There is little information on the distribution and diversity of Apicomplexan protozoal infections in the tissues of raptors in the United States. Protozoan encephalitis caused by Sarcocystis species and Toxoplasma gondii is being increasingly reported in raptors from various locations in the United States. To better determine the exposure of raptors to these Apicomplexan parasites, we examined breast and heart muscle tissue of raptors from the Carolina Raptor Center for the presence of Sarcocystis species, T. gondii and Neospora caninum via histology, Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) using DraI and HinfI enzymes (Sarocystis only). Of 187 available HandE stained tissue sections, 33 contained sarcocysts. Nineteen of these slides had a matching DNA sample to compare via PCR. Nine of these 19 were positive for Sarcocystis via ITS PCR. Using ITS PCR, we detected Sarcocystis DNA in 24 of 114 birds (21.1%). Further molecular differentiation using JNB primers showed that 9 of the 24 birds were positive for either S. neurona or S. falcatula. RFLP analysis of these 9 indicated that 4 were S. falcatula samples, and 3 were S. falcatula Arg samples that cut with both enzymes. Our Sarcocystis positive samples were also tested for S. calchasi, S. columbae and Sarcocystis sp. Ex. A. nisus using PCR primers designed for these species. These species are emerging in Europe and have already shown an expansion of their distribution. Two samples (14567 and 15203) suggestive of Sarcocystis sp. Ex. A. nisus were identified, as well as one sample (14567), which suggested the presence of S. columbae. None of these samples were confirmed by sequencing the amplicons and the other 22 samples were all negative for these parasites. Recent reports have demonstrated DNA of S. falcatula in the brain and muscles of great horned owls (Bubo virginianus), golden eagles (Aquila chrysaetos), and bald eagles (Haliaeetus leucocephalus) with encephalitis in rehabilitation centers in Indiana, Minnesota, and Virginia using PCR. DNA of S. calchasi has been found in CNS tissue of several species of birds suffering encephalitis in an aviary in California. Hawks (Accipiter species) are believed to be the source of infection. The prevalence of T. gondii was 18.4% (21 of 114) in these birds by PCR, but none were positive by histopathology. N. caninum prevalence in raptors has been poorly discussed in the literature. This parasite uses canids as the definitive host in its life cycle, and is considered to have a much more restricted host range than T. gondii. Thirty-five of 114 birds (30.7%) were found to be PCR positive for N. caninum, but no tissue cysts of N. caninum were observed in histological sections. Co-infection of 2 or all 3 species was detected in 16 of 114 birds (14%). This study demonstrates that there may be a higher prevalence of S. falcatula in raptors than was previously known, including more, as yet unknown, species of Sarcocystis capable of infecting raptors as intermediate hosts. Our PCR prevalence for T. gondii is similar to the serological prevalence for this parasite in raptors. The high PCR prevalence of N. caninum needs to be confirmed by sequencing the amplicons and the use of additional PCR primers. Information from the present study may help to inform zoos, aviaries and wildlife rehabilitation centers about parasite host diversity and reinforce the importance of preventative measures, such as making sure opossums (S. falcatula and S. falcatula-like), feral cats (T. gondii), and wild raptors (S. calchasi) do not have access to facilities. Insect control should also be emphasized because of their ability to serve as phoretic hosts and carry oocysts/sporocysts into zoos, aviaries, and rehabilitation centers. / Master of Science

<I>Neospora hughesi</I> is a recently described cause of equine protozoal myeloencephalitis (EPM). In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of <I>N. hughesi</I>. Only the gamma-IFNKO mice developed severe clinical disease following infection with <I>N. hughesi</I>. The most severe lesions were in the hearts of these mice. Two dogs fed the brains of mice, shown to contain <I>N. hughesi</I> tissue stages by cell culture and g-IFNKO mouse bioassay, did not shed <I>N. hughesi</I> oocysts over a 23 day observation period. We report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of <I>N. hughesi and N. caninum</I>. The newly defined proteins of <I>N. hughesi</I> are referred to as NhGRA6 and NhGRA7. From analysis of the sequences we found that there is a 14.8% difference in deduced amino acid sequence between NhGRA7 and NcGRA7, and a 4% difference between NhGRA6 and NcGRA6 in areas that could be compared. This thesis supports the identification of <I>N. hughesi</I> as a separate species from <I>N. caninum</I> and describes novel methods of distinguishing between the two. / Master of Science

Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission. / Ph. D.

