Made available in DSpace on 2014-07-29T15:13:40Z (GMT). No. of bitstreams: 1 TESE Bruno Medrado.pdf: 1863730 bytes, checksum: 142fb795d040f53ab90b6225b9c56530 (MD5) Previous issue date: 2010-04-10 / The main economic activity of the state of Tocantins, in Brazilian Amazon is cattle farms, with extensive breeding. Looking for contribution to cattle sanity, this study was devoted to the prevalence of antibodies against Leptospira in livestock of those farms, as production animals (cattle and equids), dogs and animal workers, from a rural governmental settlement in Aragominas, in the northwestern part of Tocantins. The statistically proofed sample was composed by 242 cows, 78 equids, 59 dogs and 41 animals workers, distributed in 38 small properties. All sampling was performed after informed consent, written in the case of human beings. For the diagnosis of leptospirosis, microscopic seroagglutination was performed in the Laboratório de Diagnóstico de Leptospirose do Setor de Medicina Veterinária Preventiva da Escola de Medicina Veterinária da UFG, Goiânia-GO. The seroprevalence for Leptospira spp in cattle was 76,5% [70,7% 81,7%], with serovar predominance of Hardjo (26,2%), followed by Wolffi (23,4%) Hebdomadis (14,1%), Castellonis (11,7%), Grippotyphosa (9,1%) e Pyrogenes (4,8%). In equids the seroprevalence was 79,3% [68,9% 87,4%], with agglutinins more intense to wild life serovars Castellonis (24,4%), Grippotyphosa (13,7%), Patoc (13,1%), Butembo (8,9%), Pomona (7,1%), Hardjo (6,6%), Pyrogenes (6,6%) e Wolffi (6,6%). Dogs presented seroprevalence of 30,5% [19,2 43,9], prevailing Canicola (26,3%), Hardjo (13,3%), Bratislava (10,0%) and Pyrogenes (10,0%). Human leptospirosis seroprevalence in animal workers was 31,7% [18,1%-48,1%], cwith detections of serovars Hardjo (26,3%), Grippotyphosa (15,8%), Pyrogenes (10,5%), Wolffi (10,5%), Autumnalis (10,5%) e Bratislava (10,5%). Looking for association with environmental and breeding conditions, the seroprevalence was associated in cattle to Bos indicus cattle (OR=7,51; [0,99-56,97]), in equids to the use of antihelminths (OR=7,64[0,95 61,50]) and for dogs with use for shepherd cattle (OR=4,44[1,35 14,58]). These data point to endemicity of Leptospira infection in the area and are highly suggestive of extensive environmental contamination with wildlife and production animal serovars. These results also emphasize the importance of the control of livestock leptospirosis, lowering environmental contamination and allowing better animal sanitation, with measures that could be implemented in new adequate settlements. / A exploração pecuária bovina constitui-se em uma atividade fundamental para o Estado do Tocantins, que tem sua economia pautada no agronegócio. Visando contribuir para a sanidade bovina na região, objetivou-se neste estudo determinar a prevalência de anticorpos anti-Leptospira spp. em animais de interesse econômico (bovinos e equinos), em cães e em humanos que tinham contato direto com animais, oriundos de assentamento rural do município de Aragominas, Tocantins, Brasil. A amostragem estatisticamente representativa foi constituída por 242 bovinos, 78 equídeos, 59 cães e 41 humanos, distribuídos em 38 propriedades. As colheitas de sangue dos animais e a aplicação de questionários foram realizadas após o aceite dos proprietários e, no caso dos humanos, após a leitura e assinatura do termo de consentimento livre e esclarecido. Para diagnóstico da leptospirose foi empregada a técnica de soroaglutinação microscópica (SAM) realizada no Laboratório de Diagnóstico de Leptospirose do Setor de Medicina Veterinária Preventiva da Escola de Medicina Veterinária da UFG, em Goiânia-GO. A prevalência de infecção por Leptospira spp. em bovinos foi de 76,5% [70,7% 81,7%], com predominância de anticorpos aos sorovares Hardjo (26,2%), seguido do Wolffi (23,4%), Hebdomadis (14,1%), Castellonis (11,7%), Grippotyphosa (9,1%) e Pyrogenes (4,8%); em equídeos foi de 79,3% [68,9% 87,4%], com maior detecção de aglutininas para os sorovares Castellonis (24,4%), Grippotyphosa (13,7%), Patoc (13,1%), Butembo (8,9%), Pomona (7,1%), Hardjo (6,6%), Pyrogenes (6,6%) e Wolffi (6,6%). Já em cães, foi detectada soroprevalência de 30,5% [19,2 43,9], com maiores respostas aos sorovares Canicola (26,3%), seguido Hardjo (13,3%), Bratislava (10,0%) e Pyrogenes (10,0%) e em humanos constataram-se 31,7% [18,1%-48,1%] de reagentes, com detecção de anticorpos para os sorovares Hardjo (26,3%), Grippotyphosa (15,8%), Pyrogenes (10,5%), Wolffi (10,5%), Autumnalis (10,5%) e Bratislava (10,5%). Dentre os fatores avaliados, a prevalência mostrou-se associada na espécie bovina à raça zebu (OR=7,51; [0,99-56,97]), nos equídeos ao uso de vermífugo (OR=7,64[0,95 61,50]) e para cães a lida com gado (OR=4,44[1,35 14,58]). Os resultados encontrados apontam para uma situação de endemicidade e são sugestivos de alta contaminação ambiental por sorovares que possuem como hospedeiro natural animais de produção e silvestres; evidenciando a necessidade de controle da infecção animal, com a finalidade de diminuir a contaminação ambiental e a consequente infecção em seres humanos.

