Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós–Graduação em Oceanografia Biológica, Instituto de Oceanografia, 2011. / Submitted by Cristiane Gomides (cristiane_gomides@hotmail.com) on 2013-12-16T13:10:23Z No. of bitstreams: 1 rubens.pdf: 37071078 bytes, checksum: e7a36c0d198f25aa295e0031caf953d7 (MD5) / Approved for entry into archive by Sabrina Andrade (sabrinabeatriz@ibest.com.br) on 2013-12-18T18:04:16Z (GMT) No. of bitstreams: 1 rubens.pdf: 37071078 bytes, checksum: e7a36c0d198f25aa295e0031caf953d7 (MD5) / Made available in DSpace on 2013-12-18T18:04:16Z (GMT). No. of bitstreams: 1 rubens.pdf: 37071078 bytes, checksum: e7a36c0d198f25aa295e0031caf953d7 (MD5) Previous issue date: 2011 / No estuário da Lagoa dos Patos são escassos os trabalhos sobre a comunidade fúngica. O objetivo deste estudo foi determinar a ocorrência de leveduras em duas áreas de marismas e os fatores abióticos que afetam sua distribuição espacial e temporal. O estudo foi realizado durante o inverno (Agosto) de 2009 e o verão (Fevereiro) de 2010 em duas enseadas rasas no estuário da Lagoa dos Patos, com diferentes níveis de eutrofização. As amostras foram coletadas em marismas dominadas por duas espécies macrófitas emersas: Juncus kraussii e Scirpus olneyi. Nos locais de coleta foram analisados os parâmetros abióticos e coletadas amostras de sedimento, água intersticial e de vegetação. Foi realizado o isolamento das leveduras do sedimento através de diluição em meio de cultivo. A identificação dos isolados foi feita através da análise de caracteres morfológicos e testes fenotípicos de metabolismo através do Sistema Vitek II. Foram isoladas 73 leveduras classificadas em 09 gêneros, com predominância de Ascomycotas. Entretanto, 10 destes isolados de leveduras não identificados e 02 isolados leveduriformes foram identificados como microalgas do gênero Prototheca. Dentre os gêneros de leveduras encontrados, houve uma predominância de leveduras do gênero Candida (n=20), seguidos por Rhodotorula (n=14), Yarrowia (n=07), Cryptococcus (n=06), Clavispora (n=04), Issatchenkia (n=03), Pichia (n=03), Torulaspora (n=03) e Kluyveromyces (n=01). O maior número de leveduras foi isolado em temperatura mais alta, no ambiente menos eutrofizado, em condições com maiores concentrações de nutrientes dissolvidos e no sedimento dominado por detrito de J. kraussii. / In the Patos Lagoon estuary few studies have been conducted on the fungal community. The aim of this study was to determine the occurrence of yeasts and the environments factors that affect their spatial and temporal distribution. The study was conducted during the winter (August) of 2009 and summer (February) 0f 2010 in two shallow bays of the Patos Lagoon estuary with different eutrophication level. The samples were collected in salt marsh areas dominated by two emerged macrophytes species: Juncus kraussii and Scirpus olneyi. Abiotic parameters were analyzed in the field and sediment samples, interstitial water and vegetation were collected. Yeasts were isolated from the sediment by dilution using the culture medium method. The identification of isolates was performed according to morphological characteristics and through metabolic tests using the Vitek II system. Seventy and three yeasts were isolated and classified in 09 genera with the predominance of Ascomycota. However, 10 of these isolated yeasts were unidentified and 02 yeast-like organisms, were identified as microalgae of the genera Prototheca. Among the identified genera of yeasts, there was a predominance of the genera Candida (n=20), followed by Rhodotorula (n=14), Yarrowia (n=07), Cryptococcus (n=06), Clavispora (n=04), Issatchenkia (n=03), Pichia (n=03), Torulaspora (n=03) e Kluyveromyces (n=01). Highest number of isolates was found in higher temperature, less polluted environment, in water with higher dissolved nutrient concentrations and in sediment dominated by detritus of Juncus kraussii. Key-words:

