Histomorphological structure of the Bursa cloacalis (Bursa of Fabricius) in young Leiothrix lutea: a comparative study using light microscopy and transmission electron microscopy / Estructura histomorfológica de la Bursa cloacalis (Bursa de Fabricius) en jóvenes Leiothrix lutea: un estudio comparativo utilizando microscopía óptica y microscopía electrónica de transmisión

SUMMARY: This study aimed to investigate the microstructure and ultrastructure of the Bursa cloacalis (Bursa of Fabricius) (BC) in young Leiothrix lutea at various days of age (a few days after hatching) using light microscopy and transmission electron microscopy. The bird BC was sampled at 1, 5, 7, and 9 days of age. Immediately after dissection, the structure and integrity of the BC (not degenerative) were retained and the specific temporal features could be visualized precisely. After hematoxylin-eosin staining and uranyl acetate/lead citrate staining, the microstructure and ultrastructure of the BC, respectively, could be observed clearly. The microscopic observations revealed the following: in addition to change in the size of BC or lymphoid follicles, many cavities were found in the BC; the distribution of the lymphoid follicles in Leiothrix lutea was different from that in other birds; and the segregating line between the bursal cortex and medulla became increasingly clear as the age increased. In conclusion, the structural data obtained in this study provides a better understanding of the specific immunological function of the BC in Leiothrix lutea.

RESUMEN: Este estudio tuvo como objetivo investigar la microestructura y ultraestructura de la Bursa cloacalis (BC) en Leiothrix lutea joven unos días después de la eclosión, utilizando microscopía óptica y microscopía electrónica de transmisión. La BC se muestreó a los 1, 5, 7 y 9 días de edad del Leiothrix lutea. Inmediatamente después de la disección, se observó la estructura y la integridad de la CB (no degenerativa) y se pudo visualizar con precisión las características temporales específicas. Después de la tinción con hematoxilina-eosina y con acetato de uranilo /citrato de plomo, pudimos observar claramente la microestructura y ultraestructura de la BC. Las observaciones microscópicas revelaron el cambio en el tamaño de la CB o de los folículos linfoides y además, se encontraron numerosas cavidades en la CB; la distribución de los folículos linfoides en Leiothrix lutea era diferente a la de otras aves; y la línea de segregación entre la corteza bursal y la médula se hizo cada vez más clara a medida que aumentaba la edad. En conclusión, los datos estructurales obtenidos en este estudio proporcionan una mejor comprensión de la función inmunológica específica de la Bursa cloacalis en Leiothrix lutea.

Identification of genogroups of Infectious Bursal Disease Virus in poultry farms at Colombia / Identificación de genogrupos del virus de la enfermedad de gumboro en granjas avícolas en colombia

RESUMEN El virus de la enfermedad de Gumboro (IBDV) es un avibirnavirus con genoma dsARN que presenta altas tasas de mutación y recombinación. A pesar del efecto inmunosupresor en aves y la frecuencia con que ocurre la infección por este agente en el país son pocos los estudios que caracterizan los cuadros clínicos y se desconoce cuáles son los genogrupos circulantes. Esta investigación tuvo como objetivo determinar la frecuencia de lesiones histopatológicas en órganos del sistema inmune e identificar los genogrupos del IBDV en aves comerciales de Colombia. Para determinar la frecuencia de presentación de lesiones en órganos del sistema inmune se analizaron 381 casos clínicos de las bases de datos del Laboratorio de Patología Aviar (LPA) de la Universidad Nacional de Colombia, sede Bogotá (periodo 2016-2018). Asimismo, se secuenciaron los productos de RT-PCR del gen que codifica para la proteína viral VP2 provenientes de 35 muestras de bursas de Fabricio. Como resultado se encontró evidencia de lesiones microscópicas compatibles con procesos de inmunodepresión en órganos del sistema inmune (bursa de Fabricio, timo, bazo y médula ósea) en el 25 % (97) de los casos analizados y se identificaron los genogrupos 1, 2 y 4 en la siguiente proporción: genogrupo 1-69 % (virus clásicos), genogrupo 2-25 % (variantes) y genogrupo 4-6 % (identificado en Suramérica). Estos hallazgos demuestran la presencia de lesiones en órganos del sistema inmune y la existencia de los genogrupos 1, 2 y 3 del IBDV circulando en aves comerciales en Colombia. Esta es la primera investigación en el país con este sistema de clasificación que permite evidenciar con mayor precisión los cambios en el genoma del IBDV. Lo anterior señala la necesidad de continuar con este tipo de estudios para tener una mejor comprensión de la infección en campo y orientar el diseño e implementación de estrategias de control.

