Os papilomavírus constituem uma extensa família de vírus associados a lesões epiteliotrópicas, assim como acometem o tecido mucoso onde podem causar tumores benignos que podem evoluir para a malignização. A papilomatose bovina é uma doença economicamente relevante, já que há a desvalorização dos animais a serem comercializados devido à aparência e depreciação do couro. Em humanos, a identificação dos tipos de papilomavírus (HPV) envolvidos bem como o relato de co-infecções são bem estabelecidos. Já foram descritos na literatura mais de 200 tipos de HPVs, o que ainda é pouco explorado na área da Medicina Veterinária, em que somente 21 tipos de papilomavírus bovino (BPV) foram caracterizados até o momento. A presente dissertação visou investigar a diversidade de BPV presentes em um papiloma de um bovino oriundo do Estado de Rondônia. A partir do DNA extraído da lesão, foi realizada a rooling circle amplification (RCA) seguida do sequenciamento de última geração (Ilumina MiSeq). O emprego dessas metodologias culminou na identificação do BPV13, que atualmente é bem caracterizado, e de três prováveis novos tipos de BPVs que haviam sido descritos em um único estudo recente. Estes resultados confirmam a presença de co-infecção em lesões de papilomatose bovina e demonstram que estas técnicas possibilitaram a detecção de espécies que não são identificadas pelos métodos convencionais. Este conhecimento sobre a diversidade de BPV servirá de base para o melhor entendimento da biologia do vírus e para a geração de vacinas profiláticas ou terapêuticas eficazes. / Papillomaviruses are an extensive family of viruses associated with epitheliotropic lesions, as well as affecting mucosal tissue where they can cause benign tumors that may progress to malignancy. Bovine papillomatosis is an economically relevant disease, since there is a devaluation of the animals to be marketed due to the appearance and depreciation of the leather. In humans, the identification of the types of papillomavirus (HPV) involved as well as the reporting of co-infections is well established. More than 200 types of HPV have been described in the literature, which is still little explored in the area of Veterinary Medicine, in which only 21 types of bovine papillomavirus (BPV) have been characterized. This study aimed to investigate the diversity of BPV present in a papilloma of a bovine animal from the State of Rondônia. From the DNA extracted from the lesion, the rooling circle amplification (RCA) was performed followed by the last generation sequencing (Ilumina MiSeq). The use of these methodologies culminated in the identification of BPV13, which is currently well characterized, and of three putative new BPVs types that had been described in a single recent study. These results confirm the presence of co-infection in bovine papillomatosis lesions and demonstrate that these techniques enabled the detection of species that are not identified by conventional methods. This knowledge on BPV diversity will serve as a basis for a better understanding of the biology of the virus and for the generation of effective prophylactic or therapeutic vaccines.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-20Bitstream added on 2014-06-13T19:55:44Z : No. of bitstreams: 1 sobrinho_lsv_me_araca.pdf: 921356 bytes, checksum: 29d3e58899a35e5de14b32e263c2fe77 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi determinar em uma população de 302 gatos provenientes de área endêmica para leishmaniose visceral, a prevalência da infecção por Leishmania spp. e a presença de coinfecção pelo Toxoplasma gondii, vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV). Foram evidenciadas formas amastigotas de Leishmania spp. em 9,93% (30/302) dos animais. A prevalência da leishmaniose observada por meio dos métodos de ELISA-proteína A, ELISA-IgG ou exame parasitológico direto foi de 21,85% (66/302), sendo 13,64% (9/66) positivos no exame parasitológico direto e sororeagentes nas técnicas de ELISA indireto. Doze animais (70,59%) foram sororeagentes para o FIV e a Leishmania spp., enquanto 17 (25,76%) apresentaram anticorpos anti-Toxoplasma gondii e anti-Leishmania spp. e cinco (71,43%) apresentavam infecção pelos três agentes. Não foi observada coinfecção entre Leishmania spp. e o FeLV. Houve associação estatisticamente significante entre a coinfecção por Leishmania spp. e pelo vírus da imunodeficiência felina, bem