O objetivo foi avaliar a vulnerabilidade do HIV de detentas da P.F.B.. Utilizou-se técnicas sorológicas e TAT. 290 detentas entrevistadas com questionário sobre comportamentos e hábitos destas mulheres e parceiros sobre: promiscuidade sexual; UDI; DST. Avaliou-se com detentas: mitos, crenças, idade, escolaridade e conhecimento HIV. A soroprevalência para HIV foi obtida e relacionada com os comportamentos de risco por análise univariada, com a utilização de tabelas de contingência entre variáveis de risco e resultado de sorologias (HIV, HVC e sífilis para as detentas) para obtenção de Odds Ratio e com a utilização de testes estatísticos de qui quadrado e exato de Fisher para um nível de significância de 5%. A positividade HIV-13,85%; anti-pallidum -22,76% ; anti-HCV -16,21%. 46 detentas fizeram o TAT, interpretados num único TAT-perfil típico destas mulheres / In order to evaluate the vulnerability by HIV, 290 female prisoners of F.B.P. were interviewed and bled for serological analysis and submited to TAT techniques. The questions were related to behavior and habits of those women and their partner, according to: promiscuous sexual relationships, use of injectec ilegal drugs and sexually transmitted diseases. We also analysed the prisoners myths, beliefs, age, school degree and general knowledge of HIV. The seroprevalence of HIV was associated to risky behaviors by means of univariate, Qui-Squared or Exact of Fisher tests. We found 13.85% of seropositives to HIV, 22.76% to anti-pallidum and 16.21% to anti-HCV. 46 prisoners were submitted to TAT, whose life stories were elucidated through one summarised, typic profile of these women

A infecção pelo herpesvírus humano tipo 8 (HHV-8) tem sido associada ao sarcoma de Kaposi (SK) iatrogênico, que acomete pacientes imunossuprimidos e/ou transplantados renais. Em populações consideradas saudáveis, a soroprevalência para o HHV-8 varia de 1% a 8%. O presente trabalho buscou: determinar a prevalência e os títulos de anticorpos anti-HHV-8 em pacientes com insuficiência renal crônica (IRC), submetidos ou não à terapia renal substitutiva (TRS) do Hospital do Rim e Hipertensão e Casa da Diálise da UNIFESP e da Santa Casa de Misericórdia de São Paulo e, comparar os resultados obtidos com outras populações da mesma região geográfica, porém de outras categorias de risco para adquirir doenças infecciosas. Soros de 805 pacientes: 295 em hemodiálise, 54 em diálise peritoneal e 456 em acompanhamento ambulatorial, sem TRS, foram testados quanto à presença de anticorpos anti-HHV-8, de fase latente e lítica da replicação viral, por meio de técnicas de imunofluorescência indireta (IFI) LANA e Lítico, padronizadas na Seção de Imunologia do Instituto Adolfo Lutz. Os resultados obtidos foram analisados em relação a dados clínicos, epidemiológicos e laboratoriais usando o teste do qui-quadrado ou exato de Fisher para as variáveis categóricas e os testes de Mann Whitney ou Kruskal Wallis para as variáveis contínuas. Foi encontrada soropositividade ao HHV-8 em 18,0% dos pacientes com IRC, dos quais 18,3% nos pacientes em TRS e 17,7% nos pacientes sem TRS, não havendo diferença significante entre os grupos. As variáveis que estiveram relacionadas à sorologia positiva ao HHV-8 foram: transplante prévio (p<0,001) e doenças sexualmente transmissíveis (p=0,003), com destaque para a sífilis (p=0,021). As demais variáveis não mostraram associação estatística embora tenha havido maior número de amostras HHV-8 soropositivas com o avançar da idade. Em relação ao tipo e ao título de anticorpos detectados, houve mais amostras com sorologia positiva para anticorpos Lítico e maiores títulos de anticorpos LANA. A comparação dos resultados dos pacientes com IRC e outras populações de São Paulo revelou taxa semelhante de prevalência de anticorpos anti-HHV-8 na população com HIV/Aids (20,4%), considerada de alto risco para esta infecção viral. Por outro lado, a prevalência detectada na população com IRC (18,0%) foi inferior às obtidas em pacientes com SK epidêmico (89,3%), SK clássico (100,0%) e SK endêmico (87,5%), e superior a outras populações sem SK: pacientes com deficiência mental e/ou física (1,6%) e profissionais da área da saúde (1,1%). Em todos os grupos analisados houve maior número de amostras com sorologia positiva para HHV-8 de fase lítica, e maiores títulos de anticorpos LANA, exceção feita aos profissionais da área da saúde. Maiores títulos de anticorpos LANA foram detectados nos pacientes com SK. Não foram encontradas outras associações significantes. Os resultados obtidos permitem concluir que os pacientes com IRC têm alta prevalência de anticorpos anti-HHV-8, comparável aos indivíduos com HIV/Aids dessa região geográfica. Ainda, sugerem que se devam acompanhar os pacientes HHV-8 soropositivos com vistas a monitorar os títulos de anticorpos LANA e verificar se estes têm valor prognóstico. Caso isto venha a ser confirmado, sugere-se a introdução da sorologia para o HHV-8 na bateria de exames do pré-transplante renal. / Human herpesvirus 8 (HHV-8) infection is frequently associated with Kaposi\'s sarcoma (KS) in immunodeficient and renal transplanted patients. The HHV-8 seroprevalence in healthy populations varies from 1% to 8%. The present study aimed to determine the HHV-8 seroprevalence and antibodies titers in chronic kidney disease (CKD) patients with or without substitutive kidney therapy (SKT) attended at Hospital do Rim e Hipertensão and Casa da Diálise of UNIFESP, and at Santa Casa de Misericórdia de São Paulo. Secondarly, to compare the serological results with those obtained from populations of the same geographic region, presenting other risk factors for acquiring infectious diseases. Serum samples were collected from 805 CKD patients: 295 under hemodialysis, 54 under peritoneal dialysis, and 456 in ambulatorial assistance without SKT. Latent and Lytic HHV-8 antibodies were searched using indirect immunofluorescence assays that were standardized at Immunology Department of Instituto Adolfo Lutz. Chi-Square test and/or Fisher\'s exact test were performed for comparing categorical variables including epidemiological, clinical and laboratorial data, and HHV-8 serum status. Continuos variables associated with HHV-8 antibodies titers were compared using Mann Whitney or Kruskal Wallis tests. An overall HHV-8-seropositivity of 18.0% was detected in CKD patients: 18.3% in patients under SKT and 17.7% in patients without SKT. Since no difference was detected in HHV-8-seropositivity among patients, they were considered as a unique group for subsequent analysis. A strong association between HHV-8-seropositivity and previous transplant was detected (p<0.001), along with an association with others sexually transmitted diseases (p=0.003), with emphasis for syphilis (p=0.021). In addition, no other data was associated with HHV-8-seropositivity, although higher proportions of HHV-8-seropositivity were detected in samples from elderly persons. In addition, more HHV-8 Lytic antibodies positive samples, and higher titers of LANA antibodies were detected. HHV-8 seroprevalence obtained from CKD patients was similar to the HHV-8 prevalence detected among HIV/Aids patients (20.4%), who were considered a high-risk group for this viral infection. On the other hand, the HHV-8 seroprevalence of CKD patients (18.0%) was lower than the prevalence of patients with epidemic KS (89.3%), classic KS (100.0%) and endemic KS (87.5%), and higher than the patients with mental and/or physical deficiency (1.6%) and health professionals (1.1%). All analyzed groups had more HHV-8-seropositive samples for Lytic antibodies and higher titers of LANA antibodies, with exception for the health professionals. The highest LANA antibodies titers were found among KS patients groups. No other association was found. In conclusion, the obtained results points out CKD patients as a high prevalent population for HHV-8 infection, similar to HIV/Aids patients from the same geographic area. As far, it suggests that HHV-8 seropositive CKD patients should be followed up in order to verify whether LANA antibodies titers have prognostic value. In confirming this hypothesis, it may propose to include the use of HHV-8 serology in the screening testing in kidney pre-transplant.

