 3) 8 necrópsias de controles sem SEP. Foi realizado acompanhamento clínico, exame laboratorial das fezes, teste de absorção de D-xilose, avaliação da composição nutricional e digestibilidade da dieta, estudo anatomopatológico, incluindo avaliação semiquantitativa e análise morfométrica do jejuno de sagüis que foram a óbito naturalmente por SEP e dos controles. Os resultados alcançados permitiram caracterizar o perfil dos animais acometidos no nosso meio; os sinais clínicos maiores e menores da síndrome; identificar esteatorréia; o comprometimento da função digestiva e absortiva do intestino delgado dos sagüis com SEP; caracterizar o quadro histopatológico como uma enterite com atrofia semelhante à doença celíaca humana. A associação dos resultados clínicos, laboratoriais e histológicos permitiu definir a SEP como processo de má-absorção, por perda de superfície absortiva de intestino delgado, decorrente de enterite crônica imunomediada, de padrão celíaco-like que leva a progressiva e grave desnutrição secundária dos animais acometidos.
Wasting marmoset syndrome (WMS) is an important cause of morbidity and mortality of marmosets and tamarins kept in captivity. The etiology of this syndrome has not been established and its main features are progressive weight loss, diarrhea, colitis, anemia, hind limb paralysis, and alopecia. The aims of this research were to demonstrate that WMS is a malabsorption process, and to analyze the underlying histological lesion of the intestine and to characterize the local immune response of the small intestine. The sick marmosets (n=40) were compared to live normal controls (n=9) or to necropsied marmosets that died of other diseases than WMS (n=8), regarding clinical follow up, fecal analysis, D-xylose absorption test, evaluation of the nutritional composition and digestibility of the diet, gross and histological examination and morphometric approach of the jejune of wasters and control marmosets. These data revealed general features of WMS under our general captivity conditions, major and minor clinical signs of waster marmosets, impaired absorptive and digestive function of small intestine with steatorrhea and atrophic enteritis similar to celiac disease. The clinical and laboratory data associated with pathology examination demonstrated that WMS is a malabsorption process due to loss of absorptive surface area that results in progressive secondary malnutrition of the waster marmosets. The major immunologic mechanism underlying the celiac-like enteritis of WMS is a T-cell immune mediated response that affects intestine architecture

A Síndrome Multissistêmica do Definhamento do Suíno (SMDS) é uma doença emergente e mundialmente distribuída, que tem trazido sérios prejuízos econômicos para a indústria suinícola. O Circovírus Suíno tipo 2 (PCV2), agente causal da doença, provoca lesões principalmente nos tecidos linfóides, e sugere-se que produza imunossupressão, predispondo o hospedeiro a infecções virais, bacterianas e fúngicas secundárias. Neste trabalho, é descrito um estudo da prevalência e bacteriologia das otites purulentas em suínos apresentando a SMDS, bem como em animais de baixo desenvolvimento e de crescimento normal. No total, foram examinados 385 suínos com idades entre 60 e 130 dias. De 242 animais com a SMDS, 57 (23,5%) apresentaram lesões purulentas no ouvido médio. Dentre 119 animais de baixo desenvolvimento, apenas 1 (0,7%) apresentou a lesão. Não foram detectadas lesões macroscópicas no ouvido médio dos 24 animais com crescimento normal (controles). Os agentes isolados com maior freqüência das lesões foram Arcanobacterium pyogenes, Streptococcus α– hemolíticos e Pasteurella multocida, encontrados em, respectivamente, 37 (43%), 32 (37,2%) e 24 (27,9%) dos 86 ouvidos submetidos à bacteriologia. A alta prevalência de lesões purulentas no ouvido médio de animais com a SMDS sugere que a infecção pelo PCV2 pode tornar o suíno mais suscetível às otites bacterianas. Por outro lado, a prevalência reduzida das lesões em suínos de baixo desenvolvimento sugere que a otite não representa uma causa importante de mau desempenho em suínos nas fases de crescimento e terminação. O isolamento do A. pyogenes, de Streptococcus α- hemolíticos e da P. multocida na maioria das lesões está de acordo com relatos anteriores, confirmando a importância desses organismos como agentes causais da otite média em suínos.
Postweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease disseminated globally that causes severe losses to the pig industry. Porcine circovirus type 2 (PCV2) is the causal agent of the disease and causes lesions mainly in lymphoid tissue and it is suggested that it can cause immunosuppression, predisposing the host to viral, bacterial and mycotic infections. In the present work we describe a study on prevalence and bacteriology of purulent otitis in pigs with PMWS, as well as in pigs with attrition and pigs with normal growth. A total amount of 385 animals were examined, with ages ranging from 60 to 130 days. Among 242 pigs with PMWS, 57 (23,5%) showed purulent lesions in the middle ear. Among 119 pigs with attrition, only 1 (0,7%) presented the lesion. In 24 control pigs, middle ear lesions were not detected. The agents most frequently isolated from the lesions were Arcanobacterium pyogenes, α–hemolytic Streptococci and Pasteurella multocida, found respectively in 36 (43%), 32 (37,2%) and 24 (27,9%) of 86 ears bacteriologically examined. The high prevalence of purulent lesions found in middle ear of PMWS affected pigs suggests that PCV2 infection can increase susceptibility of swine to bacterial otitis. On the other hand, the small prevalence of lesions in piglets with attrition suggests that otitis does not represent a significant cause for depressed growth in pigs from growing and finishing ages. The isolation of A. pyogenes, α-hemolytic Streptococci and P. multocida from most lesions agrees with previous reports, confirming the importance of these organisms as causal agents in the etiology of otitis media in pigs.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen.
Ph. D.

A síndrome de emagrecimento progressivo (SEP) é responsável por elevada morbidade e mortalidade de calitriquídeos mantidos em cativeiro em diferentes instituições. Essa síndrome representa um desafio aos médicos veterinários por suas características ainda pouco esclarecidas e são poucos os estudos multidisciplinares que visam à avaliação dos diferentes sistemas, como os órgãos linfo-hematopoiéticos. O objetivo foi caracterizar a evolução e duração da SEP, associando os dados clínicos, laboratoriais e anatomopatológicos dos órgãos linfo-hematopoiéticos de saguis naturalmente acometidos por SEP no cativeiro. Foram analisadas as fichas clínicas, necroscópicas e os resultados das amostras de sangue e urina de 47 saguis doentes, Callithrix spp., machos e fêmeas, que foram a óbito devido a SEP e eram provenientes de dois criadouros diferentes do estado de São Paulo. Os fragmentos dos órgãos linfo-hematopoiéticos, intestino delgado e do fígado foram processados e avaliados. Os resultados caracterizaram que a SEP acomete calitriquídeos de espécies diferentes, adultos, sem predisposição sexual, mantidos sob condições estáveis de manejo por em média 42 meses e a duração clínica varia de 41 dias a 1 ano e 7 meses. As características clínicas na fase inicial foram predominantemente sinais gastrintestinais e na fase terminal, sinais gastrintestinais e extra-intestinais. A anemia macrocítica normo ou hipocrômica com policromasia, esferocitose, presença de corpúsculos de Heinz e hemoglobinúria foi a alteração hematológica mais frequente. As lesões dos órgãos linfo-hematopoiéticos foram características de anemia hemolítica ou foram inespecíficas e reacionais caracterizadas por hiperplasia ou depleção das células da medula óssea, baço e linfonodo e lesões degenerativas no fígado. Na SEP, a associação clínica, laboratorial e anatomopatológica possibilitou a caracterização da evolução e duração clínica, da anemia e das alterações dos órgãos linfo-hematopoiéticos, cujas lesões foram consideradas secundárias à desnutrição crônica e progressiva decorrente da severa enterite atrófica.