como entre a presença de Leishmania spp., do vírus da imunodeficiência felina e do Toxoplasma gondii. A sensibilidade e a especificidade dos métodos de ELISA-proteína A, ELISA-IgG e reação de imunofluorescência indireta para o diagnóstico de leishmaniose felina foram de 56,6% e 89,47%, 55,55% e 90,96% e 54,55% e 96,80%, respectivamente. As concordâncias entre a RIFI e as técnicas de ELISA-proteína A e ELISA-IgG foram fracas. No entanto, houve boa concordância entre as duas últimas técnicas. O presente estudo verificou que gatos residentes em área endêmica para leishmaniose visceral são predispostos à coinfecção por Leishmania spp. e vírus da imunodeficiência felina, e que parte deles desenvolvem sintomas inespecíficos e devem ser investigados em um diagnóstico diferencial / The aim of this study was to determine, in a population of 302 cats from an endemic area for visceral leishmaniasis, the prevalence of the infection by Leishmania spp. and the presence of co-infection by Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Amastigote forms of Leishmania spp were evidenced in 9.93% (30/302) of the animals. Prevalence of leishmaniasis by ELISA-prot A, ELISA-IgG or direct parasitological examination was 21.85% (66/302), being 13.64% (9/66) positive in both direct parasitological examination and ELISA. Twelve animals (70.59%) were seroreagent for FIV and Leishmania spp., while 17 (25.76%) showed antibodies against Toxoplasma gondii and Leishmania spp. and five (71.43%) showed antibodies against those three agents. Co-infection was not observed between Leishmania spp. and FeLV. There was statistically significant correlation between the co-infection by Leishmania spp. and by the immunodeficiency virus, as well as among the present of Leishmania spp, feline immunodeficiency virus and Toxoplasma gondii. The susceptibility and the specificities of (the methods) ELISA-prot A, ELISA-IgG and reaction of indirect immunofluorescence for the diagnosis of feline leishmaniasis were 56.6% and 89.47%, 55.55% and 90.96% and 54.55% and 96.80%, respectively. The agreements between RIFI and ELISA-prot A and ELISA-IgG techniques were weak. However, there was a good agreement between the last two techniques. This study verified that cats from endemic areas for visceral leishmaniasis are predisposed to co-infection by Leishmania spp. and feline immunodeficiency virus, and that part of them developed nonspecific symptoms and should be investigated in a differential diagnosis

Orientador: Andréa Baptista Rossit / Banca: Daisy Nakamura Sato / Banca: Silvia Helena Vendramine / Resumo: São José do Rio Preto (SJRP), localizada na região Noroeste do Estado de São Paulo, Sudeste do Brasil, é considerada Município prioritário pelo Programa Nacional de Controle da Tuberculose e da AIDS. O objetivo deste trabalho foi avaliar retrospectivamente pacientes infectados pelo HIV com pelo menos um isolamento de Mycobacterium sp., atendidos em unidades de saúde de referência de SJRP e região, bem como descrever seus aspectos clínicos e sócio-demográficos. Foram avaliados no período de janeiro de 2000 a dezembro de 2006, 198 indivíduos soropositivos para o HIV com culturas positivas no Instituto Adolfo Lutz de SJRP. Houve uma correlação positiva entre a tuberculose e o registro de detenção (p=0.021). O uso do tabaco reduziu o tempo de vida entre o diagnóstico e o óbito (p=0.05). Houve associação entre o isolamento de M. tuberculosis (MT) e os níveis de linfócitos TCD4+ bem como o achado difuso para RX de tórax (p=0.014 e 0.000, respectivamente). Aproximadamente 11% de todas as cepas de MT mostraram resistência a pelo menos uma droga, enquanto 3.1% foram multiresistentes. Micobactérias não tuberculosas (MNT) totalizaram 35.19% de todos os isolamentos e a maioria das espécies pertence ao complexo Mycobacterium avium (MAC; 22.3%), seguido por M. fortuitum (5.2%) e M.gordonae (3.1%). Conclui-se que a população HIV estudada tem alta prevalência de colonização por MNT. Em um país com extensão continental como o Brasil, o conhecimento das diferenças regionais na distribuição de MNT em populações infectadas pelo HIV pode contribuir para o controle e tratamento dessas infecções oportunistas. / Abstract: São José do Rio Preto city (SJRP), Northwestern São Paulo State, Southeast Brazil, is considered "priority" by the National Programs of Tuberculosis and AIDS Control. Our purpose was to retrospectively