Einleitung: Eimeria (E.) spp. und Toxoplasma (T.) gondii sind intrazelluläre Protozoen aus dem Phylum Apicomplexa und stellen bedeutende Pathogene dar. Infektionen mit Eimeria spp. sind Auslöser der Kokzidiose, welche eine der ökonomisch bedeutsamsten Erkrankungen in der Geflügelproduktion darstellt. Die Toxoplasmose des Huhnes hingegen verläuft in der Regel subklinisch. Da beide Parasiten, Eimeria spp. und Toxoplasma gondii, weltweit verbreitet sind, können natürliche Koinfektionen auftreten. Prävalenzstudien mit Hilfe molekularer und serologischer Untersuchungsmethoden sind geeignet, um die Vorkommen, Verbreitung, und Speziesidentität von einzelnen Apicomplexa zu untersuchen, was zu besserer Diagnose, Kontrolle, und Bestandsüberwachung beiträgt. Trotz vorliegender Hinweise, dass bei Koinfektionen zu einer Interaktion zwischen Eimeria und Toxoplasma kommt, existieren Erkenntnislücken hinsichtlich der Häufigkeit und der Bedeutung von natürlichen Koinfektionen des Huhnes unter Feldbedingungen, insbesondere mit Augenmerk auf verschiedene Nutzungstypen, Produktions- und Haltungsbedingungen. Ziele der Untersuchungen: Die Ziele der Untersuchungen waren die Erhebung der Prävalenzen von verschiedenen Eimeria spp. und von T. gondii bei Hühnern, sowie die Häufigkeit ihres Auftretens als Einzel- und Koinfektionen unter Feldbedingungen unter Berücksichtigung verschiedener Nutzungstypen und Haltungsbedingungen in Griechenland. Tiere, Material und Methoden: Die Auswahl der für diese Feldstudie untersuchten Hühnerbestände erfolgte auf Basis der Anzahl von kommerziellen Hühnerhaltungen in drei Hauptregionen Griechenlands. Nach offiziellen Angaben des griechischen Ministeriums für Ländliche Entwickung und Lebensmittel konzentriert sich die griechische Geflügelindustrie hauptsächlich auf Epirus (im Nordwesten), Zentralmazedonien, und Zentralgriechenland. Die Probenentnahme erfolgte in kommerziellen und Hinterhofhaltungen, wobei die Probenanzahl proportional zur Anzahl entsprechender Haltungsformen in der jeweiligen Region gewählt wurde (n = 50). Es wurden Legehennen (n = 21), langsam wachsende Broiler (n = 15) und Hinterhofhühner beprobt (n = 14), welche verschiedenen Produktions- und Haltungsbedingungen zugeordnet waren (Haltung in ausgestalteten Käfigen, Bodenhaltung mit Einstreu und Biohaltung). Konventionelle intensive Broileraufzuchten wurden nicht berücksichtigt. Für die Erhebung der Häufigkeiten von Eimeria spp.-Infektionen wurden aus allen ausgewählten Beständen Einstreuproben gesammelt (n = 756). Die Broilerherden wurden zu einem Zeitpunkt bei der Schlachtung erneut beprobt, indem die Därme der Einzeltiere gesammelt wurden (n = 162). Die Einstreu-/Darminhaltsproben wurden quantitativ mittels McMaster-Verfahrens auf den Eimeria spp.-Oozystengehalt (als Oozysten pro Gramm Kot, OPG) untersucht. Die Eimeria-Oozysten aus positiven Proben wurden für weitere molekulare Analysen aufgereinigt und aufbewahrt. Die Eimeria-Speziesbestimmung erfolgte durch spezies-spezifische DNA-Nachweise, und die Quantifizierung der einzelnen Arten erfolgte semiquantitativ. Um die Häufigkeit von T. gondii-Infektionen zu bestimmen, wurden Blutproben (n = 1,021) von Einzeltieren zur serologischen Untersuchung mittels TgSAG1 ELISA entnommen, zeitgleich zur o. g. Beprobung der Bestände für die Einstreuproben. Für Hinterhofhaltungen mit Tieren variablen Alters wurden ältere Tiere bevorzugt beprobt, und in kommerziellen Legehennenhaltungen wurden die Tiere im Alter von ca. 10 Monaten beprobt. In beiden Fällen wurden von T. gondii-seropositiven Tieren bei der Schlachtung die Herzen gewonnen (n = 322). In Broilerhaltungen wurden ebenfalls Blutproben und das Herz während des Schlachtungsprozesses zum T. gondii-Nachweis entnommen. Das Herzgewebe wurde in einer magnetic capture Polymerasekettenreaktion (mc-PCR) sowie einem Mausbioassay auf Präsenz und Genotyp einzelner Isolate untersucht. Parallel zu den Laboruntersuchungen wurden Daten zu Haltungsbedingungen, Biosicherheitsmaβnahmen, Lage, Bestandsgröβe, Krankheitsgeschichte etc. mittels standardisierter Fragebögen erfasst, um potenzielle Risikofaktoren für Eimerien- und/oder T. gondii-Infektionen zu bestimmen. Die Datenanalyse erfolgte durch Multilevel-Modelling (generalized linear mixed modelling fit by maximum likelihood (Laplace approximation)) mittels dem R-Programm (https://www.r-project.org, Version 4.0.2, Paket lme4), dem Kruskal-Wallis-Test und bivariaten Spearman-Korrelationen im PSPP-Prgramm (GNU PSPP 1.3.0). Ergebnisse: Insgesamt lag die Nachweisrate für Eimeria spp.-Infektionen bei 85,7 %. Alle sieben Hühnereimerienspezies wurden identifiziert, wobei E. acervulina (79,3 %) und E. tenella (65,5 %) die höchste Prävalenz aufwiesen. Infektionen mit mehreren Eimeria-Arten (79,3 %) waren deutlich häufiger anzutreffen als Einzelinfektionen (20,7 %). Als Risikofaktoren wurden Herdengröβe, Art des Auslaufs und Produktionssystem identifiziert. Zwischen respiratorischen Erkrankungen und mittlerer OPG wurde in Broilerhaltungen eine sehr starke Korrelation beobachtet (P < 0.001). Biohaltungen zeigten eine höhere Prävalenz von E. tenella (P = 0,023). Nutzung einer bewachsenen Auslauffläche war stark mit der Präsenz von E. brunetti korreliert (P < 0,001). Die T. gondii-Seroprävalenz über alle untersuchten Tiere betrug 9,5 %. Dabei testeten 41,2 % aller Hinterhofhühner seropositive. In 70 % der Bio- und Freilaufhaltungen wurde mindestens ein Tier seropositiv getestet. Es wurden keine T. gondii-seropositiven Broiler gefunden, obwohl mit Hilfe der mc-PCR positive DNA-Nachweise erfolgten. Dies belegt die hohe Sensitivität der mc-PCR und ihre potenzielle Eignung für die Detektion früher Infektionen bei Hühnern. Die T. gondii-Isolate, welche im Mausbioassay gewonnen wurden, wurden als Typ II (ToxoDB#3) genotypisiert, was durch Mikrosatellitenanalyse bestätigt wurde. Für T. gondii-Infektionen wurden Produktionssystem und Futterautomatisierung als Risikofaktoren