A mastite bovina destaca-se negativamente no cenário mundial em decorrência aos prejuízos ocasionados pelo seu dispendioso tratamento, sendo considerada a principal doença que acomete a pecuária leiteira. Como alternativa ao uso de antibióticos, a terapia fotodinâmica se apresenta como uma opção terapêutica no tratamento de infecções locais, que atua sem que haja seleção de micro-organismos resistentes. Seu mecanismo de ação resulta da interação entre uma substância fotossensibilizadora e luz em comprimento de onda ressonante, promovendo a formação de espécies reativas de oxigênio que apresentam ação citotóxica contra micro-organismos. O objetivo deste estudo foi investigar a habilidade da terapia fotodinâmica, in vitro, para inativar cepas causadoras da mastite bovina. Culturas de Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis e Prototheca zopfii suspensas em PBS foram estimadas por meio da turbidez da suspensão correspondendo à concentração de 107 UFC/mL, caracterizando desta forma, oito grupos avaliados: controle (C), luz LED vermelho (L) =660nm/2 minutos de irradiação (12,22J/cm2), AM (azul de metileno - 1mM), PDT 5 segundos (0,50J/cm2), PDT 10 segundos (1,01J/cm2), PDT 30 segundos (3,05J/cm2), PDT 1 minuto (6,11J/cm2) e PDT 2 minutos (12,22J/cm2) de irradiação. Os grupos tratados com AM e Luz isoladamente não apresentaram ação citotóxica sobre os micro-organismos avaliados. S. dysgalactiae, S. aureus, C. bovis foram inativados sob 30 segundos de irradiação (3,05J/cm2); S. agalactiae e P. zopfii foram inativados sob 2 minutos de irradiação (12,22J/cm2). / Bovine mastitis stands out negatively on the world scenario due to the damage caused by its expensive treatment, being considered the main disease affecting dairy cattle. As an alternative to antibiotics, photodynamic therapy is presented as a therapeutic option in the treatment of local infections, which acts without any selection of resistant microorganisms. Its mechanism of action results from the interaction between a photosensitizing substance and light in the resonant wavelength, promoting the formation of reactive oxygen species, which have cytotoxic activity against micro -organisms. The aim of this study was to investigate, in vitro, the ability of photodynamic therapy to inactivate strains that cause bovine mastitis. Cultures of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis and Prototheca zopfii suspended in PBS were estimated by the turbidity of the suspension corresponding to a concentration of 107 CFU / mL, characterizing eight study groups: control (C), light - red LED (L) = 660nm / 2 minutes of irradiation (12.22J/cm2), BM (methylene blue - 1 mM ), PDT 5 seconds (0.50 J/cm2 ), PDT 10 seconds (1.01J/cm2), PDT 30 seconds (3.05 J/cm2 ), PDT 1 minute (6.11 J/cm2) and PDT 2 minutes (12.22 J/cm2) of irradiation. The groups treated with methylene blue and light alone had no cytotoxic effect on microorganisms evaluated. S. dysgalactiae, S. aureus, C. bovis were inactivated after 30 seconds of irradiation (3.05 J/cm2 ); S. agalactiae and P. zopfii were inactivated after 2 minutes of irradiation (12.22 J/cm2).