evaluate Mycobacterium sp. isolated from HIV-infected patients attending the HIV/TB reference health care units from SJRP and region, as well as to describe their clinical and socio-demographic aspects. One hundred and ninetyeigth HIV-seropositive individuals provided 287 positives cultures from January 2000 to December 2006. There was a positive correlation between tuberculosis and prison record (p=0.021) and tobacco use reduced the mean lifetime from tuberculosis diagnosis to obit (p = 0.05). TCD4+ levels and a diffuse chest X-ray finding were associated to Mycobacterium tuberculosis (MT) isolation (p = 0.014 and 0.000, respectively). Approximately eleven percent of all MT strains showed resistance to at least one drug while 3.1% were multidrug resistant. Non-tuberculous mycobacteria (NTM) totalized 35.19% of all species and the most frequently isolated ones were Mycobacterium avium complex (MAC; 22.3%), M. fortuitum (5.2%) and M. gordonae (3.1%). We conclude that the HIV-infected population studied has a high prevalence of NTM colonization. In a wide country like Brazil, regional differences on NTM distribution in HIV-infected individuals must be further evaluated in order to improve control and treatment of these opportunistic infections. / Mestre

Os papilomavírus constituem uma extensa família de vírus associados a lesões epiteliotrópicas, assim como acometem o tecido mucoso onde podem causar tumores benignos que podem evoluir para a malignização. A papilomatose bovina é uma doença economicamente relevante, já que há a desvalorização dos animais a serem comercializados devido à aparência e depreciação do couro. Em humanos, a identificação dos tipos de papilomavírus (HPV) envolvidos bem como o relato de co-infecções são bem estabelecidos. Já foram descritos na literatura mais de 200 tipos de HPVs, o que ainda é pouco explorado na área da Medicina Veterinária, em que somente 21 tipos de papilomavírus bovino (BPV) foram caracterizados até o momento. A presente dissertação visou investigar a diversidade de BPV presentes em um papiloma de um bovino oriundo do Estado de Rondônia. A partir do DNA extraído da lesão, foi realizada a rooling circle amplification (RCA) seguida do sequenciamento de última geração (Ilumina MiSeq). O emprego dessas metodologias culminou na identificação do BPV13, que atualmente é bem caracterizado, e de três prováveis novos tipos de BPVs que haviam sido descritos em um único estudo recente. Estes resultados confirmam a presença de co-infecção em lesões de papilomatose bovina e demonstram que estas técnicas possibilitaram a detecção de espécies que não são identificadas pelos métodos convencionais. Este conhecimento sobre a diversidade de BPV servirá de base para o melhor entendimento da biologia do vírus e para a geração de vacinas profiláticas ou terapêuticas eficazes. / Papillomaviruses are an extensive family of viruses associated with epitheliotropic lesions, as well as affecting mucosal tissue where they can cause benign tumors that may progress to malignancy. Bovine papillomatosis is an economically relevant disease, since there is a devaluation of the animals to be marketed due to the appearance and depreciation of the leather. In humans, the identification of the types of papillomavirus (HPV) involved as well as the reporting of co-infections is well established. More than 200 types of HPV have been described in the literature, which is still little explored in the area of Veterinary Medicine, in which only 21 types of bovine papillomavirus (BPV) have been characterized. This study aimed to investigate the diversity of BPV present in a papilloma of a bovine animal from the State of Rondônia. From the DNA extracted from the lesion, the rooling circle amplification (RCA) was performed followed by the last generation sequencing (Ilumina MiSeq). The use of these methodologies culminated in the identification of BPV13, which is currently well characterized, and of three putative new BPVs types that had been described in a single recent study. These results confirm the presence of co-infection in bovine papillomatosis lesions and demonstrate that these techniques enabled the detection of species that are not identified by conventional methods. This knowledge on BPV diversity will serve as a basis for a better understanding of the biology of the virus and for the generation of effective prophylactic or therapeutic vaccines.