ABSTRACT Infectious Bursal Disease Virus (IBDV) is an avibirnavirus with a dsRNA genome, which has a high mutation and recombination rates. Despite the immunosuppressive effect in poultry and the frequency of infections by this agent, few studies in Colombia that characterize the clinical signs and identify the circulating genogroups have been reported. This study aimed to determine the frequency of histopathological lesions in immune organs and to identify IBDV genogroups in poultry from Colombia. In this way, the Laboratorio de Patología Aviar (LPA) databases from the Universidad Nacional de Colombia (period 2016-2018) were analyzed in order to identify the frequency of lesions present in immune organs. 35 samples of the bursa of Fabricius, positive by RT-PCR, were sequenced to the gene that codes for the VP2 protein. 97 (25 %) cases showed microscopic lesions in the immune organs. The genogroups identified and the frequencies were: genogroup 1-69 % (classical viruses), genogroup 2-25 % (antigenic variants), and genogroup 4-6 % (identified in South America). These findings demonstrate the lesions of immune organs and the presence of different genogroups circulating in commercial birds in Colombia. This indicates the need to continue with these studies in order to have a better understanding of the infection in the field and to guide the design and implementation of control strategies.

Cryptosporidium spp. in bursa of Fabricius of broiler chickens from Uruguay / Cryptosporidium spp. em bursa de Fabrícius de frangos de corte no Uruguai

The presence of Cryptosporidium spp. was detected in broiler chickens of 32-40 days of age, colonizing the bursa of Fabricius. The histologic study with haematoxylin and eosin staining was done on specimens of trachea, complete intestinal tract and bursa. Samples of intestinal content were also studied using Kinyoun staining technique. All birds with signs of illness were positive to the presence of parasite, showing a hypertrophic bursa of Fabricius, filled with caseous content. The bursal epithelial cells were full of different developmental stages of Cryptosporidium spp. This note describing the presence of Cryptosporidium spp. in industrial flocks of broiler chickens, is the first report of this parasite in Uruguay.

A presença de Cryptosporidium spp. foi detectada em frangos de corte de 32-40 dias de idade, colonizando a bursa de Fabrícius. O estudo histológico com hematoxilina e eosina foi feito em amostras de traqueia, trato intestinal completo e bursa. As amostras de conteúdo intestinal também foram estudadas utilizando a técnica de coloração de Kinyoun. Todas as aves com sinais de doença foram positivas para a presença do parasita, mostrando uma bursa de Fabrícius hipertrófica com grande quantidade de conteúdo caseoso. As células epiteliais bursais estavam repletas de diferentes estágios de desenvolvimento de Cryptosporidium spp. Esta nota, que descreve a presença de Cryptosporidium spp. em lotes industriais de frangos de corte, é o primeiro relato deste parasita no Uruguai.

Effect of Diclazuril on the Bursa of Fabricius Morphology and SIgA Expression in Chickens Infected with Eimeria tenella

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.

Desempenho e resposta imune de frangos de corte alimentados com dietas suplementadas com cromo na forma orgânica / Immune response and performance by broiler chickens dietarysupplemented with chromium in organic form

O experimento foi conduzido com o objetivo de avaliar o efeito da suplementação de cromo-orgânico, sobre o desempenho, biometria dos órgãos digestivos e linfoides, histomoformetria da bolsa cloacal e rendimento de carcaça e cortes nobres de frangos de corte de 1 a 41 dias de idade, naturalmente submetidos a estresse por calor cíclico. Foram utilizados 400 pintos de corte da linhagem Cobb, em um delineamento experimental inteiramente casualizado, com cinco tratamentos: 0; 350; 700; 1050 e 1400 ppb de cromo na ração. Os valores do índice de temperatura de globo e umidade foram respectivamente, 83,39 e 80, para fase de crescimento e terminação. A suplementação do cromo orgânico influenciou (P 0,01) de forma quadrática a conversão alimentar dos animais segundo a equação Y=1,67-0,00016x+0,00000012x² (r² 0,976), na fase de 1 a 21 dias de idade. Os níveis de suplementação do cromo orgânico proporcionaram melhor índice de eficiência produtiva e maior percentual de parênquima cortical nas aves aos 21 dias. Na fase de 1 a 41 dias de idade, verificou-se que o peso de penas foi influenciado positivamente com os níveis de cromo orgânico e que não houve efeito para o desempenho dos frangos. Conclui-se que a suplementação da ração com cromo orgânico melhora desempenho e a resposta imune de frangos de corte frente a condições de estresse por calor.