A mastite bovina destaca-se negativamente no cenário mundial em decorrência aos prejuízos ocasionados pelo seu dispendioso tratamento, sendo considerada a principal doença que acomete a pecuária leiteira. Como alternativa ao uso de antibióticos, a terapia fotodinâmica se apresenta como uma opção terapêutica no tratamento de infecções locais, que atua sem que haja seleção de micro-organismos resistentes. Seu mecanismo de ação resulta da interação entre uma substância fotossensibilizadora e luz em comprimento de onda ressonante, promovendo a formação de espécies reativas de oxigênio que apresentam ação citotóxica contra micro-organismos. O objetivo deste estudo foi investigar a habilidade da terapia fotodinâmica, in vitro, para inativar cepas causadoras da mastite bovina. Culturas de Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis e Prototheca zopfii suspensas em PBS foram estimadas por meio da turbidez da suspensão correspondendo à concentração de 107 UFC/mL, caracterizando desta forma, oito grupos avaliados: controle (C), luz LED vermelho (L) =660nm/2 minutos de irradiação (12,22J/cm2), AM (azul de metileno - 1mM), PDT 5 segundos (0,50J/cm2), PDT 10 segundos (1,01J/cm2), PDT 30 segundos (3,05J/cm2), PDT 1 minuto (6,11J/cm2) e PDT 2 minutos (12,22J/cm2) de irradiação. Os grupos tratados com AM e Luz isoladamente não apresentaram ação citotóxica sobre os micro-organismos avaliados. S. dysgalactiae, S. aureus, C. bovis foram inativados sob 30 segundos de irradiação (3,05J/cm2); S. agalactiae e P. zopfii foram inativados sob 2 minutos de irradiação (12,22J/cm2). / Bovine mastitis stands out negatively on the world scenario due to the damage caused by its expensive treatment, being considered the main disease affecting dairy cattle. As an alternative to antibiotics, photodynamic therapy is presented as a therapeutic option in the treatment of local infections, which acts without any selection of resistant microorganisms. Its mechanism of action results from the interaction between a photosensitizing substance and light in the resonant wavelength, promoting the formation of reactive oxygen species, which have cytotoxic activity against micro -organisms. The aim of this study was to investigate, in vitro, the ability of photodynamic therapy to inactivate strains that cause bovine mastitis. Cultures of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis and Prototheca zopfii suspended in PBS were estimated by the turbidity of the suspension corresponding to a concentration of 107 CFU / mL, characterizing eight study groups: control (C), light - red LED (L) = 660nm / 2 minutes of irradiation (12.22J/cm2), BM (methylene blue - 1 mM ), PDT 5 seconds (0.50 J/cm2 ), PDT 10 seconds (1.01J/cm2), PDT 30 seconds (3.05 J/cm2 ), PDT 1 minute (6.11 J/cm2) and PDT 2 minutes (12.22 J/cm2) of irradiation. The groups treated with methylene blue and light alone had no cytotoxic effect on microorganisms evaluated. S. dysgalactiae, S. aureus, C. bovis were inactivated after 30 seconds of irradiation (3.05 J/cm2 ); S. agalactiae and P. zopfii were inactivated after 2 minutes of irradiation (12.22 J/cm2).

Infecções microbianas podem ser causadas por bactérias, fungos, vírus, parasitas e algas. O presente estudo avaliou a atividade in vitro de formulações de lípides catiônicos contra bactérias multirresistêntes (MRs), fungos e microalgas de interesse médico humano e veterinário. A atividade biocida de bicelas de brometo de dioctadecildimetilamônio (DBB) foi avaliada contra bactérias gram-negativas produtoras de β-lactamases de amplo espectro e carbapenemases mundialmente disseminadas. Para fungos (Candida albicans, Trichophyton rubrum) e microalgas (Prototheca zopfii), a atividade biocida de DBB, sem e com a associação a drogas antifúngicas (ex., miconazol, terbinafina e anfotericina B), foi avaliada determinando-se adicionalmente a atividade sinérgica. Nanodispersões catiônicas de DBB exibiram atividade bactericida, fungicida e algicida, mesmo para patógenos MRs, sendo que a sua associação com outros antimicrobianos resultou em uma atividade sinérgica que poderia favorecer a produção de formulações com potencial para aplicação clínica humana e veterinária. / Microbial infections can be caused by bactérias, fungi, virus, parasites and algae. This study evaluated in vitro activity from cationic lipds formulations against multiresistant bacterias (MRs), fungi and e microalgae from human and veterinary. The Biocidal activity from bromide dioctadecildimetilammonium biceles (DBB) was evaluated against gram-negative bactérias β-lactamases producers and carbapenemases from broad spectrum with wide spread dissemination. For fungi (Candida albicans, Trichophyton rubrum) and microalgae (Prototheca zopfii), the biocidal activity from DBB wuth and without antifungal drugs (ex., miconazole, terbinafine e amphotericin B), was also evaluated by synergistic activity. Cationic nanodispertions of DBB has shown bactericidal, fungicidal and algicidal activity, even for MRs patogens, thus , the association with the others antimicrobials resulted in a synergistic activity wich could foment the production of the formulations with a potential application in human and veterinary medicine.