Orientador: Louis Bernard Klaczko / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T14:49:11Z (GMT). No. of bitstreams: 1 Martins_AyanadeBrito_M.pdf: 905842 bytes, checksum: 4ad0bbbbe049319318ad9fdd6e725632 (MD5) Previous issue date: 2010 / Resumo: Elementos citoplasmáticos egoístas (ECEs) sao bactérias que apresentam transmissão predominantemente vertical e se mantém na população hospedeira através do aumento relativo da produção de fêmeas infectadas. Nesses casos a seleção natural favorece mecanismos de manipulação da reprodução do hospedeiro (feminização, partenogênese, mortalidade precoce de machos e incompatibilidade citoplasmática) e, por isso, os ECEs sao denominados parasitas reprodutivos. A mortalidade precoce de machos (em inglês early male-killing) refere-se a morte da prole masculina devida a presença de um ECE e seus agentes causadores sao denominados agentes androcidas. No caso de indutores de feminizarão, partenogênese e incompatibilidade citoplasmática, o próprio mecanismo de manipulação resulta no aumento relativo na produção de fêmeas infectadas, o que pode explicar a manutencao desses elementos na população hospedeira. Entretanto, para agentes androcidas, o mecanismo de manipulação não resulta automaticamente no aumento da produção de fêmeas infectadas. Uma das hipóteses para explicar a manutencao de agentes androcidas e a realocação de recursos: a morte precoce dos machos libera recursos preferencialmente para as suas irmãs. Dado que não sao conhecidos os mecanismos que explicam a manutencao de agentes androcidas em espécies de Drosophila, este mestrado teve como objetivos estimar a prevalência de agentes androcidas em populações de D. melanogaster e analisar fatores que podem estar envolvidos com a manutencao desses agentes nessas populações: (i) presença de outros organismos transmitidos verticalmente; (ii) evidencias de realocação de recursos em populações naturais e em linhagens no laboratório; e (iii) efeitos em diferentes componentes do valor adaptativo. A prevalência do fenótipo androcida em populações de D. melanogaster, estimada pela contagem de proles, variou entre próxima a 0 e 17,7% e esteve fortemente associada a presença de Spiroplasma, detectada por PCR. As razoes sexuais das proles de fêmeas infectadas foram heterogêneas, o que sugere variação na expressão do fenótipo androcida. Não foi detectada associação entre o agente androcida Spiroplasma e Wolbachia, outro ECE que coinfecta populações de D. melanogaster. Foram encontradas evidencias consistentes com a hipótese de realocação de recursos em D. melanogaster: (i) em experimentos no laboratório, fêmeas infectadas por agente androcida apresentaram menor tempo de desenvolvimento do que fêmeas não infectadas; (ii) e fêmeas do campo infectadas produziram mais filhas em um repique de quatro dias no laboratório. Não houve diferença na produção de filhas entre fêmeas infectadas e não infectadas de uma estirpe padrão do laboratório. E possível que o efeito de Spiroplasma em populações naturais esteja associado a composição genética da população hospedeira. / Abstract: Selfish cytoplasmic elements (SCEs) are maternally inherited bacteria which increase the net production of infected females. Due to a genetic conflict between the SCE and the host genomes, different mechanisms of reproductive manipulation (feminization, parthenogenesis, male-killing and cytoplasmic incompatibility) are favored through natural selection. For feminization, parthenogenesis and cytoplasmic incompatibility, the reproductive manipulation by itself results in a greater net production of daughters by the infected females, which may explain the persistence of these elements in the host population. However, this net difference does not hold for male-killer infected and uninfected females. One of the mechanisms that has been proposed to explain the adaptiveness of the male-killing trait is the resource reallocation from dead males to female hosts. Considering that it is still unclear how male-killers