identifiziert, wobei Auslaufbeweidung die Wahrscheinlichkeit für T. gondii-Infektionen erhöht. Die Gegenwart von Katzen stellte hingegen keinen nachweisbaren Risikofaktor für T. gondii-Seropositivität auf Bestands- oder Einzeltierniveau dar. Koinfektionen mit beiden Protozoen wurden in 87% aller untersuchten Hühnerbestände nachgewiesen, wobei Hinterhof-, Bio-, und Freilandhaltungen am häufigsten betroffen waren. In den moisten Fällen von Koinfektionen wurde E. acervulina nachgewiesen. Schlussfolgerungen: Die Prävalenz sowohl von Eimeria spp. als auch von T. gondii war generell hoch und auf einem vergleichbaren Niveau mit den Ergebnissen früherer Studien in anderen Ländern. Faktoren wie Produktionssystem, Haltungs- und Managementbedingungen sind mit dem Risiko von Mono- oder Koinfektionen verbunden. Die gewonnenen Erkenntisse erlauben die gezielte Planung zukünftiger Studien hinsichtlich sich ändernder Haltungsbedingungen, z. B. dem Trend zur verstärkten Bio- und tierfreundlichen Hühnerhaltung uns dem Einsatz langsam wachsender Rassen. Es besteht auch in Griechenland ein Bedarf an nachhaltiger Kontrolle von Kokzidieninfektionen, einschlieβlich der Minimierung zoonotischer Erreger wie T. gondii in Nutztierbeständen. Die verwendeten serologischen und molekularen Methoden können nach den Studienerkenntnisse ausserdem zur frühzeitigen Überwachung von T. gondii–Infektionen in Hühnerbeständen beitragen.:Chapter 1 - Introduction Chapter 2 - Literature Review 2.1 Eimeria spp. 2.1.1 Life cycle 2.1.2 Diagnosis and control of Coccidiosis in chickens 2.1.3 Economic Impact of Coccidiosis 2.1.4 Prevalence of Eimeria spp. in chicken farms 2.1.5 Data of chicken coccidiosis from Greece 2.2 Toxoplasma gondii 2.2.1 Life cycle 2.2.2 Toxoplasmosis and Zoonotic Importance 2.2.3 Prevalence in poultry and risk factors 2.2.4 T. gondii data from Greece 2.3 Co-existence of Eimeria spp. and Toxoplasma gondii in chickens Chapter 3 - Overview of own scientific work 3.1 Aims 3.2 Presentation of own scientific work: Publications 3.2.1 Publication 1: Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors. 3.2.2 Publication 2: Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp. Chapter 4 - Overreaching Discussion Chapter 5 - Conclusions Chapter 6 - Summary Chapter 7 - Zusammenfassung Chapter 8 - References Acknowledgements / Introduction: Eimeria spp. and Toxoplasma gondii are intracellular Apicomplexan protozoa and represent important pathogens for chickens. Coccidiosis, caused by Eimeria spp. is one of the most notable diseases in chickens having a high economic impact on the poultry industry worldwide, while toxoplasmosis is usually subclinical in these hosts. As both Eimeria spp and Toxoplasma gondii have a broad worldwide distribution, natural co-infections in chickens can occur. Prevalence studies using molecular and serological techniques have proven a very useful approach to study the diversity and distribution of these parasites’ mono-infections, further contributing to more efficient diagnosis, control and monitoring. Despite existing indications that these two parasites interact when the host is infected simultaneously, there are still knowledge gaps regarding the frequency and impact of naturally occurring co-infections under field conditions, particularly in different types of poultry, production and housing systems. Objective: Determination of the level of occurrence of Eimeria spp. and Toxoplasma gondii mono- and co-infections under field conditions, in different types of chickens and farm profiles in Greece. Animals, materials and methods: Selection of poultry operations was based on the number of commercial farms in three major Greek regions, as poultry farms in Greece are highly concentrated in Epirus (in North-Western Greece), Central Macedonia, and Central Greece, based on the data from the Hellenic Ministry of Rural Development and Food. Sampling from both commercial operations and backyard farms was conducted proportionately to their frequency (n=50) and type of flocks included in the study were layers (n=21), slow-growing broilers (n=15) and backyard chickens (n=14), under different production and housing systems (enriched cages, floor-housed in litter, free-range and organic systems). Conventional intensively reared broilers were not included in the study. To record Eimeria spp. occurrence, faecal samples were collected from the litter of the chicken housing (n=756). Broiler flocks were followed up to the slaughterhouse for a second sampling where the whole gut was collected (n=162). Samples were quantitatively examined by a modified McMaster technique to calculate oocysts per gram (OPG) of faeces, followed by collection and purification of the oocysts for further molecular analysis. Species identification was performed by multiple PCR assays using species-specific primers and PCR bands were categorized by intensity semiquantitavely. To record Toxoplasma gondii infections, simultaneously to faecal sampling, blood samples (n=1,021) were also collected from individual animals for serological T. gondii detection via TgSAG1 ELISA. In our sampled broiler flocks, animals were sampled at slaughter, where blood samples and the heart were collected for T. gondii detection. In backyard flocks, blood samples were taken from the older animals and in layers from individual animals (at the age of approximately 10 months). Toxoplasma positive animals were followed up to slaughter to collect heart tissue (n=322), further processed for bioassay in KO mice and mc-PCR in order to characterize Toxoplasma gondii isolates. In parallel, potential risk factors and impact regarding mono- and co-infection of both parasites were investigated through an obtained questionnaire containing additional information about farm management, biosecurity status, location, production rate and diseases history. For the data analysis a multilevel-modelling (generalized linear mixed modelling fit by maximum