Infecções microbianas podem ser causadas por bactérias, fungos, vírus, parasitas e algas. O presente estudo avaliou a atividade in vitro de formulações de lípides catiônicos contra bactérias multirresistêntes (MRs), fungos e microalgas de interesse médico humano e veterinário. A atividade biocida de bicelas de brometo de dioctadecildimetilamônio (DBB) foi avaliada contra bactérias gram-negativas produtoras de β-lactamases de amplo espectro e carbapenemases mundialmente disseminadas. Para fungos (Candida albicans, Trichophyton rubrum) e microalgas (Prototheca zopfii), a atividade biocida de DBB, sem e com a associação a drogas antifúngicas (ex., miconazol, terbinafina e anfotericina B), foi avaliada determinando-se adicionalmente a atividade sinérgica. Nanodispersões catiônicas de DBB exibiram atividade bactericida, fungicida e algicida, mesmo para patógenos MRs, sendo que a sua associação com outros antimicrobianos resultou em uma atividade sinérgica que poderia favorecer a produção de formulações com potencial para aplicação clínica humana e veterinária. / Microbial infections can be caused by bactérias, fungi, virus, parasites and algae. This study evaluated in vitro activity from cationic lipds formulations against multiresistant bacterias (MRs), fungi and e microalgae from human and veterinary. The Biocidal activity from bromide dioctadecildimetilammonium biceles (DBB) was evaluated against gram-negative bactérias β-lactamases producers and carbapenemases from broad spectrum with wide spread dissemination. For fungi (Candida albicans, Trichophyton rubrum) and microalgae (Prototheca zopfii), the biocidal activity from DBB wuth and without antifungal drugs (ex., miconazole, terbinafine e amphotericin B), was also evaluated by synergistic activity. Cationic nanodispertions of DBB has shown bactericidal, fungicidal and algicidal activity, even for MRs patogens, thus , the association with the others antimicrobials resulted in a synergistic activity wich could foment the production of the formulations with a potential application in human and veterinary medicine.

Made available in DSpace on 2014-07-29T15:07:41Z (GMT). No. of bitstreams: 1 Eveline_Xavier.pdf: 537505 bytes, checksum: f46fc6f4fae888045c26180cb3ce26b0 (MD5) Previous issue date: 2008-08-05 / Mastitis is the most frequent disease in dairy cows, it causes great economic losses in herds and dairy industries and represents a hazard for public health. Among the etiologic agents identified, there is a predominance of bacteria species. However, several other groups of microorganisms may be present, one of those is a unicellular alga of the Prototheca genus, the Prototheca zopfii, an environmental agent of bovine mastitis. The infected cows usually have granulomatous changes in mammary tissue. Moreover microorganisms do not respond to routine therapy, resulting in the elimination of the infected animals. The main purposes of this study was to determine the occurrence of Prototheca zopfii on bulk tank milk in the state of Goiás and identify the limit of detection for Prototheca zopfii by two different approaches. We also wanted to know the samples pre-incubated with small concentration of Protoheca zopfii permits us to obtain positive growth in microbiological culture. We analyzed 473 samples from farms with high somatic cell count patterns but we did not detect Protoheca zopfii in any of those samples. The limit of sensitivity of the Prototheca zopfii was analyzed, in the approach of isolation the limit of detection was between 130 to 3,400 UFC/mL. In the approach of tally the limit was between 25 to 300 UFC/mL. It wasn t observed significant difference (p>0,05) between the approaches analyzed. However, significant statistical difference (p<0,05) occurred between the five types analyzed. After the pre-incubation at 37ºC/24h, the limit of sensibility was analyzed by the approach isolation, the limit was 30 UFC/mL, what means that the pre-incubation is so important to decrease the possibility of occurrence of false negative / Entre as enfermidades de grande preocupação para os bovinos leiteiros encontram-se as mastites, incluindo aquelas causadas por Prototheca spp., um agente ambiental, relacionado à mastite bovina que destaca-se pela gravidade das lesões que