persist in Drosophila populations, the present study aimed to assess the male-killer prevalence in D. melanogaster populations and to analyze different factors which may explain their persistence in these population: (i) interaction with other vertically transmitted elements; (ii) evidence supporting the resource release hypothesis in natural populations and in laboratory strains; and (iii) direct fitness effects. The incidence of the male-killing phenotype in D. melanogaster, obtained counting the laboratory raised broods of collected females, ranged from close to 0 to 17.7% and was strongly associated with Spiroplasma infection, assessed by PCR. The sex ratio of female biased trains had a bimodal distribution which suggests variation in the expression of the male-killing phenotype. No evidence of positive or negative interaction between male-killing Spiroplasma and Wolbachia (other SCE coinfecting D. melanogaster) infections was found. We found evidence consistent with the resource reallocation hypothesis in D. melanogaster: (i) infected females had a shorter generation time in laboratory experiments; (ii) and field females produced more daughters in their first brood in the laboratory. No difference in number of daughter was detected between infected and uninfected females in an experiment using flies from a standard laboratory strain. The effect of male-killing Spiroplasma in natural populations may be conditioned to the host's genetic background. / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Magister Curationis - MCur / Globally, tuberculosis (TB) and AIDS are the leading causes of morbidity and mortality of people, particularly in many developing countries. South Africa is among those countries with a high double burden of TB and HIV infections in the world. Although policy guidelines have been developed to mitigate the problems of TB and HIV coinfection, there are still challenges with their implementation. The aim of this study was to analyse the implementation of the integrated TB and HIV policy in a rural sub-district of the Western Cape. The study applied a descriptive survey method to collect data from 60 respondents selected using an all-inclusive sampling strategy. A self-reporting questionnaire was used, and data were analysed using the Statistical Package for Social Sciences (SPSS) program version 23. Descriptive statistics are presented with graphs and percentages. All ethical principles were adhered to. The results showed that 86% of the respondents were female nurses, 59% were registered nurses, and the rest were enrolled and assistant nurses. The general view of participants was that implementation of the integrated TB and HIV policy was poor, as only 25% (11) felt that they have sufficient knowledge and skills to implement it, while 50% (22) did not feel equipped to implement the integrated policy. These perceptions of inadequacy were justified by a lack of sufficient training, with only 32% of the respondents having attended training on integrated TB and HIV management. The nurses’ responses on actual practices ranged from poor to sufficient, and example being the management of HIV-positive TB patients with CD4 count of <100/μl, where 27.9% were unsure when to initiate antiretroviral therapy and 44.7% were unsure when isoniazid prophylaxis could not be given to HIVpositive patients. However, the findings indicated that TB and HIV policy guidelines were 86% and 85.7% accessible respectively. The study concluded that nurses do not implement the integrated TB and HIV policy guidelines sufficiently, mainly due to lack of adequate training which resulted in limited knowledge thus poor practice. It is recommended that a continuous staff capacity development programme, which includes suitable pre-service and in-service training in TB and HIV/AIDS management be developed and implemented as it has the potential to address the current knowledge and skills gaps which impact on implementation of the integrated TB and HIV policy.