likelihood (Laplace approximation)) was performed using R (https://www.r-project.org) version 4.0.2, by applying the package lme4, as well as Kruskal-Wallis tests and bivariate correlations in PSPP statistical program (GNU PSPP 1.3.0). Results: Overall Eimeria spp. positivity level was 85.7%. All seven Eimeria species were identified with E. acervulina (79.3%) and E. tenella (65.5%) being the most prevalent ones. Mixed infections (79.3%) were more common than single-species (20.7%) Significant identified risk factors were flock size, type of outdoor area, and production system. A very strong correlation (p < 0.001) was found between the presence of respiratory disease and the average OPG level in broiler farms. Organic flocks showed higher prevalence of E. tenella (p = 0.023), while presence of vegetation at the outdoor area correlated strongly with E. brunetti (p < 0.001). Toxoplasma gondii seroprevalence was 9.5%. 41.2% of the backyard chickens sampled were seropositive and 70% of the organic and free-range layer farms had at least one Toxoplasma gondii seropositive hen. No serologically positive broilers were found, however mc-PCR revealed positive samples, stressing out the high sensitivity and potential contribution of this method in early detection of the parasite. Toxoplasma gondii isolates obtained by mouse bioassay were genotyped and found to belong to type II (ToxoDB#3) as confirmed also by microsatellite typing. The most significant risk factors were production system and feeding system automation, with free-grazing practices increasing the likelihood for Toxoplasma infections. Presence of cats showed no association with Toxoplasma gondii seropositivity on a farm and individual animal level. The two protozoan parasites were found to co-exist in 87% of the studied poultry operations, with backyard, organic and free-range farms showing the highest occurrence. E. acervulina was the species identified in most of the co-existence cases. Conclusions: Prevalence of both Eimeria spp. and Toxoplasma gondii is overall high and comparable with findings from similar studies in other countries. Production system, husbandry and management conditions relate to increased risk of both mono- and co-infections, giving useful insights and indications for future studies, particularly in the light of increasing application of slow-growing, organic and “higher welfare” poultry farming practices. It could be shown that also in Greece there is a need for sustainable coccidiosis control, both in terms of Eimeria spp. infection control and of a minimization of T. gondii introduction to operations. A combination of the serological and molecular methods used in this study can contribute to earlier and more accurate diagnosis of Toxoplasma gondii infections in chickens, which is a crucial and sensitive subject for safeguarding transmission to humans.:Chapter 1 - Introduction Chapter 2 - Literature Review 2.1 Eimeria spp. 2.1.1 Life cycle 2.1.2 Diagnosis and control of Coccidiosis in chickens 2.1.3 Economic Impact of Coccidiosis 2.1.4 Prevalence of Eimeria spp. in chicken farms 2.1.5 Data of chicken coccidiosis from Greece 2.2 Toxoplasma gondii 2.2.1 Life cycle 2.2.2 Toxoplasmosis and Zoonotic Importance 2.2.3 Prevalence in poultry and risk factors 2.2.4 T. gondii data from Greece 2.3 Co-existence of Eimeria spp. and Toxoplasma gondii in chickens Chapter 3 - Overview of own scientific work 3.1 Aims 3.2 Presentation of own scientific work: Publications 3.2.1 Publication 1: Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors. 3.2.2 Publication 2: Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp. Chapter 4 - Overreaching Discussion Chapter 5 - Conclusions Chapter 6 - Summary Chapter 7 - Zusammenfassung Chapter 8 - References Acknowledgements

Les arboviroses représentent un fardeau sanitaire considérable et croissant à l’échelle mondiale. La complexité des facteurs biotiques et abiotiques qui interviennent dans la transmission de ces arbovirus pose un défi de taille aux scientifiques qui tentent de comprendre, de modéliser ou d’anticiper leur transmission ainsi qu’aux intervenants de santé publique qui ont la responsabilité de surveiller, d’évaluer et gérer le risque que posent les arbovirus pour la santé des populations. Cette étude visait à estimer et caractériser le risque d’exposition à plusieurs arbovirus suspectés d’être actifs et émergents au Québec mais dont la distribution avait peu ou n’avait pas été étudiée au Québec : le virus du Nil occidental (VNO), le virus de l’encéphalite équine de l’est (VEEE) et deux virus du sérogroupe de la Californie (VSGC), le virus de Jamestown Canyon (VJC) et le virus du lièvre d’Amérique (VLA). Basée notamment sur l’hypothèse selon laquelle les animaux d’espèces différentes qui partagent un environnement commun partagent également un risque environnemental commun, cette étude visait également à évaluer si les populations d’animaux de compagnie pouvaient aider à estimer et caractériser le risque d’infection arbovirale chez l’humain. L’échantillonnage sérologique de populations humaines, canines et équines du sud-ouest du Québec a permis d’évaluer et de comparer la séroprévalence aux arbovirus étudiés chez chacune de ces trois espèces. Les estimations de séroprévalence ont révélé un niveau d’activité arbovirale significative pour chacun des arbovirus. Des différences ont été remarquées quant au pourcentage de sujets séropositifs chez chacune des espèces. Les facteurs environnementaux ayant une influence sur le risque d’infection par le VEEE ont été modélisés à partir de données sérologiques et cliniques chez les chevaux. Les milieux humides boisés ont été identifiés comme les principaux environnements à risque pour le VEEE au Québec alors que les zones agricoles ont été identifiées comme des environnements protecteurs. Les facteurs environnementaux ayant un impact sur le risque d’infection par le VNO ont été modélisés à partir des