causam no tecido mamário e pela resistência a ações terapêuticas. Objetivou-se com o presente estudo determinar a ocorrência de Prototheca zopfii no leite cru refrigerado no estado de Goiás, identificar o limite de detecção deste patógeno pelos métodos de semeadura para isolamento, e semeadura para contagem e também verificar se a incubação de amostras de leite com baixo número de células da Prototheca zopfii permite a obtenção de resultados positivos na cultura microbiológica. Foram analisadas 473 amostras originárias de propriedades com histórico de elevada contagem de células somáticas e procedeu-se a avaliação do limite de sensibilidade para o agente por meio do método de semeadura para isolamento, cujo limite de detecção correspondeu à faixa de 130 a 3.400 UFC/mL. Quanto ao método de semeadura para contagem o limite estabelecido foi 25 a 300 UFC/mL. Portanto houve diferença estatística (p<0,05) entre os métodos analisados, porém, não houve diferença estatística (p>0,05) entre as cinco cepas analisadas. Após a incubação das amostras por 24 horas a 37ºC verificou-se que o limite de sensibilidade pelo método semeadura para isolamento diminui para 30 UFC/mL, diminuindo-se assim a possibilidade de ocorrência de falso negativo. Em nenhuma das amostras analisadas foi detectada a presença da Prototheca zopfi

Die Protothekenmastitis des Rindes ist eine therapieresistente, weltweit vorkommende Infektionskrankheit. Das ätiologische Agens, die farblose Alge Prototheca (P.) zopfii, kommt ubiquitär in feuchten Habitaten vor und verursacht fakultativ akute bis chronische Entzündungen des Rindereuters. Es gibt Hinweise auf das Vorkommen eines speziellen, Mastitis-assoziierten Biotyps von P. zopfii, der sogenannten Variante II. Durch die oft zu beobachtende endemische Ausbreitung in Milchviehbeständen sowie durch die nachhaltige Therapieresistenz, welche oft zum wirtschaftlichen Totalverlust der betroffenen Milchkühe führt, stellen Protothekenmastitiden beim Rind ein großes ökonomisches Problem für den betroffenen Betrieb dar. Da bisher nur sehr beschränkte Erkenntnisse zur lokalen und systemischen Immunantwort sowie zur Erregerausscheidung im Verlaufe der Protothekenmastitis des Rindes vorlagen, wurden die verschiedenen klinischen Stadien dieser Infektion serologisch, kulturell sowie durch Bestimmung der Zahl der somatischen Zellen in der Milch charakterisiert. Zu diesem Zwecke wurden drei verschiedene ELISA-Systeme entwickelt, die anschließend auch auf ihren möglichen Einsatz bei der Diagnostik der Protothekenmastitis hin untersucht wurden. Dies geschah in einem hochgradig an Protothekenmastitis erkrankten Milchviehbestand. Darüber hinaus wurden verschiedene Isolate von P. zopfii auxanographisch, biochemisch, serologisch und genetisch untersucht, um eine Differenzierung innerhalb der Algenspezies P. zopfii vornehmen zu können. Anhand der auxanographischen, biochemischen, serologischen und genetischen Untersuchungen war eine eindeutige Differenzierung von drei verschiedenen Bio-, Sero- und Genotypen innerhalb der Algenspezies P. zopfii möglich. Alle untersuchten Mastitisisolate konnten eindeutig der Variante II von P. zopfii zugeordnet werden, womit dieser Variante eine besondere epidemiologische Bedeutung bei der Entstehung der Protothekenmastitis des Rindes zu zukommen scheint. Die Untersuchungen dieser Arbeit zeigen, dass akut infizierte Tiere sowohl die höchsten Antikörperaktivitäten an IgG im Blutserum sowie an IgA und IgG1 im Milchserum als auch die höchsten Gehalte an somatischen Zellen in der Milch aufweisen. Chronisch infizierte Milchkühe weisen zum Teil sehr hohe Antikörperaktivitäten in der Milch auf und unterschieden sich nicht signifikant von akut infizierten Tieren. Demgegenüber weisen diese chronisch infizierten Tiere signifikant höhere IgG-Aktivitäten im Blutserum sowie IgA- und IgG1-Aktivitäten in der Milch auf als nicht infizierte Tiere. Somit ist eine eindeutige Differenzierung zwischen infizierten und nichtinfizierten Kühen möglich. Die ELISAs zum Nachweis von spezifischem IgA und IgG1 im Milchserum erwiesen sich als besonders geeignet, um infizierte Kühe zu identifizieren. Beide serologischen Testsysteme wiesen Sensitivitäten von 96,3 % für IgA sowie 92,6 % für IgG1 und Spezifitäten von 94,4 % (IgA) und 