Wasting marmoset syndrome (WMS) causes high morbidity and mortality of marmosets and tamarins kept in captivity in different colonies. WMS challenges the veterinarian due to its unclear and not established features and there are few multidisciplinary studies that carried out an evaluation of different systems, such the lymphohematopoietic system. The aim was described the duration and evolution of illness based on an association of clinical, laboratory and pathological aspects of WMS. Medical record, laboratory data and pathological findings were analyzed of 47 Callithrix spp., males and females, sick marmosets that died due to WMS in two different colonies in São Paulo state. Tissue samples of small intestine, lymphohematopoietc system and liver were histological processed and evaluated. The results showed that WMS affects adult marmosets of different species; there are no sex-related differences, and the marmosets are at least 42 months under similar general management at colony. The clinical duration of WMS is from 41 days to 1 year and 7 months. The clinical features were gastrointestinal symptoms in the beginning and extra-gastrointestinal and gastrointestinal signs in the end. Normochromic or hypochromic macrocytic anemia with polychromasia, spherocytes, Heinz bodies, and hemoglobinuria is the common hematological result. The lymphohematopoietic system lesions were the common findings of hemolytic anemia or unspecific and reacting features such as hyperplasia or depletion of cell numbers of bone marrow, spleen and lymph node, and degenerative lesions of liver. The clinical, laboratory and pathological association allowed the characterization of evolution and duration of the WMS, the anemia and the lesions of lymphohematopoietic organs which lesions were considered secondary to chronic and progressive malnutrition as a result of severe atrophic.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2016
A hiponatrémia é um distúrbio hidro-electrolítico comum em doentes com patologia aguda do sistema nervoso central, sendo frequentemente atribuída ao síndrome da secreção inapropriada de hormona anti-diurética (SIADH). Esta entidade é caracterizada por uma hiponatrémia hipoosmolar, com urina hiperconcentrada, e evidência de normo ou hipervolémia. Porém, existe uma outra entidade prevalente nesta população, o salt wasting syndrome (SWS), cuja diferença é cursar com hipovolémia. Apresenta-se um caso de um homem de 89 anos, internado por quadro de recusa alimentar, prostração e diarreia com 24 h de evolução, sendo que analiticamente, destacava-se hiponatrémia hipoosmolar sem sinais óbvios de desidratação, colocando-se a duvida no diagnóstico diferencial entre SWS e SIADH. A problemática da correcta distinção entre ambos os síndromes prende-se com a natureza divergente da terapêutica a instituir: restrição hídrica no SIADH e reposição hidrossalina no SWS. A determinação do grau de volémia do doente nem sempre é uma tarefa fácil com recurso apenas ao exame objectivo. A utilização de Scores de decisão clínica pode revelar-se útil, ao conjugar todos os dados imprecisos obtidos no exame objectivo e análises mais correntes, de modo a obter uma estimativa relativamente fiável. O estudo da fracção de excreção de ácido úrico ou do balanço cumulativo do sódio, podem apresentar-se como alternativas ao modelo clássico de distinção das hiponatrémias assente na estimativa da volémia.
Hyponatremia is a common electrolyte disorder in patients with acute disease of the central nervous system, often being assigned to the syndrome of inappropriate secretion of central anti-diuretic hormone (SIADH). This syndrome is characterized by a hypoosmolar hyponatremia with a concentrated urine and evidence of euvolemia or hypervolemia. However, there is another prevalent syndrome in this population, the salt wasting syndrome (SWS), whose only difference is present of hypovolemia. Presenting a case of a 89 year-old man hospitalized for anorexia, prostration and diarrhea, plus an hypoosmolar hyponatremia with no obvious signs of dehydration, placing the doubt in the differential diagnosis between SWS and SIADH. The problematic of the proper distinction between the two is related to the divergent nature of the therapy to use: fluid restriction on SIADH and hydrosaline reposition on SWS. Assessment of the patient’s volume status trough the physical examination isn’t always an easy task. The use of Clinical Decision Scores can be useful, by combining all the inaccurate data obtained in the physical examination and routine laboratory exams, it’s theoretically possible to create a relatively reliable estimation of the patient’s volume status. The study of uric acid excretion fraction or the cumulative balance to sodium, which has been proposed as alternatives to the classic model of distinguishing the hyponatremias based on the estimation of blood volume.

Protein wasting is a complication of glucocorticoid (GC) therapy. It causes substantial morbidity and there is no treatment. This thesis investigates the metabolic mechanisms underlying GC-induced protein wasting and the potential for anabolic hormones to reverse protein loss. The models of GC excess were Cushing's syndrome and GC therapy. Whole body protein metabolism was assessed using the leucine turnover technique and body composition by dual-energy X-ray absorptiometry to estimate lean body mass (LBM) and fat mass (FM). As previous studies demonstrated that LBM and FM influenced rates of protein metabolism, the magnitude of body compositional abnormality in Cushing's syndrome was determined. After accounting for the greater FM (30%) and lesser LBM (15%), protein metabolism in Cushing's syndrome was characterised by a significant increase in protein oxidation, an abnormality that leads to irreversible protein loss. Successful treatment of Cushing's syndrome normalised protein oxidation. Studies of the acute and chronic effects of therapeutic GCs revealed a time-dependent effect on protein metabolism. GCs acutely increased protein oxidation. However, the rate of protein oxidation during chronic therapy at a similar dose was not significantly different to untreated control subjects. This time-dependent change suggests that GC-induced stimulation of protein oxidation does not persist and could represent a metabolic adaptation to limit protein loss. This finding contrasts with that in Cushing's syndrome, where protein oxidation is persistently elevated. This difference may represent a dose effect. Studies in GH-deficient subjects revealed that GH induced a fall in protein oxidation that was significantly correlated with a subsequent gain in LBM. This suggests that the anabolic potential of a therapeutic substance can be predicted by its ability to suppress protein oxidation acutely. Finally, the potential for GH and androgens to reverse the metabolic effects of GCs was assessed. A preliminary study in GC users revealed that a GH dose of 0.8 mg/d was effective in reducing protein oxidation. In a subsequent study, the GH-induced reduction in protein oxidation in women on GCs was enhanced by combined treatment with dehydroepiandrosterone, an androgen. In summary, GCs induce protein loss by stimulating protein oxidation. GH reverses this effect and this action is enhanced by coadministration of androgens. GH and androgens may be used therapeutically to prevent protein loss induced by GCs.