données sérologiques chez le chien et des données cliniques agrégées chez l’humain. Cette modélisation a suggéré un risque singulièrement plus élevé en zone agricole chez le chien et un risque plus faible en zone forestière chez l’humain, des facteurs rarement identifiés dans la littérature Nord-Américaine. Les facteurs environnementaux et individuels ayant un impact sur le risque d’infection par les VSGC chez l’humain et le chien ont par la suite été modélisés à partir des données sérologiques chez ces deux espèces. D’après nos modèles, le risque d’infection par ces virus serait supérieur en zone forestière et le degré d’exposition aux piqures de moustiques serait un facteur déterminant du risque d’infection chez les deux espèces. Cette étude a permis de bonifier de façon substantielle le portrait de l’activité arbovirale au Québec. Elle a permis de caractériser la distribution du risque et a fourni des données probantes pouvant soutenir la recherche ainsi que la planification des interventions en santé publique. La méthodologie utilisée dans le cadre de cette étude supporte la pertinence de l’approche « One Health » pour l’étude des maladies vectorielles émergentes. / Arboviral infections represent a considerable and growing health burden globally. The complexity of biotic and abiotic factors involved in the transmission of these arboviruses pose a challenge to scientists trying to understand, model or anticipate arboviral transmission as well as to public health authorities who have the responsibility to monitor, assess and manage the public health risk posed by arboviruses. This study aimed at estimating and characterizing the risk of exposure to several arboviruses suspected of being active and emerging in Québec but whose distribution had not been studied thoroughly in Québec: West Nile virus (WNV), eastern equine encephalitis virus (EEEV) and two viruses of the California serogroup (CSG), Jamestown Canyon virus (JCV) and Snowshoe hare virus (SHV). Based on the assumption that animals of different species sharing a common environment also share similar environmental risk, this study also aimed to assess whether some populations of domestic animals could help to estimate and characterize the risk of arboviral infection in humans. Serological sampling of human, canine and equine populations from southwestern Québec was used to evaluate and compare the seroprevalence to the selected arboviruses in each of these three species. Seroprevalence estimates showed a significant level of arboviral activity for all arboviruses. Differences were noted in the percentage of seropositive individuals in each species. Environmental factors that influence the risk of infection by EEEV were modeled based on serological and clinical data in horses. Wooded wetlands were identified as the main risk environments for EEEV in Québec while agricultural areas were identified as protective environments. Environmental factors affecting the risk of WNV infection were modeled based on serological data in dogs and aggregated clinical data in humans. These models suggested a higher risk in agricultural areas in dogs and a lower risk in forest areas in humans, two factors rarely identified in the North American literature. Environmental and individual factors affecting the risk of infection by CSGV in humans and dogs have subsequently been modeled based on serological data in these two species. According to our models, the risk of infection with these viruses would be higher in forested areas and the degree of exposure to mosquito bites would be a risk factor for infection in both species. This study substantially enhanced the comprehension of arboviral activity in Québec. It allowed for characterizing the distribution of risk and provided evidence that may support research and planning of public health interventions. The methodology used in this study supports the relevance of the "One Health" approach for the study of emerging vector-borne diseases.

Ο ιός του αιμορραγικού πυρετού Κριμαίας-Κογκό (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), καθώς και οι χανταϊοί (hantaviruses) προκαλούν στον άνθρωπο αιμορραγικό πυρετό. Αυτοί παρουσιάζουν ευρεία γεωγραφική κατανομή και αποτελούν απειλή για τη δημόσια υγεία, λόγω του υψηλού ποσοστού θνητότητας που σημειώνουν και της απουσίας αποτελεσματικής θεραπευτικής αγωγής. Παρότι επιδημιολογικές μελέτες δείχνουν την παρουσία αντισωμάτων στον ελληνικό πληθυσμό, περιορισμένες είναι οι αναφορές κλινικών περιστατικών CCHF και HFRS στην Ελλάδα. Σκοπός της παρούσας μελέτης είναι να προσδιορίσει τον επιπολασμό της μόλυνσης με τον CCHFV και τους χανταϊούς στον Ν. Αχαΐας, που αν και παρουσιάζει ευνοϊκές συνθήκες για την κυκλοφορία των δύο ιών, δεν έχει μελετηθεί στο παρελθόν. Σχεδιάσθηκε διατμηματική μελέτη και συγκεντρώθηκαν προοπτικά 207 δείγματα ορού φαινομενικά υγιών ατόμων-κατοίκων της περιοχής, τα οποία εξετάστηκαν με τη μέθοδο ELISA και έμμεσου ανοσοφθορισμού για την ύπαρξη αντισωμάτων έναντι του CCHFV και των χανταϊών. Ο επιπολασμός για τη μόλυνση με CCHFV βρέθηκε 3,4% και 9,7% για τη μόλυνση με χανταϊούς, ενώ κανένα από τα οροθετικά άτομα δεν ανακαλούσε συμπτώματα παρόμοια με αυτά του CCHF ή του HFRS. Για τον CCHFV, βρέθηκε ότι η ηλικία, η αγροτοκτηνοτροφική ενασχόληση, η κατοχή/εκτροφή αιγοπροβάτων, το ιστορικό νύγματος κρότωνα, η μόνιμη διαμονή σε υψόμετρο ≥400μ., η μόνιμη διαμονή σε μη αρδευόμενες αρόσιμες εκτάσεις ή σε αγροτικές εκτάσεις με σημαντικό ποσοστό φυσικής βλάστησης, καθώς και η μόνιμη διαμονή σε αγροτική περιοχή είναι σημαντικοί παράγοντες κινδύνου. Από αυτούς, το νύγμα κρότωνα, η αγροτοκτηνοτροφική ενασχόληση και η μόνιμη διαμονή σε υψόμετρο ≥400μ. βρέθηκαν να προβλέπουν καλύτερα την οροθετικότητα ενός ατόμου. Επίσης, βρέθηκε ότι παράγοντες που σχετίζονται με τη μόλυνση με χανταϊούς είναι: η ηλικία, η θέαση τρωκτικών σε ακτίνα <200μ. γύρω από την οικία και η ιδιοκτησία υπόγειας αποθήκης. Από αυτούς, μόνο η ηλικία βρέθηκε