96,3 % (IgG1) auf. Demgegenüber wies der ELISA zum Nachweis von spezifischem IgG im Blutserum bei einer Spezifität von 100 % nur eine Sensitivität von 81,5 % auf. Die sehr gute Reproduzierbarkeit der Tests wurde durch Intra-Assay-Variationen von 6,08 % für den Nachweis von IgA im Milchserum und 7,20 % für IgG1 sowie durch die geringe Inter-Assay-Variation von 6,32 % (IgA) und 9,74 % (IgG1) belegt. Der Einsatz dieser Testsysteme bei der Sanierung eines hochgradig mit P. zopfii infizierten Milchviehbestandes zeigte, dass die serologische Diagnostik dem bisher gebräuchlichen kulturellen Erregernachweis bei der Identifikation intermittierender Erregerausscheider überlegen ist. Es wurde deutlich, dass 70,5% der infizierten Tiere die Erreger über einen Zeitraum von 12 Monaten permanent ausschieden und mindestens weitere 4,9 %, wahrscheinlich jedoch wesentlich mehr, dieser infizierten Tiere intermittierende Erregerausscheider waren. Somit scheint der serologische Erregernachweis für die Diagnostik der Protothekenmastitis des Rindes besser geeignet zu sein als die kulturelle Diagnostik. Dabei wurde die höchste Sensitivität durch die Kombination des Nachweises von spezifischem IgA und IgG1 im Milchserum erzielt. / Protothecosis is a severe, often endemic mastitis in cattle caused by colorless algae of the genus Prototheca. Only little and insufficient knowledge about the organism itself, and the host immune response to this infection existed. Therefore, the aim of this thesis was to characterize the local and systemic immune response and the possible elimination or persistence of the pathogen in the host. To gain more information on the specific immune response, different clinical stages of infection were characterized serologically, culturally, and by determination of the number of the somatic cells in milk. Three different ELISA systems were developed, which were also examined for their diagnostic application potential. For the investigations, a dairy herd highly infected with Prototheca zopfii and severe clinical manifestation of protothecal mastitis was used. The ELISA was evaluated using serum and whey from animals with different clinical stages of infection. As antibody isotypes, IgG in serum, and IgA and IgG1 in whey were used. In addition, different isolates of P. zopfii were biochemically, serologically, and genetically examined in order to allow a differentiation of individual isolates within the species P. zopfii. The biochemical, serological and genetic investigations allowed a clear differentiation of the three known Variants of P. zopfii. All examined mastitis isolates could be assigned to variant II of P. zopfii. Therefore, it can be concluded that this variant has a particular epidemiological significance in the etiology of bovine protothecal mastitis. The serological investigations showed high antibody activities during acute and chronic stage of infection. The antibody activity was low in chronically infected, but presently cultural negative animals and also in uninfected animals. A strong correlation was observed between whey IgA and whey IgG1 antibody activity and the count of somatic cells in milk. Whereas, only a weak correlation exists to the number of algae cells excreted with the milk. A sensitivity of 96 % and a specificity of 94 % were calculated for the ELISA based on IgA levels. The ELISA for detection of specific IgG1 in whey shows a sensitivity of 92,6 % and a specificity of 96,3 %. Intra-assay and interassay variations were calculated to be at 6.08 % and 6.32 %, respectively. Based on these data, these ELISAs are suitable for discrimination between infected and uninfected animals, and might therefore be used for the screening of affected herds. When used in the remediation of a high-grade infected dairy herd the serological showed clear advantages in the identification of intermittent shedders. By culturing of Prototheca from milk, it was shown that 70.5% of the infected animals were permanent shedders, whereas 4.9 % were intermittent shedders. Since intermittent shedders could be clearly identified serologically, but might not be recognized by culturing, it can be assumed that serological diagnostics is more suitable for the identification of inapparently infected, intermittent shedders.