碩士
國立臺灣大學
獸醫學研究所
94
Post-weaning multisystemic wasting syndrome (PMWS), first described in Canada in 1991, has been now reported in most swine-producing countries and has become a significant problem for the pig industry. Porcine circovirus type 2 (PCV2) is the necessary cause of PMWS in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. The aim of this study is to find out if other co-infection is necessary in the PMWS cases in Taiwan. The PMWS cases were diagnosed by three criteria described in previous study: (1) the presence of compatible clinical signs, (2) the presence of the characteristic microscopic lymphoid lesions, and (3) the detection of PCV2 within these tissues by PCR. Also, we detected Porcine circovirus typee I (PCV1), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Classic swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella from different biological specimens of growth retarded pigs by RT-nPCR or PCR. In 62 pigs examined, 55 pigs were PCV2 positive (88.71%), 29 pigs were PPV positive (46.77%), 25 pigs were PRV positive (40.32%), 6 pigs were PCV1 positive (9.68%), 36 pigs were Mycoplasma hyopneumoniae positive (58.06%), 11 pigs were APP positive (17.74%), 42 pigs were PRRSV positive (67.74%), 10 pigs were CSFV positive (16.13%), 0 pigs were Salmonella positive and there were 30 PMWS cases. After statistic analysis, we found that co-infection with CSFV (p=0.04), PRRSV (p=0.0027), and PPV (p=0.0047) can increase the probability to induce PMWS. The nucleotide sequences of full-length genome of 8 Taiwan isolates were also analyzed by PCR and sequencing. After comparing nucleotide sequences of Taiwan strains published on GenBank, the PCV2 strains shared 93.1% to 99.8% identity. Comparing the amino acid sequences translated from ORF2 of PCV2 among the Taiwan strains, their amino acid sequences shared 88.5% to 100% similarity. The nucleotide sequences of PCV2 isolates shared 93.4% to 99.8% identity with other isolates world wide.

Yes
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparpfl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparpfl/flCreAlb) and whole-body (Tiparpfl/flCreCMV; TiparpEx3−/−) Tiparp null mice. Tiparpfl/flCreAlb and TiparpEx3−/− mice given a single injection of 10 μg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparpfl/flCreAlb and TiparpEx3−/− mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparpfl/flCreAlb and TiparpEx3−/− mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparpfl/flCreAlb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.
This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), CIHR New Investigator Award, an Early Researcher Award from the Ontario Ministry of Innovation (ER10-07-028), an unrestricted research grant from the DOW Chemical Company, the Johan Throne Holst Foundation, Novo Nordic Foundation and the Norwegian Cancer Society to J.M.

This thesis explores the experience of living with cancer-related cachexia (CRC) from the patient perspective. Critique of the literature indicates few examples where patients have had the opportunity to speak. Following a challenging recruitment process, six people living with the syndrome were interviewed to elicit their narrative. Their stories were examined and themes identified relating to their personal feelings and how these affected social interactions. Thematic analysis was applied to produce what is a rich qualitative description of the experience from this small sample. Living with CRC requires development of strategies to survive. Emergent themes included the loss of sense of self and a changing relationship to the social world, social isolation and dissatisfaction with truth-telling by health professionals. Recommendations are made to mitigate the suffering of patients by empowering them through better information and acknowledgement of their condition. The balance between nutrition and wellbeing is re-examined, calling for a reorientation of perspective from a focus on intake towards a focus on quality of life. This clearly falls within the nurse-as patient-advocate paradigm and the relevance and meaning of this research to the nursing profession is explored. Potential areas for further research in regards to both patient experience and nursing practice are extrapolated.