Neospora caninum (Apicomplexa) é o agente causador da neosporose, descrita como a principal causa de aborto parasitário em gado bovino .Parasitos desse filo interagem e invadem as células hospedeiras através da secreção coordenada de proteínas do complexo apical, formado por diversas organelas, dentre elas as roptrias (ROPs), que desempenham papel fundamental no processo de infecção, associado à formação do vacúolo parasitóforo (PV), sobrevivência no ambiente intracelular e virulência do parasito. Essas proteínas podem ser caracterizadas em: proteínas de roptrias de pescoço (RONs), e proteínas de roptrias (ROPs). Além disso, algumas ROPs podem se diferenciar e, dessa maneira, constituem uma grande família de quinases e pseudo-quinases, denominada família de roptrias quinase (ROPKs). O objetivo deste estudo foi caracterizar as proteínas de roptrias NcROP15B (NcLIV_011700) e NcROP55 (NcLIV_031550) de N. caninum. As sequências de NcROP15B e NcROP55 foram alinhadas com homólogos de alguns microrganismos, incluindo apicomplexas, ao BLAST. NcROP15B apresentou identidade de 19% com as sequencias de ROP15 de T. gondii e Hammondia hammondi. NcROP55 mostrou identidade de 14% com a ROP37 de T. gondii e com ROP28 de Hammondia hammondi, e 15% com a ROP28 de Neospora caninum. Foram detectados domínios pertencentes à família de proteínas quinase para NcROP15B e NcROP55, e peptídeo sinal apenas para NcROP15B. Os primers foram delineados para amplificar regiões de cDNA de ambos os genes com diferentes tamanhos, denominados NcROP15B maior, NcROP15B menor, NcROP55 maior e NcROP55 menor. Os insertos foram subclonados (pGEM) e posteriormente ligados em plasmídeo de expressão pET28 em E. coli BL21(DE3) e a expressão recombinante induzida por IPTG. As formas recombinantes foram expressas com 30 kDa e 16 kDa (NcROP15B fragmento Maior e Menor, respectivamente) e NcROP55 Maior e Menor gerou um peso molecular de 19.9 kDa e 15 kDa, respectivamente. Após purificação, NcROP15B e NcROP55 foram utilizadas para obtenção de soros policlonais. Anti-ROP15B e anti-ROP55 reagiram com extrato de N. caninum em Western Blot 1D, tendo NcROP15B sido detectada com 35 kDa, próximo ao predito (32 kDa) e NcROP55 com aproximadamente 35 KDa, porém abaixo do predito (47.9 kDa). Na imunofluorescência confocal, NcROP-15B e NcROP55 exibiram padrão de localização na região perinuclear de taquizoítos de N. caninum. Os anticorpos anti-NcROP15B e anti-NcROP55 apresentaram, individualmente, capacidade limitada para inibir o processo de adesão/invasão de N. caninum, sendo 16% e 6,43% respectivamente. Quando os soros anti-NcROP15B e anti-NcROP55 foram associados, a a inibição da invasão aumentou para 62%. As proteínas NcROP-15B e NcROP55 podem representar quinases importantes no metabolismo de N. caninum, e podem estar relacionadas ao processo de invasão e proliferação do parasito. Dessa maneira, são possíveis alvos para se considerar no estudo de medidas preventivas, sendo necessários mais estudos para avaliar suas funções na sobrevivência intracelular e virulência de N. caninum. / Neospora caninum (Apicomplexa) is the causative agent of neosporosis, described as the main responsible of parasitic abortion in cattle. Parasites of this phylum interact and invade the host cells through a coordinated secretion of proteins of the apical complex, formed by organelles like rhoptries, which play a key role in the infection process, associated to the formation of the parasitophorous vacuole (PV), intracellular survival and parasite virulence. These proteins can be characterized in: neck rhoptry proteins (RONs), and rhoptry proteins (ROPs). Some ROPs can differentiate and thus constitute a large family of kinases and pseudo-kinases, called the kinase rhoptry family (ROPKs). The aim of this study was to characterize N. caninum NcROP15B (NcLIV_011700) and NcROP55 (NcLIV_031550) rhoptry proteins. The amino acid sequences of NcROP15B and NcROP55 were aligned with homologous proteins from some microorganisms, including apicomplexan on BLAST. NcROP15B showed a 19% identity with the ROP15 of T. gondii and Hammondia hammondi. NcROP55 had 14% of identity with ROP37 of T. gondii and with ROP28 of Hammondia hammondi, and 15% with ROP28 of Neospora caninum. Domains were