Die Protothekenmastitis des Rindes ist eine therapieresistente, weltweit vorkommende Infektionskrankheit. Das ätiologische Agens, die farblose Alge Prototheca (P.) zopfii, kommt ubiquitär in feuchten Habitaten vor und verursacht fakultativ akute bis chronische Entzündungen des Rindereuters. Es gibt Hinweise auf das Vorkommen eines speziellen, Mastitis-assoziierten Biotyps von P. zopfii, der sogenannten Variante II. Durch die oft zu beobachtende endemische Ausbreitung in Milchviehbeständen sowie durch die nachhaltige Therapieresistenz, welche oft zum wirtschaftlichen Totalverlust der betroffenen Milchkühe führt, stellen Protothekenmastitiden beim Rind ein großes ökonomisches Problem für den betroffenen Betrieb dar. Da bisher nur sehr beschränkte Erkenntnisse zur lokalen und systemischen Immunantwort sowie zur Erregerausscheidung im Verlaufe der Protothekenmastitis des Rindes vorlagen, wurden die verschiedenen klinischen Stadien dieser Infektion serologisch, kulturell sowie durch Bestimmung der Zahl der somatischen Zellen in der Milch charakterisiert. Zu diesem Zwecke wurden drei verschiedene ELISA-Systeme entwickelt, die anschließend auch auf ihren möglichen Einsatz bei der Diagnostik der Protothekenmastitis hin untersucht wurden. Dies geschah in einem hochgradig an Protothekenmastitis erkrankten Milchviehbestand. Darüber hinaus wurden verschiedene Isolate von P. zopfii auxanographisch, biochemisch, serologisch und genetisch untersucht, um eine Differenzierung innerhalb der Algenspezies P. zopfii vornehmen zu können. Anhand der auxanographischen, biochemischen, serologischen und genetischen Untersuchungen war eine eindeutige Differenzierung von drei verschiedenen Bio-, Sero- und Genotypen innerhalb der Algenspezies P. zopfii möglich. Alle untersuchten Mastitisisolate konnten eindeutig der Variante II von P. zopfii zugeordnet werden, womit dieser Variante eine besondere epidemiologische Bedeutung bei der Entstehung der Protothekenmastitis des Rindes zu zukommen scheint. Die Untersuchungen dieser Arbeit zeigen, dass akut infizierte Tiere sowohl die höchsten Antikörperaktivitäten an IgG im Blutserum sowie an IgA und IgG1 im Milchserum als auch die höchsten Gehalte an somatischen Zellen in der Milch aufweisen. Chronisch infizierte Milchkühe weisen zum Teil sehr hohe Antikörperaktivitäten in der Milch auf und unterschieden sich nicht signifikant von akut infizierten Tieren. Demgegenüber weisen diese chronisch infizierten Tiere signifikant höhere IgG-Aktivitäten im Blutserum sowie IgA- und IgG1-Aktivitäten in der Milch auf als nicht infizierte Tiere. Somit ist eine eindeutige Differenzierung zwischen infizierten und nichtinfizierten Kühen möglich. Die ELISAs zum Nachweis von spezifischem IgA und IgG1 im Milchserum erwiesen sich als besonders geeignet, um infizierte Kühe zu identifizieren. Beide serologischen Testsysteme wiesen Sensitivitäten von 96,3 % für IgA sowie 92,6 % für IgG1 und Spezifitäten von 94,4 % (IgA) und 96,3 % (IgG1) auf. Demgegenüber wies der ELISA zum Nachweis von spezifischem IgG im Blutserum bei einer Spezifität von 100 % nur eine Sensitivität von 81,5 % auf. Die sehr gute Reproduzierbarkeit der Tests wurde durch Intra-Assay-Variationen von 6,08 % für den Nachweis von IgA im Milchserum und 7,20 % für IgG1 sowie durch die geringe Inter-Assay-Variation von 6,32 % (IgA) und 9,74 % (IgG1) belegt. Der Einsatz dieser Testsysteme bei der Sanierung eines hochgradig mit P. zopfii infizierten Milchviehbestandes zeigte, dass die serologische Diagnostik dem bisher gebräuchlichen kulturellen Erregernachweis bei der Identifikation intermittierender Erregerausscheider