O objetivo do presente estudo foi avaliar os efeitos tóxicos da Senna occidentalis (So) sobre o sistema linfo-hematopoiético em ratos recém-desmamados ou expostos pré-natalmente. Avaliou-se nestes animais: o consumo de água e ração, o ganho de peso; parâmetros hematológicos, padrões histopatológicos, além da resposta imunológica específica e não específica. Inicialmente, o estudo foi realizado em ratos recém-desmamados, os quais foram expostos a diferentes concentrações de sementes de So na ração a saber: 1% (So1), 2% (So2) e 4% (So4) durante 14 dias ou que receberam So4 durante 28 dias. Os animais do grupo peer-feeding (PF) receberam a mesma quantidade de ração consumida pelos animais expostos a So4, porém isentas da planta. Após 14 dias de exposição, os resultados obtidos mostraram uma diminuição significante nos parâmetros de consumo de ração, de ganho de peso e da celularidade da medula óssea e do peso relativo no timo nos grupos So2 e So4 e um aumento no peso relativo do baço nos grupos So2 e So4. Entretanto, os animais dos grupos So4 e PF também apresentaram diminuição da celularidade da medula óssea. Na avaliação da resposta imune não especifica todos grupos expostos às sementes da planta, bem como o grupo PF, apresentaram redução na porcentagem de fagocitose por neutrófilos; porém, apenas o grupo So4 mostrou redução do burst basal por neutrófilos. O estudo morfológico do baço mostrou proliferação hematopoiética extramedular e aumento de megacariócitos multinucleados nos ratos do grupo So4. Apenas nos experimentos com animais do grupo So4 expostos por 28 dias, verificou-se similaridade entre resultados dos parâmetros acima descritos, além da ocorrência de anemia microcítica e hipocrômica. Num segundo momento avaliou-se as proles de ratas que receberam sementes de S. occidentalis a 4% na ração, do 6º ao 20º dia de gestação, assim como as proles das ratas do grupo peer-feeding (PF). Os filhotes provenientes de mães do grupo So4 e PF apresentaram um aumento do burst oxidativo e da fagocitose por neutrófilos. Os animais da prole do grupo PF apresentaram hemograma indicativo de anemia megaloblástica. Já no estudo morfológico do baço verificou-se hematopoiese extramedular nos filhotes das mães do grupo So4. Portanto, a presente pesquisa mostrou que a S. occidentalis pode comprometer alguns parâmetros do sistema imunológico de ratos expostos às sementes da planta durantes diferentes fases do desenvolvimento. Além disso, verificou-se que as sementes desta planta também promovem efeitos tóxicos sobre eritrócitos. A inclusão do grupo PF permitiu verificar que os efeitos observados não são decorrentes de possíveis alterações nutricionais promovidas pela redução do consumo de ração, mas sim relacionados ao efeito tóxico direto da S. occidentalis. / The aim of present study was to determinate the Senna occidentalis (So) toxic effects on lymphohematopoetic system in rats during the growth and pre-natal period. The effects were evaluated on the basis of food consumption, weight gain, hematological and immunological parameters, as well as histopathology analysis. Initially, the study was done in growing rats exposed to S. occidentalis seeds in different concentrations: 1% (So1), 2% (So2) and 4% (So4) in feed during 14 or 28 days. Peer feeding-group of rats (PF) was also evaluated; this group received the same amount of feed of those from So4-group, however, free of S. occidentalis seeds. S. occidentalis 14 days exposure decreased food consumption, weight gain, thymus relative organ weight and bone marrow cellularity and increased the spleen relative weight of rats from So2 and So4-group. However, So4- and PF- groups also presented a decreased bone marrow cellularity. All seed exposed-groups and also PF-group had a decrease on neutrophil phagocytosis percentage; however, only rats from So4-group had a decreased neutrophil basal burst. Spleen morphologic analysis indicated the presence of extramedular hematopoietic proliferation and increased multinucleated megakariocytes on So4-group. Similar results were found for all the parameters described after S. occidentalis exposure during 28 days. Furthermore, the rats of So-4 group presented microcytic and hypochromic anemia. On a second moment, rats offspring exposed to S. occidentalis at 4% in feed during the 6th to 20th gestational day and rats offspring of PF group were evaluated. The So4- and PF- rats offspring groups presented an increased neutrophil oxidative burst and phagocytosis, however PF-rats offspring group also had an altered complete blood count compatible with megaloblastic anemia. Moreover spleen morphologic analysis indicated the presence of extramedular hematopoiesis on So4 rats offspring group. Therefore, the present study showed that S. occidentalis can compromise some immunological parameters in rats exposed to seeds during different development periods. This exposure also promotes toxic effects on erythrocytes. The PF group allowed us to verify that the observed effects are related to direct S. occidentalis toxic effects and not due a possible nutritional alteration caused by the reduced feed ingestion.

In the research reported here, the pathology, virulence, and strain differences of Taura syndrome virus (TSV) was studied. Initial studies on TSV pathogenesis compared the survival of juveniles of a highly Taura syndrome (TS) susceptible line of Penaeus vannamei, a line of TS resistant P. vannamei, and an innately TS resistant P. stylirostris line following TSV challenge by feeding (per os) or injection methods, in the absence of horizontal transmission via cannibalism and/or absorption from the water. Per os_TSV challenge resulted in I00% survival in P. stylirostris, but challenge by per os exposure produced significant mortality commencing on about the same post-exposure day in both SPF and SPR P. vannamei (P < 0.001), suggesting that P. stylirostris is significantly (P < 0.001) more resistant toper os TSV infection and presentation of TS disease than either SPF or SPR P. vannamei. The potential roles of the cuticular lining of the stomach and hindgut and unlined portions of the gut in TSV resistance in penaeid shrimp are discussed as factors where an innate resistance mechanism was postulated to explain the observed differences between the different species and populations of shrimp in TSV susceptibility. To investigate apparent TSV strain differences, three geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris) and one TSV isolate from Belize, Central America (BLZ02TSV from P. vannamei) were analyzed and compared to the reference TSV isolate (HI94TSV) by selected TSV diagnostic and genetic analysis methods. The results show that screening of penaeid shrimp broodstock and postlarvae by MAb I Al testing will not detect all TSV isolates, possibly leading to false negative results, further spread of TSV and re-emergence of TS in regions where it has been eradicated. The putative VP1 antigenic epitope recognized by TSV MAb 1A1 is identified, with SIN98TSV and BLZ02TSV having 70.0% and 80.0% AA homology, respectively, within the 10 AA region. There are three distinct electropherotypes and 'serotypes' of TSV, with electropherotype A (TSV Etype-A) and serotype A (TSV-A) representing those TSV isolates conforming to VP1 properties of the Hawaiian 1994 TSV isolate, electropherotype B (TSV Etype-B) and serotype B (TSV-B) representing those TSV isolates conforming to the VP1 properties of the Sinaloan 1998 TSV isolate, and electropherotype C (TSV Etype-C) and TSV serotype C (TSV-C), representing those TSV isolates conforming to the VP1 properties of the Belize 2002 TSV isolate. In a parallel activity, the University of Arizona (UAZ) Aquaculture Pathology Diagnostic Laboratory (APL) Case Database (DB) and the UAZ Aquaculture Pathology Diagnostic Laboratory Client Address Book Database (AB), relational databases, were created using FileMaker Pro software, are used to keep an up to date and accurate record of all UAZAPL diagnostic and research case and client information and may be searched and sorted to find case data and/or client information of interest.

Clostridium difficile-associated disease (CDAD) in neonatal pigs has emerged as a serious economic concern for swine producers throughout North America. The disease has been diagnosed clinically and reproduced in experimental inoculation trials in pigs, but little is known of the epidemiology or pathogenesis of the disease in pigs. Strain characteristics and distribution of C. difficile isolates from pigs, calves, dogs, horses, and humans were assessed by PCR-ribotyping. Porcine and bovine isolates were dominated by a single ribotype. This ribotype was uncommon among isolates from other host species; it was particularly uncommon from humans, suggesting there is little transfer of isolates between humans and calves or pigs. The reason for a single common ribotype circulating among distinct bovine and porcine populations is unknown. The intragastric inoculation of newborn pigs demonstrated their sensitivity to C. diffcile toxins. Toxin B (TcdB) surprisingly resulted in more severe lesions than Toxin A (TcdA). The two toxins together acted synergistically. Colon explants were sensitive to TcdA in a dose-dependent manner. However, TcdB did not cause significant lesions in the explants, nor was there any synergism with TcdA. Electron microscopy of colon explants treated with TcdA revealed severe, ultrastructural lesions that accrued in a dose-dependent manner by two h post infection. Direct immunohistochemistry assays demonstrated specific binding of biotinylated TcdA throughout the gastrointestinal tract of neonatal pigs. The density of bound toxin in different segments correlated with the severity of lesions in those segments from pigs gavaged with TcdA. TcdB did not bind any tissues, though it was fully active in cell-culture assays. A monoclonal antibody to Galalpha1-3beta1-4GlcNAc-R (alpha-Gal epitope), a putative receptor for TcdA in pigs, specifically bound the brush border of enterocytes, but the distribution of binding did not correlate with the distribution of TcdA binding. Specific TcdA binding to the plasmallema of microvilli was also confirmed by immunoelectron microscopy. By five min post inoculation some toxin was already visible in endosomes or free in the cytoplasm. TcdA localized to the mitochondria of epithelial cells and, less frequently, to the nuclei. Endothelial cells and leucocytes in the superficial lamina propria were similarly labeled by toxin.

The genome of a Korean isolate of Hepatopancreatic parvovirus (HPV) was partially cloned, sequenced and characterized. Random PCR amplification of viral DNA was combined with conventional cloning methods to generate three clones named HPV8 (2,136 bp insert), HPV3 (1,560 bp insert), and CP1139 (413 bp insert). DNA sequencing demonstrated overlapping regions between HPV8 and HPV3 and between HPV3 and CPII39. The combined sequence of these three clones encompass approximately 3,350 bp of the total 5,000 bp estimated for the HPV genome. A large open reading frame (1,692 bp) was found within clones HPV3/CPII39 encoding a polypeptide of 549 residues (∼60 kDa) whose amino terminus shows 100% homology with the first 12 residues sequenced from an apparently single 54 kDa (by SDS-PAGE) structural protein found in a Korean isolate of HPV. Two new gene probes EC.592 (592 bp) and EC.350 (350 bp) were developed by PCR amplification of previously constructed HPV (Korean) clones and labeled with DIG11-dUTP. These probes recognize different regions of the HPV genome. The specificity of both probes was confirmed by in situ hybridization using HPV-infected Penaeus chinensis (Korean) as a positive control and specific-pathogen free P. vannamei and IHHNV-infected P. stylirostris, as negative controls. Both probes were used in in situ hybridization to compare their reaction to HPV-type lesions detected by conventional H&E histology in 7 species of HPV-infected shrimp, and one of freshwater prawn, from 13 countries. The results of this comparison strongly suggest the existence of genomic differences among these geographic isolates. A post-embedding in situ hybridization assay at the electron microscope level was developed to detect HPV nucleic acids on HPV-infected hepatopancreata from P. chinensis and P. monodon . Hybridized probe (EC.592) was detected with an anti-DIG sheep antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal was observed within HPV-infected hepatopancreatic cells, which was specifically located within intranuclear viral inclusions, cytoplasm, microvillous border, and associated to necrotic debris within the lumen of hepatopancreatic tubules. A non-destructive method, based on the PCR, was developed to detect HPV by examination of crude fecal samples from HPV-infected shrimp.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-30Bitstream added on 2014-06-13T20:04:34Z : No. of bitstreams: 1 ragazani_avf_dr_jabo.pdf: 399859 bytes, checksum: f93ad8981a9e4aaa051ed6bff03ccacc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium... / The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below)

Orientador: Ruben Pablo Schocken-Iturrino / Banca: Maria Luiza Poiatti / Banca: Alessandra Aparecida Medeiros / Banca: Hélio José Montassier / Banca: Antônio Carlos Monteiro / Resumo: Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below) / Doutor

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / Toll-like receptors (TLR) are instrumental in discriminating between pathogenic and commensal bacteria and act as mediators, along with downstream chemokines, of subsequent innate and adaptive immune responses. However, little is known about the expression and regulation of TLR or chemokines in swine. The objectives of the experiments described herein were to characterize the expression of porcine TLR and to identify regulatory patterns in these receptors in the presence of live Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The first two experiments evaluated the in vivo and in vitro expression of TLR2, 4, 5 and 9. Our results indicate that TLR2, 4, 5 and 9 are constitutively expressed in vitro in a porcine jejunal epithelial cell line (IPEC-J2), porcine mononuclear phagocytes (pMPs) and in vivo in the distal ileum. In IPEC-J2 cells, ST elicited an increase in TLR2 mRNA (P < 0.05), and both ST and SC increased TLR2 mRNA in pMPs (P < 0.05). In vivo, oral challenge with ST increased (P < 0.05) both TLR2 and TLR4 mRNA in the distal ileum. In addition, the second experiment evaluated interleukin 8 (IL8) and CC chemokine ligand 20 (CCL20) expression in IPEC-J2 cells in response to ST or purified bacterial flagellin (Flag). TLR5 was constitutively expressed in the ileum and in IPEC-J2 and pMP cells. Interestingly, IL8 and CCL20 mRNA and protein were increased (P < 0.05) by ST and Flag, even in the absence of changes in TLR5. In the third experiment, the expression of TLR and chemoattractive mediators were evaluated in a panel of tissues obtained from pigs challenged with ST and SC. All genes of interest were constitutively expressed; however, the effects of treatment were limited to isolated tissues and genes. Taken together, the data indicate that TLR and chemoattractive mediators are expressed in porcine tissues and cells and that the observations described represent novel evidence that pig pathogens may regulate TLR expression and activate chemokine secretion.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Enteric pathogens are responsible for major economic losses in the swine industry. In the U.S., Salmonella enterica subspecies enterica serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for essentially all cases of salmonellosis in swine. Previous studies documented that oral ST eroded growth and produced unmistakable changes in the endocrine stress and somatotropic axis of young growing pigs. However, these effects occurred in the absence of elevated systemic inflammatory cytokines that were previously thought to accompany disease-associated growth retardation. In the current study, it was hypothesized that SC would produce very different systemic inflammatory cytokine responses compared to ST given the likelihood of SC to produce systemic disease in pigs. Weaned pigs were housed two per pen with free access to feed and water during a 14 d experiment. On d 0, pigs were fed either 108 CFU SC or 108 ST, and bacteria were re-fed twice weekly through the course of the experiment. Control pigs were fed dough without bacteria. Serum was collected on d 0, 7, and 14 for determination of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and insulin-like growth factor-I (IGF-I) were determined. Rectal temperatures (RT) were monitored daily beginning 2 d prior to challenge with bacteria and until 7 d following the first bacterial feeding. Pigs were weighed initially, and at the conclusion of the study. Daily body weight gain was reduced by 25.4% in pigs fed SC (P<.0001) compared to control, while growth was similar between control pigs and those fed ST. Pigs fed SC had increased RT beginning on d 2 and continuing though d 7 (P < 0.05) with the greatest elevation spike on d 3 (P < 0.001) when compared to controls. On d 7, pigs fed SC had reduced IGF-I when compared to both control (P < 0.01) and ST pigs (P = 0.01). Despite the obvious febrile response, and the reductions in body weight gain and serum IGF-I, circulating TNFα and IL-1β were not affected by treatment. It was concluded that elevated TNFα and IL-1β are not obligatory correlates of SC-induced pathology and growth retardation in weaned pigs.

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