να προβλέπει καλύτερα την οροθετικότητα ενός ατόμου. Επιπλέον, παρατηρήθηκε ότι σχεδόν το 75% των θετικών ατόμων για αντισώματα έναντι των χανταϊών παρουσίαζαν ήπια επηρεασμένη νεφρική λειτουργία. Εντοπίστηκαν, επίσης, ενδημικές εστίες των ιών στον νομό: ο Δ. Ερυμάνθου για τον CCHFV και ο Δ. Δυτικής Αχαΐας για του χανταϊούς. Λαμβάνοντας υπόψη τα παραπάνω αποτελέσματα, θα πρέπει οι κλινικοί γιατροί της περιοχής να συμπεριλαμβάνουν τον CCHF και τον HFRS στη διαφορική διάγνωση εμπύρετων νοσημάτων, ιδίως όταν αυτά συνοδεύονται από θρομβοπενία ή επηρεασμένη νεφρική λειτουργία. / Crimean-Congo hemorrhagic fever virus (CCHFV) and hantaviruses cause to humans fever with hemorrhagic manifestations. These viruses present wide geographic distribution and represent major threats for public health, because of the high fatality rate that they present and the lack of appropriate treatment. Although seroprevalence studies show the presence of antibodies against CCHFV and hantaviruses in the greek population, only some reports of human cases have been reported to date in Greece. The aim of the present study is to estimate seroprevalence for CCHFV and hantaviruses in humans in the prefecture of Achaia, where the local conditions potentially favor the circulation of these viruses and which has not been previously studied. A cross-sectional study was designed and 207 human sera were collected from apparently healthy individuals living in Achaia, which were tested for CCHFV and hantaviruses IgG antibodies by ELISA and by indirect immunofluorescence assay (IFA). Seroprevalence for CCHFV infection was estimated at 3.4%, whereas for hantaviruses at 9.7%; none recalled any illness resembling CCHF or HFRS. For CCHFV, it was found that age, agro-pastoral occupation, tending sheep and/or goats, tick bite, living in areas at an altitude of ≥400m., living at rural areas, living on non-irrigated arable land or on land principally occupied by agriculture, with significant areas of natural vegetation are significantly related to seropositivity. Among them, tick bite, agro-pastoral occupation and living in areas at an altitude of ≥400m. better predict seropositivity of an individual. For hantaviruses, it was found that age, rodent sighting around home and the ownership of an underground shed are significantly related to seropositivity. Among them, it seems that only age can predict seropositivity of an individual. Moreover, it was observed that almost 75% of the seropositive for hantaviruses individuals presented mild renal dysfunction. In this study, endemic foci were also detected: the municipality of Erimanthos for CCHFV and the municipality of Western Achaia for hantaviruses. Clinicians should include CCHF and HFRS in the differential diagnosis of an acute febrile case, especially when thrombocytopenia or impaired renal function is encountered.

Circulation des Phlébovirus en Turquie, l'Iran et l'Algérie a été étudiée. L'isolement, la caractérisation génomique, les relations phylogénétiques de six virus ont été présentées: le virus Adana (ADAV), deux souches de virus Toros (TORV), le virus Zerdali (ZERV) de la Turquie; le virus Dashli (DASHV) de l'Iran; le virus Toscana (TOSV) d'Algérie. Cette étude a commencé avec la collection de 38,131 phlébotomes de la nature. La méthode de séquençage de nouvelle génération (NGS) à haut débit nous à été utilisée pour l’analyse des génomes complet des virus isoles. En conclusion, cette étude a d'importantes contributions sur phlébovirus négligées. Voici quelques-unes des contributions significatives; (i) ZERV et TORV qui sont étroitement apparentés au virus Tehran (THEV) et le virus Corfou (CFUV), respectivement, ont été isolés depuis 56 et 30 ans des premiers isolements de THEV et CFUV, respectivement, (ii) Détection du virus ADAV un animal domestique et sur quelques sérums humain par test de neutralisation. Ce virus ADAV constitue avec le virus le virus (SALV), le virus Arbia (ARBV), et le virus (ADRV) le groupe Salehabad. Seul le virus ADRV a été détectée dans le liquide cérébro-spinal auparavant landais que avec les autres, aucune preuve pathogène n’a été détectée, (iii) Nous avons découvert la plus récente circulation phlébovirus en Iran après 56 années, (iv) TOSV a été isolé en Algérie pour la première fois et la circulation a été confirmée par séropositivités dans le sérum humain. / Sandfly-borne phlebovirus circulation in Turkey, Iran, and Algeria was investigated. The isolation, genomic characterization, phylogenetic relationships of 6 viruses was presented: Adana virus (ADAV), two strains of Toros virus (TORV), Zerdali virus (ZERV) from Turkey; Dashli virus (DASHV) from Iran; Toscana virus (TOSV) from Algeria. This study has begun with the collection of 38,131 sandflies from nature. The well established, high-throughput methodology was applied for the discovery of viruses including PCR tools and cell culture methods. Next generation sequencing (NGS) technology facilitated to perform complete genome analysis of the isolated viruses. In conclusion, this study has contributions to the neglected sandfly-borne phlebovirus group and filled some gaps about the circulation of these agents in Turkey, Iran, and Algeria. Following are some significant contributions; (i) ZERV and TORV which are closely related to Tehran virus (THEV) and Corfou virus (CFUV), respectively were isolated after 56 and 30 years of the first isolations of THEV and CFUV, respectively, (ii) There was no evidence of the pathogenicity of Salehabad virus (SALV) and Arbia virus (ARBV) except the detection of Adria virus (ADRV) in CSF until ADAV which belongs to the Salehabad serocomplex was detected in domestic animal and very few human sera by neutralization assay, (iii) We have discovered the most recent sandfly-borne phlebovirus circulation in Iran after 56 years, (iv) TOSV was isolated in Algeria for the first time and circulation was confirmed by seropositivities in human sera.