Magister Public Health - MPH / The study was conducted among TB patients aged 18 years and older who were registered from January to June 2010. The findings showed a high level of knowledge on TB/HIV prevention and a low level of knowledge on TB/HIV treatment and care. The study also showed that respondents who did not know their HIV status had a positive attitude towards VCT services, while those who knew their status had a positive attitude towards HIV/TB prevention and care programmes. Finally, the findings also showed that most respondents trusted conventional medicine more than traditional medicine. All these findings suggested that respondents had good knowledge of HIV and TB co-infection, had a positive attitude and practiced favourable behaviour towards programmes related to the prevention and care of this co-infection. / South Africa

Introduction: Tuberculosis (TB) pericarditis is a relatively rare form of tuberculosis which has been on the decline. However, the advent of the human immunodeficiency virus (HIV) pandemic has brought about the resurgence of tuberculosis pericarditis and an even poorer prognosis for patients with HIV co-infection. Objectives: The aim is to describe the baseline characteristics of tuberculous pericarditis patients and to assess the impact of HIV on the clinical presentation of this disease. Methods: The study describes baseline data from a randomised clinical trial which explored the use of adjunctive corticosteroids in management of TB pericarditis then went on to compare HIV co-infected patients versus those without HIV co-infection using logistic regression. Results: There were 1394 patients enrolled into the study, 64% were HIV positive, 19% were HIV negative and 17% had an unknown HIV status. Forty four percent of the participants were female and age had a positively skewed distribution with median 36 years (IQR: of 29-46). HIV co-infected patients were younger with OR 0.97(95% CI: 0.96-0.98), more likely to have previously had TB with OR 2.15(95% CI: 1.25-3.72), had a more acute illness with OR 0.99(95% CI: 0.99-1.00), had lower hemoglobin with OR 0.72(95% CI: 0.67-0.78), lower White Cell Count, (WCC) with OR 0.90(95% CI: 0.86-0.96) and higher globulin with OR 1.07(95% CI: 1.05-1.09). Conclusion: HIV co-infected participants are younger, more likely to have been previously diagnosed with TB, have a more acute illness, lower haemoglobin, lower WCC and higher globulin. / Dissertation (MSc)--University of Pretoria, 2013. / School of Health Systems and Public Health (SHSPH) / unrestricted

In-vitro-Koinfektionsstudien mit Toxoplasma gondii und Eimeria tenella in primären Hühnermakrophagen Institut für Parasitologie der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Feburar 2020 91 Seiten, 3 Publikationen, 2 eingreichte Manuskripte, 17 Abbildungen, 5 Tabellen, 188 Literaturangaben Schlüsselwörter: Toxoplasma gondii, Eimeria tenella, Koinfektion, Makrophagen, in vitro, Wirt-Parasit-Interaktion, Parasitenwachstum, Wirtsimmunantwort, Cell-imaging Einleitung: Toxoplasma gondii und Eimeria tenella sind intrazelluläre apikomplexe Parasiten, die in Hühnern weit verbreitet sind. Während Infektionen mit dem zoonotischen Erreger T. gondii beim Geflügel im Allgemeinen subklinisch verlaufen, kann E. tenella in Hühnerbeständen schwere Erkrankungen und wirtschaftliche Verluste verursachen. Aufgrund der hohen Seroprävalenz beider Parasiten bei Hühnern sind Koinfektionen wahrscheinlich. Hühnermakrophagen sind wichtig für die Wirtsimmunantwort gegen diese beiden Protozoen. Es ist jedoch wenig über die Wirt-Parasit- sowie Parasit-Parasit-Interaktion in Hühnermakrophagen während einer Koinfektion bekannt. Ziele der Arbeit: Ein geeignetes In-vitro-Koinfektionsmodell für T. gondii- und E. tenella-Infektionen in primären Hühnermakrophagen wurde erstellt und angewendet, um die Makrophagenantwort auf beide Parasitenarten