Dissertations / Theses on the topic 'Feline infectious peritoniti'

O coronavírus felino (FCoV) ocorre sob uma grande diversidade gênica de amostras e é classificado em dois patotipos: o coronavírus felino entérico (FECoV) e o vírus da peritonite infecciosa felina (FIPV). O patotipo FIPV é altamente virulento e responsável pelo desenvolvimento de uma doença altamente fatal, denominada de peritonite infecciosa felina (PIF). Já o FECoV apresenta-se amplamente disseminado na população felina e é responsável na maioria das vezes por infecção assintomática. Atualmente, nenhum marcador gênico conhecido é capaz de diferenciar os patotipos FECoV de FIPV. O presente estudo foi dividido em dois capítulos. No primeiro capítulo, objetivou-se avaliar a diversidade molecular do gene da membrana (M) em 190 amostras provenientes de 5 gatos sem manifestações de PIF (PIF-) e de 10 gatos com manifestações clínicas e histopatológicas de PIF (PIF+). Com esse estudo, conclui-se que tanto a hipótese de mutação in vivo do FECoV para FIPV, quanto a hipótese de transmissão entre gatos do patotipo FIPV são plausíveis. No segundo capítulo, com o objetivo de avaliar a diversidade dos genes 3a-c, E e M foram sequenciados clones de amplicons para estes genes obtidos, de 6 gatos PIF+ e 2 gatos PIF-. Os genes 3a-c, E e M apresentaram diversidade gênica que confere a constituição das quasiespécies de coronavírus felino com probabilidade de emergência do patotipo de alta virulência, mas de um modo hospedeiro-específico. Com o segundo estudo, conclui-se que as linhagens FIPV de coronavírus felino apresentaram a proteína 3c truncada, sendo o gene 3c o único marcador de patotipo dos FCoVs observado dentre os genes estudados.
Feline coronavirus (FCoV) occurs as a large genic diversity of strains and is classified as two pathotypes: feline enteric coronavirus (FECoV) and feline infectious peritonitis virus (FIPV). The FIPV pathotype is highly virulent and responsible for the onset of a highly fatal disease named feline infectious peritonitis (FIP), while the FECoV pathotype is widely disseminated in feline populations leading mostly to asymptomatic infections. No genic marker is currently known to differentiate the FECoV and FIPV pathotypes. This study has been divided in two chapters. In the first chapter, the aim was to evaluate the molecular diversity of the membrane (M) gene in 190 samples from 5 cats without FIP (FIP-) and 10 cats with clinical and histopathological evidence of FIP (FIP+). The conclusion of this study is that both the in vivo mutation hypothesis in the FECoV-to-FIP direction and the hypothesis of FIPV transmission amongst cats are plausible. In the second chapter, aimed to evaluate the diversity of genes 3a-c, E and M, clones of amplicons for these genes were obtained and sequenced from samples from six FIP+ and 2 FIP- cats. Genes 3a-c, E and M show a genic diversity that results in a quasispecies constitution of FCoV that leads to the probability of the emergence of the highly virulent pathotype in a host-specific way. The conclusion of this second study is that FIPV lineages show a truncated form of the 3C protein, making the 3c gene the only pathotype marker for FCoV observed amongst the genes studied herein.

Dissertação de Mestrado Integrado em Medicina Veterinária
A peritonite infeciosa felina (PIF) é uma doença patogénica e fatal, causada pelo coronavírus felino (FCoV), capaz de induzir uma reação inflamatória sistémica. A PIF é associada a falhas no sistema imunitário, nomeadamente à inibição da resposta humoral e celular, havendo um conjunto de fatores genéticos, ambientais e do hospedeiro que predispõem para o seu aparecimento. Estudos recentes sugerem novos fármacos, promissores na cura da PIF. O GS-441524 revela-se muito promissor para todas as formas clínicas de PIF, relevando altas taxas de remissão. Contudo, não se encontra comercialmente disponível e são ainda necessários estudos para o licenciamento deste medicamento. O presente estudo teve como objetivo caracterizar gatos diagnosticados com PIF na Unidade de Isolamento de Doenças Infeciosas do Hospital Veterinário da Faculdade de Medicina Veterinária da Universidade de Lisboa, entre outubro de 2013 e novembro de 2019. Com o auxílio dos médicos responsáveis por este departamento foi possível a obtenção de dados, não só relativamente ao historial dos animais, como resultados de exames complementares. O diagnóstico foi realizado através de necropsia ou por deteção de coronavírus através de PCR em tempo real a partir de líquido de derrame torácico ou abdominal. A partir de janeiro de 2019 o diagnóstico foi otimizado recorrendo-se à deteção de mutações do coronavírus por PCR em tempo real em diferentes amostras (sangue, líquido de derrame, granulomas ou linfonodos). Analisando os dados disponíveis, foi possível identificar na presente população os seguintes fatores de risco: idade, fator raça, origem e a presença de pelo menos um fator de stress antes do aparecimento dos sinais clínicos. Perante os desafios económicos que esta doença carece desenvolveu-se um fluxograma para direcionar os recursos financeiros de forma a obter um diagnostico definitivo ante-mortem, PCR em tempo real para deteção de mutações, e quando não é possível, post-mortem.
ABSTRACT - Feline Infectious Peritonitis: casuistry and clinical forms - Feline infectious peritonitis (FIP) is a fatal and pathogenic disease caused by the feline coronavirus, which is responsible for inducing an inflammatory reaction. FIP is responsible for a number of faults in the immune system, namely the inhibition of humoral and cellular response. A set of genetic, environmental and host factors can predispose its appearance. Recent studies have shown promising new drugs in the treatment of FIP. GS-441524 has been reported as a very promising drug in the treatment of all clinical forms of FIP, with high rates of remission. However, it is not yet commercially available and studies are required to its licence. The present study aimed to study cats diagnosed with FIP, in the Infectious Diseases Isolation Unit of the Veterinary Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, between October 2013 and November 2019. With the help of the doctors responsible for this department, it was possible to collect data regarding the animal’s history, complementary test results. The diagnosis of FIP was made through autopsy or real time PCR positive for feline coronavirus in thoracic or abdominal fluid. Since January of 2019, the diagnosis was improved by using the real time PCR detection of mutation of feline coronavirus in different samples (blood, effusion fluid and granuloma or lymph nodes). With the analysis of the available data, it was possible to identify in the present population the following risk factors: age, being of breed, origin and the presence of at least one stress factor before the appearance of clinical signs. Facing the economic challenges of FIP, a flowchart has been developed to direct the financial resources in order to obtain a definitive ante-mortem diagnosis, real time PCR with detection of mutations, and if not possible, post-mortem.
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Dissertação de Mestrado Integrado em Medicina Veternária
A infecção pelo coronavírus felino (FCoV) em gatos, não só leva à excreção do vírus após infecção intestinal mas também à infecção sistémica, podendo manter-se assintomática ou manifestar-se como Peritonite Infecciosa Felina (PIF), uma doença fatal. Estima-se que 80 a 90% da população felina esteja infectada por este vírus mas apenas 5 a 12% desenvolvem a doença. O FCoV divide-se em dois subtipos, o tipo I e o tipo II, sendo o primeiro o responsável pela maioria das infecções. A patogénese da doença é complexa e não é totalmente conhecida, não estando claramente identificado qual o papel do sistema imunitário no seu estabelecimento e manutenção. Considera-se que uma forte resposta imunitária celular pode prevenir a doença e, pelo contrário, que uma forte resposta imunitária humoral pode levar ao estabelecimento da doença, na sua forma exsudativa ou não exsudativa. De forma a conhecer o perfil imunitário de animais naturalmente infectados pelo FCoV foram determinados os níveis de transcrição do mRNA das citoquinas IL-10, IL-4, IL-12p35, TNF-α e IFN-γ em amostras de sangue dos animais em estudo, através de RTqPCR. Estes foram divididos em três grupos para comparação dos resultados: o primeiro sem sintomatologia associada a PIF (I); o segundo com sintomatologia gastrointestinal suspeita de PIF (II) e o terceiro com sintomatologia neurológica e ocular suspeita de PIF (III). Foi ainda realizada a subtipificação viral e determinada a carga viral em líquido de derrame e em zaragatoas rectais e/ou amostras de fezes, avaliando o nível de excreção viral destes animais na tentativa de associar ao perfil de citoquinas. Apesar da tentativa de realizar o estudo com grupos com a maior homogeneidade possível, observou-se muita variabilidade nos níveis de transcrição. A carga viral entre os três grupos revelou-se sem diferenças significativas pelo que fica por responder o seu efeito no desenvolvimento e manutenção da doença. Já nos perfis de citoquinas, o TNF-α revelou-se inesperadamente elevado em animais naturalmente infectados e assintomáticos e diminuído nos dois grupos naturalmente infectados mas com sintomatologia suspeita de PIF. Os perfis da expressão de citoquinas aparentemente demonstram uma resposta predominantemente celular nos grupos I e III e mista no grupo II, apesar de estatisticamente não se terem verificado diferenças entre os grupos.
ABSTRACT - Evaluation of feline coronavirus viral load and its relation with the expression profile of immune mediators on healthy carriers and on animals with Feline Infectious Peritonitis - Infection with feline coronavirus (FCoV) in cats, not only leads to virus elimination on faeces after intestinal infection but also to systemic infection that may remain asymptomatic or manifest as Feline Infectious Peritonitis (FIP), a fatal disease. It is estimated that 80 to 90% of the cat population is infected with this virus, but only 5 to 12% develop the disease, one of the most important viral diseases in cats. FCoV can be divided into two types, type I and type II, the former being responsible for the majority of infections. The pathogenesis of the disease is complex and the role of the immune system in the establishment and maintenance of disease remains unclear. It is considered that a strong cellular immune response can prevent the disease, and, conversely, that a strong humoral immune response may lead to the establishment of the disease whether on in its exudative or non-exudative form. In order to learn about the immune profile of naturally infected cats with FCoV, the mRNA transcription levels of IL-10, IL-4, IL-12p35, TNF-α and IFN-γ cytokines was measured by RTqPCR in blood samples. The animals were divided into three groups for comparison purposes: the first had no symptoms associated with PIF (I); the second had gastrointestinal symptoms suspected of FIP (II) and the third had ocular and neurologic symptoms suspected of FIP (III). Furthermore viral subtyping was performed and viral load was determined in effusions and in rectal swabs and/or faecal samples, assessing the level of viral shedding of these animals in an attempt to associate with the cytokine profile. Despite the attempts to arrange the study groups with the greatest possible homogeneity, there was significative variability on the transcription levels. The viral load between the three groups revealed no significant differences, leaving the effect of viral load in the development and maintenance of the disease unanswered. The TNF-α profile proved unexpectedly high in naturally infected and asymptomatic cats and decreased in both naturally infected with suspected symptoms of FIP groups. The expression profiles of cytokines apparently show a predominantly cellular response in groups I and III and mixed in group II, although there have been no statistically differences between groups.

La peritonite infettiva felina (FIP) è una patologia letale dei felidi, a patogenesi virale ed immunomediata. La propensione dell’agente eziologico alle mutazioni – coronavirus felino, FCoV – e la sintomatologia spesso aspecifica, rendono questa patologia complessa sia dal punto di vista diagnostico che patogenetico. Non si è ancora a conoscenza, infatti, della mutazione virale responsabile della patologia, non esiste ancora un gold standard per la diagnosi intra vitam e non è ancora disponibile una terapia valida. Lo scopo di questa tesi è di tentare di chiarire alcuni di questi aspetti. I primi due obiettivi (studio I e II) erano diretti allo sviluppo di un test per la diagnosi di FIP in forma effusiva. In particolare, è stata valutata l’accuratezza diagnostica del valore ottenuto dal rapporto tra le due conte leucocitarie fornite dal Sysmex XT-2000iV (Δ total nucleated cell count – TNCC) su campioni di versamento FIP indotto. Successivamente, è stato valutata la possibilità di effettuare lo stesso test su surnatanti di versamenti dopo aggiunta di sangue intero felino, in modo da poter ottenere questo valore su campioni congelati o con risultati dubbi ad altri esami. Il terzo scopo (studio III) era volto a confermare o smentire la presenza di pattern elettroforetici atipici in corso di FIP, come registrato nel nostro laboratorio negli ultimi anni. E’ stato quindi svolto uno studio retrospettivo per confrontare pattern elettroforetici in due periodi di tempo diversi. Il quarto obiettivo (studio IV) si prefissava di valutare l’accuratezza diagnostica di diversi test, sia clinico patologici che molecolari, per trovare il miglior test o la miglior combinazione di test per la diagnosi di FIP in vivo. Nello stesso studio è stato anche valutato il sequenziamento del gene spike (S), ultimamente proposto come discriminante, quando mutato, tra i due patotipi del FCoV. Il quinto scopo (studio V) era di mettere a punto una metodica molecolare isotermica (loop isothermal amplification method – LAMP) per il rilevamento del FCoV. Questa metodica, essendo veloce ed economica, potrebbe facilitare l’identificazione dei gatti eliminatori del FCoV o, per alcuni campioni, la diagnosi di FIP. Durante il mio percorso di dottorato ho anche partecipato ad un progetto sotto la supervisione della prof. Séverine Tasker e della dott.ssa Emi Barker dell’università di Bristol. Questo progetto (studi VI e VI.1) ha lo scopo di scoprire la vera prevalenza, in un ampio numero di tessuti, fluidi e feci ottenute da gatti affetti e non affetti da FIP, di due mutazioni del gene spike considerate ultimamente come co-responsabili della FIP. Gli studi I e II hanno dimostrato che il ΔTNC può essere usato per diagnosticare la FIP con buona accuratezza. Lo studio III ha confermato che i pattern elettroforetici tipici di FIP sono meno frequenti negli ultimi anni, possibilmente per modificazioni nella patogenicità dei FCoVs. Dallo studio IV si evince che i test molecolari possono confermare la diagnosi di FIP, ma che solo l’AGP puo’ escluderla; l’esame citologico dei versamenti dovrebbe essere il test di scelta sui versamenti, mentre sui tessuti il sequenziamento del gene S dovrebbe essere usato per confermare la diagnosi, mentre la PCR 3’ UTR PCR quando la FIP è meno probabile. La metodica LAMP sviluppata nello studio V si è rivelata molto specifica ma poco sensibile, dimostrandosi un buon test per confermare la presenza di FCoV in campioni biologici, ma non per escluderla. Infine, lo studio VI ha messo in evidenza la presenza di coronavirus mutati anche nelle feci di gatti non affetti da FIP, mostrando che i successivi progressi in questo studio metteranno in evidenza nuovi aspetti della patogenesi della FIP.
Feline infectious peritonitis (FIP) is a deadly disease of felids with a viral and immune-mediated pathogenesis. The nature of the etiological agent – feline coronavirus, FCoV – and the non specific clinical presentation make this disease particularly challenging both from a pathogenetic and a diagnostic point of view. Many aspects still represent an issue, like not knowing the mutation(s) responsible for the development of FIP, the lack of a gold standard for the diagnosis of FIP in vivo and the absence of an effective treatment. This thesis was aimed to clarify some of these aspects, specifically: a novel test on effusions was developed (namely, Δ total nucleated cell count – ΔTNC – i.e. the ratio between the two white blood cell count provided by the Sysmex XT-2000iV analyzer) (studies I and II); the frequency of atypical serum protein electrophoresis (SPE) patterns in cats with FIP, anecdotally reported during our diagnostic activity (study III) was investigated, a comparison of clinico-pathological and molecular tests for the diagnosis of FIP (study IV) was performed, a loop isothermal amplification method (LAMP) for the detection of FCoV was developed(study V) and an investigation on the prevalence of two mutations of the spike (S) protein gene in a wide number of samples from FIP and non-FIP cats was carried out (this latter study developed during an externship at the University of Bristol in collaboration with Prof. Séverine Tasker and Dr. Emi Barker) (study VI). The results of studies I and II demonstrated that the ΔTNC is a reliable method to support the diagnosis of FIP either on fresh or on frozen effusions. Study III confirmed that SPE profiles consistent with FIP are less frequent in recent years than in the past, possibly due to changes in the pathogenic characteristics of the FCoVs. However, study IV demonstrated that: on blood molecular tests may support a clinical diagnosis of FIP but none of the test, except the measurement of a1 acid-glycoprotein (AGP) may rule out this disease; cytology should be preferred on effusions either to exclude or confirm the disease and, on tissues, S gene sequencing should be preferred when histology is highly consistent with FIP while 3’ UTR PCR when FIP is less likely; the LAMP method developed in study V may be used to confirm the presence of FCoVs in the samples but is poorly sensitive and cannot exclude the presence of FCoVs. Finally, pyrosequencing of FCoV performed in study VI demonstrated the presence of gene S mutations also in FCoVs from fecal samples. The analysis of sequences recorded in this latter study, however, is still ongoing and future results may provide new insights on the pathogenesis and diagnosis of FIP.

Feline coronavirus (FCoV) and feline calicivirus (FCV) commonly infect domestic cats, and are an important cause of morbidity and mortality. There are currently no effective antiviral agents for these pathogens. Studies reported herein attempt address this therapeutic shortfall through the testing of a panel of small molecule compounds and specifically designed small interfering RNAs (siRNAs) for antiviral effects against both viruses. Initial compound screening identified chloroquine, mefloquine, and hexamethylene amiloride as effective inhibitors of FCoV, whilst mefloquine effectively inhibited FCV. Efficacy at low micromolar concentration was confirmed with orthogonal testing, albeit with relatively narrow selective indices. Preliminary experiments performed to inform the antiviral mechanism of the compounds against FCoV demonstrated all three compounds acted at an early stage of viral replication. For FCV, mefloquine exhibited potent inhibition of a panel of recent field isolates and demonstrated additive effects in combination with recombinant feline interferon omega. For both FCoV and FCV, a number of siRNAs demonstrated potent and specific inhibition of viral replication. These were effective at low nanomolar concentrations, when used in combination, and against high viral loads. A structural siRNA variant, Dicer-substrate siRNA, demonstrated similar or better efficacy, depending on the target, over canonical siRNAs directed at the same FCoV motif. Limitations of antiviral siRNAs in terms of antiviral resistance were investigated. FCoV serially passaged through siRNA treated cells rapidly acquired resistance, however combination therapy with three siRNAs was able to delay this considerably. For FCV, siRNAs effective against the reference strain were broadly efficacious against field isolates, although some variability was noted. Taken together these results provide important information regarding potential antiviral therapies against these important pathogens.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Feline infectious peritonitis (FIP) is a highly contagious, progressive and invariably fatal viral disease of cats, and occasionally of wild felids, which results from antibody-mediated hypersensitivity reactions (types III and IV) in individuals incapable to produce a cell-mediated immune response. Although the prevalence of FIP is high worldwide, recent anatomopathological studies about this disease are scarce. Furthermore, the microscopic characteristics of the bone marrow of FIP-affected cats do not exist in the available literature. Based on this, the purpose of this dissertation is to describe possible bone marrow lesions seen in spontaneous cases of FIP. Therefore, the bone marrow collected systematically from the femoral diaphysis of 16 cats necropsied in the LPV-UFSM (Santa Maria, RS, Brazil), between January 2000 and June 2017, with a definitive diagnosis of FIP, were evaluated phenotypically (histopathology [hematoxylin and eosin] and histochemistry [Perls reaction]) and immunophenotypically (immunohistochemistry using anti-myeloid [MAC387] and anti-lymphoid [CD79αcy and CD3] markers). The results showed, regardless of the clinicopathological form of the disease (“dry” [noneffusive] or “wet” [effusive]), myeloid hyperplasia; erythroid hipoplasia; megakaryocytic dysplasia (dismegakaryocytopoiesis); and medullary plasmacytosis. In cases of “dry FIP”, but not in those of “wet PIF”, there was bone marrow and hepatic hemosiderosis. These lesions allowed establishing that cats with FIP develop myelodysplasia, a myeloproliferative lesion very similar to that reported in HIV-infected humans. It is suggested that, based on the findings described here, myelodysplasia is considered to be the main cause of hematological abnormalities observed in FIP, especially for non-regenerative anemia and thrombocytopenia, frequently developed by patients.
Peritonite infecciosa felina (PIF) é uma doença marcadamente contagiosa, progressiva e invariavelmente fatal de gatos, ocasionalmente de felideos selvagens, que decorre de uma reação de hipersensibilidade (tipos III e IV) em um indivíduo incapaz de montar uma resposta imune celular adequada. Apesar da prevalência da peritonite infecciosa felina (PIF) ser alta em praticamente o mundo todo, estudos anatomopatológicos recentes acerca dessa doença são escassos. Não obstante, as características microscópicas da medula óssea de gatos com PIF inexistem na literatura consultada. Com base nisso, o objetivo deste estudo é descrever possíveis alterações medulares ósseas vistas em casos espontâneos de PIF. Para isso, as medulas ósseas colhidas sistematicamente da região diafisária dos fêmures de 16 gatos necropsiados no LPV-UFSM (Santa Maria, RS, Brasil), entre janeiro de 2000 e junho de 2017, e que tiveram diagnóstico definitivo de PIF, foram avaliadas fenotípica (histopatologia [hematoxilina e eosina] e histoquímica [reação de Perls]) e imunofenotipicamente (imuno-histoquímica utilizando marcadores anti-mieloide (MAC387) e anti-linfoide (CD79 αcy e CD3). Os resultados permitem afirmar que, independentemente da apresentação clinicopatológica da doença (seca ou úmida), ocorre: 1) hiperplasia mieloide; 2) hipoplasia eritroide, 3) displasia megacariocítica (dismegacariocitopoiese) e 4) plasmocitose medular. Nos casos de PIF seca, mas não naqueles de PIF úmida, há hemossiderose medular óssea e hepática. Essas alterações permitem estabelecer que gatos com PIF desenvolvem mielodisplasia, uma lesão mieloproliferativa muito semelhante àquela relatada em humanos infectados pelo HIV. Sugere-se que a partir dos achados aqui descritos, mielodisplasia seja considerada a principal responsável pelas alterações hematológicas observadas na PIF, especialmente pela anemia e trombocitopenia arregenerativas tão frequentemente desenvolvidas pelos pacientes com essa doença.

O presente relatório foi realizado no âmbito do estágio curricular do Mestrado Integrado em Medicina Veterinária da Universidade de Évora, encontrando-se dividido em três partes. A primeira parte corresponde à análise da casuística acompanhada durante o estágio curricular, realizado no Laboratório Veterinário INNO, entre seis de outubro de 2020 e 26 de março de 2021. A segunda parte consiste numa revisão bibliográfica sobre a Peritonite Infeciosa Felina (PIF), uma doença infeciosa, inflamatória e imunomediada. Por fim, a terceira parte apresenta um estudo retrospetivo acerca da contribuição do rácio A/G e ΔTCN das efusões cavitárias para o diagnóstico de PIF. Apesar de não existirem caraterísticas bioquímicas e celulares nas efusões que permitam o diagnóstico definitivo da PIF, os resultados do estudo permitiram averiguar que este torna-se mais provável quando as efusões apresentam rácio A/G inferior a 0,88 e ΔTCN superior a 4,9; Abstract: Laboratorial Diagnosis in Companion Animals The present report refers to the internship for the completion of the Master Degree in Veterinary Medicine of the University of Évora, and is divided in three parts. The first part includes a statistical description of the followed procedures during the curricular internship, which was held at Laboratório Veterinário INNO, between October of 2020 and March of 2021. The second part consists of a bibliographic review on Feline Infectious Peritonitis (FIP), an infectious, inflammatory and immunomediated disease. Finally, the third part presents a retrospective study about the contribution of the A/G and ΔTCNN ratio of cavitary effusions to the diagnosis of FIP. Although there are no biochemical and cellular characteristics in effusions that allow the definitive diagnosis of PIF, the results of the study allowed to verify that it becomes more likely when effusions have an A/G ratio of less than 0,88 and ΔTCN greater than 4,9.

Il microbiota intestinale (insieme dei microrganismi che si trovano all’interno dell’apparato gastroenterico) svolge diverse funzioni e, tra queste, di particolare interesse è il suo rapporto con il sistema immunitario. Infatti, diversi studi hanno evidenziato la presenza di disbiosi non solo in corso di patologie gastroenteriche, ma anche autoimmuni ed infettive. Gli studi in medicina veterinaria sull’argomento sono ancora pochi e proprio per questo motivo, all’interno di questo progetto, è stato scelto di indagare la possibile relazione tra il microbiota intestinale e due particolari patologie infettive (la peritonite infettiva felina -FIP- e la leishmaniosi canina) la cui patogenesi è fortemente influenzata dal tipo di risposta immunitaria sviluppata dall’ospite. Gli scopi di questo progetto sono quindi stati: la valutazione del microbiota intestinale in gatti affetti o meno da FIP (studio I). Dal momento che diagnosi in vivo di FIP risulta spesso difficoltosa, è stato valutato il potenziale, come biomarker di FIP, della paraoxonasi-1, una proteina di fase acuta negativa fortemente influenzata da importanti stati ossidativi (studi II e III). Per lo stesso motivo è stata valutata la correlazione tra le performance diagnostiche di istopatologia, immunoistochimica e RT-PCR su differenti organi (studio IV). Infine, è stata indagata la composizione del microbiota intestinale in cani infetti o meno da Leishmania spp., correlando i risultati ottenuti con le differenti popolazioni leucocitarie valutate mediante citofluorimetria (studi V e VI). I risultati ottenuti da questo progetto hanno fornito delle indicazioni preliminari sulla composizione del microbiota intestinale in gatti affetti da FIP o positivi per Coronavirus, che necessitano però un approfondimento su un gruppo di studio più ampio (studio I). È stato possibile determinare gli intervalli di riferimento della paraoxonasi-1 nel gatto ed evidenziare le sue buone performance come marker diagnostico in corso di FIP (studi II e III). Nonostante l’immunoistochimica rimanga il gold standard per la diagnosi di FIP, l’associazione con RT-PCR potrebbe ridurre gli errori diagnostici, vista la buona correlazione tra le due metodiche (studio IV). Infine, la valutazione della composizione del microbiota e delle popolazioni leucocitarie in cani affetti da leishmaniosi ha messo in luce delle differenze significative sia rispetto ai cani sani, che agli esposti asintomatici. Questi risultati sono incoraggianti e possono fungere da punto di partenza per ulteriori indagini (studi V e VI).
The gut microbiota (consortium of all the microorganisms that inhabit the gastrointestinal tract) plays different roles in the host. Among these, its relationship with the immune system has been of great interest in the last few years. Indeed, several studies highlight the presence of dysbiosis not only in gastrointestinal diseases, but also during autoimmune or infectious diseases. Literature about this topic is scarce in veterinary medicine. Thus, in this project, the possible relationship between gut microbiota and two specific diseases (feline infectious peritonis -FIP- and canine leishmaniasis) was investigated. These diseases were chosen due to the pivotal role of the immune response in their pathogenesis. The aims of this projects were: the evaluation of gut microbiota of cats with and without FIP (study I). Since in vivo diagnosis of FIP is quite challenging, the potential role of paroxonase-1 (a negative acute phase protein strongly influenced by oxidation) as a biomarker of FIP was investigated (studies II-III). For the same reason, the diagnostic agreement among histopathology, immunohistochemistry and RT-PCR on different organs was evaluated (study IV). Finally, the gut microbiota composition in dogs infected or not by Leishmania spp. was investigated. The results were correlated with the leukocyte populations studied by flow cytometry (studies V-VI). Results obtained in this project provided preliminary data about gut microbiota composition in cats affected by FIP or only Coronavirus positive. This achievement needs to be further investigated on a bigger sample size (study I). Paraoxonase-1 reference interval and its good performance as a diagnostic biomarker of FIP were determined (studies II-III). Despite the immunohistochemistry is still the gold standard for FIP diagnosis, the good diagnostic agreement obtained in the study suggested that a possible association with RT-PCR could minimize diagnostic errors (study IV). Finally, the gut microbiota composition and leukocyte populations of leishmaniotic dogs highlighted some significant differences compared with both healthy and exposed asymptomatic dogs. These promising results could be a starting point for further researches (studies V-VI).

Dissertação de Mestrado Integrado em Medicina Veterinária
O Derrame Pleural (DP) é uma afeção que resulta da acumulação excessiva de líquido na cavidade pleural. O DP é mais comum em gatos do que em cães e manifesta-se frequentemente através de dispneia, taquipneia, posição ortopneica e respiração abdominal. Este trabalho destina-se a sensibilizar para um diagnóstico definitivo da causa subjacente ao DP pois a sua apresentação clínica pode ser bastante semelhante independentemente da sua origem. Com este trabalho, pretendeu-se detetar qual a etiologia mais frequente do DP e com que tipo de derrame mais se relaciona, de acordo com a classificação laboratorial. Também foram avaliados os fatores de risco para cada etiologia e o prognóstico expectável para estes casos. Esta dissertação incluiu um estudo em 73 gatos com DP, observando-se uma maior expressão de DP secundários a neoplasia, representando 49.3% da amostra seguido de DP secundário a Insuficiência cardíaca congestiva (ICC), que representou 34.2%. Num menor número de casos, foram identificados 4 gatos com Piotórax, 4 com Peritonite infeciosa felina (PIF), 2 com Pneumonia e 2 com Quilotórax Idiopático (QI). Nos DP secundários a ICC, a principal causa encontrada foi a cardiomiopatia hipertrófica (CMH). Em relação aos casos de neoplasia, 44% do grupo tinha carcinoma mamário e 33% linfoma mediastínico. Neste estudo verificou-se a exatidão de alguns fatores de risco como guias para a deteção da etiologia do DP, por exemplo: na relação entre animais jovens e/ou positivos a FeLV que desenvolveram linfoma mediastínico; a maior prevalência de gatas a partir dos 9 anos de idade e que não tinham sido esterilizadas ou que tinham sido depois dos 2 anos de idade e que desenvolveram carcinoma mamário; a relação entre gatos machos e/ou da raça Persa com ICC. Por outro lado, observou-se para uma dada etiologia o mesmo tipo de DP, segundo a classificação do laboratório, ou seja, a presença de transudado modificado mais relacionado com ICC, o exsudado não séptico mais relacionado com causa neoplásica e o desenvolvimento de quilotórax mais relacionado com doença cardíaca do que com QI. O mau prognóstico de DP, na grande maioria dos casos, revelou que este é um sinal de avançado estado da doença subjacente, pelo que 74.5% dos animais faleceram ou foram sujeitos a eutanásia em menos de 6 meses após o respetivo diagnóstico. Para os casos de doença cardíaca e neoplasia o diagnóstico mais precoce poderia retardar algumas complicações como o DP e isso poderia manifestar-se num aumento da sobrevida desses gatos.
ABSTRACT - Pleural effusion (PE) is the abnormal accumulation of fluid within the pleural cavity. PE is more common in cats than in dogs, and manifests itself through dyspnea, tachypnea and abdominal breathing. The goal of this study is to highlight the importance of making a definitive diagnosis of the underlying cause of PE, because its clinical presentation can be quite similar regardless of the cause. This study intended to detect the most frequent etiology of PE and make a link with the type of effusion with which it is most closely related, according to the laboratory classification. The risk factors for each etiology and expected prognosis for these cases were also evaluated. This dissertation included a study of 73 cats with PE. Of the total number of cats 49.3% had PE due to neoplasia and in 34.2% it was due to congestive heart failure (CHF). In a smaller number of cases, PE was found in 4 cats with piothorax4 cats with Feline infectious peritonitis (FIP), 2 cats with pneumonia and 2 with IQ. In the cases of PE secondary to CHF, the main cause was hypertrophic cardiomyopathy (HCM). Regarding the cases of neoplasia, 44% had mammary carcinoma and 33% mediastinal lymphoma. In this study we verified the existence of some risk factors regarding the etiology of PE. For example: the link between young and / or FeLV positive animals that developed mediastinal lymphoma; the high prevalence of female cats aged 9 years and older who had not been sterilized or who had been so after 2 years of age and developed breast carcinoma; the relationship between male and / or Persian cats with CHF was also verified. Futhermore, according to the laboratory classification, the same type of PE was observed for a particular etiology, ie, modified transudate was found to be more related to CHF, nonseptic exudate was more related to neoplastic causes and chylothorax was more related with cardiac disease than with IQ. In general the poor prognosis of PE was due to the advanced stage of the underlying illness: 74.5% of the animals died or were euthanized in less than 6 months after their diagnosis. For cases of cardiac pathology and neoplasia early diagnosis could delay some complications such as PD and this could manifest it self as an increase in the survival rate of these cats.
N/A

Feline infectious peritonitis (FIP) is a systemic, fatal, viral-induced immune-mediated disease that affects cats globally. The disease is caused by virulent biotypes of feline coronaviruses (FCoV), known as the feline infectious peritonitis virus (FIPV). Traditionally, the treatment of FIP has centered on the use of immunosuppressive therapies. However, although some immunomodulatory therapeutic agents temporarily dampen clinical signs, there are no treatments that address the underlying problem of viral replication. Recent in vitro studies seeking an efficacious FCoV treatment investigated 19 candidate compounds used commonly in the treatment of other coronavirus infections. One of these compounds, mefloquine, a drug typically used in the treatment and prophylaxis of malaria in humans, showed great promise in that it substantially reduced the viral load of FIPV in infected Crandell Rees feline kidney cells without cytotoxic effects at low mefloquine concentrations. This evidence suggests that mefloquine should be considered for further investigation as a potential treatment option for FIP. However, the careful consideration of the idiosyncrasies of feline hepatic metabolism is required for any proposed treatment in cats in order to minimise any potential for harm. In cats, some therapeutic agents are known to undergo delayed phase II glucuronidative metabolism, resulting in toxicity. As there are no pharmacokinetic studies on the use of mefloquine in the cat, the overall aim of this project is to use an in vitro model of feline hepatic metabolism to ascertain whether mefloquine is likely to accumulate in the cat.

博士
國立臺灣大學
獸醫學研究所
103
Feline coronavirus (FCoV) is an ubiquitous viral pathogen in the cat populations. The infection of FCoV is usually asymptomatic. However, less than 5% of the seropositive cats develop a highly lethal immune-mediated disease, feline infectious peritonitis (FIP). Until now, neither effective treatment nor protective vaccine is available. Euthanasia is suggested once the disease was diagnosed, as all FIP cats eventually died from this disease. To date, several questions remain unsolved regarding roles of the virus and the host genetic factors contributing to the development of FIP. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. However, an FIP outbreak from a private animal shelter that causes 13 out of 46 cats died from FIP was confirmed in our lab recently. Sequence analysis of the S and 3c gene showed that the FIPVs of the outbreak were from the same origin and the results indicate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment. Serotype II FCoV was suggested to be significantly correlated with FIP and an outbreak caused by this type of FCoV has been confirmed. A Baculovirus-expressed type-specific spike proteins based assay was used fro the seroprevalence study of two types of FCoV in the past eight years in Taiwan was firstly carried out. Type I FCoV was found to be predominant compared to type II virus. Results derived from serotyping and genotyping support our current understanding of the evolution of disease-related FCoV and the transmission of FIP. With an immune-mediated disease entity, host genetic variant was suggested to influence the occurrence of FIP. The association between the single nucleotide polymorphisms (SNPs) of tumor necrosis factor-α (fTNFA), DC-SIGN (CD209), and the five FIP-associated SNPs identified from Birman cats, and the outcome of FCoV infection was determined. Among the 57 SNPs identified, five of them, including one in the promoter region of fTNFA, one in the coding region of extracellular domain of DC-SIGN, and three in the introns of CD209 were associated with the outcome of FCoV infections. None of the five Birman FIP cat-associated SNPs showed significant differences between our FIP and non-FIP groups. As disease resistance is multifactorial, the five genetic traits identified in this study should facilitate in the future breeding of the disease-resistant animal to reduce the occurrence of cats succumbing to FIP.

Orientação: Daniel Murta
A peritonite infecciosa felina (PIF) é das doenças infecciosas mais frequentes na espécie felina, afectando gatos domésticos (Felis catis) e selvagens. Esta doença é provocada por uma estirpe virulenta do coronavírus felino (FeCoV), e é sempre fatal após manifestação dos sintomas, sendo normalmente descrita como sendo uma doença do indivíduo, fortemente dependente não só da virulência viral, mas também de factores ambientais e do hospedeiro, nomeadamente da sua resposta imunitária. O presente estudo retrospectivo focou-se numa população de 122 gatos seguidos entre os anos 2010 e 2014 no Hospital Veterinário Vasco da Gama em Lisboa, para os quais foi realizada a titulação de anticorpos anti-FeCoV, sendo o objectivo principal relacionar o título de anticorpos anticoronavírus (anti-FeCoV) com a anamnese, sinais clínicos e parâmetros laboratoriais apresentados pelos animais suspeitos de PIF, avaliando assim se a sua determinação contribui para um correcto diagnóstico da PIF. Para além de se ter encontrado uma relação estatisticamente significativa entre um título positivo de anticorpos anti-FeCoV e a ocorrência de líquidos de derrame (especialmente ascite), a hiperglobulinémia e a hiperbilirrubinémia, também foi encontrada uma relação estatisticamente significativa entre um título negativo de anticorpos anti-FeCoV e gatos sem contacto regular com outros gatos e a presença de sinais clínicos neurológicos. Todas as relações identificadas estão de acordo com o descrito na bibliografia, com a excepção dos sinais clínicos neurológicos associados a títulos negativos de anticorpos anti-FeCoV, que geralmente estão presentes em animais afectados por PIF, esperando-se por isso que a incidência destes sintomas em animais com títulos anti-FeCoV positivos fosse predominante. Nesta população a titulação de anticorpos anti-FeCoV foi realizada essencialmente como meio complementar de diagnóstico clínico de PIF e não como meio de avaliar a ocorrência de uma exposição prévia ao FeCoV, não tendo sido detectada uma diferença significativa entre os títulos de anticorpos anti-FeCoV entre ambos os grupos. Assim sendo, e com base nos resultados obtidos neste estudo, a repetição periódica da titulação em animais sem sintomatologia de PIF mas que poderão ter estado em contacto com animais infectados com FeCoV durante um período de um ano deverá ser realizada para avaliar a evolução da situação, de acordo com o preconizado pelo laboratório DNATech. Já no caso dos animais com sintomatologia suspeita de PIF, a titulação deverá ser realizada e o resultado analisado tendo em conta a anamnese, sinais clínicos e parâmetros laboratoriais apresentados pelos animais, continuando a histopatologia associada à imunohistoquímica a ser actualmente o método de diagnóstico de PIF com maior valor diagnóstico.
Feline infectious peritonitis (FIP) is one of the most frequent feline infectious diseases, affecting both domestic (Felis Catis) and wild cats. This disease is caused by a virulent strain of feline coronavirus and is always fatal upon clinical manifestation, being usually described as a disease that affects single animals and whose outcome dependens not only on the virulence of a given viral strain, but also on environmental factors and on host characteristics, namely its immune response. The main aim of the present study was to find a connection between the anti-feline coronavírus (FeCoV) antibody titers and the anamnesis, clinical signs and laboratory values from animals suspected of FIP, thus evaluating the contribution of its determination for a correct FIP diagnosis. For this purpose, a group of 122 cats followed between 2010 and 2014 at the Vasco da Gama Veterinary Hospital in Lisbon, for whom anti-FeCoV antibody titer determination was performed, was chosen. Besides finding a statistically significant association between a positive anti-FeCoV antibody titer and the presence of effusions (mainly ascitis), hyperglobulinemia and hyperbilirrubinemia, a statistically significant association between a negative anti-FeCoV antibody titer and cats that have no contact with other cats and neurologic symptoms has also been identified. All these associations are in accordance with what was previously described in the literature, with the exception of the last one, since neurological symptoms are very often identified in cats affected by FIP, thus an association with a positive titer would be more likely. Finally, in this population this test was performed essentially as a complementary diagnostic tool in FIP-suspected cases and not to evaluate the occurrence of a previous FeCoV exposure; no difference in FeCoV antibody titers between the two studied groups was found. Based on the presented results, it is advisable to repeat the anti-FeCoV titration in animals whithout FIP symptoms that have been exposed to FeCoV-infected animals during 1 year, in accordance with DNATech lab recommendations. If the animal has FIP-related symptoms, the titration result must always be analysed having into account the anamnesis, clinical signs and laboratory values presented by the affected animals. However, a definitive FIP diagnosis can only be obtained after performing the FIP golden standard technique histopathology, preferably in association with immunohistochemistry.

碩士
臺灣大學
獸醫學研究所
98
Feline infectious peritonitis (FIP) is a fatal, immune-mediated disease in felids caused by feline coronavirus (FCoV). FCoV are widespread, however, only 5-12 % of seropositive cats develop FIP. Most infections are asymptomatic or result to mild enteritis. Up to present, there is no virological assay that can distinguish virulent from avriulent virus, moreover no effective vaccine or therapy is available for FIP. The objectives of this study were to perform a clinical trial for antiviral agents recently identified to be effective against FCoV infection in vitro, and to correlate the integrity of 3c gene of FCoV with FIP. Nine clinically healthy FCoV shedding cats from three multi-cat families were monitored. After taking Nelfinavir 6.25~50 mg/kg for up to 24 weeks, one infected cat stop shedding virus and two infected cats were no longer viremia, however most of the cats continue to shed FCoV from enteric tract. Through assessment of clinical signs and blood chemistry, no significant side effect was found. Deletions of FCoV 3c gene were reported to associate with viral virulence. In this study, 3c genes of FCoV were analyzed from 19 asymptomatic and 11 FIP cats. Of the 19 sequences analyzed, most are intact except one (5.3%) with a 3-nucleotide in frame deletion. Among the 3c genes from 11 FIP cats, only 2 were intact; the majority (81.8%) exhibited serious aberrations, including various deletions (1-595nt), insertion and point mutation. All these mutations result in premature termination of translation and truncation of the 3c polypeptide. Through statistical analysis, mutation of 3c gene demonstrates a significantly higher correlation with FIP (p<0.01).The preliminary results from clinical trial indicates that Nelfinavir when administrated alone can not eliminate viral shedding from chronic FCoV shedders. As the associating mutation of with FIP has been confirmed base on this finding, it is possible to develop an diagnostic assay for FIP in the near future.

Orientação : Andreia Santos ; co-orientação : Luís Resende
A Peritonite Infecciosa Felina (PIF) é uma das mais importantes doenças infecciosas fatais em gatos. Trata-se de uma doença imunomediada causada pelo Coronavírus Felino (FCoV) que afecta principalmente animais jovens e para a qual ainda não existe um tratamento eficaz. O objectivo deste estudo foi avaliar os aspectos epidemiológicos e clínico-patológicos presentes numa amostra populacional de 20 felídeos diagnosticados com PIF no Hospital Veterinário Principal entre 2006 e 2014, a fim de identificar eventuais factores de risco e as alterações clínicas e laboratoriais mais frequentes. Foi ainda avaliado o tratamento instituído e o tempo médio de vida após o diagnóstico. O presente estudo demonstrou que a PIF é uma doença que ocorre em felídeos jovens, com uma média de idades de 17 meses, tendo-se verificado uma proporção significativamente maior de gatos machos (70%) com PIF comparativamente a fêmeas (30%). Os principais sinais clínicos apresentados foram perda de peso (80%), anorexia (80%), piréxia (75%) e distensão abdominal (75%) e as principais alterações hematológicas foram trombocitopénia (82,4%), linfopénia (64,7%), anemia (52,9%), hiperproteinémia (61,5%) e hipoalbuminémia (61,5%). A forma efusiva de PIF foi encontrada em 15 gatos e a forma não-efusiva em 5 gatos. As principais alterações encontradas no líquido de derrame nos gatos com PIF efusiva foram uma coloração amarela, proteínas totais entre 4,5-6,2 g/dL e ratio Albumina: Globulina < 0,4. No que concerne à terapêutica instituída, a prednisolona foi o fármaco mais utilizado no tratamento de PIF, tendo sido utilizada em 84,6% dos animais submetidos a tratamento. O interferão ómega recombinante felino apenas foi utilizado em 38,5% dos animais, aos quais também foi administrada concomitantemente prednisolona. Verificou-se a existência de diferenças estatisticamente significativas no tempo de sobrevida entre gatos tratados com interferão ómega felino e gatos tratados com outros fármacos (p=0,013). O tempo de sobrevida dos felinos submetidos a tratamento variou de um mínimo de 7 dias a um máximo de 240 dias, sendo a média de 43,7 dias, não se tendo verificado a existência de diferenças estatisticamente significativas no tempo de sobrevida entre gatos com PIF efusiva e gatos com PIF não efusiva (p=1,000).
Feline Infectious Peritonitis (FIP) is one of the most important fatal infectious diseases on cats. It is an immune-mediated disease caused by feline coronavirus (FCoV) that mainly affects young animals and for which there is still no effective treatment. The aim of this study was to evaluate the epidemiological and clinicopathological aspects present on a population sample of 20 cats diagnosed with FIP at the Hospital Veterinário Principal from 2006 to 2014, in order to identify possible risk factors and the most frequent clinical and laboratory changes. It was also assessed the established treatment and the average survival time after diagnosis. The present study demonstrated that FIP is a disease that occurs in young felines, with a mean age of 17 months, and it was found a significantly greater proportion of male cats (70%) with FIP compared with females (30%). The main clinical signs presented were weight loss (80%), anorexia (80%), pyrexia (75%) and abdominal distension (75%) and the main haematological changes were thrombocytopenia (82,4%), lymphopenia (64,7%), anaemia (52,9%), hyperproteinaemia (61,5%) and hypoalbuminaemia (61,5%). The effusive form of FIP was found in 15 cats and non-effusive form in 5 cats. The main changes found in effusion fluid in cats with effusive FIP were a yellow color, total proteins between 4,5-6,2 g/dL and Albumin: Globulin ratio < 0,4. As far as therapeutic approach is concerned, prednisolone was the most commonly used drug in the treatment of FIP, and it had been used in 84,6% of animals subjected to treatment. The recombinant feline interferon omega was used in only 38,5% of the animals, to which was concomitantly administered prednisolone. It has been found that there are significant differences in survival time between cats treated with feline interferon omega and cats treated with other drugs (p=0,013). The survival time of treated cats ranged from 7 days to a maximum of 240 days, with a mean of 43,7 days, and there was no statistically significant differences in survival time between cats with effusive FIP and cats with non-effusive FIP (p=1,000).