überlegen ist. Es wurde deutlich, dass 70,5% der infizierten Tiere die Erreger über einen Zeitraum von 12 Monaten permanent ausschieden und mindestens weitere 4,9 %, wahrscheinlich jedoch wesentlich mehr, dieser infizierten Tiere intermittierende Erregerausscheider waren. Somit scheint der serologische Erregernachweis für die Diagnostik der Protothekenmastitis des Rindes besser geeignet zu sein als die kulturelle Diagnostik. Dabei wurde die höchste Sensitivität durch die Kombination des Nachweises von spezifischem IgA und IgG1 im Milchserum erzielt. / Protothecosis is a severe, often endemic mastitis in cattle caused by colorless algae of the genus Prototheca. Only little and insufficient knowledge about the organism itself, and the host immune response to this infection existed. Therefore, the aim of this thesis was to characterize the local and systemic immune response and the possible elimination or persistence of the pathogen in the host. To gain more information on the specific immune response, different clinical stages of infection were characterized serologically, culturally, and by determination of the number of the somatic cells in milk. Three different ELISA systems were developed, which were also examined for their diagnostic application potential. For the investigations, a dairy herd highly infected with Prototheca zopfii and severe clinical manifestation of protothecal mastitis was used. The ELISA was evaluated using serum and whey from animals with different clinical stages of infection. As antibody isotypes, IgG in serum, and IgA and IgG1 in whey were used. In addition, different isolates of P. zopfii were biochemically, serologically, and genetically examined in order to allow a differentiation of individual isolates within the species P. zopfii. The biochemical, serological and genetic investigations allowed a clear differentiation of the three known Variants of P. zopfii. All examined mastitis isolates could be assigned to variant II of P. zopfii. Therefore, it can be concluded that this variant has a particular epidemiological significance in the etiology of bovine protothecal mastitis. The serological investigations showed high antibody activities during acute and chronic stage of infection. The antibody activity was low in chronically infected, but presently cultural negative animals and also in uninfected animals. A strong correlation was observed between whey IgA and whey IgG1 antibody activity and the count of somatic cells in milk. Whereas, only a weak correlation exists to the number of algae cells excreted with the milk. A sensitivity of 96 % and a specificity of 94 % were calculated for the ELISA based on IgA levels. The ELISA for detection of specific IgG1 in whey shows a sensitivity of 92,6 % and a specificity of 96,3 %. Intra-assay and interassay variations were calculated to be at 6.08 % and 6.32 %, respectively. Based on these data, these ELISAs are suitable for discrimination between infected and uninfected animals, and might therefore be used for the screening of affected herds. When used in the remediation of a high-grade infected dairy herd the serological showed clear advantages in the identification of intermittent shedders. By culturing of Prototheca from milk, it was shown that 70.5% of the infected animals were permanent shedders, whereas 4.9 % were intermittent shedders. Since intermittent shedders could be clearly identified serologically, but might not be recognized by culturing, it can be assumed that serological diagnostics is more suitable for the identification of inapparently infected, intermittent shedders.