Department of Microbiology / PhD (Microbiology) / Human herpes virus type 8 (HHV-8), also known as Kaposi’s sarcoma associated herpes virus (KSHV), is the etiologic agent of Kaposi’s sarcoma (KS), and AIDS related Kaposi’s sarcoma (AIDS-KS). HHV-8 which is a member of the Herpesviridae family, exhibits extensive genetic diversity globally. In endemic regions, infection with HHV-8 occurs very early on in life, which is an indication of both environmental and vertical routes of transmission. The advent of HIV leads to the classification of an AIDS-KS defining condition in HIV infections. This suggests that in regions where HIV and HHV-8 are endemic, KS may become common in a mature HIV epidemic. Just like the prevalence of HIV in Northern South Africa is generally high as in most regions of the country, as the HIV epidemic matures in South Africa, it is important to understand the burden and distribution of HHV-8 infection, and the likely genotypes infecting the population. The main objective of the thesis was to establish the epidemiology and infecting genotypes of HHV-8 in Northern South Africa (Limpopo Province), where no data exists. First, a systematic review of the literature was carried out for the entire African continent to determine the seroprevalence and genotype distribution of HHV-8 in all African countries (n=53). In this review, Sudan and South Sudan were considered as one country. Articles were searched using the PRISMA guideline and exported using an article grid. More than two-thirds (64%) of the studies reported on seroprevalence, 29.3% on genotypes; and 9.5% were on both seroprevalence and genotypes. About 45% (24/53) of the African countries had data on HHV-8 seroprevalence exclusively, and more than half (53%) had data on either seroprevalence or genotypes. Almost half (47%) of the countries had no data on HHV-8 infection. There was high heterogeneity in the types of tests and interpretation algorithms used in determining HHV-8 seropositivity across the different studies. Generally, seroprevalence ranged from 2.0% in a group of young children in Eritrea to 100% in a small group of individuals with KS in the Central Africa Republic and a larger group of KS in individuals in Morocco. Approximately, 16% of all the studies reported on children. The difference in seroprevalence across the African region was not significant (95% CI, X2 =0.86; p =0.35), although specifically, a relatively significant ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|VIII level of infection was observed in HIV-infected children. About 38% of the countries had data on K1 genotypes A, A5, B, C, F and Z which occurred at frequencies of 5.3%, 26.3%, 42.1%, 18.4%, 5.3% and 2.6% respectively. Twenty-three percent of the countries had data for K15 genotypes, whereas genotypes P, M and N occurred at frequencies of 52.2%, 39.1% and 8.7% respectively. Data on HHV-8 inter-genotype recombinant is scanty. Our finding suggests that HHV-8 is endemic on the entire African continent, and in HIV endemic regions, but there is need for a harmonized testing protocol for better understanding of HHV-8 seropositivity. HHV-8 genotype A5 and B for K1 gene and genotype P and M for K15 gene are the most predominant genotypes in Africa. The review, for the first time, has provided information on HHV-8 burden on the entire African continent, and suggests that vaccine development efforts for Africa should focus on genotypes B and P. The second component of the investigation focused on the burden of HHV-8 in an HIV population in Northern South Africa (Limpopo Province). Plasma from 3501 HIV infected individuals from 5 districts in Limpopo Province were assessed for antibodies to both the lytic antigen (ORFK8.1) and the latent antigen (ORF73). The distribution of infection was analyzed based on demographic, socioeconomic, and immunological parameters. Statistical inferences for significant differences were determined by Chisquare at a confidence interval of 95%. P-values less than 0.05 were considered significant. About 19.0% of the study population was positive for antibodies to either the lytic or latent antigens or both. Prevalence of antibodies to the lytic antigen was significantly higher than prevalence of antibodies to the latent antigen (17.3% vs 4.1%; p=0.0001). Significant differences were observed for age groups, racial population groups, districts and year of sample collection (p=<0.0001, p=<0.0001, p=<0.0001 and p=0.0385) respectively. Associations were found between both antigens in comparison to the different variables such as age group, racial population groups and districts (R2 value ranging between 0.886 and 1.0). The burden of HHV-8 has now been established for the first time in Northern South Africa. The third aspect of the investigation was a meta-analysis of HHV-8 seroprevalence in Southern Africa in order to understand the impact of geographical location (urban vs rural) on infection. The analysis revealed a significant association between urban settings and HHV-8 infection (p=0.0001). ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|IX The fourth component of the thesis examined the detection of HHV-8 antigen through polymerase chain reaction (PCR) in 534 participants in HIV infected and HIV noninfected populations. A selection of mouthwash DNA samples were subjected to Next Generation Sequencing (NGS) for subsequent genotype inference. Mouth wash samples were obtained from each consenting individual before eating or smoking, and their DNA was purified. A 233bp fragment of the ORF26 gene of HHV-8 was amplified by PCR. HHV-8 was detected in 150 of the 534 participants (28.1%). A significant difference in detection was observed for gender, HIV status, district and the level of education (p=0,0003; p=0.0094; p=0.0002 and p=0.0095) respectively. Consensus sequences were derived from NGS reads for 13 samples. The genotyping results revealed that genotype Q, B, E and N are the genotypes predominant in the study population. As such no mixed infections were detected. Therefore, from the investigations foregoing have demonstrated for the first time the following: (1) HHV-8 is endemic in the entire African continent, which suggest a coendemicity in regions already endemic for HIV; (2) HHV-8 is endemic in Northern South Africa; (3) Urban settings in Southern Africa are associated with high HHV-8 infection; (4) HHV-8 genotypes Q, B, E and N may be predominant in Northern South Africa, with B and P common on the entire African continent. Hence, studies should focus on the generation of full length HHV-8 genomes of the common genotypes to support the selection of genes for vaccine design and development. / NRF