und Koinfektionen zu untersuchen. Tiere, Material und Methoden: Von Monozyten abgeleitete Makrophagen wurden aus Hühnerblut isoliert und aufgereinigt. Eine Koinfektion mit zwei Tachyzoiten des RH-Stammes von T. gondii und zwei Sporozoiten des E. tenella-Houghton-Stammes pro Zelle wurde gleichzeitig oder nacheinander in vitro durchgeführt. Morphologische Veränderungen der Makrophagen und die Parasitenentwicklung wurden mittels Konfokalmikroskopie (CLSM) 2, 6, 12, 24, 48 und 72 Stunden nach Infektion (hpi) visualisiert. Die Parasitenvermehrung wurde evaluiert, indem die Expression des 529-bp-Fragments von T. gondii und des ITS-1-Genfragments von E. tenella durch qPCR bewertet wurden. Die Makrophagen-Phagozytose wurde durch Exposition gegenüber pH-sensitiven fluoreszierenden Biopartikeln stimuliert und durch ein dreidimensionales Modell unter Verwendung von CLSM und Imaris®-Software 2, 6, 12 und 24 hpi quantifiziert. Weiterhin wurden während der Infektion relevante Zytokine (IL-6, IL-10, IL-12, iNOS, TNF-α und IFN-γ) durch Genexpressionsanalyse 24, 48 und 72 hpi untersucht. Zusätzlich wurden die Zellinvasion durch und das Überleben von T. gondii im Verlauf einer sequentiellen Infektion evaluiert, indem Makrophagen zuvor der Infektion mit E. tenella ausgesetzt waren. Die Motilität invasiver Tachyzoiten wurde innerhalb von 20 hpi durch Live-Cell-Imaging verfolgt. Ergebnisse: Die Makrophagen zeigten eine deutliche immunologische Reaktion und Phagozytoseaktivierung ab 2 hpi. Signifikante Veränderungen der Morphologie mit erhöhter Zellvakuolisierung und -ablösung wurden ab 24 hpi während der Koinfektion beobachtet. Bei zuvor E. tenella-exponierten Makrophagen fiel auf, dass T. gondii bei hoher Motilität über 4 Stunden an der Makrophagenmembran anhaftete, bevor es zu einer Penetration kam. Ab 24 hpi vermehrten sich T. gondii in koinfizierten Makrophagen besser als E. tenella. Eine Koinfektion hemmte die Phagozytoseaktivität von Makrophagen nach 2 hpi erheblich, so dass Biopartikel nicht aufgenommen wurden (12 hpi). Mittels qPCR wurde gezeigt, dass bei sequenzieller Koinfektion 2 hpi circa 4-fach weniger T. gondii überlebten als bei Monoinfektion (P < 0,05). Die Anzahl beider Parasiten nahm während der simultanen Koinfektion zu, aber bei der sequentiellen Koinfektion war die Vermehrung von T. gondii im Vergleich zur Monoinfektion bis 24 hpi auf etwa die Hälfte reduziert. Bis 72 hpi verdoppelte sich die Anzahl von E. tenella, während T. gondii bei Koinfektion in diesem Zeitraum auf dem gleichen Niveau wie 48 hpi blieb. Die mRNA-Expression von iNOS (48 hpi), TNF-α (72 hpi) und von IL-10 (48 hpi und 72 hpi) war während der Koinfektion im Vergleich zur E. tenella Monoinfektion signifikant (P < 0,05) erhöht. Schlussfolgerungen: In dem Koinfektionsmodell wurde eine Interaktion zwischen T. gondii und E. tenella beobachtet. Zusätzlich zu den morphologischen Veränderungen und der Phagozytosehemmung der Makrophagen unterschieden sich die Parasitenvermehrung sowie die Zytokinexpression zwischen Koinfektion und Monoinfektion. E. tenella beeinträchtigt das aktive Eindringen von T. gondii in Wirtszellen, wie dies erfolgt, ist derzeit noch nicht geklärt. / In vitro co-infection studies on Toxoplasma gondii and Eimeria tenella in primary poultry macrophages Institute of Parasitology, Faculty of Veterinary Medicine, University of Leipzig Submitted in February 2020 91 pages, 3 publications, 2 submitted manuscripts, 17 figures, 5 tables, 188 references Keywords: Toxoplasma gondii, Eimeria tenella, co-infection, macrophages, in vitro, host-parasite interaction, parasite growth, host immune response, cell imaging. Introduction: Toxoplasma gondii and Eimeria tenella are intracellular apicomplexan parasites that are widely distributed in chicken. Whereas infections with the zoonotic pathogen T. gondii are generally subclinical in poultry, E. tenella may cause severe