碩士
國立臺灣大學
獸醫學研究所
93
Feline coronavirus (FCoV) infection in cats may cause peritonitis (FIP) or a milder form of enteritis. The study is to investigate cats from private clinic and National Taiwan University Animal Hospital, with correlation of a nested reverse transcription polymerase chain reaction (RT-nPCR) assay to detect feline coronavirus highly conserved 3’ untranslated region in clinical specimens. A total of 40 cats from June 2000 to April 2004 were screened to be feline coronavirus positive and died with the diagnosis as effusive FIP . Among these 40 cats, FCoV can be detected in all of the effusion specimens. Most of the cats were purebred, with 75% (30/40)aged from 3 month to 3 year. Among them 20 with blood test data were analysed, 70% (14/20)appeared to have anemia, 90% (18/20)with lymphopenia and 70% (14/20) with A/G ratio lower than 0.7. Virus isolation were attempted using body effusion collected from positively diagnosed animals. Limited amount of coronavirus can only be detected up to the third passage by RT-nPCR. Serologic assay revealed that FCoV type I might be predominant in Taipei area. Sequence comparison were made from 475bp of 7a/7b gene, and the result shows high degree of homology to several other known FCoV strains, including FIPV strain NOR-15 isolated in the USA, which are 95.4% identical in this region.

碩士
國立臺灣大學
獸醫學研究所
99
Feline infectious peritonitis, FIP, is an immune-mediated disease caused by feline coronavirus. Many combinations of drugs, including antiviral drugs, immune-modulator and immunosuppressive agents, were used to control the disease. However, euthanasia is still the most suggested method. The aim of this research is to evaluate of different therapeutic strategies in treatment of FIP and to state a potential therapeutic strategy. One hundred nineteen FIP highly suspected diseased cats, presented at National Taiwan University Animal Teaching Hospital between June 2003 and May 2011, were enrolled in this study. Sixty-four cats were confirmed to have FIP via pathological confirmation after death and another two cats were confirmed with positive IFA and PCR tests, one of the two cats were further confirmed with biopsy and another is still alive. Thirteen-five of the 66 cats were selected with the criteria of survival over 2 weeks and algorithm of the disease staging and therefore included in the analysis. Antiviral drug (nelfinavir): there was no difference in hematological changes and survival time between the subgroup1a (w/o nelfinavir) and subgroup1b (w/ nelfinavir). These may indicate that nelfinavir had no additional effect in this combination. Interferon: there was no difference in hematological changes and survival time between the subgroup2a (w/ feline interferon-omega) and subgroup2b (w/ human recombinant interferon-alpha). These may indicate that different origin of interferon had no effect in this combination and no obvious side effect. ACEI (angiotensin converting enzyme inhibitor, benazepril) was first time used in treatment of FIP. We compared group1 (w/benazepril) and subgroup2a+2b (w/o benazepril), increasing of albumin and potassium were observed in subgroup 2a+2b. These may indicated that ACEI conduced to the treatment by improvement of vasculitis caused by immune-complex. The drug-combinations of group1 included prednisolone, ACEI, human recombinant interferon-alpha, and with/out nelfinavir. The other combinations were assigned to group2. The PCV, Hb, RBC counts and albumin were significantly higher and total bilirubin was lower in group1 than in group2 at 6th weeks after treatment. These indicated that the combination of group1 had better effect in improvement of anemia and leakage of protein. Besides, 3 cats with effusive FIP in group1 became non-effusive FIP during therapy indicated the potential therapeutic strategy fro FIP. Fifty percentage of group1 survived over than 14 weeks and only 11.54% of group2, respectively. The medians of survival time were 74.5days and 27days of group1 and group2, respectively. Both Kaplan-meier survival analysis and cumulative survival rate were significant higher in group1 than in group2 (p&lt;0.05). In effusive FIP, the medians survival time of group1 and group2 were 74.5days and 22days, respectively. Both Kaplan-meier survival analysis and cumulative survival rate were significant higher in group1 than in group2 (p&lt;0.05). In conclusion, improvement of anemia and albumin and increasing of Kaplan-meier survival analysis and cumulative survival rate after treatment of the combinations, including prednisolone, ACEI, human recombinant interferon-alpha, and with/out nelfinavir, showed a potential therapeutic strategy, However, more cases and further study were still needed.

碩士
國立臺灣大學
臨床動物醫學研究所
97
Fifty-one naturally infected feline infectious peritonitis (FIP) diseased cats were enrolled in this study for staging of the course of FIP disease. Effusive FIP was found in 30/51 cats (58.8%), whereas non-effusive FIP was diagnosed in 12/51 cats (23.5%), and nine cats (9/51, 17.6%) were mixed type. The age ranged from 3 month-old to 7 year-old. Most of them were less than 1-year-old (38/51, 74.5%). Twenty-five of 51 (49%) cats with FIP were male while 26 (51%) were female. There was no gender prevalence. Anorexia (55.6~75%), fever (50~77.8%), and depression (33.3~60%) were the major clinical signs at presenting. Higher detection rate of coronavirus by RT-PCR was found in pleural effusion (9/9, 100%), ascites (17/18, 94.4%), and rectal swabs (66.7% in non-effusive FIP, 60% in effusive FIP). The clinicopathological changes include anemia, lymphopenia, hyperbilirubinemia, hypoalbuminemia, hyperproteinemia, elevation of ALT and AST, hyponatremia, and hypokalemia. Highly significant (p<0.01) decreasing of hematocrit (26.5±7.0% to 17.0±4.6%), elevation of total bilirubin (0.7±1.0 mg/dL to 2.5±1.3 mg/dL), and decreasing of albumin (2.5±0.3 g/dL to 2.2±0.4 g/dL) were noted in both effusive and non-effusive FIP at presenting and 0-3 days before death. Meanwhile, significant (p<0.05) decreasing of total protein (8.1±1.4 g/dL to 7.3±1.7 g/dL), elevation of ALT (61±69 U/L to 160±177 U/L) and AST(170±215 U/L to 562±348 U/L) were also observed. In the serial blood examinations in effusive group, variation of anemia (25.8±6.3% to 17.2±4.3%, p<0.01), increasing of total bilrubin (0.5±0.4 mg/dL to 2.8±1.2 mg/dL, p<0.01) and AST (103±63 U/L to 673±322 U/L, p<0.01) were observed between 2 weeks and 0 to 3 days before death. Total bilirubin (1.0±0.9 mg/dL to 2.8±1.2 mg/dL, p<0.01) and AST (141±48 U/L to 673±322 U/L, p<0.01) increased dramatically between 1 week and 0 to 3 days before death. The correlation coefficients were 0.45557 (P=0.0017) between TP and survival time after diagnosis and 0.42981 (P=0.0032) between globulin and survival time after diagnosis. Patients with higher TP and globulin at presenting could be survival longer. In conclusion, clinicopathological finding could provide an objective evaluation in the course of disease. The change of hematocrit, bilirubin and AST were more precise to forecast the course of FIP.

碩士
國立臺灣大學
獸醫學研究所
105
Abstract Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. So far, it remains incurable, and there is no effective vaccine commercially available. The compound diphyllin, a novel vacuolar ATPase blocker, has been shown to inhibit the intracellular endosomal acidification, thus interfering the infection of influenza virus and dengue virus. This study aims to investigate the antiviral activity of diphyllin against the type II feline infectious peritonitis virus (FIPV) strain NTU156. The results show that diphyllin dose-dependently inhibited endosomal acidification in Fcwf-4 cells. Treatment with diphyllin altered the cellular susceptibility to FIPV and inhibited the downstream virus replication. Furthermore, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting Galanthus nivalis agglutinin demonstrated enhanced cell protection. In the in vitro model of antibody-dependent enhancement of FIPV infection, diphyllin also showed a significant antiviral activity against FIPV in Fcwf-4 cells and U937 cells. In addition, nanoparticulate diphyllin further demonstrated a decreased cytotoxicity and improved inhibitory effect against FIPV. These results collectively suggest that diphyllin possesses therapeutic potential for the treatment of FIP. Keywords: Feline infectious peritonitis virus, Diphyllin, Vacuolar ATPase inhibitor, Antibody-dependent enhancement, Nanoparticles

碩士
國立臺灣大學
獸醫學研究所
97
Feline infectious peritonitis (FIP) is an immune-mediated disease manifesting clinically as effusive or non-effusive form. The causative agents, feline coronaviruses (FCoVs), are serologically classified into type I and II. In order to understand the differences in the distribution pattern of FCoV-infected cells within the lesions between effusive and non-effusive FIP, cats presented to the Veterinary Hospital at National Taiwan University with the final diagnosis of FIP were selected. Tissue samples from five typical non-effusive cases and eight typical effusive cases were subjected to immunohistochemistry staining. FCoV-infected cells were found in nearly all kinds of the examined organ samples. The FCoV-positive rates of kidney (80%) and brain (100%) in non-effusive FIP were higher than those of effusive FIP (37.5% and 40%, respectively). As comparing to effusive cases, a tendency that the lesions associated with FCoV were more abundant and severe in non-effusive ones was noted. To correlate different type of FCoVs with the development of FIP, in situ amplification of FCoV nucleic acid in affected tissues was attempted using type-specific primers via direct in situ RT-PCR. As the process of digestion might greatly influence the test results, optimization of the assay condition was first established. For F96-156, a FIP case that infected with type I and II FCoVs, incubation with 50 μg/mL of proteinase K at 37℃ for one hour in liver sections, and 2 mg/mL of pepsin at room temperature for four hours in lung sections were found to be optimal. Under these optimized conditions, type II FCoV-infected cells were detected in both liver and lung.

博士
國立臺灣大學
獸醫學研究所
102
Feline infectious peritonitis (FIP) is an immune mediated highly lethal disease in all domestic and non-domestic Felidae. The causative pathogen of FIP is feline coronavirus (FCoV), which belongs to the Alphacoronavirus in the family Coronaviridae. The infection of FCoV is distributed widely around the world, including Taiwan. Most of the infection is asymptomatic and only mild enteritis could be observed in some kittens. However, there are about 5-12% seropositive cats will develop FIP. Once the clinical signs appeared, diseased cats usually died within one month. Hence, FIP is one of the most important diseases in cats. As a disease with nearly 100% of mortality, no preventive vaccine and effective therapeutic agents are currently available for FIP. Several questions regarding the virus and its related disease remain to be answered. According to the outcome of infection, FCoV could be further categorized into two biotypes: feline enteric coronavirus (FECV), which leads to the enteric infection and is widely distributed in cat populations especially in multicat environment, and feline infectious peritonitis virus (FIPV), which induces the highly lethal FIP. In addition, these two biotypes of FCoV showed a difference in infection kinetics in vitro and in disease manifestation as well. FIPV infects the target cell, macrophages, more efficiently, spread to other susceptible cells faster, and sustained the infection better than FECV. Despite several virulence related genes, i.e., spike, membrane, 3c, and 7b, have been proposed, the actual role of the mutation of these genes is unclear. In this study, full length of accessory gene 3c was cloned and the effect of the expression of 3C protein on the FCoV harboring a truncated accessory gene 3c is assessed. The 3C protein is confirmed to play an inhibitory role in the viral replication, which may be associated with the low virulence of FECV, and this effect is independent to the autophagy pathway. Due to the lack of vaccine and therapy, the suggested clinical management of FIP is euthanasia that causes an enormous financial, emotional and ethical impact. To search the agents with potential therapeutic effect, the anti-FCoV activity of 16 commercially available compounds belongs to four categories, i.e., nucleoside analogue, protease inhibitor, reverse transcriptse inhibitor, and compounds with other activities, were examined. Among all the compounds tested, nelfinavir, a protease inhibitor, and Galanthus nivalis agglutinin, a mannose-binding lectin, were found to exhibit a significant anti-FCoV effect. In addition, the combination of these two compounds were demonstrated to complete inhibit the viral replication, synergistically. Despite the antiviral compounds with therapeutic potential were identified, as FIP cats presented in the clinics are usually at the late stage of the disease, the effect of therapy is often very limited. In order to prevent the occurrence of disease more efficiently, the identification of the disease-associated genetic polymorphism in cats was attempted. Expression of several cytokines was shown to be significantly altered in FIP cats when compared to the FCoV-infected non-FIP cats. Among the cytokines that have been studied, the transcription level and plasma concentration of IFN-γ is consistently decreased in FIP cats. Since the polymorphisms of IFN-γ gene in human have been demonstrated to be associated with various virus infections and immune-mediated diseases. In order to identify the possible genetic factor associated with the occurrence of FIP, a survey of feline IFN-γ gene were conducted in 63 FIP cats and 82 asymptomatic cats from our teaching hospital during the past six years. Sixteen single nucleotide polymorphisms were successfully identified in the intron 1, and three of them, i.e., polymorphisms at position +401, +408, and +428, were found significantly associated with the occurrence of FIP. In addition, polymorphism at position+428 also showed a clear correlation with the plasma concentration of IFN-γ in FIP cats. Since the disease-associated polymorphism could be used as a marker for the selection of FIP-resistant cats, three genotyping methods using amplification refractory mutation system polymerase chain reaction and restriction fragment length polymorphism for fIFNG+401, +408, and +428 were established in this study. These selection markers could be utilized for the further identification of FIP-resistant and -susceptible cats, and decrease the occurrence of FIP in cats populations.