Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

Im Graduiertenkolleg „Schlachttierbelastung und Produktsicherheit“ der Veterinärmedizinischen Fakultät der Universität Leipzig sollten in interdisziplinärer Zusammenarbeit Erkenntnisse zum Verhalten transportbelasteter Schlachtschweine in Bezug auf bakterielle Translokationsprozesse erarbeitet werden. Es wurden Mastschweine aus herkömmlichen Mastbetrieben definierten Belastungssituationen ausgesetzt , geschlachtet und untersucht. Dabei sollten physiologische, pathologisch-anatomische, immunologische, lebensmittel- und fleischhygienische, bakteriologische, virologische sowie ethologische Fragestellungen bearbeitet werden. Ziel war es festzustellen, ob eine Belastung der Tiere (u.a. durch den Transport) Auswirkungen auf die Produktqualität hat und ob durch eine Translokation pathogener Erreger ein Risiko für die Gesundheit des Verbrauchers besteht. Im Teilprojekt des Institutes für Virologie wurde untersucht, ob Rotavirus-Infektionen von Schlachtschweinen unter der Problematik der Belastung ein mögliches Infektionsrisiko für den Menschen darstellen. Um die zu erwartende niedrige Konzentration von Rotaviren in Organen von Schlachtschweinen nachweisen zu können, wurde eine kompetitive RT-PCR zum Nachweis von Rotaviren der Gruppe A verschiedener Spezies entwickelt. Dazu wurde ein sogenannter Kompetitor synthetisch hergestellt, welcher als interne oder externe Reaktionskontrolle eingesetzt wurde. Zum einen diente er der Überprüfung des ordnungsgemäßen Verlaufes einer RT-PCR, zum anderen wurde er zur Herstellung von Standards verwendet. Die RT-PCR wurde anschließend in eine „Real time“ RT-PCR umgewandelt. Sowohl mit der herkömmlichen als auch mit der „Real-time“ RT-PCR konnten 10 spezifische RNA-Moleküle in einer Probe nachgewiesen werden. In einer SPF-Schweineherde, welche einer Belastung infolge eines Tiertransports ausgesetzt war, konnten mit Hilfe der RT-PCR klinisch gesunde intermittierende Rotavirus-Ausscheider entdeckt werden. Bei einigen dieser Tiere gelang der Nachweis der Virusausscheidung über einen Zeitraum von drei Monaten. Nach der Schlachtung wurden in Organen des lymphatischen Systems bei zwei Schweinen sehr geringe Konzentrationen an rotavirus-spezifischer RNA detektiert. Infektiöses Virus konnte daraus allerdings nicht isoliert werden. Auch in einer Mastschweineherde konnte bei einigen Tieren Rotavirus-spezifische RNA im Kot nachgewiesen werden. Ein Infektionsversuch dieser Tiere mit Salmonella typhimurium konnte keine Reaktivierung der Rotavirus-Infektion auslösen. Aufgrund des Zoonose-Potentials von Rotaviren kann nach den Untersuchungen ein Infektionsrisiko für den Verbraucher durch eine endogene Kontamination von Schlachttieren mit Rotaviren nicht sicher ausgeschlossen werden. Die Untersuchungen zeigten auch, dass intermittierende Rotavirus-Ausscheider ein Infektionsrisiko für den Verbraucher darstellen, wenn z.B. bei der Schlachtung der Tiere oder bei der Verarbeitung des Fleisches dieser Tiere hygienische Grundregeln verletzt werden. Besonders gefährdet wären hierbei Neugeborene, Kinder, Senioren und immunsupprimierte Personen. Ein Ort einer Viruspersistenz in Organen konnte auch nach diesen Untersuchungen nicht gefunden werden. Dennoch scheint es, dass Rotaviren in der Natur oder in einer Population von Menschen oder Tieren persistieren. Durch ständige Neuinfektionen bzw. Reinfektionen empfänglicher Organismen haben Rotaviren so ihre Erhaltung gesichert. / Within the graduate programme “Schlachttierbelastung und Produktsicherheit” of the Veterinary Faculty of the University of Leipzig, the behaviour of slaughter swine exposed to the stress of transport was observed in an interdisciplinary collaboration concerning the translocation processes of bacteria. Fattened pigs from conventional pig fattening units were exposed to particular stress situations, and then slaughtered and examined. The following aspects of this process were investigated: physiology, pathological-anatomy, immunology, food and meat hygiene, bacteriology, virology and ethology. The aim of this study was to verify whether exposing the animals to stress situations (such as transport) influences the quality of the product and whether the translocation of pathogens represents a risk for consumer health. Within the sub-project of the Institute for Virology, analyses were made to verify whether rotavirus infections of slaughter swine exposed to stress situations represents a potential contamination risk for humans. In order to detect the expected low concentration of rotaviruses in the organs of slaughtered pigs, a competitive RT-PCR was developed as a test of rotaviruses for various group A species. To do this, a so-called competitor was synthetically created, which was used as an internal and external reaction control. On one hand, it was used to verify the regular course of an RT-PCR reaction, and on the other hand, it was implemented to develop standards. RT-PCR was then modified by means of a real time RT-PCR. Both with the conventional and with the real time RT-PCR, it was possible to detect 10 specific RNA molecules/ sample.With this new very sensitive and specific amplification process, it was possible to detect rotavirus-specific RNA in the excrement of people and of pigs, cows, horses, rabbits and monkeys. the evidence of the virus excretion was produced over a time period of three months. After slaughtering, low amounts of rotavirus specific RNA were found in the organs of the lymphatic system. There were no indications that any of these organs were infectious. Rotavirus specific RNA was also found in the excrement of some fattened pigs. An attempt to infect these animals with Salmonella typhimurium was unable to cause any reactivation of the rotavirus infection. An infection risk for the consumer through an endogenous rotavirus contamination of fattened pigs cannot be excluded with any degree of certainty on the basis of these analyses due to the zoonotic potential of rotaviruses. The analyses also showed that intermittent rotavirus excretors represent an infection risk for the consumer, if for example basic hygiene rules are broken during the slaughter or meat processing of these animals. At special risk may be new-borns, children, youth, the elderly and people suffering from immunodeficiency. These examinations could not find a specific place in the organs where the virus persists. Nevertheless, it seems that rotaviruses persist in the environment or in a population of people or animals. With constant new infections or re-infections of receptive organisms, rotaviruses seem to have assured their survival.

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

OLIVEIRA, S. B. Vírus sincicial respiratório humano em pneumonias adquiridas na comunidade em dois anos consecutivos de vigilância (2010 e 2011) em um hospital infantil de Fortaleza. 2014. 66 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2014. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-06-07T12:34:14Z No. of bitstreams: 1 Dissertação Sabrynna Brito Oliveira.pdf: 1150049 bytes, checksum: 97017c28c16ded6006029fc59fc82a08 (MD5) / Rejected by Erika Fernandes (erikaleitefernandes@gmail.com), reason: Corrigir nome do arquivo on 2017-06-07T14:00:45Z (GMT) / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-06-07T14:07:36Z No. of bitstreams: 1 2014_dis_sboliveira.pdf: 1069633 bytes, checksum: a05458c3bad0f74f614f7e949d8ef1e0 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-06-07T14:11:46Z (GMT) No. of bitstreams: 1 2014_dis_sboliveira.pdf: 1069633 bytes, checksum: a05458c3bad0f74f614f7e949d8ef1e0 (MD5) / Made available in DSpace on 2017-06-07T14:11:46Z (GMT). No. of bitstreams: 1 2014_dis_sboliveira.pdf: 1069633 bytes, checksum: a05458c3bad0f74f614f7e949d8ef1e0 (MD5) Previous issue date: 2014-01-17 / The Human Respiratory Syncytial Virus (HRSV) is the leading cause of acute respiratory infection, lower respiratory tract infections in children under five years old viral agent. The example of these infections can quote the Community Acquired Pneumonia (CAP) is the leading cause of death among infectious diseases in this age group. Correct identification of the causative agent of CAP is important for the institution of correct treatment, thus avoiding the misuse of antibiotics and preventing hospital infections. The objectives of this study were to assess the percentage of detection of HRSV in cases of CAP in the city of Fortaleza, Ceará - Brazil, in the years 2010 and 2011 through the immunofluorescence assay (IFA) and polymerase chain reaction preceded transcription reverse viral RNA (RT-PCR), and check for significant differences in the epidemiology and seasonality of HRSV compared the results between techniques. Of the 483 samples of CAP, 195 (40.37 %) were diagnosed in 2010 and 288 (59.63 %) in 2011. Of the total, 55 samples (11:38 %) were positive for HRSV by IFA technique and 97 (20.8%) by RT - PCR. A significant increase of 43.29 % in total samples positive for HRSV by RT-PCR when compared to the IFA technique (p = 0.0000). RT - PCR showed a sensitivity, specificity, agreement and positive and negative predictive value of 94.54 %, 89.48 %, 62.82 %, 53.60 % and 99.22 % respectively. It was observed that HRSV did not follow the same seasonal pattern when comparing the years 2010 and 2011. Positive cases 2011 occurred mainly in the months of March and April , three months before the 2010 positive cases , which occurred in the months of May, June , July and August (p = 0.0000). It was observed that the peak positive was correlated with the rainy season, but not necessarily all peaks of rainfall of the year in which the positive and even between both techniques showed peaks at different months. The occurrence of HRSV in Fortaleza showed a seasonal pattern associated with the rainy season for both techniques. The RT -PCR was more sensitive in detecting cases of CAP caused by HRSV compared to the IFI , enabling better identification of the viral agent and allowing a better characterization of the extent of the epidemic virus periods. / O Vírus Sincicial Respiratório Humano (VSRh) é o principal agente viral causador de infecções respiratórias agudas do trato respiratório inferior em crianças de até cinco anos de idade. A exemplo dessas infecções pode-se citar a Pneumonia Adquirida na Comunidade (PAC) que representa uma das principais causas de óbito entre as doenças infecciosas nessa faixa etária. Os objetivos desse estudo foram verificar o percentual de detecção de VSRh em casos de PAC na cidade de Fortaleza, Ceará – Brasil, nos anos de 2010 e 2011 através das técnicas de imunofluorescência indireta (IFI) e da reação em cadeia da polimerase precedida de transcrição reversa do RNA viral (RT-PCR), além de verificar se há diferença significativa na epidemiologia e sazonalidade do VSRh quando comparados os resultados entre as técnicas utilizadas. Das 483 amostras de PAC, 195 (40.37%) foram diagnosticadas em 2010 e 288 (59.63%) em 2011. Do total, 55 amostras (11.38%) foram positivas para VSRh pela técnica de IFI e 97 (20.08%) pela técnica de RT-PCR. Houve um aumento significativo de 43.29%no total de amostras positivas para VSRh por RT-PCR, quando comparado com a técnica de IFI (p=0,0000).A RT-PCR apresentou sensibilidade, especificidade, concordância e valor preditivo positivo e negativo de 94.54%, 89.48%, 62.82%, 53.60% e 99.22% respectivamente. Observou-se que VSRh não seguiu o mesmo padrão sazonal quando comparados os anos de 2010 e 2011. Os casos positivos de 2011 aconteceram principalmente nos meses de março e abril, três meses antes dos casos positivos de 2010, que ocorreram nos meses de maio, junho, julho e agosto (p=0,0000). Observou-se que o pico de positividade esteve correlacionado com o período chuvoso, mas não necessariamente com todos os picos de chuvas do ano correspondente e ainda que a positividade entre as duas técnicas apresentou picos em meses distintos.A ocorrência do VSRh na cidade de Fortaleza mostrou um padrão sazonal associado ao período chuvoso por ambas as técnicas. A técnica de RT-PCR mostrou-se mais sensível na detecção de casos de PAC por VSRh quando comparada à IFI, possibilitando uma melhor identificação do agente viral e permitindo uma melhor caracterização da extensão dos períodos epidêmicos do vírus.

No description available.

Samples of different varieties grapevine from Małopolska and Podkarpacie collected on spring 2016 were tested for Grapevine Pinot gris virus (GPGV) by RT-PCR. 16 out of 65 plants were found positive for GPGV. Two set of primers were used for detection, targeting partial movement protein and coat protein, and RdRp domain, both of those regions were sequenced. Phylogenetic analyses settled the Polish isolates to described before asymptotic group. Also GPGV positive samples were tested by multiplex and simplex RT- PCR for multiple infections, and 18,5% of all samples were GPGV and GFkV positive. This was first survey of GPGV in Poland.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Among sheep producers, the parasitic nematode Haemonchus contortus is a major animal health concern. Caribbean hair sheep are more resistant than conventional wool breeds to this blood-feeding, abomasal parasite. Our objective was to determine differences in the immune response associated with parasite-resistant hair and susceptible wool lambs infected with 10,000 H. contortus and in uninfected controls. Animals were sacrificed and abomasum and lymph node tissues were collected at 3 or 27 days post-infection (PI), and for controls on day 17, 27, or 38 relative to d 0 of infected animals. Blood and fecal samples were collected throughout the study. Lower fecal egg counts, higher packed cell volumes, and heavier lymph nodes of infected hair compared to wool lambs, suggests hair lambs have increased parasite resistance. Greater tissue infiltration of eosinophils (P < 0.05) was observed in hair compared to wool sheep by 3 days PI, with no breed differences in globule leukocytes. Total serum IgA and IgE were greater in control hair versus wool sheep (P < 0.05). After 3, 5, and 21 of infection, total serum IgA (P< 0.05), total lymph node IgE (P < 0.01), but not total serum IgE were greater in hair sheep compared to wool sheep. Gene expression was measured between hair and wool lambs for abomasal and lymph node tissues using bovine cDNA microarrays and real-time RT-PCR. Microarray analysis revealed cell survival, endosome function, gut motility, and anti-coagulation pathways are important in abomasal and lymph node tissues during H. contortus infection. Immune genes, including IL-4, IL-4 Ra, IL-12 Rb1, and IL-12 Rb2, are also highly represented in abomasal or lymph node tissue of infected animals. Eleven genes were evaluated using real-time RT-PCR and included TH1 and TH2 cytokines, cytokine receptors, and IgE. Parasite infection leads to increased expression of IL-13 and IgE in both tissues and breeds when compared to control animals. Breed comparison of gene expression shows resistant hair sheep produce a stronger modified TH2-type immune response during infection. Differential cell infiltration, antibody production, and regulation of TH2 cytokines between breeds may be partially responsible for differences in parasite resistance. / Ph. D.

Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries, and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. It has been speculated that HEV replicates in sites other than the liver. Since HEV is presumably fecal-orally transmitted it is unclear how the virus reaches the liver and extrahepatic replication could be a possible explanation. The recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication in a swine model. We experimentally infected specific-pathogen-free (SPF) pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were each inoculated intravenously with swine HEV, nineteen pigs (group 2) with the US-2 strain of human HEV, and seventeen pigs (group 3) as uninoculated controls. To identify the potential extrahepatic sites of HEV replication using the swine model, two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days post inoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive strand HEV RNA in each tissue collected during necropsy at different DPIs. A negative strand-specific RT-PCR was standardized and used to detect the replicative, negative-strand of HEV RNA from tissues that tested positive for the positive strand RNA. As expected, positive strand HEV RNA was detected in almost every type of tissue at some time point during viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine and human HEV inoculated pigs. However, replicative, negative strand of HEV RNA was detected primarily in the small intestine, lymph nodes, colon, and liver. Our results demonstrate for the first time that HEV replicates in tissues other than the liver and that the gastrointestinal tract is also the target of virus infection. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV. / Master of Science

O objetivo deste trabalho foi caracterizar astrovírus em amostras fecais coletadas de crianças com e sem diarréia, em São Paulo, Brasil, e divididas em dois grupos, EPM e HU, de acordo com a origem. A detecção foi realizada utilizando-se RT-PCR, com primers específicos. Os resultados para as amostras EPM mostram que 66/234 (28,2%) foram positivas para astrovírus. Para as amostras HU, 18/187 (9,6%) foram positivas. A genotipagem foi realizada com a técnica de nested/RT-PCR. De 66 amostras positivas (EPM), 19 (28,7%) foram caracterizadas como HAstV-1, 4 (6,0%) como HAstV-2, 2 (3,0%) como HAstV-3, 1 (1,5%) como HAstV-5 e 3 (4,5%) como HAstV-8. Das 18 positivas do HU, 1 (5,5%) amostra foi caracterizada como HAstV-1, 7 (38,8%) como HAstV-2 e 1 (5,5%) como HAstV-8. As amostras genotipadas em ambos os grupos foram submetidas ao seqüenciamento de nucleotídeos para confirmação dos resultados. Detecção e genotipagem de astrovírus em casos de diarréias pediátricas são técnicas são importantes e descrevem como esse vírus está circulando em São Paulo, Brasil. / The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo city, Brazil, and grouped into two distinct groups, EPM and HU. Detection was carried out using RT-PCR with specific primers. Results for EPM set showed that 66/234 (28,2%) were positive. In the HU set of samples, 18/187 (9,6%) were positive for astrovirus. Genotyping was carried out with nested/RT-PCR. Out of 66 astrovirus positive EPM samples, 19 (28,7%) were characterized as HAstV-1, 4 (6,0%) as HAstV-2, 2 (3,0%) as HAstV-3, 1 (1,5%) as HAstV-5 and 3 (4,5%) as HAstV-8. Among 18 astrovirus positive HU samples, 1 (5,5%) was characterized as HAstV-1, 7 (38,8%) as HAstV-2 and 1 (5,5%) as HAstV-8. Genotyped samples were confirmed by nucleotide sequencing. Detection and genotyping of astrovirus in pediatric diarrhea are important and describes how this virus is circulating in São Paulo, Brazil.