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Neospora caninum is a protozoan which can cause abortions in caprines. However, information regarding the humoral immune response and the occurrence of reproductive disorders is scarce. This is the first study in which the kinetics of antibodies is studied in pregnant goats naturally infected by N. caninum, as well as their respective conceptuses. The subclasses of IgG (IgG1 and IgG2) were also evaluated in pregnant goats. Reproductive problems related to neosporosis (abortion and stillbirth) occurred in 15.38% of the goats. There was a statistically significant association between the increased titres of maternal IgG in the second half of the gestational period with the occurrence of endogenous transplacental transmission. The rate of congenital transmission was 77%. During the gestational period of the seropositive goats, there was mainly a predominance of the subclass IgG2, although mixed patterns of IgG2–IgG1 and the IgG1 pattern were also observed. These results indicate that N. caninum is responsible for the occurrence of important alterations in the humoral immune response of naturally infected goats, and is also a potential causative agent for reproductive disorders in goats. The high proportion of infected conceptuses reinforces the suggestion that congenital infection is one of the main routes of parasite transmission in goats.

Um surto de compactação primária de abomaso associada ao consumo de silagem de girassol ocorreu num rebanho bovino leiteiro do estado de Minas Gerais, do qual seis de 21 vacas secas em fase final de gestação e que recebiam silagem de girassol morreram. Os bovinos que adoeceram apresentaram distensão abdominal, fezes ressecadas e escassas e, nos bovinos que morreram, eram pastosas, escuras e fétidas; nesses havia também desidratação acentuada e palidez de mucosas. Três bovinos foram necropsiados e os achados patológicos eram constituídos principalmente por distensão acentuada do abomaso associada a grande quantidade de conteúdo alimentar ressecado coberto por coágulos de sangue. No abomaso havia úlceras, com perfuração da parede em um dos bovinos. A alimentação de bovinos de maior exigência nutricional com silagem de girassol de características indesejáveis como única fonte de volumoso foi o principal fator para a compactação de abomaso nos casos apresentados. An outbreak of primary abomasal impaction associated with consumption of sunflower silage occurred in a dairy cattle herd from the state of Minas Gerais. Six of 21 dry cows in late pregnancy fed with sunflower silage died. Cattle that became ill presented abdominal distension and scant and dried feces. In the animals that died, severe dehydration, pale mucous membranes, dark, sticky and stinking feces were seen. Three cows were necropsied and the main pathological findings consisted of severe abomasal distension associated with a large quantity of dried alimentary content covered by bloody clots. Ulcers were found in abomasum, with perforation in one cow. Feeding cattle with high nutritional needs, with sunflower silage of undesirable characteristics as unique source of roughage was the main cause for the abomasal impaction.

Foram analisados 14 equídeos (13 equinos e 1 muar) com diagnóstico clínico e histológico de raiva provenientes de quatro regiões do Brasil. O curso clínico médio foi de quatro dias de evolução, incluindo incoordenação motora, paralisia dos membros pélvicos, paresia dos membros torácicos e decúbito. Os achados histopatológicos caracterizaram-se por meningoencefalite e meningomielite não supurativa com infiltrado perivascular linfoplasmocitário. Corpúsculos de Negri foram observados em 64,28% (9/14) dos casos, principalmente na medula espinhal cervical e nos neurônios de Purkinje do cerebelo. Em 55,55% (5/9) dos equídeos analisados tiveram resultado positivo pela técnica de imunofluorescência direta para raiva. Todos os casos foram positivos na imuno-histoquímica para raiva, cujas reações foram mais evidentes no córtex temporal, córtex occipital e medula espinhal cervical. A técnica de imuno-histoquímica foi essencial para confirmar todos os casos de raiva nos equídeos. Thirteen horses and one mule from four regions of Brazil with clinical and histological diagnosis of rabies were analyzed. The clinical course lasted four days, with motor incoordination, paresis and paralysis of the legs, and decubitus. The main histopathological findings were lymphoplasmacytic meningoencephalitis and non-suppurative meningomyelitis associated with characteristic Negri bodies in 64.28% (9/14) cases, mainly in the cervical spinal cord and in Purkinje neurons of the cerebellum. In 55.55% (5/9), the animals examined were positive by for the fluorescent antibody test for rabies. All animals were positive in immunohistochemistry for rabies, whose immunolabeling were more evident in the temporal cortex, occipital cortex and cervical spinal cord. Immunohistochemistry procedure was essential to confirm all cases of rabies in the equids.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:56:07Z No. of bitstreams: 1 S0102-09352008000400009.pdf: 616367 bytes, checksum: 6d3fc9513e6dd65af61791f8c233eeba (MD5) Made available in DSpace on 2013-08-22T18:56:07Z (GMT). No. of bitstreams: 1 S0102-09352008000400009.pdf: 616367 bytes, checksum: 6d3fc9513e6dd65af61791f8c233eeba (MD5) Previous issue date: 2008-08-01 Made available in DSpace on 2013-09-30T19:49:42Z (GMT). No. of bitstreams: 2 S0102-09352008000400009.pdf: 616367 bytes, checksum: 6d3fc9513e6dd65af61791f8c233eeba (MD5) S0102-09352008000400009.pdf.txt: 14243 bytes, checksum: 0e3857dd785e6b0949fcbf388696951f (MD5) Previous issue date: 2008-08-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:15:16Z No. of bitstreams: 2 S0102-09352008000400009.pdf: 616367 bytes, checksum: 6d3fc9513e6dd65af61791f8c233eeba (MD5) S0102-09352008000400009.pdf.txt: 14243 bytes, checksum: 0e3857dd785e6b0949fcbf388696951f (MD5) Made available in DSpace on 2014-05-20T13:15:16Z (GMT). No. of bitstreams: 2 S0102-09352008000400009.pdf: 616367 bytes, checksum: 6d3fc9513e6dd65af61791f8c233eeba (MD5) S0102-09352008000400009.pdf.txt: 14243 bytes, checksum: 0e3857dd785e6b0949fcbf388696951f (MD5) Previous issue date: 2008-08-01 Uma cadela da raça Yorkshire Terrier apresentou hematêmese, distensão abdominal, poliúria e polidipsia. Após o exame clínico do paciente, estabeleceu-se a suspeita clínica de nefropatia. Os resultados dos exames laboratoriais revelaram anemia normocítica normocrômica e concentrações séricas de uréia (306mg/dl) e de creatinina (3,6mg/dl) acima dos valores de referência. Ao ultra-som bidimensional observaram-se áreas císticas hipoecóicas e anecóicas, padrão renal hipercogênico e perda da relação corticomedular. À necropsia, a região medular apresentou grande quantidade de cistos pequenos em meio a tecido conjuntivo fibroso. A lesão tubulointersticial cortical foi a responsável pela insuficiência renal resultante. Firmou-se diagnóstico de nefrite tubulointersticial. A Yorkshire Terrier bitch presented hematemesis, abdominal distention, poliuria, and polidipsia. After clinical examination, it was established a clinical suspect of nephropathy. The complementary exams showed normochromic and normocytic anemia and serum concentrations of ureia (306mg/dl) and creatinine (3.6mg/dl) above the references values. At two-dimensional ultrasound hipoechoic and anechoic cystic areas were observed, as well as hiperechogenic renal pattern and corticomedullary ratio loss. At necropsy, the medullary region presented many small cysts in a fibrous connective tissue. Cortical tubulointesticial lesion was responsible to the resulting renal failure. It was established a tubulointersticial nephritis diagnosis. UNESP Faculdade de Ciências Agrárias e Veterinárias UFLA Departamento de Medicina Veterinária UFMG EV UNESP Faculdade de Ciências Agrárias e Veterinárias

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:23:48Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:35:22Z : No. of bitstreams: 1 2-s2.0-78149443638.pdf: 1599546 bytes, checksum: a8b7d0c3dd7c2b4c34b170ba23d97ba3 (MD5) Made available in DSpace on 2014-05-27T11:23:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-01 Baccharis coridifolia is one of the most important poisonous plants to cattle in the South of Brazil. The plant provokes necrotic lesions in the lymphoid tissues and in the gastrointestinal tract of cattle. Experimental administration to mice produces most of the lesions seen in the lymphoid tissues of cattle. This study was conducted to search possible differences in the susceptibility of T and B lymphocyte subpopulations. Lymph nodes, spleen, thymus and gut-associated lymphoid tissue (GALT) of cattle and mice experimentally poisoned were evaluated. The results were evaluated based on cell populations affected or remaining in the organs. Immunostaining for B lymphocytes (anti-BLA-36) identified the germinal center of follicles of the lymph node, spleen and GALT in both species. Immunostaining for T lymphocyte (anti-CD3) identified the paracortical area of the germinal centers of the lymph nodes and GALT, the periarteriolar area of the spleen, and the whole thymus both in cattle and mice. Experimentally poisoned cattle and mice shows necrosis of the germinal center of secondary follicles of the lymph nodes, spleen and GALT, where necrotic cells were immunostained for B and less often for T lymphocyte. Necrotic cells in the paracortical region of the lymph node were less often and were immunostained. Necrotic lesions of the thymus were seen only in mice, with positively stained for T lymphocyte. The distribution of the lesions in the lymphoid tissues and the immunostaining in necrotic cells suggested that the active principles of the plant are cytotoxic to B and T cells. Departamento de Medicina Veterinária Universidade Federal de Lavras, Lavras, MG Departamento de Patologia Veterinária Universidade Estadual Paulista, Jaboticabal-SP Departamento de Patologia Veterinária Universidade Estadual Paulista, Jaboticabal-SP

The poisonous plant Baccharis coridifolia causes necrosis in lymphoid tissues and the gastrointestinal tract of cattle, horses, sheep and rabbits. An experimental poisoning of mice was undertaken to establish an experimental model in a laboratory animal specie. A single 5 to 8-g/kg dose of a suspension of the plant was administered by gavage to 11 mice. To 3 other control mice, the same volume of water was administered. Plant-dosed mice manifested clinical effects after 12 h: tachipnea, trembles, dehydration and prostration. Most of the dosed mice died 14 to 33 h after plant administration--3 survived for 12 d. Six mice had remarkable necrosis of the germinative center of secondary follicles in lymph nodes and spleen; 3 mice had necrosis of lymphoid tissues in intestine and thymus. Mice reproduce most of the lesions observed in naturally poisoned cattle and the use of this specie as an experimental model is valid.

Relata-se um caso de porfiria eritropoética congênita em um bovino Holandês Preto e Branco, fêmea, de 9 meses de idade, proveniente da cidade de Machado, MG. O quadro clínico caracterizava-se pela presença de lesões crônicas de fotossensibilizaçâo observadas nas partes não pigmentadas do corpo, que apareceram após a exposição do animal à luz solar, aos 4 meses de idade. Além da fotossensibilizaçâo, as alterações macroscópicas mais evidentes foram coloração marrom-avermelhada dos ossos e marrom-rosada dos dentes, causadas pela deposição de porfirina. A case of bovine congenital erythropoietic porphyria in a 9-month-old Holstein female, from Machado, Minas Gerais, Brazil is described. The calf showed chronic cutaneous photosensitization, limited to white-haired areas, when exposed to sun-light. Reddish-brown discoloration of bones and brownish-pink discoloration of teeth, caused by deposition of porphyrin, were observed.

Um caso de "lechiguana" bovina, é relatado em bovino proveniente de Fama, MG, que apresentou aumento de volume de aproximadamente 30x8cm, de consistência firme e com superfície ulcerada, localizado no lado esquerdo da região toracolombar. As lesões microscópicas caracterizaram-se por paniculite piogranulomatosa proliferativa, com linfangite e microabscessos eosinofilicos. Em alguns abscessos havia rosetas (Splendore-HoeppIi) associadas a bactérias Gram-negativas. Após a biópsia o animal foi tratado com 3g diárias de cloranfenicol, por 5 dias. Houve total recuperação dentro de 30 dias. A case of a disease known as bovine "lechiguana"' in a cow fron Fama, Minas Gerais is described. The cow presented a 30x8 cm hard swelling covered by ulcerated skin and located at the left side of the dorsum. Histologically there was proliferative piogranulomatous paniculitis with lymphamgitis and eosinophilic microabscesses. In some of the abscessos there were rosettes (Splendore-HoeppIi) associated with Gram-negative bacteria. After a biopsy was performed the animal was treated with 3g/day cloranphenicol for 5 days with complete recovery within 30 days.

RESUMO É descrito um surto de aflatoxicose em suínos Duroc de 2 a 6 meses, que consumiam ração contaminada por 519 ppb de aflatoxinas. A morbidade foi de 97%. A mortalidade foi alta, mas não determinada com exatidão. Os sinais clínicos incluíam recusa do alimento, pouco ganho ou perda de peso, diarreia e icterícia. Lesões subagudas e crônicas foram observadas em 4 porcos necropsiados. Nas lesões subagudas o fígado era escuro, com áreas centrolobulares hemorrágicas deprimidas, havia edema da parede da vesícula biliar e bile viscosa. Nos casos crônicos o fígado apresentava-se firme e amarelo-alaranjado. Havia líquido citrino nas cavidades e edema no cólon espiral. Havia avermelhamento da mucosa gástrica da região fúndica. Alterações subagudas eram necrose e hemorragias centrolobulares, hepatomegalocitose, fibrose e hiperplasia ductal. Nos casos crônicos havia fibrose, hiperplasia ductal, megalocitose e degeneração gordurosa hepatocelular. O diagnóstico de aflatoxicose foi feito baseado nos sinais clínicos, nos achados morfológicos e na presença de níveis significativos de aflatoxina na ração dos porcos (B1 = 484,3ppb, B2 = 110,8ppb, G1 = 343,7ppb e G2 = 99,2ppb). Frangos submetidos à mesma alimentação também desenvolveram lesões da toxicose. SUMMARY An outbreak of aflatoxicosis in 2 to 6 monthold Duroc swine fed on ration with 519ppb of aflatoxins is reported. Morbidity was 97% and mortality was high but not exactly determined. Clinical signs included feed refusal, poor weight gain or loss of weight, diarrnea and icterus. Subacute and chronic changes were observed in four necropsied pigs. In subacute cases the liver was dark and had depressed hemorrhagic centrilobular areas; there was edema of gallbladder and inspissated bile. In chronic cases the liver was firm orange-yellow and there was yellowish effusions in the organic cavities and edema of the spiral colon. The gastric mucosa of the fundus was dark red. Histologically the subacute changes of the liver were centrilobular necrosis and hemorrhage. hepatocellular megalocytosis, fibrosis, and ductal hyperplasia. In chronic cases there were fibrosis, biliary hyperplasia, hepatomegalocytosis and hepatocellular fatty change. Aflatoxicosis was confirmed up on the clinical signs and lesions, and up on the detection significant levels of aflatoxin on the ration fed to pigs (B1 = 84.3ppb, B2 = 110.8ppb, G1 = 343.7ppb, and G2 = 99.2ppb). Chicks fed the same ration developed aflatoxicosis.

The Brazilian tannery industry produces annually an enormous amount of scraps and leather shavings impregnated with chromium due to the tanning process. Technologies have been developed to remove chromium from this material. The resultant residue from the chromium removal process is high in protein with a high digestibility, suggesting that this residue may be used to feed monogastric animals. However, due to the nature of this residue, there is a risk of chromium poisoning and the introduction of botulism. The aim of this work was to establish the risks of adding tannery residues to animal feeds, using rats as an experimental model. Forty-eight Wistar rats were placed under eight treatments in a 2 by 4 factorial arrangement. The rats were fed over 60 days with a standard AIN-93 diet and the treatments consisted of replacing part of the diet with 0, 25, 37.5 or 50% of the chromium-tanned leather residue, in natura, known as ‘wet blue’, or the same percentages of this residue after chromium extraction. Industrial processing was able to remove 70–80% of the chromium from the residue. This high chromium level had a negative effect on the weight gain of the animals and caused lesions in the kidneys especially due to the chromium extraction material, suggesting that the removal process increases the biological activity of chromium, making it nefrotoxic. The gravity of this effect was directly proportional to the inclusion level. The results of this study indicate that until industrial processing is refined so that more of the chromium residue is removed, the use of the tannery residues in animal feeds is not safe.

O presente estudo avaliou a participação de agentes bacterianos e virais em abortos em bovinos de propriedades rurais do sul de Minas Gerais. Foi realizada análise histopatológica e imuno-histoquímica dos casos de aborto recebidos pelo Setor de Patologia Veterinária da Universidade Federal de Lavras no período de 1999 a 2013. De 60 fetos analisados, em 30 (50%) foram observadas lesões microscópicas. Destes, oito apresentavam lesões compatíveis com infecção por agentes bacterianos e três apresentaram lesões sugestivas de agentes virais. Dos abortos bacterianos, um feto tinha lesões compatíveis com leptospirose, caracterizadas por icterícia e colestase, nefrite intersticial linfoplasmocítica e nefrose tubular. Sete fetos apresentaram pneumonia ou broncopneumonia purulenta; num deles havia também pleurite e peritonite fibrinosas; e em dois desses fetos houve imunomarcação para Brucella abortus. Dos três fetos com lesões sugestivas de aborto viral ocorreu imunomarcação anti-Herpesvírus bovino em um. Os resultados demonstram a ocorrência de abortos de origem bacteriana e viral na Região do estudo e que medidas profiláticas devem ser adotadas nas propriedades. O trabalho demonstra também que a imuno-histoquímica (IHQ); associada à histopatologia; é uma ferramenta útil e viável para o diagnóstico, especialmente quando provas microbiológicas e/ou sorológicas não estão disponíveis.

Neospora caninum has been described as a parasite that sporadically causes reproductive problems in goats. Several aspects of the pathogenesis of neosporosis in naturally infected goats remain to be established. The aims of the present study were to characterize the placental lesions in goats naturally infected by N. caninum and to evaluate several diagnostic techniques for effective detection of this protozoan in the goat placenta. Some placentas in this study originated from abortion and stillbirth in which there were severe lesions. The lesions were characterized mainly by necrosis involving the mesenchyme of the chorionic villi and trophoblast cells often alongside mononuclear inflammation and in some cases with neutrophilic infiltration. N. caninum DNA was detected in these placentas, but parasite structures were not visualized through immunohistochemistry (IHC). However, five of 11 placentas from N. caninum-infected goats that gave birth to healthy kids had histological lesions characterized by mononuclear inflammation. Of these 11 placentas, N. caninum DNA was detected in seven, and N. caninum tachyzoites were detected in only one of these seven placentas using IHC. The present study demonstrates that severe lesions in the placenta are associated with abortion and stillbirth in caprine neosporosis and the placental alterations are likely involved in abortion pathogenesis. Moreover, the results highlight the importance of using more than one diagnostic technique for the detection of the protozoan in placentas because N. caninum cannot be reliably detected by histological and immunohistochemical tests.

Background Neospora caninum is an apicomplexan protozoan that is considered one of the main agents responsible for abortion in ruminants. The lesions found in the central nervous system (CNS) of aborted fetuses show multifocal necrosis, gliosis, and perivascular cuffs of mononuclear cells, but the inflammatory and glial cells have not been immunophenotypically characterized. The lesions in the CNS of infected adult animals have rarely been described. Therefore, in this study, we characterized the lesions, the immunophenotypes of the inflammatory and glial cells and the expression of MHC-II and PCNA in the CNS of goats infected with N. caninum. The CNS of eight aborted fetuses and six adult male goats naturally infected with N. caninum were analyzed with lectin histochemistry (RCA1) and immunohistochemistry (with anti-CD3, −CD79α, −GFAP, −MHC-II, and -PCNA antibodies). All animals were the offspring of dams naturally infected with N. caninum. Results The microscopic lesions in the CNS of the aborted fetuses consisted of perivascular cuffs composed mainly of macrophages (RCA1+), rare T lymphocytes (CD3+), and rare B lymphocytes (CD79α+). Multifocal necrosis surrounded by astrocytes (GFAP+), gliosis composed predominantly of monocytic-lineage cells (macrophages and microglia, RCA1+), and the cysts of N. caninum, related (or not) to the lesions were present. Similar lesions were found in four of the six male goats, and multinucleate giant cells related to focal gliosis were also found in three adult goats. Anti-GFAP immunostaining showed astrocytes characterizing areas of glial scarring. Cysts of N. caninum were found in three adult male goats. The presence of N. caninum was evaluated with histopathology, immunohistochemistry, and PCR. Immunohistochemistry demonstrated anti-PCNA labeling of macrophages and microglia in the perivascular cuffs and the expression of MHC-II by microglia and endothelial cells in the CNS of the aborted fetuses and adult male goats. Conclusions Macrophages and microglia were the predominant inflammatory cells in the CNS of aborted fetuses and healthy adult male goats infected with N. caninum. Activated astrocytes were mainly associated with inflamed areas, suggesting that astrocytes were involved in the resolution of the lesions.

O presente trabalho compreende o Estudo de diferentes cepas de Neospora caninum do Brasil e da Escócia quanto às suas características genéticas, avaliação de patogenicidade, virulência e possibilidade de isolamento. O primeiro capítulo, consiste de um artigo publicado no periódico internacional Veterinary Parasitology e descreve uma tentativa de isolamento de cepas do parasito utilizando tecido placentário de cabras naturalmente infectadas, em um bioensaio utilizando gerbis. Apesar dos gerbis serem descritos na literatura como sendo altamente susceptíveis à infecção por N.caninum, somente foi obtido sucesso na infecção de animais após imunossupressão com altas doses de metilpredinisolona. Animais imunossuprimidos apresentaram sinais clínicos de emagrecimento, pelos arrepiados e alterações de propriocepção como Head tilt. Análises histopatológicas revelaram reações inflamatórias no encéfalo, consistindo de infiltrados inflamatórios mononucleares multifocais associados à manguitos perivasculares e estruturas parasitárias semelhantes a cistos e taquizoítos, juntamente com áreas de necrose associadas à infiltrado inflamatório de macrófagos e reação de cicatrização em musculatura esquelética. Apesar do sucesso na infecção dos gerbis, não foi possível obter o isolamento do parasito em cultivo celular em uma primeira tentativa, o que levou à segunda parte do trabalho. O capítulo dois consiste de um artigo publicado no periódico intenracional Experimental Parasitology. Foram utilizados tecidos dos gerbis infectados descritos anteriormente e novas amostras de sistema nervoso central de caprinos naturalmente infectados em um bioensaio em camundongos C57BL6 Knockout para o gene Interferon gamma (camundongos knockout- KO). Os camundongos Knockout por serem incapazes de desenvolver uma resposta imune adequada ao N.caninum, permitiram a rápida replicação de taquizoítos principalmente em macrófagos peritoneais, os camundongos apresentaram acentuada perde de peso, letargia e ascite 15 a 30 dias após inoculação, quando foram eutanasiados e o líquido peritoneal utilizado para infecção de monocamadas de células VERO. A grande quantidade de taquizoítos no líquido peritoneal de camundongos KO permitiu uma rápida adaptação e crescimento dos mesmos em cultura de células, dando origem a dois isolados de cabras nomeados NC-Goat1 e NC-Goat2. Os dois isolados foram testados quanto à sua Patogenicidade em um modelo de infecção experimental em camundongos BALB/c, amplamente utilizado na literatura. As cepas não apresentaram alta patogenicidade e virulência no modelo de infecção de camundongos, apesar das duas cepas terem sido associadas a doença clínica em caprinos, com abortos, natimortos e encefalite em caprinos jovens e adultos. O terceiro capítulo apresenta resultados de um estudo genético de genotipagem dos isolados NC-Goat1 e NC-Goat2, amostras clínicas de caprinos e bovinos do sul de Minas Gerais e amostras de bovinos da região de Dumfries e Galloway na Escócia. Utilizando a técnica de genotipagem por microssatélites, análises filogenéticas evidenciaram que as cepas de N.caninum presentes nos isolados e nas amostras clínicas de caprinos tinha relação próxima com os isolados brasileiros NC-SP1, NC-Bahia e NC-Goiás, enquanto que as amostras de bovinos de minas gerais não se correlacionaram proximamente às outras. Analisando as amostras provenientes de rebanhos escoceses, todos tenderam a se relacionar proximamente entre elas e com outras amostras escocesas descritas anteriormente na literatura. Análises adicionais utilizando marcadores microssatélites descritos pelo grupo de pesquisa do Instituto Moredun, evidenciaram genótipos mistos associados ao isolado padrão de N. caninum (NC-1), sugerindo que o isolado poderia ter sido composto por múltiplos genótipos ao isolamento e à medida que foi replicado em cultura celular convergiu em uma população mais homogênea de parasitos. The present work comprises the study of different strains of Neospora caninum, from Brazil and Scotland, regarding their genetic characteristics, pathogenicity, virulence and attempts at isolation. The first chapter consists of an article published in the journal Veterinary Parasitology and describes an attempt to isolate parasite strains using placental tissue from naturally infected goats in a bioassay using gerbils. Although gerbils are described in the literature as highly susceptible to N. caninum infection, successful infection of animals has only been achieved after immunosuppression with high doses of methylpredinisolone. Immunosuppressed animals showed clinical signs of weight loss, fur and proprioception changes such as Head tilt. Histopathological analysis revealed inflammatory reactions in the brain consisting of multifocal mononuclear inflammatory infiltrates associated with perivascular cuffs, cyst-like and tachyzoite-like parasitic structures, along with areas of necrosis associated with macrophage inflammatory infiltrate and skeletal muscle scar formation. Despite the success in gerbil infection, it was not possible to obtain isolation of the parasite in cell culture in a first attempt, which led to the second part of the work. The chapter two consists of an article published in the journal Experimental Parasitology. Tissues from the previously described infected gerbils and new samples from the central nervous system of naturally infected goats were used in a bioassay using C57BL6 interferon gamma Knockout mouse (KO mice). Knockout mice, being unable to develop an adequate immune response to N. caninum, allowed the rapid replication of tachyzoites mainly in peritoneal macrophages, showing marked weight loss, lethargy and ascites 15 to 30 days after inoculation, when they were euthanized, and the peritoneal fluid used for infection of VERO cell monolayers. The large amount of tachyzoites in the peritoneal fluid of KO mice allowed their rapid adaptation and growth in cell culture, resulting in two goat isolates named NC- Goat1 and NC-Goat2. Both isolates were tested for pathogenicity in a widely used experimental BALB/c mouse infection model. The strains did not show high pathogenicity and virulence in the mouse infection model, although both strains were associated with clinical disease in goats, with abortions, stillbirths and encephalitis in young and adult goats. The third chapter presents the results of a genetic genotyping study of the isolates NC- Goat1 and NC-Goat2, clinical samples from goats and cattle from southern Minas Gerais and samples from cattle from the Dumfries and Galloway region of Scotland. Using the microsatellite genotyping technique, phylogenetic analyzes showed that N. caninum strains present in the isolates and clinical samples of goats had a close relationship with the Brazilian isolates NC-SP1, NC-Bahia and NC-Goiás, while the samples from Minas Gerais cattle did not correlate closely to the others. Analysing samples from Scottish herds, all tended to relate closely to each other and to other Scottish samples previously described in the literature. Additional analyses using microsatellite markers described by the Moredun Institute research group showed mixed genotypes associated with the standard N. caninum isolate (NC-1), suggesting that the isolate could have been composed of multiple genotypes on isolation and as it was replicated in cell culture they converged to a more homogeneous population of parasites.

A icterícia é a coloração amarelada decorrente da deposição de bilirrubina em tecidos ricos em elastina, devido à elevada concentração plasmática. A icterícia ocorre por alterações no metabolismo ou na excreção de bilirrubina, sendo classificadas em pré-hepática, hepática ou pós-hepática. A icterícia pré-hepática ocorre quando há hemólise intra- ou extravascular de causa infecciosa ou imunomediada. A icterícia hepática decorre de lesão hepática extensa. A icterícia pós-hepática pode ser secundária à obstrução parcial ou completa do ducto colédoco por colelitíases ou neoplasias. O objetivo desse estudo foi identificar, avaliar e classificar as causas de icterícia em cães necropsiados entre 2014 e 2017 associando as lesões macroscópicas, histológicas e exames complementares. Foram avaliados macro- e microscopicamente 84 cães com diferentes graus de icterícia, sendo classificados 22 (26,2%) cães com icterícia pré-hepática, 48 (57,1%) cães com icterícia hepática, 13 (15,5%) com icterícia pré-hepática e hepática e um (1,2%) com icterícia pós-hepática. Nos exames hematológicos, a alteração mais frequente foi anemia moderada a acentuada com leucocitose e trombocitopenia. Na bioquímica sanguínea, a maioria dos cães estava com azotemia e alteração nos níveis séricos das enzimas hepáticas, como fosfatase alcalina (FA), aspartato aminotransferase (AST) e alanina aminotransferase (ALT). Foram identificadas várias etiologias associadas à icterícia, dentre elas pode-se destacar, agentes infecciosos (47/84), neoplasias (13/84), processos degenerativos (12/84), crônicos (6/84), processos septicêmicos (5/84) e obstrutivos (1/84). Dentre as causas infecciosas, as mais frequentes foram leptospirose (40/84) e erliquiose (7/84). Das neoplásicas, foram colangiocarcinoma (5/84), hemangiossarcoma (4/84) e (2/84) linfoma. Dos processos degenerativos, os mais frequentes foram degenerações gordurosa (9/84) e glicogênica (3/84). Dos processos crônicos os mais frequentes foram fibrose hepática (cirrose) (6/84) e dos obstrutivos foi diagnosticado somente um caso de colelitíase (1/84). A PCR foi utilizada para o diagnóstico confirmatório da infecção por Leptopira interrogans e Ehrlichia canis. O DNA de Leptospira interrogans foi amplificado em 37 cães, e destes, quatro estavam co-infectados com Ehrlichia canis. A classificação e a identificação das causas de icterícia em cães são de relevância devido a frequência de doenças que apresentam essa alteração, muitas vezes sem diagnóstico ante-mortem. Icterus is a yellowish discoloration developed from the bilirubin deposition, in tissues abundant in elastin, due to its high plasmatic concentration. It occurs in metabolism changes or bilirubin excretion and it is classified in pre-hepatic, hepatic and post-hepatic. Pre-hepatic icterus is a result of intra- or extravascular hemolysis by infectious or immune-mediated forms. The hepatic jaundice is a consequence of extensive liver damage. Post-hepatic form is usually secondary to partial or complete bile duct obstruction in cholelithiasis or neoplasms. The aim of this study was to evaluate and classify different icterus causes in dogs necropsied between 2014 and 2017, associating macroscopic and histologic changes as well as ancillary tests. Eighty four dogs were analyzed macro- and microscopically and separated in groups of icterus causes; 22 (26,2 %) dogs had pre-hepatic icterus, 48 (57,1%) hepatic, 13 (15.5%) pre-hepatic and hepatic and one (1.2%) with post-hepatic icterus. The hematological exam detected moderate anemia with severe leukocytosis and thrombocytopenia. The biochemical exam detected azotemia in most of the dogs with high increase in the sera levels of the hepatic enzymes, as alkaline phosphatase (AP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Many factors were identified as the cause of icterus and the main factors were infectious agents (47/84), neoplasms (13/84), degenerative changes (12/84), chronic changes (6/84) and obstructive cause (1/84). The most frequent infectious causes were leptospirosis (40/84) and ehrlichiosis (7/84). Neoplastic causes include cholangiocarcinoma (5/84), hemangiosarcoma (4/84) and lymphoma (2/84). Degenerative changes were lipidosis (9/84) and glycogen (3/84) degeneration. Hepatic fibrosis (cirrhosis) (6/84) was the most frequent chronic changes and cholelithiasis (1/84) was diagnosed once as obstructive cause. PCR was performed to confirm Leptopira interrogans e Ehrlichia canis infections. DNA of Leptospira interrogans was amplified in 37 dogs, which four had coinfection with Ehrlichia canis. The classification and identification of icterus causes in dogs are very important due to the number of diseases with this alteration, frequently without ante-mortem diagnosis.

O envenenamento por aranhas do gênero Loxosceles provoca um conjunto de sinais e sintomas, chamado de loxoscelismo, que na maioria dos casos manifesta-se através do quadro dermonecrótico. O modelo mais utilizado para o estudo do loxoscelismo cutâneo é o coelho. No entanto, o desenvolvimento de um modelo animal de menor porte, fácil manuseio, menor custo e manutenção são necessários para o estudo da patogênese do loxoscelismo. A inflamação provocada pelo veneno bruto de Loxosceles similis, foi avaliada considerando a ativação de neutrófilos e macrófagos, vasodilatação, hiperemia, edema, hemorragia e a produção do TNF e VEGF utilizando o modelo murino de implante de esponjas. Trinta e dois camundongos Swiss, machos (6-8 semanas) foram implantados subcutaneamente com discos de esponjas de poliéster-poliuretano. Quatorze dias após a implantação os animais foram separados em dois grupos: (1) grupo controle: 16 camundongos que receberam intraimplante 30 L de solução salina; (2) grupo tratado: 16 camundongos que receberam a injeção intra-implante de 30 L (0.5 g) do veneno bruto de L. similis. Os animais foram eutanaziados com xilazina/quetamina 1h e 4h pós-injeção intra-implante. Microscopicamente, os implantes dos grupos tratados apresentaram inflamação aguda caracterizada por: infiltrado neutrofílico, edema, vasodilatação, hiperemia e hemorragia intensa. Alguns vasos apresentaram-se com as suas paredes rompidas. Através da análise morfométrica foi possível observar que a área de vasos foi significativamente maior nos grupos tratados. Os parâmetros bioquímicos como: conteúdo de hemoglobina, níveis das citocinas fator de necrose tumoral (TNF), fator de crescimento endotelial (VEGF), atividade das enzimas n-acetil- - glucosaminidase (NAG) e mieloperoxidase (MPO) foram significativamente maiores nos grupos tratados. Os efeitos do veneno de Loxosceles no tecido de granulação do implante em camundongos foram similares ao que se observa no loxoscelismo cutâneo em outras espécies (humanos ou coelhos). Consequentemente, o modelo murino de implante de esponjas promove um novo método para investigação de mecanismos celulares e moleculares associados com o loxoscelismo cutâneo.O envenenamento por aranhas Loxosceles conduz a um conjunto de sinais e sintomas, denominado loxoscelismo, que na maioria dos casos se manifesta sob o quadro dermonecrótico. Neste estudo, utilizou-se o modelo de implante subcutâneo de esponja em camundongo para descrever os efeitos do veneno de Loxosceles similis, com ênfase em 24 h após a injeção intraimplante e para avaliar a patogenia do loxoscelismo 1, 4 e 24 horas após a injeção do veneno. O presente trabalho tem como objetivos: (1) caracterizar histologicamente os efeitos do veneno bruto de L. similis no implante subcutâneo de esponjas; (2) quantificar os mastócitos do implante e mensurar sua atividade de desgranulação; (3) quantificar os subtipos de colágeno tipo I e III; (4) verificar, quantificar e avaliar a importância da apoptose no implante 1, 4 e 24 horas após a injeção do veneno na patogenia do loxoscelismo. Trinta camundongos Swiss (6-8 semanas, machos) foram implantados subcutaneamente com esponjas de poliéster-poliuretano. Após o 14º dia de implante, os animais foram divididos em seis grupos (n=5 para cada grupo): três grupos controles (C1h, C4h e C24 h), no qual receberam a injeção intraimplante de 30L de solução salina e três grupos tratados (T1h, T4h e T24 h), no qual receberam a injeção intraimplante de 30L (0,5 g) do veneno de L. similis. Após os intervalos de 1, 4 e 24 horas os animais foram eutanaziados, os implantes foram colhidos e processados para microscopia de luz e eletrônica. À microscopia de luz observouse, nos implantes injetados com veneno, inflamação aguda com intenso edema, presença de trombos ocludentes, vasculite, aumento dos índices de mastócitos e de desgranulação de mastócitos e de apoptose de células gigantes. Além disso, observou-se degradação de colágeno dos tipos I e III. As análises das ultraestruturas evidenciaram-se vários tipos celulares em apoptose. Os presentes resultados sugerem que a apoptose, a desgranulação de mastócitos e a degradação do colágeno são importantes na patogenia do loxoscelismo e sua ocorrência em alguns tipos celulares pode explicar a dificuldade no reparo da úlcera que comumente se observa no loxoscelismo. Envenomation by Loxosceles spider bite leads to a set of signs and symptoms, called loxoscelism, which in most cases manifests through the dermonecrotic frame. The development of a smaller size animal model, of easy handling and maintenance, and lower cost is needed to study the loxoscelism pathogenesis. The inflammatory effects of the Loxosceles similis crude venom was evaluated considering neutrophil and macrophage activation, vasodilatation, hyperhaemia, edema, hemorrhage and TNF- and VEGF production using the murine sponge implant model. Thirty two, male, Swiss mice (68 weeks old) were implanted subcutaneously with polyetherpolyurethane sponge discs. Fourteen days post implantation, animals were separated into two groups: (1) control group16 mice received 30 L of saline intra-implant; (2) treated group-sixteen mice injected with 0.5 g/30 L of L. similis crude venom intra-implant. The animals were euthanized with xylazine/ketamine after 1 and 4 h post-injection. Microscopically, implants of the treated groups presented an acute inflammation characterized by: neutrophilic infiltrate, edema, vasodilatation, hyperhaemia, and severe hemorrhage. Some vessels presented ruptured walls. Under morphometric analysis, vessel area was bigger in the treated groups compared with the control ones. The biochemical parameters, hemoglobin content, levels of the cytokines tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF), inflammatory enzyme activities n-acethyl--glucosaminidase (NAG) and myeloperoxidadase (MPO) were also significantly higher in the venom-treated groups. The effects of Loxosceles venom in the granulation tissue of the implant in mice were similar to those observed in cutaneous loxoscelism in other species (human and rabbits). Consequently, the murine sponge implant model provides a new method to investigate cellular/molecular mechanisms associated with cutaneous loxoscelism.Envenomation by the Loxosceles spider causes loxoscelism, a pattern of signs and symptoms that primarily manifests in the dermonecrotic form. Our studies have shown that a mouse subcutaneous sponge implantation model may be useful in evaluating the effects of Loxosceles similis venom. This model provides an ideal microenvironment in which to study loxoscelism; however, it is still important to evaluate its pathogenesis and to observe the effects of L. similis venom for longer time periods than those in previous studies of this model. The aims of this study are: (1) to histologically characterize the effects of L. similis crude venom in a subcutaneous sponge implant; (2) to quantify the mast cells present in the implant and to measure their degranulation activity; (3) to quantify collagen subtypes I and III; and (4) to verify, quantify, and evaluate the effects of apoptosis in the implant on the pathogenesis of loxoscelism at 1h, 4h, and 24 h after injecting the venom. Thirty Swiss mice (6-8 weeks, male) were subcutaneously implanted with polyester-polyurethane sponge discs. Fourteen days post-implantation, the animals were divided into six groups (5 animals per group): three control groups (C1h, C4h, and C24h), in which the mice received 30 l injections of intra-implant saline, and three treated groups (T1h, T4h, and T24h), in which the mice received 30 L (0.5 g) injections of L. similis crude venom at 1h, 4h, and 24 h intervals. After each time interval, the animals were euthanized, and the implants were harvested and processed for light and electron microscopic analyses. The following results were observed in the implants harvested from the treated groups: acute inflammation with marked edema, thrombus, and vasculitis, as well as increased levels of mast cells and mast cell degranulation, and apoptosis in giant cells. Furthermore, degradation of collagen types I and III was observed. An analysis of the ultrastructure revealed apoptosis in various cell types. The present results suggest that apoptosis in some cell types associated with an increase in mast cell degranulation and the degradation of collagen fibers are important in the pathogenesis of loxoscelism therefore may explain the difficulty in repairing the ulcer is commonly observed in severe cases of loxoscelism cutaneous in humans.

A enteropatia proliferativa causada pela bactéria intracelular, Lawsonia intracellularis, tem sido relatada em eqüinos na Austrália, Estados Unidos, Canadá e em países na Europa (Suíça e Bélgica). No entanto, até o momento, a doença não foi diagnosticada em nenhum país da América Latina. A prevalência da doença em eqüinos é desconhecida. Pesquisou-se a ocorrência da enteropatia proliferativa em eqüinos no Estado de Minas Gerais, Brasil, através de estudo retrospectivo no arquivo do Setor de Patologia da Escola de Veterinária da Universidade Federal de Minas Gerais (UFMG) e da Universidade Federal de Lavras (UFLA), e através da aplicação da técnica de PCR em fezes e sorologia. O estudo retrospectivo foi realizado a partir de amostras em blocos de parafina, de 51 casos compilados de equinos com desordens entéricas provenientes na Escola de Veterinária da UFMG, no período de 1970 a 2006, e de nove casos provenientes da UFLA no período de 2000 a 2007. Cortes histológicos em lâminas silanizadas dos blocos contendo fragmentos de intestino e estômago foram submetidos a imunoistoquímica (IHQ) utilizando o método da Streptavidina Marcada, anticorpo policlonal contra L. intracellularis e solução de cromógeno de AEC (Amino Etil-carbazol). Não foi identificada nenhuma amostra positiva à IHQ. Para realização da pesquisa por PCR em fezes e sorologia foram utilizados o teste sorológico, usando a técnica de imunoperoxidase em lâminas de vidro, e a técnica de PCR em amostras fecais coletadas de 223 eqüinos provenientes de 14 haras e de eqüinos hospitalizados na escola de veterinária da UFMG. Das 223 amostras avaliadas sorologicamente, foram identificados vinte e um eqüinos com titulação de 1:60, sendo considerados positivos. À técnica de PCR em fezes para detecção de DNA da L. intracellularis identificou sete equinos positivos. Soropositividade e detecção de bactérias nas fezes de potros na população de equinos do Estado de Minas Gerais, Brasil, indica a presença do agente e a possibilidade de doença associada a infecção, sendo um alerta para os clínicos como possível diagnóstico diferencial de enterite em eqüinos The proliferative enteropathy caused by intracellular bacteria, Lawsonia intracellularis, has been described in equine in Australia, United States, Canada and in countries of Europe, (Switzerland and Belgium). Currently, the disease has not been diagnosed in any country of Latin America. The predominance of the disease in equine is still unknown. A retrospective study was conducted using paraffin blocks of equine cases from the veterinarian schools of Universidade Federal de Minas Gerais (UFMG) and Universidade Federal de Lavras (UFLA). Also a prospective study using serology and PCR for detecting antibodies and L. intracellularis shedding in fecal samples was conducted in different equine herds in the state of Minas Gerais, Brasil. Fifty-one cases of equines with enteric disorders from the period of 1970 to 2006 from UFMG and nine cases of UFLA were compiled and selected from the period of 2000 to 2007. Histological cuts of the blocks containing fragments of intestine and stomach were prepared in signalized glass-slides, and were submitted to IHQ using the method of Streptavidin, polyclonal antibody against L. intracellularis and ACE (Amino Etil-carbazol) cromogen solution. No positive sample in IHQ was identified. For the longitudinal study, sera and fecal samples from 223 animals from 14 horse farms and veterinarian schools of UFMG a serological test using the imunoperoxidase technique in glass sheets and PCR were used. From the 223 samples serologically tested, eight equine had 1:60 titer, and were considered positive. The PCR technique in feces for bacterias DNA detection identified seven positive equine for L. intracellularis. Seropositivity and detection of bacteria in colts feces in equine population in the State of Minas Gerais, Brasil, indicates the presence of the agent and the possibility of the disease associated with the infection. This should be an alert to clinics as possible differential diagnosis of enteric disease equine