detected belonging to the protein kinase family for NcROP15B and NcROP55, and signal peptide only for NcROP15B. Primers were designed to amplify cDNA regions of both genes, opting for fragments of different sizes, names as NcROP15B major, NcROP15B minor, NcROP55 major and NcROP55 minor. The inserts were subcloned (pGEM) and then ligated into pET28 expression plasmid in E. coli BL21 (DE3) and IPTG-induced recombinant expression. The recombinant forms were expressed with 30 kDa and 16 kDa (NcROP15B Major and Minor, respectively) and Major and Minor NcROP55 had molecular weights of 19.9 kDa and 15 kDa, respectively. After purification, NcROP15B and NcROP55 were used to obtain polyclonal serum. Anti-ROP15B and anti-ROP55 reacted with N. caninum extract in Western Blot 1D, with NcROP15B being detected at 35 kDa, near predicted (32 kDa) and NcROP55 at approximately 35 KDa, but below the predicted (47.9 kDa). At confocal immunofluorescence, NcROP-15B and NcROP55 exhibited a localization pattern at the perinuclear region of N. caninum tachyzoites. Anti-NcROP15B and anti-NcROP55 antibodies had, individually, limited ability to inhibit the N. caninum adhesion/invasion process (16 and 6,43%, respectively). When associated, anti-NcROP15B and anti-NcROP55 sera inhibition of invasion increased to 62%. NcROP-15B and NcROP55 proteins might represent important kinases in the metabolism of N. caninum with a possible role in the parasite invasion and proliferation process. Thus, they represent possible targets for preventive measures, but further studies are necessary to evaluate their functions in intracellular survival and virulence of N. caninum.

Neospora caninum é um parasita intracelular obrigatório do filo Apicomplexa, intimamente relacionado a Toxoplasma gondii e responsável por abortamento e perda da fertilidade em bovinos, o que acarreta prejuízos significativos na pecuária mundial. Como parte de seu ciclo intracelular, a primeira interação do parasita com a célula alvo é realizada por proteínas de superfície conhecidas como superfamília SRS (Surface Antigen Glycoprotein - Related Sequences). Proteínas SRS ou SAG tem sido alvo de intensas pesquisas devido ao seu padrão imunodominante, exibindo grande potencial como ferramenta de diagnóstico e/ou candidatos vacinais. Atualmente existem cinco genes pertencentes à extensa família de proteínas SRSs descritos na literatura científica para N. caninum, dos quais dois foram caracterizados de taquizoítas por serem altamente reconhecidos por soros de animais infectados: NcSRS29B (SAG1) e NcSRS29C (SRS2). Diante disso, este trabalho foi realizado com o objetivo de caracterizar as proteínas de superfície SRS, NcSRS57 e NcSRS67. Além disso, foi obtido um panorama geral de proteínas ancoradas por GPI de N. caninum na linhagem Nc-1. Dentre os homólogos apicomplexas, NcSRS67 apresentou maior identidade e similaridade com Hammondia hammondi (HHA_450490), enquanto NcSRS57 revelou maior identidade e similaridade com Toxoplasma gondii (TgSRS57). NcSRS67 e NcSRS57 apresentaram a terceira maior semelhança entre os homólogos envolvidos no alinhamento estrutural. Estas duas proteínas SRS possuem doze resíduos de cisteína conservados que por predição formam seis pontes dissulfeto distribuídas em dois domínios SRS (D1 e D2), formando sanduiches de folhas ? e ? hélices associadas entre si. A sequência codificadora de NcSRS67 (sem o peptídeo sinal e sem a âncora GPI) foi clonada e expressa constitutivamente no plasmídeo pCR-Blunt II-TOPO-His6x. A forma nativa de NcSRS67 apresentou massa molecular de 35 kDa (predito 30.6 kDa sem peptídeo sinal e sem âncora GPI). A sequência rNcSRS57 (sem o peptídeo sinal e sem a âncora GPI) foi clonada em pET32, entretanto apenas um fragmento de 92 pb foi traduzido em relação a sequência clonada de 1074pb, devido a presença de stop códon oculto. Este evento gerou rNcSRS57 com massa molecular abaixo do esperado (19,5 kDa). NcSRS57 nativa apresentou massa de 43 kDa (predito sem peptídeo sinal e sem âncora GPI 31.14 kDa). Os efeitos inibitórios dos anticorpos policlonais anti-rNcSRS67, anti-rNcSRS57 e a associação destes sobre a adesão/invasão de taquizoítas foram investigados in vitro, resultando em uma inibição de 20% para o anticorpo anti-rNcSRS67, 16% para o anticorpo anti-rNcSRS57 e 11% para a associação destes dois anticorpos. NcSRS67 foi localizada sobre parte da superfície de taquizoítas, ao contrário de NcSRS57, que abrangeu toda a área da superfície destes parasitas. Apesar das inúmeras tentativas, as formas nativas de NcSRS67 e NcSRS57 obtidas por eletroforese 2D não foram identificadas por MS/MS. O tratamento de taquizoítas de N. caninum com a enzima fosfolipase C fosfatidilinositol (PI-PLC) específica, seguido de análises por MS/MS também gerou a identificação de proteínas de N. caninum, ii dentre elas as proteínas mais abundantes já identificadas no secretoma de N. caninum, NcSRS29B (SAG1) e NcSRS29C (SRS2). Dessa forma, os resultados obtidos neste estudo agregam conhecimentos sobre o parasita N. caninum e revelam-se úteis na busca e seleção de novos alvos a serem investigados contra a neosporose. / Neospora caninum is an obligate intracellular parasite of the Apicomplexa phylum, closely related to Toxoplasma gondii and responsible for abortion and loss of fertility in cattle, resulting in significant losses in the worldwide livestock. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed by surface proteins known as SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SRS or SAG proteins have been subject of intensive research due to their immunodominant pattern, exhibiting great potential as a diagnostic tool and/or vaccine candidates. Currently there are five genes belonging to the SRS family of proteins described in the scientific literature for N. caninum. Two of these genes were isolated from tachyzoites due to their high sera reactivity of infected animals: NcSRS29B (SAG1) and NcSRS29C (SRS2). Therefore, this work was carried out with the aim of characterizing SRS surface proteins, NcSRS57 and NcSRS67. In addition, we have performed an overview of N. caninum GPI anchored proteins in the Nc-1 lineage. Our results showed that; among the apicomplexan homologues, NcSRS67 presented higher identity and similarity with Hammondia hammondi (HHA_450490), while NcSRS57 revealed greater identity and similarity with Toxoplasma gondii (TgSRS57). NcSRS67 and NcSRS57 presented the third major similarity between the homologues involved in the structural alignment. These two SRS proteins have twelve conserved cysteine residues predicted to form six disulfide bonds distributed in two SRS domains (D1 and D2), forming ?-sheet sandwiches and ?-helices associated with each other. The coding sequence of NcSRS67 (without the signal peptide and without the GPI anchor) was cloned and constitutively expressed in the plasmid pCR-Blunt II-TOPO-His6x. The native form of NcSRS67 has a molecular mass of 35 kDa (predicted 30.6 kDa without signal peptide and without the GPI anchor). The rNcSRS57 sequence (without the signal peptide and without the GPI anchor) was cloned into pET32, however only a 92 bp fragment was translated in contrast to the cloned sequence of 1074 bp, due to the presence of a hidden stop codon. This event generated rNcSRS57 with molecular mass lower than expected (19.5 kDa). Native NcSRS57 has 43 kDa mass (predicted without signal peptide and without GPI anchor 31.14 kDa). The inhibitory effects of the anti-rNcSRS67 polyclonal antibodies, anti-rNcSRS57 and the association of both on the adhesion/invasion of tachyzoites were investigated in vitro, resulting in a 20% inhibition for the anti-rNcSRS67 antibody, 16% rNcSRS57 and 11% for the association. NcSRS67 was localized on part of the surface of tachyzoites, unlike NcSRS57, which covered the entire surface area of these parasites. Despite of the iv numerous attempts, native forms of NcSRS67 and NcSRS57 obtained by 2D electrophoresis were not identified by MS/MS. The treatment of N. caninum tachyzoites with the specific phospholipase C phosphatidylinositol (PI-PLC) enzyme, followed by MS/MS analysis also generated the identification of N. caninum proteins, among them the most abundant proteins already identified in the secretome of N. caninum, NcSRS29B (SAG1) and NcSRS29C (SRS2). Thus, the results obtained in this study increase the knowledge of the parasite N. caninum and demonstrate to be useful in the search and selection of new targets to be investigated against neosporosis.

O Neospora caninum é um protozoário filogeneticamente relacionado a vários coccídeos de importância em medicina veterinária e humana, além de ser um parasita intracelular obrigatório capaz de causar abortamentos em bovinos e paralisia neuromuscular em cães. O estudo de antígenos e proteínas recombinantes de N. caninum gerou uma série de informações para um melhor diagnóstico e diferenciação deste protozoário e demais agentes a ele relacionados. No presente trabalho, propomos a expressão do clone do gene da proteína interna Nc56 de N. caninum em sistema procarioto (Escherichia coli), para avaliar sua antigenicidade frente a soros de bovinos imunoreativos contra Neospora caninum e Toxoplasma gondii. Para tanto, estabelecemos as condições ideais de expressão da proteína (indução por 4 horas e concentração de L-arabinose de 0,2%) e posteriormente avaliamos duas formas de purificação do produto recombinante (cromatografia de afinidade por metal imobilizado e eluição direta do gel de SDS-PAGE), constatando que a eluição de proteínas recombinantes diretamente do gel de SDS-PAGE mostrou-se o procedimento mais adequado para evitar a presença de proteínas contaminantes advindas do sistema procarioto de expressão. Foi selecionado um painel de soros bovinos (animais de campo), previamente testados pela reação de imunofluorescência indireta (RIFI) para avaliar sua reatividade para Neospora caninum e Toxoplasma gondii. Utilizamos o soro de um bezerro macho da raça Holandesa recém-nascido, colhido antes da administração de colostro materno (mãe soronegativa pela RIFI-Nc e RIFI-Tg) como controle negativo. O controle positivo foi obtido do mesmo bezerro após inoculação experimental de taquizoítos viáveis de N. caninum, quando este animal já apresentava seis meses de idade. O ELISA indireto foi realizado com microplacas de poliestireno com 96 cavidades de fundo chato sensibilizadas com 10 &mu;g/mL de proteína recombinante purificada Nc56 e apresentou altas densidades ópticas tanto para soros bovinos positivos na RIFI para N. caninum quanto para T. gondii, quando comparadas com a densidade óptica do soro controle negativo. A proteína recombinante purificada e o extrato bruto de proteínas solúveis em tampão desnaturante foram submetidos ao teste de Western-blotting, para avaliação de antigenicidade frente a soros de animais positivos e negativos para N. caninum e T. gondii, previamente testados por RIFI. Os resultados apresentados nas duas técnicas (ELISA e Western-blotting) demonstram que a proteína recombinante purificada Nc56 de N. caninum possui reatividade cruzada entre os dois coccídeos, não se prestando, portanto, para uso em sorodiagnóstico da neosporose. / Neospora caninum, a protozoa phylogenetically related to other coccidea, is an obligatory intracellular parasite, able to cause abortions in bovine and neuromuscular paralysis in dogs. Researches with Neospora caninum antigens and recombinant proteins leads to numerous information to a better diagnosis and differentiation of several coccidea. The purposes of this study were expressing the Neospora caninum inner protein Nc56 in a prokaryotic system (Escherichia coli) and evaluate its antigenicity with i>Neospora caninum and Toxoplasma gondii positive bovine sera. It was established the ideal conditions to express the protein (4 hour induction and 0,2% L-arabinose concentration) and, after that, two protein purification systems were assayed (immobilized metal affinity chromatography and elution from polyacrylamide gel) resulting in a better protein recuperation the elution method. A panel of previous RIFI-tested bovine sera was selected to evaluate the immune reactivity of Nc56 protein with Neospora caninum and Toxoplasma gondii positive sera. The negative control was obtained from a Neospora caninum and Toxoplasma gondii negative newborn male bovine and the positive control was obtained from the same animal (6 month old), after the inoculation of live Neospora caninum tachyzoits. The indirect ELISA assay was performed in 96-wells microtiters plates, coated with purified Nc56 recombinant protein (10 µg/mL), resulting in higher optical densities for Neospora caninum and Toxoplasma gondii positive sera, when compared with negative control. The purified recombinant protein and the crude extract of soluble proteins in denaturing binding buffer were submitted to Western-blotting to evaluate its antigenicity with Neospora caninum and Toxoplasma gondii/i> positive sera. The results obtained in both techniques shown that Nc56 purified recombinant protein has cross reactivity between both coccidea, therefore, it is not indicated to be used as antigen in neosporosis serodiagnosis.

Neospora caninum e Hammondia heydorni são coccídeos formadores de cistos proximamente relacionados com outros organismos heteroxenos pertencentes ao filo Apicomplexa, como Toxoplasma gondii e Hammondia hammondi. Para saber se o cachorro-do-mato (Cerdocyon thous), um canídeo silvestre comumente encontrado do nordeste da Argentina ao nordeste da América do Sul, pode ser hospedeiro definitivo de N. caninum, exemplares de cachorro-do-mato foram alimentados com tecidos de bovinos e bubalinos naturalmente infectados por N. caninum. As fezes dos cachorros-do-mato foram colhidas e examinadas diariamente para pesquisa de oocistos. No primeiro experimento, masseter e cérebro (peça inteira dos dois órgãos) colhidos de dois bovinos de aproximadamente dois anos de idade, foram picados, misturados e fornecidos às quatro raposas em dois dias consecutivos. Duas raposas eliminaram oocistos sub-esféricos não esporulados medindo 10-13µm no oitavo e nono dias pós-infecção, respectivamente. Uma das raposas eliminou oocistos por cinco e a outra nove dias. Amostras de DNA extraída dos de oocistos de cada dia de eliminação foram analisadas por PCR com primers grupo-específicos PCR-ITS-1 e espécie-especificos Nc5-NPCR. Todas as amostras foram positivas por PCR-HSP-70 e PCR-ITS-1, mas negativas por Nc5-NPCR. A sequência de nucleotídeos PCR-ITS-1 revelou 100% de identidade com seqüências homólogas de H. Heydorni. Concluímos que H. heydorni também utiliza o cachorro-do-mato (Cerdocyon thous) como hospedeiro definitivo. O cachorro-do-mato é usualmente reportado em contato com rebanhos em diversas regiões do Brasil. Então, é razoável inferir que estes carnívoros possam exercer um importante papel nos ciclos silvestre e doméstico da infecção por H. heydorni. Em um segundo experimento, sete exemplares de cachorro-do-mato (Cerdocyon thous) foram alimentados com tecidos de 3 búfalos (Bubalus bubalis) naturalmente infectados por N. caninum. As fezes das raposas foram analisadas 30 dias antes e trinta depois da infecção experimental e nenhuma das raposas eliminou oocistos N. caninum-like nas fezes. Estes resultados sugerem que o cachorro-do-mato (Cerdocyon thous) não é hospedeiro definitivo de N. caninum, entretanto estudos adicionais são necessários. / Neospora caninum and Hammondia heydorni are two cyst forming coccidians closely related to other apicomplexans, such as Toxoplasma gondii and Hammondia hammondi with a two host life-cycle. To clarify whether crab-eating fox (Cerdocyon thous), a wild carnivore commonly found from northern Argentina to northern South America, can be final hosts of N. caninum, foxes were fed on tissues of cows and water buffaloes naturally infected with N. caninum. Faeces were examined from 30 days before to 30 days after infection. In experiment 1, the whole masseter muscle and brain from two-year old bovines are collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered for four fox, in two consecutive days. Two foxes shed unsporulated oocysts subspherical in shape measuring 10-13µm, after eight and nine days post-infection, respectively. One of the foxes eliminated oocysts for five days, while the other fox shed oocysts for nine days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by a PCR carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S coding genes (PCR-ITS-1). These samples were also submitted to a N. caninum specific gene (Nc-5nPCR). All of them were positive by PCR-ITS-1 and negative by Nc-5nPCR. The PCR-ITS-1 nucleotide sequence revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. In experiment 2, seven fox consumed tissues from three N. caninum naturally infected water buffaloes. Feaces were examined from 30 days before to 30 days after infection and none fox shed N. caninum-like oocysts. These results suggest that the crab-eating fox cannot be definitive host of N. caninum; however additional studies will be necessary.

Os objetivos desse estudo foram avaliar a transmissão transplacentária por N. caninum em diferentes fases da gestação (Exp I) e avaliar diferentes tecidos de bovinos como meio de transmissão de N. caninum em cães jovens e adultos (Exp II). No Exp I, Três cadelas foram inoculadas com 108 taquizoítos de N. caninum na 3ª semana de gestação, três na 6ª semana e uma permaneceu como controle. Todas as cadelas infectadas, e pelo menos um de seus filhotes, apresentaram soroconversão a anticorpos anti-N. caninum pela Reação de Imunofluorescência Indireta. Verificou-se presença do parasita pela coloração de Hematoxilina-Eosina em Sistema Nervoso Central e pela PCR ITS-1 e RFLP em linfonodo, cérebro, coração e fígado. No Exp II, cães jovens e adultos receberam diferentes tecidos de bovinos naturalmente infectados com N. caninum, sendo coração, cérebro, masseter e fígado. Não houve soroconverteu. Apenas os cães jovens eliminaram oocistos de N. caninum ao ingerirem masseter (2 cães, 40%), coração (2 cães, 40%), fígado (1 cão, 33%) e cérebro (3 cães, 75%). / The objectives of this study were to evaluate transplacental transmission of N. caninum in different stages of pregnancy (Exp I) and evaluate different bovine tis.sues as means of transmission of N. caninum in young dogs and adults (Exp II). In Exp I, Three dogs were inoculated with 108 tachyzoites of N. caninum in the third week of gestation, three at 6 sixth week and one remained as a control. All infected dogs, and at least one of their offspring, seroconverted to anti-N. caninum antibodies by Immunofluorescence Assay. There was presence of the parasite by Hematoxylin- Eosine exam in Central Nervous System and by PCR and RFLP ITS-1 in lymph node, brain, heart and liver. In Exp II, young and adult dogs received different tissues of cattle naturally infected with N. caninum: heart, brain, liver and masseter. None seroconverted. Only the young dogs shed oocysts of N. caninum by eating masseter (2 dogs, 40%), heart (2 dogs, 40%), liver (one dog, 33%) and brain (3 dogs, 75%).

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-02-09T13:28:20Z No. of bitstreams: 1 PGCA15MA194.pdf: 586318 bytes, checksum: 3ad784366608195f04409160c5d88990 (MD5) / Made available in DSpace on 2018-02-09T13:28:20Z (GMT). No. of bitstreams: 1 PGCA15MA194.pdf: 586318 bytes, checksum: 3ad784366608195f04409160c5d88990 (MD5) Previous issue date: 2015-12-10 / FUMDES / In order to investigate the prevalence of antibodies against N. caninum in beef cattle in the mountainous region of Santa Catarina, during the months of January 2013 to September 2015 were harvested 507 cattle blood samples from 16 of 18 counties affiliated with the Association of Municipalities of the Mountain Region (AMURES), for execution the Reaction of immunofluorescence (IFA) for the detection of antibodies (≥1:100) IgG against N. caninum. Data about sex, age and origin of the animals were obtained through the records in SISBOV (Brazilian Service of Production Chain Traceability of Cattle and Buffaloes), and were tabulated for statistical analysis (Exact Test Fisher and Chi-Square, P≤0.05) to correlate the serological results with the analyzed variables. The survey revealed that of the 507 samples tested, 70 were positive, which indicated a prevalence of 13.81%. The bonds were found 1:100 (16), 1:200 (22), 1:400 (17), 1:800 (nine), 1:1600 (four) and 1:3200 (two). Of the positive samples 32.86% (23/70) were males and 67.14 %% (47/70) female. Statistical analysis showed no significant difference regarding the variables gender (P=0.1072), age (P=0.4116) and county (P=0.6838). Correlation was observed (P<0.05) between serology and the gender and age when examined together. In 13 of the 16 municipalities at least one positive sample was observed and in all age groups assessed positive animals were identified. Although the seroprevalence observed in this study was relatively low, the infection by N. caninum in beef cattle is widespread in the region studied / Com o objetivo de pesquisar a prevalência de anticorpos contra N. caninum em bovinos de corte na Região Serrana de Santa Catarina, entre os meses de Janeiro de 2013 a Setembro de 2015 foram colhidas 507 amostras de sangue de bovinos, provenientes de 16 dos 18 municípios afiliados à Associação dos Municípios da Região Serrana (AMURES), para a realização da Reação da Imunofluorescência Indireta (RIFI) para a detecção de anticorpos (≥1:100) IgG contra N. caninum. Dados acerca do sexo, idade e procedência dos animais foram obtidos por meio dos registros no SISBOV (Serviço Brasileiro de Rastreabilidade da Cadeia Produtiva de Bovinos e Bubalinos), e foram tabulados para a análise estatística (Testes exato de Fisher e do Qui-Quadrado, P≤0,05) para correlacionar os resultados da sorologia com as varáveis analisadas. O exame revelou que das 507 amostras analisadas, 70 foram positivas, o que indicou uma prevalência de 13,81%. Os títulos encontrados foram de 1:100 (16), 1:200 (22), 1:400 (17), 1:800 (nove), 1:1600 (quatro) e 1:3200 (dois). Das amostras positivas 32,86% (23/70) foram de machos e 67,14%% (47/70) de fêmeas. A análise estatística não demonstrou diferença significativa com relação às variáveis sexo (P=0,1072), idade (P=0,4116) e município (P=0,6838). Foi observada correlação (P<0,05) entre a sorologia e as variáveis sexo e idade quando analisadas conjuntamente. Em 13 dos 16 municípios ao menos uma amostra positiva foi observada e em todas as faixas etárias avaliadas foram identificados animais positivos. Embora a soroprevalência observada no presente estudo foi relativamente baixa, a infecção por N. caninum em bovinos de corte está amplamente distribuída na região estudada

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T13:47:39Z No. of bitstreams: 1 DISS_2014_Ricardo Vilas Boas.pdf: 6795713 bytes, checksum: 1ad10c8b43fdf390a1ec7efe5f9c145d (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-07T15:53:07Z (GMT) No. of bitstreams: 1 DISS_2014_Ricardo Vilas Boas.pdf: 6795713 bytes, checksum: 1ad10c8b43fdf390a1ec7efe5f9c145d (MD5) / Made available in DSpace on 2017-11-07T15:53:07Z (GMT). No. of bitstreams: 1 DISS_2014_Ricardo Vilas Boas.pdf: 6795713 bytes, checksum: 1ad10c8b43fdf390a1ec7efe5f9c145d (MD5) Previous issue date: 2014-02-28 / A neosporose é uma enfermidade parasitária considerada uma das principais causas de abortamento em bovinos em todo o mundo. Objetivou-se pesquisar a prevalência de anticorpos anti-Neospora caninum em fêmeas bovinas e possíveis fatores de risco para a infecção desses animais. Para isso, foram analisados 621 amostras de soro sanguíneo procedentes de 63 propriedades com rebanho leiteiro de agricultura familiar no município de Ji-Paraná, principal bacia leiteria do estado de Rondônia, as quais foram submetidas a Reação de Imunofluorescência Indireta (RIFI), sendo considerados bovinos positivos amostras com titulo 100 para N. caninum. Do total de amostras analisadas(66/621), encontrou-se uma prevalência de 10,61% (IC 95% 8,04-13,86%). Os títulos variaram de 100 a 1600, assim distribuídos: 22 (33,33%) amostras de soro apresentaram título de 100; 21 (31,82%) título de 200; 16 (24,24%) título de 400, três (4,55%) título de 800 e quatro (6,06%) amostras apresentaram título de 1600. A prevalência de anticorpos anti-N. caninum por propriedade foi de 60,31% (38/63), sendo que apenas as variáveis ocorrências de aborto e nascimento de bezerros fracos tiveram associação com a ocorrência de propriedades soropositivas para N. caninum. Conclui-se que as fêmeas bovinas leiteiras na região estão expostas à infecção por N. caninum e que a presença de determinados problemas reprodutivos, como aborto e nascimentos de bezerros fracos, estão associados à ocorrência de neosporose no rebanho da região. / The neosporosis is considered a parasitic disease leading cause of abortion in cattle worldwide. The aim of this study was to investigate the prevalence of antibodies anti- Neospora caninum of dairy cows and associate to possible risk factors infection. 621 serum samples taken from dairy cows on 63 familiar agriculture farms in the county of Ji-Paraná, in the main milk producing region of Rondônia, Brazil, were evaluated by means Immunofluorescence Assay (IFAT). Samples were considered positive with a titer 100. Among all the samples analyzed (66/621), the prevalence was 10.61% (IC 95% 8,04-13,86%). The titers of antibodies ranging between 100 and 1600, distributed as follows: 22 (33.33%) serum samples had titers of 100, 21 (31.82%) title 200; 16 (24.24%) title 400, three (4.55%) title 800 and of four (6.06%) samples had titers of 1600. The prevalence of anti-N. caninum in farms was 60.31% (38/63), It is concluded that the dairy cows in the area studied are exposed to infection by N. caninum and that the presence of reproductive problems, such as abortion, and weak calves born are associated with the occurrence of neosporosis in cattle in the county.