disease and economical loss in chicken herds. Due to the high seroprevelences with both parasites in chicken, co-infections are likely to exist. Chicken macrophages are essential in the host innate immune response against these two protozoa. However, little is known about the host-parasite and parasite-parasite interaction in chicken macrophages during co-infection. Objectives: A suitable in vitro model for both T. gondii and E. tenella infection in chicken primary macrophages was established and applied to study the course of infection in mono- or co-infection. Animals, materials and methods: Monocyte-derived macrophages were isolated and purified from chicken blood. Co-infection with two T. gondii RH strain tachyzoites and two E. tenella Houghton strain sporozoites per cell were performed simultaneously or sequentially in vitro. Morphologic alterations of macrophages and parasite development were visualized by confocal laser scanning microscopy (CLSM) at 2, 6, 12, 24, 48 and 72 hours post infection (hpi). Parasite growth was evaluated by assessing expression of the 529-bp fragment of T. gondii and ITS-1 gene fragment of E. tenella by qPCR in parallel. Macrophage phagocytosis was stimulated by exposure to pH sensitive fluorescent bioparticles and quantified by a three-dimensional model using CLSM and Imaris® software at 2, 6, 12 and 24 hpi. Furthermore, infection-related cytokines (IL-6, IL-10, IL-12, iNOS, TNF-α and IFN-γ) were evaluated by gene expression analysis at 24, 48, 72 hpi. The course of sequential infection was evaluated to determine cell invasion and survival of T. gondii in macrophages previously exposed to E. tenella sporozoites. Motility of invasive stages was tracked at the early phase of infection (within 20 hpi) by real time life cell imaging. Results：By cell imaging, macrophages displayed distinctly immunologic activation and phagocytosis at 2 hpi and thereafter. Significant changes of morphology with increased cell vacuolation and detachment were observed on 24 hpi during co-infection. T. gondii tachyzoites adhered for more than 4 hours to host cells displaying high motility instead of cell entry during the early sequential co-infection. However, T. gondii showed better replication than E. tenella in co-infected macrophages from 24 hpi onwards. Co-infection caused inhibition of phagocytosis by macrophages and bioparticles were not incorporated into co-infected cells at 12 hpi by CLSM. By qPCR, it was shown that approximately 4-fold less T. gondii survived in sequentially co-infected cultures at 2 hpi as compared to mono-infection (P < 0.05). Replication of both parasites increased during simultaneous co-infection whereas only half numbers of replicated T. gondii was found in sequential co-infection compared to mono-infection at 24 hpi. At the end of the study period (72 hpi), E. tenella multiplication tended to double increase while T. gondii replication was not distinctly altered during co-infection compared to 48 hpi. Expression of mRNA for iNOS at 48 hpi, for TNF-α at 72 hpi and for IL-10 at 48 and 72 hpi was significantly elevated during co-infection compared to mono-infection (P < 0.05). Conclusions：Mutual interaction between T. gondii and E. tenella were observed in the selected co-infection model. Increased macrophage stress may explain vacuolization and phagocytosis inhibition. In addition to morphologic alterations of macrophages, cytokine up/down- regulation differed between co-infected and mono-infected macrophage cultures. It appears that E. tenella, impairs the active penetration of T. gondii into host cells which deserves further study. The established in vitro infection model may serve to investigate host immune response of macrophages to diverse intracellular pathogens that infect chicken.

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/-mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect.