Resumo
The study aimed to evaluate the action of aqueous extract of noni in an extender for sheep semen freezing. Treatments differed in inclusion of aqueous extract of noni in the extender: T1 Ë no addition; T2 Ë 24µg/mL; T3 Ë 72µg/mL; and T4 Ë 120µg/mL. Ejaculates were collected, diluted in the four treatments, and frozen. After thawing, the semen was subjected to a thermoresistance test and evaluated for subjective motility, vigor, membrane integrity assessment by hypo-osmotic swelling test, live-dead assay, computer-assisted sperm analysis and the status of sperm capacitation and acrosome reaction. Data were subjected to ANOVA, and then to Student Newman Keuls's test at 5% significance level. In the thermoresistance test after two hours of incubation, motility in T4 (120µg/mL) was lower than in the other treatments, with no differences in the HoS test in either diluted semen or in the semen evaluated immediately post-thawing, while for the other times, treatments showed similar responses. Regarding the motility parameters, a difference was observed for progressive motility, curvilinear velocity, average path velocity, and amplitude of lateral head displacement. As to the sperm capacitation status, a difference was observed between treatments for the sperm capacitated with intact acrosome.(AU)
Este estudo teve como objetivo avaliar a ação do extrato aquoso de noni em diluente para congelação de sêmen de carneiro. Os tratamentos diferiram quanto à inclusão de extrato aquoso de noni ao meio diluidor em: T1Ë sem adição de extrato; T2Ë 24µg/mL ; T3- 72µg/mL e 120µg/mL. Por meio de vagina artificial, 16 ejaculados foram coletados, diluídos entre os quatro tratamentos e congelados. Após o descongelamento, o sêmen foi submetido ao teste de termorresistência e avaliado quanto à motilidade subjetiva, ao vigor espermático, à integridade de membrana pelo teste hiposmótico, bem como ao teste supravital, à análise de sêmen assistida por computador (CASA) e ao status de capacitação espermática e de reação acrossomal. Os dados foram submetidos a uma análise de variância, seguida pelo teste de Student-Newman-Keuls com 5% de significância. No teste de termorresistência, após duas horas de incubação, a motilidade do T4 (120µg/mL) apresentou-se inferior aos demais tratamentos. Não houve diferença significativa no teste HOS tanto para o sêmen diluído quanto para o sêmen avaliado imediatamente pós-descongelação; para as demais horas, os tratamentos apresentaram comportamento semelhante. Para os parâmetros de cinética, foi observada diferença estatística para motilidade progressiva, velocidade curvilinear, velocidade do percurso médio e amplitude de deslocamento lateral da cabeça. Quanto ao estado de capacitação espermática, observou-se diferença entre os tratamentos para espermatozoide capacitado com acrossomo intacto.(AU)
Assuntos
Animais , Peróxidos Lipídicos/química , Preservação do Sêmen/veterinária , Ovinos/embriologia , Membrana CelularResumo
The study aimed to evaluate the action of aqueous extract of noni in an extender for sheep semen freezing. Treatments differed in inclusion of aqueous extract of noni in the extender: T1 Ë no addition; T2 Ë 24µg/mL; T3 Ë 72µg/mL; and T4 Ë 120µg/mL. Ejaculates were collected, diluted in the four treatments, and frozen. After thawing, the semen was subjected to a thermoresistance test and evaluated for subjective motility, vigor, membrane integrity assessment by hypo-osmotic swelling test, live-dead assay, computer-assisted sperm analysis and the status of sperm capacitation and acrosome reaction. Data were subjected to ANOVA, and then to Student Newman Keuls's test at 5% significance level. In the thermoresistance test after two hours of incubation, motility in T4 (120µg/mL) was lower than in the other treatments, with no differences in the HoS test in either diluted semen or in the semen evaluated immediately post-thawing, while for the other times, treatments showed similar responses. Regarding the motility parameters, a difference was observed for progressive motility, curvilinear velocity, average path velocity, and amplitude of lateral head displacement. As to the sperm capacitation status, a difference was observed between treatments for the sperm capacitated with intact acrosome.(AU)
Este estudo teve como objetivo avaliar a ação do extrato aquoso de noni em diluente para congelação de sêmen de carneiro. Os tratamentos diferiram quanto à inclusão de extrato aquoso de noni ao meio diluidor em: T1Ë sem adição de extrato; T2Ë 24µg/mL ; T3- 72µg/mL e 120µg/mL. Por meio de vagina artificial, 16 ejaculados foram coletados, diluídos entre os quatro tratamentos e congelados. Após o descongelamento, o sêmen foi submetido ao teste de termorresistência e avaliado quanto à motilidade subjetiva, ao vigor espermático, à integridade de membrana pelo teste hiposmótico, bem como ao teste supravital, à análise de sêmen assistida por computador (CASA) e ao status de capacitação espermática e de reação acrossomal. Os dados foram submetidos a uma análise de variância, seguida pelo teste de Student-Newman-Keuls com 5% de significância. No teste de termorresistência, após duas horas de incubação, a motilidade do T4 (120µg/mL) apresentou-se inferior aos demais tratamentos. Não houve diferença significativa no teste HOS tanto para o sêmen diluído quanto para o sêmen avaliado imediatamente pós-descongelação; para as demais horas, os tratamentos apresentaram comportamento semelhante. Para os parâmetros de cinética, foi observada diferença estatística para motilidade progressiva, velocidade curvilinear, velocidade do percurso médio e amplitude de deslocamento lateral da cabeça. Quanto ao estado de capacitação espermática, observou-se diferença entre os tratamentos para espermatozoide capacitado com acrossomo intacto.(AU)
Assuntos
Animais , Peróxidos Lipídicos/química , Preservação do Sêmen/veterinária , Ovinos/embriologia , Membrana CelularResumo
This study stems from the findings during the gross and histopathological exam of 3,338 cattle brains as part of the bovine spongiform encephalopathy (BSE) active surveillance program of the Brazilian Ministry of Agriculture, Livestock, and Supply from 2001 to 2005. The work was carried out in the Veterinary Pathology Laboratory of the Federal University of Santa Maria which at the time (2001-2007) was the national reference laboratory for the diagnosis of BSE and other transmissible spongiform encephalopathies. Both gross and histopathological aspects are described. Several gross aspects were annotated: anatomic normal structures not commonly recognized (non-lesions), lesions of no clinical significance, postmortem changes and artifacts; all these can amount to important pitfalls that distract the pathologist during the routine gross examination of the central nervous system (CNS). Accordingly, equivalent pitfalls were described in the histological examination. Non-lesions observed were the pineal body, embryo remnants such as the external germinal layer of the cerebellum, subependymal plates, and clusters of neuroblasts in the basal ganglia; or circumventricular structures such as area postrema, subcomisural organ, and melanosis in the leptomeninges and vessel walls. Lesions with little or no clinical importance included age-related changes as lipofuscin, hemosiderin, mineralization and hyalinization of vessel walls within the brain and meninges. Corpora amylacea and corpora arenacea were detected respectively in astrocyte processes and the pineal body. Cytoplasmic neuronal vacuolization was observed in the red nucleus and habenular nucleus. Sarcocystis sp. without a correspondent inflammatory reaction was rarely observed. Included within findings with no clinical manifestation were axonal spheroids and perivascular mononuclear cuffings. Changes in the CNS due to killing, sampling and fixation methods can obscure or distract from the more critical lesions. The ones related to the process of killing included hemorrhages caused in cattle destroyed by a captive bolt. Artifacts related to sampling and handling of CNS tissue consisted of inclusion of bone sand in the neural tissue from sawing the calvarium; dark neurons produced by excessive handling of the brain, and micro-organisms that contaminated the tissues during sampling or histological processing. Postmortem autolytic or putrefactive changes observed included vacuolar changes in the myelin sheath, clear halos surrounding neurons and oligodendrocytes, clusters of putrefaction bacilli within vessels or dispersed throughout the brain tissue associated or not to clear halos. One interesting, and somewhat frequent, postmortem autolytic change found in the bovine brain was the partial dissolution of the granule cell layer (GCL) of the cerebellum, also referred to as conglutination of the GCL or as the French denomination "état glace". Due to the shortage of comprehensive publications in the subject, this review is intended to address the main pitfalls that can be observed in the brain of cattle hoping to help other pathologists avoiding misinterpret them.(AU)
Os resultados deste estudo foram obtidos pelo exame macroscópico e histopatológico de 3.338 cérebros de bovinos examinados durante o programa de vigilância ativa da encefalopatia espongiforme bovina (BSE) do Ministério da Agricultura, Pecuária e Abastecimento (MAPA), de 2001 a 2005. O trabalho foi realizado no Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM) que, de 2001 a 2007, foi o laboratório nacional de referência para o diagnóstico da BSE e de outras encefalopatias espongiformes transmissíveis. Macroscopicamente, foram descritas estruturas anatômicas normais (não-lesões), mas que são, com frequência, interpretadas como lesões; lesões sem significado clínico; alterações pós-mortais e artefatos. Esses achados podem confundir e desviar a atenção do patologista durante o exame de rotina do sistema nervoso central (SNC). Da mesma forma, estruturas equivalentes foram descritas no exame histológico. As não-lesões observadas foram corpo pineal, remanescentes embrionários, como a camada germinativa externa do cerebelo, placas subependimárias e aglomerados de neuroblastos nos gânglios da base; ou estruturas circunventriculares, como área de postrema, órgão subcomissural e melanose em leptomeninges e paredes dos vasos. Lesões com pouca ou nenhuma importância relacionadas ao envelhecimento incluíram lipofuscina, hemossiderina, mineralização, hialinização das paredes dos vasos do encéfalo e das meninges. Corpora amylacea foram detectados em processos astrocíticos e corpora arenacea, no corpo pineal. Adicionalmente, foi observada vacuolização no citoplasma de neurônios do núcleo vermelho e do núcleo habenular. Sarcocystis sp. sem reação inflamatória correspondente foi raramente observado. Incluídos nos achados sem manifestação clínica estavam esferóides axonais e manguitos mononucleares perivasculares. Alterações no SNC causadas pelo método de abate, amostragem e fixação podem simular ou obscurecer lesões mais importantes. Aquelas relacionadas ao método de abate incluíram hemorragias causadas em bovinos dessensibilizados pelo dardo cativo ou por punção por faca da medula na articulação atlanto-occipital. Artefatos relacionados à amostragem e manuseio de tecido do SNC consistiram na inclusão de pó de osso no tecido neural em consequência do uso de serra para abrir a caixa craniana; neurônios escuros produzidos pelo manuseio excessivo do cérebro e micro-organismos que contaminaram os tecidos durante a amostragem ou processamento histológico. Alterações autolíticas pós-mortais ou de putrefação incluíram vacuolizações na bainha de mielina, halos claros em torno dos neurônios e oligodendrócitos, aglomerados de bacilos de putrefação dentro dos vasos ou dispersos em todo o tecido cerebral, relacionados ou não a halos claros. Uma alteração autolítica pós-mortal intrigante e relativamente frequente encontrada foi a dissolução parcial da camada de células granulares (CCG) do cerebelo, também referida como conglutinação da CCG ou "état glacé". Devido à escassez de publicações abrangentes neste assunto, esta revisão pretende abordar as principais ciladas que possam aparecer no cérebro de bovinos, na esperança de ajudar outros patologistas a evitar interpretá-las erroneamente.(AU)
Assuntos
Animais , Bovinos , Encéfalo/anatomia & histologia , Bovinos/anatomia & histologia , Doenças dos Bovinos/fisiopatologia , Encefalopatia Espongiforme Bovina/fisiopatologia , Autopsia/veterináriaResumo
Doença do corpúsculo de inclusão (IBD) é uma enfermidade caracterizada por corpúsculos intracitoplasmáticos em diversos tecidos, principalmente no sistema nervoso central, responsável pelos principais sinais clínicos atribuídos à doença que acomete Boas e Phytons de cativeiro; essa enfermidade tem sido uma preocupação mundial devido à alta morbidade e mortalidade. O diagnóstico é feito pela visualização dos corpúsculos causados por um Arenavírus modificado. Salmonella sp. pertence à microflora de animais de sangue frio e quente, e é um patógeno oportunista que pode causar quadros gastrointestinais ou septicêmicos. Em répteis a Salmonella sp. é a bactéria com maior frequência de citações em espondilites e osteomielites. Relata-se um caso de uma jiboia (Boa constrictor constrictor) que apresentava restrição de movimento e múltiplos granulomas dorsais nas vértebras; à radiografia evidenciaram-se regiões fraturadas. Após meses de tratamentos sem melhora clínica e o aparecimento de novas lesões o animal ficou prostrado, anoréxico, caquético e desenvolveu opistótono; optou-se pela eutanásia. À necropsia verificaram-se, nas vértebras, múltiplos focos dorsais com aumento de volume que variava de 1,7cm à 3,8cm. Ao corte as vértebras eram deformadas e exibiam conteúdo caseoso focal próximo ao canal medular, este foi coletado para microbiologia onde se identificou Salmonella sp. À microscopia as vértebras tinham um infiltrado inflamatório multifocal moderado de macrófagos e heterofilos. Algumas áreas possuíam grande quantidade de granulomas com calcificação central e inúmeras células gigantes; outros mostravam áreas de osteomalácia e fibrose. Em raros focos havia fratura do corpo vertebral e compressão da medula espinhal com leve infiltrado inflamatório invadindo o canal medular. No pulmão, principalmente no epitélio brônquico, por vezes até dentro de linfócitos do tecido linfoide bronco-associado, no intestino, fígado, vesícula biliar, nos rins e no encéfalo foram encontradas diversas estruturas eosinofílicas intracitoplasmáticas arredondadas que variavam de 1 a 10 µm. Essas estruturas acompanhavam ou não inflamações mononucleares. Os achados são compatíveis com IBD e espondilite por salmonelose. A IBD é uma enfermidade frequente em serpentes de cativeiro, de importância mundial, que provavelmente é subdiagnosticada no Brasil. Essa doença causa imunossupressão que favorece ao desenvolvimento de outras enfermidades, e é tipicamente associada a outras doenças como a espondilite encontrada no caso.(AU)
Inclusion Body Disease (IBD) is a disorder characterized by intracytoplasmic corpuscles in different tissues, mainly in the CNS, wich is responsible for the major neurological signs attributable to this disease. It affects Boas and Phytons in captivity and have been a global concern due to the high morbidity and mortality. The diagnosis is made by visualization of corpuscles caused by a modified Arenaviruses. Salmonella sp. belongs to microflora of cold and warm-blooded animals; it is an opportunistic pathogen that can causes gastrointestinal or septic disorders. In reptiles, Salmonella sp. is the bacteria most frequently quotes in spondylitis and osteomyelitis. This article describes a boa constrictor (Boa constrictor constrictor) that had restriction of movement and multiple granulomas in the dorsal vertebrae, the shadowgraph showed up fractured regions. After months of treatment without clinical improvement and the emergence of new injuries, the animal started to get prostrate, anorexic, cachectic and developed opisthotonos. It was opted for euthanasia. At necropsy it was found in multiple spots swelling of the dorsal vertebrae that ranging from mild to moderate. At the cutting vertebrae it was visible deformed and showed focal caseous content near the spinal cord, this was collected for microbiology where it was identified Salmonella sp. At microscopic evaluation the vertebrae had one to multifocal moderate inflammatory infiltrate of macrophages and heterophils. Some areas had lots of granulomas with central calcification and numerous giant cells. Other vertebras showed areas of osteomalácea and fibrosis. Rare focus had vertebral body fracture and spinal cord compression with mild infiltration entering the spinal cord canal. In the lung, especially in the bronchial epithelium, sometimes even within lymphocytes in bronchial-associated lymphoid tissue, in the intestine, liver, gall bladder, kidney and brain were found various structures of eosinophilic intracytoplasmic rounded ranging between 1 and 10 micrometers. These structures accompanied or not mononuclear inflammation. These findings are consistent with IBD and spondylitis due to salmonellosis. The IBD is a common disease in captive snakes, of world importance, is probably underdiagnosed in Brazil. This disease causes immunosuppression favoring the development of other affections, and is typically associated with other diseases such as spondylitis found in the case.(AU)
Assuntos
Animais , Salmonella/isolamento & purificação , Salmonelose Animal , Serpentes/microbiologia , Espondilite/veterinária , Corpos de Inclusão , ArenavirusResumo
Doença do corpúsculo de inclusão (IBD) é uma enfermidade caracterizada por corpúsculos intracitoplasmáticos em diversos tecidos, principalmente no sistema nervoso central, responsável pelos principais sinais clínicos atribuídos à doença que acomete Boas e Phytons de cativeiro; essa enfermidade tem sido uma preocupação mundial devido à alta morbidade e mortalidade. O diagnóstico é feito pela visualização dos corpúsculos causados por um Arenavírus modificado. Salmonella sp. pertence à microflora de animais de sangue frio e quente, e é um patógeno oportunista que pode causar quadros gastrointestinais ou septicêmicos. Em répteis a Salmonella sp. é a bactéria com maior frequência de citações em espondilites e osteomielites. Relata-se um caso de uma jiboia (Boa constrictor constrictor) que apresentava restrição de movimento e múltiplos granulomas dorsais nas vértebras; à radiografia evidenciaram-se regiões fraturadas. Após meses de tratamentos sem melhora clínica e o aparecimento de novas lesões o animal ficou prostrado, anoréxico, caquético e desenvolveu opistótono; optou-se pela eutanásia. À necropsia verificaram-se, nas vértebras, múltiplos focos dorsais com aumento de volume que variava de 1,7cm à 3,8cm. Ao corte as vértebras eram deformadas e exibiam conteúdo caseoso focal próximo ao canal medular, este foi coletado para microbiologia onde se identificou Salmonella sp. À microscopia as vértebras tinham um infiltrado inflamatório multifocal moderado de macrófagos e heterofilos. Algumas áreas possuíam grande quantidade de granulomas com calcificação central e inúmeras células gigantes; outros mostravam áreas de osteomalácia e fibrose. Em raros focos havia fratura do corpo vertebral e compressão da medula espinhal com leve infiltrado inflamatório invadindo o canal medular. No pulmão, principalmente no epitélio brônquico, por vezes até dentro de linfócitos do tecido linfoide bronco-associado, no intestino, fígado, vesícula biliar, nos rins e no encéfalo foram encontradas diversas estruturas eosinofílicas intracitoplasmáticas arredondadas que variavam de 1 a 10 µm. Essas estruturas acompanhavam ou não inflamações mononucleares. Os achados são compatíveis com IBD e espondilite por salmonelose. A IBD é uma enfermidade frequente em serpentes de cativeiro, de importância mundial, que provavelmente é subdiagnosticada no Brasil. Essa doença causa imunossupressão que favorece ao desenvolvimento de outras enfermidades, e é tipicamente associada a outras doenças como a espondilite encontrada no caso.(AU)
Inclusion Body Disease (IBD) is a disorder characterized by intracytoplasmic corpuscles in different tissues, mainly in the CNS, wich is responsible for the major neurological signs attributable to this disease. It affects Boas and Phytons in captivity and have been a global concern due to the high morbidity and mortality. The diagnosis is made by visualization of corpuscles caused by a modified Arenaviruses. Salmonella sp. belongs to microflora of cold and warm-blooded animals; it is an opportunistic pathogen that can causes gastrointestinal or septic disorders. In reptiles, Salmonella sp. is the bacteria most frequently quotes in spondylitis and osteomyelitis. This article describes a boa constrictor (Boa constrictor constrictor) that had restriction of movement and multiple granulomas in the dorsal vertebrae, the shadowgraph showed up fractured regions. After months of treatment without clinical improvement and the emergence of new injuries, the animal started to get prostrate, anorexic, cachectic and developed opisthotonos. It was opted for euthanasia. At necropsy it was found in multiple spots swelling of the dorsal vertebrae that ranging from mild to moderate. At the cutting vertebrae it was visible deformed and showed focal caseous content near the spinal cord, this was collected for microbiology where it was identified Salmonella sp. At microscopic evaluation the vertebrae had one to multifocal moderate inflammatory infiltrate of macrophages and heterophils. Some areas had lots of granulomas with central calcification and numerous giant cells. Other vertebras showed areas of osteomalácea and fibrosis. Rare focus had vertebral body fracture and spinal cord compression with mild infiltration entering the spinal cord canal. In the lung, especially in the bronchial epithelium, sometimes even within lymphocytes in bronchial-associated lymphoid tissue, in the intestine, liver, gall bladder, kidney and brain were found various structures of eosinophilic intracytoplasmic rounded ranging between 1 and 10 micrometers. These structures accompanied or not mononuclear inflammation. These findings are consistent with IBD and spondylitis due to salmonellosis. The IBD is a common disease in captive snakes, of world importance, is probably underdiagnosed in Brazil. This disease causes immunosuppression favoring the development of other affections, and is typically associated with other diseases such as spondylitis found in the case.(AU)
Assuntos
Animais , Salmonella/isolamento & purificação , Salmonelose Animal , Serpentes/microbiologia , Espondilite/veterinária , Corpos de Inclusão , ArenavirusResumo
Background The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.(AU)
Assuntos
Dobramento de Proteína , Elapidae , Venenos Elapídicos , Anticorpos , NeurotoxinasResumo
The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.(AU)
Assuntos
Animais , /análise , Neurotoxinas/classificação , Neurotoxinas/genética , Antivenenos/biossíntese , SerpentesResumo
The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.
Assuntos
Animais , Antivenenos/biossíntese , Neurotoxinas/classificação , Neurotoxinas/genética , SerpentesResumo
A inclusão de aditivos antimicotoxinas (AAMs) em rações para aves é uma das principais estratégias para reduzir os efeitos prejudiciais das micotoxinas. AAMs são substâncias capazes de adsorver, inativar, neutralizar ou biotransformar micotoxinas. O objetivo deste trabalho foi desenvolver uma metodologia ex vivo para avaliação de AAMs utilizados na produção avícola. Explantes jejunais de frangos de corte destinados ao consumo humano foram utilizados em dois estudos para testar a eficácia de AAMs frente à aflatoxina B1 (AFB1) e ao deoxinivalenol (DON); o efeito direto de DON sobre as vilosidades intestinais também foi investigado. Quatro pares de câmaras de Ussing com área de contato intestinal de 1,0 cm² foram mantidas a 37 ºC e borbulhadas com carbogênio durante 120 min. No estudo 1, seis AAMs disponíveis comercialmente (aditivo antimicotoxinas - AAM 1 a 6) tiveram sua capacidade de reduzir a absorção intestinal de AFB1 avaliada a fim de verificar eficácia. Explantes jejunais (n=4/ave) foram obtidos de 60 frangos no abate, totalizando 240 amostras (40 amostras/AAM). Os explantes foram submetidos a dois tratamentos por AAM: T1 (controle) - 2,8 mg/L de AFB1 e T2 - 2,8 mg/L de AFB1 + 0,5% AAM. Os AAMs também foram testados in vitro para avaliar a adsorção de AFB1 em fluido intestinal artificial. O estudo 2 analisou o impacto deletério de DON sobre as vilosidades intestinais e a eficácia de um AAM. Dois experimentos foram conduzidos para avaliar parâmetros histopatológicos e imunohistoquímicos: Experimento 1) T1 (controle positivo) - somente solução tampão, e T2 - 10 mg/L de DON (duas replicatas/tratamento); e Experimento 2) T1 (controle positivo) - somente solução tampão, T2 (controle negativo) - 10 mg/L de DON, T3 - somente o AAM (0,5%) e T4 - 10 mg/L de DON + 0,5% AAM. Explantes jejunais (n=4/ave) foram obtidos de 22 frangos no abate, totalizando 88 amostras (40 e 48 amostras para os experimentos 1 e 2, respectivamente). Os resultados do estudo 1 demonstraram que AAM1 ao AAM6 reduziram a absorção intestinal de AFB1 nos ensaios ex vivo em 67,11%, 73,82%, 80,70%, 85,86%, 86,28% e 82,32%, respectivamente; já nos testes in vitro, AAM1 ao AAM6 apresentaram adsorção de 99,72%, 99,37%, 99,67%, 99,53%, 99,04% e 99,15%, respectivamente. Não houve correlação entre os achados ex vivo e in vitro. Quanto ao estudo 2, DON reduziu o tamanho dos enterócitos e de seus núcleos e aumentou a vacuolização do citoplasma, o desnudamento do ápice das vilosidades e o número de células apoptóticas no Experimento 1; os parâmetros investigados no Experimento 2 revelaram que o uso do AAM mitigou os efeitos prejudiciais de DON sobre as vilosidades intestinais. A presente avaliação demonstra que o modelo ex vivo utilizando explantes intestinais de frangos de corte montados em câmaras de Ussing é uma ferramenta viável para complementar ensaios in vitro e in vivo empregados na avaliação da eficácia de AAMs e também para a triagem de novos compostos. Além disso, essa metodologia pode ser aplicada para determinar o impacto negativo de DON na integridade do epitélio intestinal de frangos de corte
Inclusion of antimycotoxins additives (AMAs) in poultry feed is one of the main strategies to reduce the harmful effects of mycotoxins. AMAs are substances capable of adsorbing, inactivating, neutralizing or biotransforming mycotoxins. This work aimed to develop an ex vivo methodology to evaluate AMAs used in poultry production. Intestinal explants from broiler chickens destined to human consumption were used in two studies to test the efficacy of AMAs against aflatoxin B1 (AFB1) and deoxynivalenol (DON); the direct effect of DON upon intestinal tissue was also investigated. Four pairs of Ussing chambers with an intestinal contact area of 1.0 cm² were used at 37°C and bubbled with carbogen gas for 120 min. In study 1, six commercially available AMAs (antimycotoxins additive - AMA 1 to 6) had their ability to reduce intestinal absorption of AFB1 evaluated in order to assess efficacy. Jejunal explants (n=4/bird) were collected from 60 broilers at slaughter, totaling 240 samples (40 samples/AMA). The explants were subjected to two treatments per AMA: T1 (control) - 2.8 mg/L of AFB1, and T2 - 2.8 mg/L of AFB1 + 0.5% AMA. The AMAs were also tested in vitro to investigate AFB1 adsorption in artificial intestinal fluid. Study 2 analyzed the detrimental impact of DON on intestinal tissue as well as the efficacy of an AMA. Two experiments were conducted to examine histopathological and immunohistochemical parameters: Experiment 1) T1 (positive control) - buffer solution only, and T2 - 10 mg/L DON (two replicates/treatment); and Experiment 2) T1 (positive control) - buffer solution only, T2 (negative control) - 10 mg/L DON, T3 - AMA (0.5%) only, and T4 - 10 mg/L DON + 0.5% AMA. Jejunal explants (n=4/bird) were taken from 22 broilers at slaughter, totaling 88 samples (40 and 48 samples for experiments 1 and 2, respectively). Results of study 1 showed that AMA1 to AMA6 decreased intestinal absorption of AFB1 in the ex vivo assays by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively; as for the in vitro tests, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. No correlation was seen between ex vivo and in vitro findings. Regarding the second study, DON reduced the size of enterocytes as well as of their nuclei and increased cytoplasmic vacuolization, apical denudation of villi and the number of apoptotic cells in Experiment 1; the parameters investigated in Experiment 2 evidenced that the AMA mitigated the harmful effects of DON upon intestinal villi. The present assessment demonstrates that the ex vivo model using intestinal explants from broiler chickens mounted on Ussing chambers is a viable tool to complement in vitro and in vivo assays employed to evaluate the efficacy of AMAs and also to screen new compounds. Moreover, this methodology may be applied to determine the burden imposed by DON on the integrity of the intestinal epithelium of broilers
Resumo
Avaliaram-se a proporção volumétrica dos constituintes do corpo lúteo e a distribuição de células lúteas com grânulos citoplasmáticos no corpo lúteo de animais Nelore ao longo da gestação e em animais não gestantes. Foram coletados ovários com corpos lúteos de 24 animais abatidos em frigorífico. Os animais foram distribuídos em dois grupos: gestantes e não gestantes. A idade gestacional foi determinada pela medição do comprimento apicocaudal do feto. Os ovários foram distribuídos segundo o trimestre gestacional: primeiro - gestação de até 90 dias; segundo - de 91 a 180 dias; e terceiro - de 181 a 270 dias. Os corpos lúteos, processados para inclusão em paraplast, foram avaliados em microscópio de luz. Na proporção volumétrica do corpo lúteo, os constituintes avaliados foram citoplasma e núcleo de células lúteas, tecido conjuntivo e fibroblastos, células endoteliais e pericitos e vasos sanguíneos. A proporção volumétrica de citoplasma de células lúteas reduziu de 46,1±2,8% para 37,9±3,5% e a de núcleo de células lúteas reduziu de 9,2±1,0% para 6,2±1,0%, do primeiro para o segundo trimestre. A proporção de tecido conjuntivo e fibroblastos aumentou de 20,9±5,4%, no primeiro trimestre, para 34,0±4,2%, no segundo trimestre. Grânulos citoplasmáticos foram evidenciados pelas técnicas histoquímicas xylidine ponceau e azul de bromofenol, que indicam sua provável composição proteica. A quantidade de células maiores com grânulos aumentou significativamente de 2,3±1,9%, no primeiro trimestre, para 25,5±20,3%, no segundo. A proporção volumétrica de citoplasma e núcleo de células lúteas diminuiu ao longo da gestação, e a de tecido conjuntivo aumentou. A quantidade de células lúteas maiores contendo grânulos também aumentou ao longo da gestação.(AU)
The purpose of this study was to evaluate, in cycling and pregnant Nelore cows, the volumetric proportion of the corpus luteum and the distribution of lutein cells with cytoplasmic granules. Twenty four ovaries with corpus luteum were collected in a slaughterhouse. The ovaries were distributed into two groups: pregnant and non-pregnant cows. The group of pregnant cows was divided according to the stage of pregnancy, by the crown-rump fetus length: 1st third - until 90 days, 2nd third - between 91 and 180 days and 3rd third - between 181 and 270 days. The corpus luteum were processed for inclusion in paraplast, and slides were stained for analysis in light microscopy. The constituents quantified in the volumetric proportion were: lutein cells cytoplasm and nucleus, connective tissue and fibroblasts, endothelial cells and pericyte and blood vessels. The volumetric proportion of lutein cells cytoplasm decreased significantly from 46.1±2.8% to 37.9±3.5%, and also decreased significantly in the nucleus from 9.2±1.0% to 6.2±1.0% between the first and the second trimester of pregnancy. The amount of connective tissue and fibroblasts increased significantly throughout gestation, with 20.9±5.4% on the first trimester and 34.0±4.2% on the second. Granules were evidenced by the histochemical technique of Xylidine Ponceau and Bromophenol blue, indicating their probable protein composition. The amount of lutein cells with granules increased throughout gestation, but significant difference was observed only between the first (2.3±1.9%) and second trimester (25.5±20.3%). The cytoplasmic and nucleus volumetric proportion decreased during pregnancy and the volumetric proportion of connective tissue increased. The amount of lutein cells with granules increased throughout pregnancy.(AU)
Assuntos
Animais , Feminino , Gravidez , Bovinos , Gravidez/fisiologia , Corpo Lúteo/anatomia & histologia , Grânulos CitoplasmáticosResumo
Avaliaram-se a proporção volumétrica dos constituintes do corpo lúteo e a distribuição de células lúteas com grânulos citoplasmáticos no corpo lúteo de animais Nelore ao longo da gestação e em animais não gestantes. Foram coletados ovários com corpos lúteos de 24 animais abatidos em frigorífico. Os animais foram distribuídos em dois grupos: gestantes e não gestantes. A idade gestacional foi determinada pela medição do comprimento apicocaudal do feto. Os ovários foram distribuídos segundo o trimestre gestacional: primeiro - gestação de até 90 dias; segundo - de 91 a 180 dias; e terceiro - de 181 a 270 dias. Os corpos lúteos, processados para inclusão em paraplast, foram avaliados em microscópio de luz. Na proporção volumétrica do corpo lúteo, os constituintes avaliados foram citoplasma e núcleo de células lúteas, tecido conjuntivo e fibroblastos, células endoteliais e pericitos e vasos sanguíneos. A proporção volumétrica de citoplasma de células lúteas reduziu de 46,1±2,8% para 37,9±3,5% e a de núcleo de células lúteas reduziu de 9,2±1,0% para 6,2±1,0%, do primeiro para o segundo trimestre. A proporção de tecido conjuntivo e fibroblastos aumentou de 20,9±5,4%, no primeiro trimestre, para 34,0±4,2%, no segundo trimestre. Grânulos citoplasmáticos foram evidenciados pelas técnicas histoquímicas xylidine ponceau e azul de bromofenol, que indicam sua provável composição proteica. A quantidade de células maiores com grânulos aumentou significativamente de 2,3±1,9%, no primeiro trimestre, para 25,5±20,3%, no segundo. A proporção volumétrica de citoplasma e núcleo de células lúteas diminuiu ao longo da gestação, e a de tecido conjuntivo aumentou. A quantidade de células lúteas maiores contendo grânulos também aumentou ao longo da gestação.(AU)
The purpose of this study was to evaluate, in cycling and pregnant Nelore cows, the volumetric proportion of the corpus luteum and the distribution of lutein cells with cytoplasmic granules. Twenty four ovaries with corpus luteum were collected in a slaughterhouse. The ovaries were distributed into two groups: pregnant and non-pregnant cows. The group of pregnant cows was divided according to the stage of pregnancy, by the crown-rump fetus length: 1st third - until 90 days, 2nd third - between 91 and 180 days and 3rd third - between 181 and 270 days. The corpus luteum were processed for inclusion in paraplast, and slides were stained for analysis in light microscopy. The constituents quantified in the volumetric proportion were: lutein cells cytoplasm and nucleus, connective tissue and fibroblasts, endothelial cells and pericyte and blood vessels. The volumetric proportion of lutein cells cytoplasm decreased significantly from 46.1±2.8% to 37.9±3.5%, and also decreased significantly in the nucleus from 9.2±1.0% to 6.2±1.0% between the first and the second trimester of pregnancy. The amount of connective tissue and fibroblasts increased significantly throughout gestation, with 20.9±5.4% on the first trimester and 34.0±4.2% on the second. Granules were evidenced by the histochemical technique of Xylidine Ponceau and Bromophenol blue, indicating their probable protein composition. The amount of lutein cells with granules increased throughout gestation, but significant difference was observed only between the first (2.3±1.9%) and second trimester (25.5±20.3%). The cytoplasmic and nucleus volumetric proportion decreased during pregnancy and the volumetric proportion of connective tissue increased. The amount of lutein cells with granules increased throughout pregnancy.(AU)
Assuntos
Animais , Bovinos , Grânulos Citoplasmáticos , Corpo Lúteo , Histologia , Citoplasma , Vasos Sanguíneos , Prenhez , Tecido ConjuntivoResumo
A case of asteroid bodies associated with granulomatous meningoencephalitis is reported in a 4-year-old female dachshund that showed intermittent tremors and blindness. Necropsy revealed leptomeninges moderately hyperemic and from the parietal cortex extending into the temporal and frontal cortex, thalamus and midbrain flattening of the gyri and a grayish and friable focally extensive area. Microscopically intense and extensive perivascular inflammatory infiltrates of lymphocytes, plasma cells, macrophages and giant cells were observed. In the cytoplasm of the giant cells, neuropil stellate structures ranging from 20 to 80 m in diameter with a central dense and homogenous zone and with amphophilic and vacuolated cytoplasm consistent with asteroid bodies were present. Special staining and transmission electron microscopy permitted the diagnosis of asteroid bodies associated with granulomatous meningoencephalitis.
Assuntos
Feminino , Animais , Cães , Corpos de Inclusão/virologia , Meningoencefalite/veterinária , Sistema Nervoso/patologia , Cistos Aracnóideos/veterináriaResumo
A case of asteroid bodies associated with granulomatous meningoencephalitis is reported in a 4-year-old female dachshund that showed intermittent tremors and blindness. Necropsy revealed leptomeninges moderately hyperemic and from the parietal cortex extending into the temporal and frontal cortex, thalamus and midbrain flattening of the gyri and a grayish and friable focally extensive area. Microscopically intense and extensive perivascular inflammatory infiltrates of lymphocytes, plasma cells, macrophages and giant cells were observed. In the cytoplasm of the giant cells, neuropil stellate structures ranging from 20 to 80 m in diameter with a central dense and homogenous zone and with amphophilic and vacuolated cytoplasm consistent with asteroid bodies were present. Special staining and transmission electron microscopy permitted the diagnosis of asteroid bodies associated with granulomatous meningoencephalitis.(AU)
Assuntos
Animais , Feminino , Cães , Meningoencefalite/veterinária , Corpos de Inclusão/virologia , Sistema Nervoso/patologia , Cistos Aracnóideos/veterináriaResumo
An adult male Amazon tree boa (Corallus hortulanus) from the Zoo-Botanical Foundation (Belo Horizonte, Brazil) died after a period of apathy and anorexia. Cachexia was the only significant gross finding. Numerous eosinophilic intracytoplasmic inclusion bodies were found in the liver, lungs, heart, testes, kidneys, and adipose tissue, compatible with the Inclusion Body Disease. The snake also had an undifferentiated metastatic sarcoma. In order to further characterize the inclusion bodies found in this case, transmission electronic microscopy was performed. Inclusion Body Disease affects boid snakes, causing regurgitation, anorexia and neurological signs that eventually lead to death. This is a challenging disease since its etiology, pathogenesis, and epidemiology are unknown, and therefore treatment is not effective. This is the first report of Inclusion Body Disease in boid snake in Brazil.
Assuntos
Animais , Boidae , Corpos de Inclusão , Sarcoma/veterináriaResumo
An adult male Amazon tree boa (Corallus hortulanus) from the Zoo-Botanical Foundation (Belo Horizonte, Brazil) died after a period of apathy and anorexia. Cachexia was the only significant gross finding. Numerous eosinophilic intracytoplasmic inclusion bodies were found in the liver, lungs, heart, testes, kidneys, and adipose tissue, compatible with the Inclusion Body Disease. The snake also had an undifferentiated metastatic sarcoma. In order to further characterize the inclusion bodies found in this case, transmission electronic microscopy was performed. Inclusion Body Disease affects boid snakes, causing regurgitation, anorexia and neurological signs that eventually lead to death. This is a challenging disease since its etiology, pathogenesis, and epidemiology are unknown, and therefore treatment is not effective. This is the first report of Inclusion Body Disease in boid snake in Brazil.(AU)
Assuntos
Animais , Boidae , Corpos de Inclusão , Sarcoma/veterináriaResumo
This research was carried out to examine cytopathological effects of Helicoverpa armigera Cytoplasmic polyhedrosis virus (HaCPV) on infected midgut cotton bollworm (Helicoverpa armigera) using transmission and scanning electron microscope. The symptoms on infected host larvae of the host, compared with healthy ones, were getting swollen with milky-white and fragile Histopathological examinations showed infection with HaCPV small polyhedral inclusion bodies (PIB) after 1 or 2 days which were observed in columnar cells of midgut. Virions were partially or completely occupied in a polyhedral matrix to form polyhedral inclusion bodies (PIB) at periphery of virogenic stroma. PIBs were measured 0.5 to 3.5 mm and virions about 46 nm in diameter. Microvilli of infected columnar cells were affected and degenerated immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 3 days after infection. In addition,PIB were found in goblet cells, 5 or 6 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organells were left. These cytopathic effects caused in the midgut by HaCPV on cotton bollworm larvae are essentially similar to those have been reported for lepidoperan and dipteran infection by CPV.
Resumo
Foi realizado estudo retrospectivo de casos de poxviroses diagnosticadas e notificadas em bovinos, no Distrito Federal (DF), entre 2015 a 2018, pelo Serviço Oficial Veterinário da Secretaria de Agricultura, Abastecimento e Desenvolvimento Rural do DF. Na maioria dos casos, foram coletadas amostras de lesões orais, cutâneas (tetos, úbere) ou podais para diagnóstico molecular por PCR. Em aproximadamente 70% dos casos, também foram coletados fragmentos para histopatologia. De 2.467 bovinos avaliados clinicamente, de 385 propriedades, 93 apresentaram lesões sugestivas e/ou compatíveis com poxviroses. Desses, foram confirmados 52 casos de poxviroses: 27 por VACV, nove por PCPV, oito por BPSV, cinco por coinfecção PCPV e BPSV e três por parapoxvírus não identificado. As enfermidades foram observadas em propriedades com diferentes tipos de exploração (corte, leite ou mista) e em nove das 30 regiões administrativas (RAs) do DF. As lesões macroscópicas consistiram de pápulas, vesículas, úlceras, crostas e cicatrizes, e variaram de tipo, intensidade e local acometido, de acordo com o(s) vírus detectado(s). Foi observado apenas um episódio de comprometimento humano em um caso de infecção por BPSV. Os achados histopatológicos foram muito similares, independente do poxvírus envolvido e incluíram inflamação linfoplasmo-histiocítica e/ou neutrofílica multifocal superficial leve a moderada com acantose e hiperqueratose paraqueratótica, usualmente associada a crostas serocelulares e espongiose. Nos casos com úlceras, havia áreas focalmente extensas de necrose com infiltrado acentuado de neutrófilos no tecido conjuntivo adjacente. Em seis casos (dois de VACV, dois de PCPV e dois de coinfecção por PCPV/BPSV), foi observada pequena a moderada quantidade de corpúsculos de inclusão eosinofílicos de 4-8 µm no citoplasma dos queratinócitos. Os dados do presente estudo demonstram que há ampla circulação de diferentes poxvírus em bovinos de diferentes categorias e em várias RAs do DF.
A retrospective study of poxviruses cases diagnosed and notified in cattle, in Distrito Federal (DF), Brazil, between 2015 and 2018, was conducted. In the most cases, samples of oral, cutaneous (teats, udder) or roof lesions were collected to molecular diagnosis by PCR. In approximately 70% of the cases, additional samples were collected to histopathology. Out of 2,467 cattle analysed of 385 farms, 93 presented lesions sugestive and/or compatible with poxviruses. Fifty-two cases of poxviruses were confirmed: 27 by VACV, 9 by PCPV, 8 by BPSV, 5 by PCPV and BPSV coinfection, and 3 by not identified parapoxvirus. The diseases were observed in farms with diferente types of exploration (beef, dairy, or mixed cattle) and in 9 of 30 administrative regions (RAs) of DF. Gross findings include papules, vesicles, ulcers, crusts, and scars, and varied of type, intensity and affected tissue according to the detected virus. Only one episode of human infection in a case of BPSV was observed. Histopathology findings were very similar in all poxviruses and consisted of mild to moderate multifocal infiltrate of lymphocytes, plasma cells, macrophages and/or neutrophils with acanthosis and parakeratotic hyperkeratosis, usually associated to crusts and spongiosis. In the ulcerated cases, there was focally extensive areas of necrosis with severe infiltrate of neutrophils in the adjacent connective tissue. Few to moderate amount of cytoplasmic eosinophilic inclusion bodies (4-8µm) were noted in six cases (two of VACV, two of PCPV and two of PCPV and BPSV coinfection). Data of the current study demonstrate that there is wide circulation of different poxvirus in cattle of different categories and in many RAs of DF.
Resumo
O ensaio toxicológico foi realizado com o objetivo de determinar o potencial tóxico do fármaco ibuprofeno através de alterações comportamentais e alterações histopatológicas em brânquias e fígados de juvenis de Rhamdia quelen submetidos a diferentes concentrações de Ibuprofeno (0,0; 0,5; 5,0 e 50,0 mg/L). As quais foram determinadas a partir de ensaios agudos com uma CL50 de 5,0mg/L. Para cada concentração foram feitas quatro réplicas, com quatro exemplares, cada. O experimento teve duração total de 120 horas. Na preparação do material foi utilizada a técnica histológica de rotina para inclusão em parafina, e para as análises as lâminas foram coradas com Hematoxilina e Eosina. Foram evidenciadas alterações nos tecidos analisados, como: Fusão incompleta de lamelas e aneurisma, entre outras, para as brânquias e vacuolização citoplasmática e necrose focal, entre outras, para os fígados, as quais foram classificadas em estágios I, II e III, quanto a severidade das lesões, determinado o Índice de Alterações Histológicas e os Valores Médios das Alterações. O fármaco apresentou potencial tóxico para esta espécie, mesmo em concentrações baixas, sendo evidenciados danos que vão de moderados a severos nos fígados e brânquias dos peixes analisados.
The toxicological test was performed to determine the toxic potential of the drug ibuprofen through histopathological eyepieces in gills and livers of Rhamdia quelen juveniles submitted to different concentrations of Ibuprofen (0,0; 0,5; 5,0 e 50,0 mg/L). These were determined from acute tests with an CL50 of 5,0mg/L. For each of them, four replicates, with four specimens each. The experiment lasted 120 hours. In preparing the material for the routine histological technique for inclusion in paraffin, and as analyzes as slides were stained with Hematoxylin and Eosin. Our analogues were tested, such as: Incomplete lamellae and aneurysm fusion, among others, for gills and vacuolization, cytoplasmic and focal necrosis, among others, for livers, which were classified in stages I, II and III for Severity of injuries, determined the Index of Historical Changes and Average Values of Changes. The drug presented toxic potential for this species, even at low concentrations, and damages ranging from moderate to severe in the livers and gills of the fish analyzed were observed.
Resumo
A torta de girassol é um coproduto da cadeia do biodiesel considerada como boa alternativa para uso na alimentação animal por apresentar, principalmente, altos teores de proteína e extrato etéreo. Assim, o objetivo desse trabalho foi avaliar os efeitos da inclusão da torta de girassol em dietas para cordeiros mestiços Santa Inês x Dorper sobre as características de carcaça e não constituintes de carcaça; qualidade da carne pelos métodos da análise físico-química e sensorial; bem como a quantificação e identificação das proteínas que obtiveram maior expressividade diante dos tratamentos, pela análise da proteômica. Foram utilizados 24 cordeiros Santa Inês x Dorper, castrados, com peso vivo inicial de 18,9 ± 2,17 kg, os quais foram distribuídos em um delineamento inteiramente casualizado com quatro tratamentos e seis repetições. As dietas experimentais foram constituídas por feno de capim tífton 85, milho em grão moído, farelo de soja, sal mineral e os seguintes níveis de inclusão da torta de girassol que correspondiam aos tratamentos: 0, 5, 10 e 15% na ração completa. Os animais foram mantidos em confinamento até atingirem, em média, 32,1 ± 2,6 kg de peso final. Foram realizadas avaliações objetivas e subjetivas na carcaça. No músculo Longissimus dorsi foi mensurado a cor, e também foram feitas as análises físico-químicas; e no músculo Semimembranosus, foi tomado o pH da carne , ainda na carcaça, 24 horas após o abate, e também foi feita a avaliação sensorial, quantificando, por intermédio de notas, os atributos de aroma, cor, dureza, suculência, sabor e avaliação global. A expressão proteica foi feita através da produção de géis pelas técnicas de eletroforese bidimensional e da espectrometria de massa. Foram realizadas, também, análises referentes à ontologia gênica e de interactoma. Os rendimentos de carcaça (quente, fria e biológico), decresceram linearmente de acordo com o aumento dos níveis de inclusão da torta. Os pesos e rendimentos dos cortes, a composição tecidual da perna, assim como os constituintes não carcaça não foram influenciados (p>0,05) pela dieta. Os parâmetros físico-químicos e sensoriais da carne de cordeiro também não foram influenciados (p>0,05) pelos níveis de inclusão da torta de girassol. Nos géis, foram observados uma média de 117 ± 16,8 spots, dos quais apenas 30 foram recortados, sendo 29 identificados, correspondendo a 23 proteínas diferentes. Destas, apenas a actina citoplasmática e ATP sintase foram diferencialmente expressas (p<0,05) entre os tratamentos. O interactoma dessas duas proteínas não mostrou nenhuma relação direta, porém, indiretamente elas se encontram associadas através do processo de contração muscular. A torta de girassol pode ser incluída em dietas à base de milho e soja, de cordeiros mestiços Santa Inês x Dorper, até o nível de 15% sem provocar alteração na maioria das características dos constituintes e não constituintes de carcaça; nas características físico-químicas e sensoriais da carne, assim como na expressão proteica do músculo Semimembranosus.
The sunflower cake is a biodiesel byproduct chain considered a good alternative for use in animal feed by presenting mainly high protein and ether extract. Thus, the objective of this study was to evaluate the effects of the inclusion of sunflower cake in diets for Santa Inês x Dorper crossbred lambs on carcass characteristics and non carcass constituents; quality of meat by methods of physical-chemical and sensory analysis; as well as the quantification and identification of the proteins that obtained greater expressiveness before the treatments, by the proteomics analysis. Twenty four lambs were used Santa Inês x Dorper, castrated, with initial weight of 18.9 ± 2.17 kg, which were distributed in a completely randomized design with four treatments and six repetitions. The experimental diets were composed of grass hay Tifton 85, grain ground corn, soybean meal, mineral salt and the following levels of inclusion of sunflower cake which corresponded to the treatments: 0, 5, 10 and 15% in the total ration. The animals were kept in feedlot until they reach an average of 32.1 ± 2.6 kg body final weight. Objective and subjective evaluations were performed on the carcass. In the Longissimus dorsi muscle the color was measured, and the physical-chemical analyzes were also performed; And in the Semimembranosus muscle, the pH of the meat was still taken in the carcass, 24 hours after the slaughter, and also the sensorial evaluation was made, quantifying, through notes, the attributes of aroma, color, hardness, juiciness, flavor and overall assessment. Protein expression was achieved through the production of gels by two-dimensional electrophoresis and mass spectrometry techniques. Analyzes were also made regarding gene ontology and interacome. Carcass yields (hot, cold and biological) decreased linearly according to the increase of the inclusion levels of the cake. The weights and yields of the cuts, the tissue composition of the leg, as well as the non-carcass constituents were not influenced (p>0.05) by the diet. The physical-chemical and sensorial parameters of lamb meat were also not influenced (p>0.05) by the inclusion levels of the sunflower cake. In the gels, an average of 117 ± 16.8 spots were observed, of which only 30 were cut out, 29 of which were identified, corresponding to 23 different proteins. Of these, only cytoplasmic actin and ATP synthase were differentially expressed (p<0.05) between treatments. The interactome of these two proteins did not show any direct relation, however, indirectly they are associated through the process of muscular contraction. Sunflower cake can be included in corn and soybean diets of Santa Inês x Dorper crossbred lambs up to the 15% level without alteration in most of the characteristics of the constituents and not carcass constituents; In the physical-chemical and sensorial characteristics of the meat, as well as in the protein expression of Semimembranosus muscle.
Resumo
O mastocitoma (MCT) é um dos tumores mais frequentes em cães, compreendendo de 16 a 21% de todas as neoplasias cutâneas nesta espécie. A graduação histológica é considerada o padrão ouro na avaliação prognóstica dos MCTs, porém seu comportamento biológico variado ressalta a necessidade de métodos complementares para uma avaliação precisa. Atualmente, identifica-se na literatura indexada uma série de estudos propondo novos marcadores moleculares como fatores prognósticos em MCTs caninos, porém poucos estão consolidados na rotina clínica e/ou diagnóstica. Assim, este trabalho teve como objetivo principal investigar o valor prognóstico de marcadores imunohistoquímicos publicados em artigos científicos através de uma revisão sistemática e meta-análise, assim como avaliar o valor prognóstico do fator de crescimento do endotélio vascular (VEGF) em MCTs caninos. O processo de revisão sistemática se iniciou com 124 estudos identificados em 5 bases de dados e, após a aplicação dos critérios de inclusão e exclusão, 7 artigos foram selecionados para a meta-análise. Os resultados demonstraram que o marcador de proliferação celular Ki67 pode ser considerado um bom fator prognóstico nos MCTs caninos. Porém, a baixa qualidade dos artigos e a insuficiência de dados publicados impossibilitaram a avaliação de outros marcadores, incluindo o receptor de tirosina quinase KIT. No estudo retrospectivo, a imunoexpressão dos marcadores KIT, Ki67 e VEGF foi avaliada em MCTs cutâneos de 43 cães. O marcador Ki67 apresentou correlação significativa com a taxa de mortalidade e sobrevida e o marcador KIT apresentou diferença significativa de sobrevida entre o padrão membranoso e citoplasmático difuso, porém não foram observadas diferenças no VEGF. Em conclusão, estes estudos validaram os marcadores de proliferação celular Ki67 e o receptor de tirosina quinase KIT como principais fatores prognósticos em MCTs caninos, enquanto o VEGF não foi indicado. Por fim, a revisão sistemática permitiu uma análise crítica da qualidade dos trabalhos publicados na área, ressaltando a importância da padronização de metodologias analíticas e da documentação completa de dados clínicos, patológicos e estatísticos empregados em estudos controlados na oncologia veterinária.
Mast cell tumor (MCT) is one of the most frequent tumors in dogs, accounting for 16 to 21% of all cutaneous tumors in this species. Histological grading is considered the standard evaluation method to access the prognostic of MCTs. However, its varied biological behavior highlights the need of complementary methods for an accurate evaluation. Currently, a number of studies proposing new molecular markers as prognostic factors in canine MCTs is identified in the indexed literature, but few are consolidated in the clinical and/or diagnostic routine. The aim of this study was to investigate the prognostic value of immunohistochemical markers published in scientific articles through a systematic review and meta-analysis, as well as to evaluate the prognostic value of vascular endothelial growth factor (VEGF) in canine MCTs. The systematic review process has started with 124 studies identified in 5 databases and, after applying the inclusion and exclusion criteria, 7 articles were selected for the meta-analysis. The results demonstrated that the cell proliferation marker Ki67 can be considered a good prognostic factor in canine MCTs. However, poor quality of the articles and insufficient published data made it impossible to evaluate other markers, including the tyrosine kinase receptor KIT. In the retrospective study, immunoexpression of KIT, Ki67 and VEGF markers was evaluated in cutaneous MCTs from 43 dogs. The Ki67 marker showed a significant correlation with the mortality and survival rates, and the KIT marker showed a significant difference in survival between the membranous and diffuse cytoplasmic patterns groups, but no differences were observed in VEGF. In conclusion, these studies validated the cell proliferation marker Ki67 and the KIT tyrosine kinase receptor as major prognostic factors in canine MCTs, whereas VEGF was not indicated. Finally, the systematic review allowed a critical analysis of the quality of the published studies in this area, emphasizing the importance of the standardization of analytical methodologies and the complete documentation of clinical, pathological and statistical data used in controlled studies in veterinary oncology.