This study was conducted to evaluate the effect of adding organic chromium dietary supplementation on performance, digestiveand lymphoid organs biometry and histomorphometry of the clocal bursa and on carcass and prime cuts performance of broilersfrom 1 to 41 days old, naturally stressed by heat cyclic. We used 400 chicks Cobb, the experimental design adopted was acompletely randomized, with five treatments: 0; 350; 700; 1050 and 1400 ppb chromium in diet. The supplementation withorganic chromium influenced (p<0,01) quadratic form on alimentary conversion the animals according to the equation Y = 1.67to 0.00016 x 0.00000012 x ² (r ² 0.976) in the period 1 to 21. The levels of supplementation of organic chromium improvementproductive efficiency and percentage of cortical parenchyma of broilers to 21 days. In phase to 1 from 41 days old, it was observedimprovement the feathers weight with levels of organic chromium, and there wasn’t influence on performance of broilers. Thechromium in the organic form improves the performance of broiler chickens and immune response by broiler chickens in cyclic heat stress.

Developmental changes in cell proliferation and apoptosis in the normal duck bursa of Fabricius

The aim of this work was to investigate developmental changes in cell proliferation and apoptosis in normal duck bursa of Fabricius using flow cytometry and immunohistochemistry. Studies were carried out on Tianfu ducks on days 24 and 27 of embryogenesis (E24 and E27) along with days 20, 70, and 200 of postnatal development (P20, P70, and P200). Results showed that the percentage of G0/G1 bursa cells significantly increased between E24 and P200 while the percentage of cells in the S phase or G2 + M phase as well as the proliferating index obviously decreased during the same period. Proliferation cell nuclear antigen was detected in lymphocyte and interfollicular epithelium. The proliferative lymphocyte density tended to decrease from E24 to P200. Apoptotic bodies in macrophages, free apoptotic bodies, or nuclei with condensed chromatin in lymphocytes in follicles were identified by transferase-mediated dUTP nick-end labeling. Both flow cytometry and microscopic analysis reveal that the proportion of apoptotic cells and apoptotic lymphocyte density increased from E24 to P20, fell on P70, then rose again on P200. Our foundings demonstrate that cell proliferation decreases and apoptosis increases with age. These changes may account for duck bursa development and involution.

Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks / Avaliação da depleção linfóide folicular da Bursa de Fabricius: uma metodologia alternativa utilizando análise digital de imagem e redes neurais artificiais

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89 percent and 96 percent, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90 percent and obtained the maximum value for the specificity of 92 percent. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

Cinquenta Bursa de Fabrícius (BF) foram examinadas através de microscopia óptica convencional e imagens digitais foram obtidas e processadas através do software Matlab® 6.5. Redes Neurais Artificiais (ANN) foram geradas com a utilização do software Neuroshell® Classifier, e os dados das análises óptica e digital foram comparados. A ANN classificou corretamente a maioria dos folículos, atingindo sensibilidade e especificidade de 89 por cento e 96 por cento, respectivamente. Quando os folículos foram agrupados de forma binária houve um aumento da sensibilidade para 90 por cento e obteve-se um valor máximo para a especificidade de 92 por cento. Estes resultados demonstram que o uso da análise digital de imagem associada à ANNé uma ferramenta bastante útil para a classificação patológica da depleção linfóide da BF. Além disso, fornece resultados objetivos que permitem medir a dimensão do erro classificatório, tornando possível a comparação entre distintos bancos de dados.

Expression patterns of five kinds of diffuse neuroendocrine system (DNES)cells in duck bursa of Fabricius / 中国兽医学报

The expression patterns of gastrin(Gas),β-endorphin(End).somatostatin(Ss),glucagons(Glu),and 5-hydroxytryptamine (5-HT)cells in the duck bursa of Fabricius were studied by the immunohiswchemicai method associated with image analysis.The resuhs showed that the Gas-positive cells with high intensity wrte seen in the follicle-associated epitllelium (FAE),the End with medium intensity in the interfollicular epithelium,the End,Gas and 5-HT with medium or high intensity in the cortex of lymphoid follicles.and the Gas with high intensity in the medulla of lymphoid follicles.Both SS and Gh showed negative reaction.The pre-sent results demonstrated that the Gas,End and 5-HT cells are differentially expressed in the different parts of the duck bursa.The positive cells distributed in the cortex of lymphoid follicles might favour exerting their influence via an endocrine, autocrine or paracrine pathways on the development of B cells.

Growth and histological development of bursa of Fabricius in ducklings / 中国兽医学报

By measuring the absolute weight and growth index of the bursa of Fabricius and using routine H.E method,the growth and histological development of the bursa in 1 to 49 days duckling were studied.The results showed that the absolute weight of the bursa increased as the ducklings grew.The growth index of the bursa reached its peak at 21 days.The height and width of both the large plica and projection formed by the follicle-associated epithelium(FAE)enlarged gradually,the same as the lymphoid follicle area and cortex width.The results indicated that the bursa of Fabricius in duckling develops slowly from 1 to 14 days,and quickly from 14 to 35 days(especially from 21 to 28 days),then steadily from 35 to 49 days.Histologically,the duckling bursa has approached its maturation by the 28th day.

Apoptose na depleção linfocitária induzida pela toxina T-2 em frangos de corte. Histomorfometria da bolsa de Fabricius

It was studied by the histomorphometry of the bursa of Fabricius the involvement of apoptosis as a mechanism of lymphoid depletion in broiler chicks, after the intake of feed containing T-2 toxin of Fusarium sporotrichioides. Forty-two one-day-old chicks were distributed randomly in three groups. The treated group (n=15) received contaminated feed with 2.64 mg/kg of the T-2 toxin. The residual group (n=12) received contaminated feed up to 7 and 14 days and noncontaminated feed during the last week. One control group (n=15) received noncontaminated feed. Five animals from each control and treated groups were killed at 7, 14 and 21 days, and six from the residual group at 14 and 21 days, and their bursa of Fabricius (BF) were collected for histomorphometric analysis and determination of apoptotic index. The apoptosis was confirmed by in situ labeling of the fragmentation of the genome (TUNEL) and by electrophoresis of DNA in agarose gel. BF of treated and residual groups showed moderate to intense depletion of the lymphoid follicles and higher apoptotic index than the control group (P 0.05). These results allowed to conclude that apoptosis is involved in the mechanism of lymphoid depletion caused by T-2 toxin in broiler chicks.

Estudou-se, pela histomorfometria da bolsa de Fabrício, o envolvimento da apoptose como mecanismo de depleção linfocitária em frangos de corte, após ingestão de toxina T-2 de Fusarium sporotrichioides veiculada pela ração. Foram utilizados 42 pintos de um dia de idade, distribuídos ao acaso em três grupos. O grupo tratado (n = 15) recebeu diariamente ração contaminada com 2,64 mg/kg da toxina T-2; o grupo residual (n = 12) recebeu ração contaminada por 7 ou 14 dias, passando a receber ração limpa (não contaminada) na semana antecedente ao sacrifício. Um grupo controle (n = 15) recebeu ração não contaminada. Aos 7, 14 e 21 dias de experimento cinco animais de cada um dos grupos controle e tratado e aos 14 e 21dias seis do grupo residual foram sacrificados, colhendo-se as bolsas de Fabrício (BF) para análise histomorfométrica e determinação do índice apoptótico. As características da apoptose foram confirmadas pela técnica de marcação in situ da fragmentação do genoma (TUNEL) e pela eletroforese do DNA, em gel de agarose. As BF nos grupos tratado e residual, aos 14 e 21 dias, apresentaram depleção linfocitária de moderada a intensa e elevado índice apoptótico, significativamente maior do que do grupo controle (P 0,05). Os resultados permitem concluir que a apoptose está envolvida no mecanismo da depleção linfocitária provocada pela toxina T-2, em frangos de corte.

Studies of the B-cell active factors in the bursa of fabricius of Peking Duck / 中国免疫学杂志

Bursa of Fabricius is the central organ of B-cell development in birds. There are some active factors in Bursa that are involved in the maturation of B lymphocytes. Bursa extracts from 2-3 month-old peking Duck, with a M. W. of 10,000 dal, can obviously increase the number of plaque-forming cells (PFCs) in normal or cyclophosphamidetreated mouse spleen. Cyclophosphamide (CP) is a kind of B-cell inhibitor. The extracts can also increase the life expectation of mouse treated with deathal dose of CP. These results show that Bursa extracts contain a factor (or some factors) which can promote the development or function of B cells. The factor is named as Bursatin. Because the PFCs-stimulating activity of the factor is susceptible to high temperature and pronase E, Bursatin is infered to be a peptide. The effect of Bursa extracts on elevating the cAMP level of mouse spleen cells suggests that Bursatin may function via cAMP. Besides, it is interesting that we have found that the extracts can also increase the rumber of E-rosett forming cells in pig thymocytes. The result shows that there are several active factors or certain factor poesses different activities in Bursa extracts.

Seperation of B-cell active factor from the bursa of fabricius of Peking Duck / 中国免疫学杂志

Bursa of Fabricius is a very important organ for B-eell development. We sot bursa extracts from 2-3 months old Peking Duck. A partially purified PFC-stimulating active factor has been seperated from the extracts by twice molecular sieve chromatography, Sephadex G-15 and Moselect G-10. The M. W. of the active factor is below 750 dal. The results of HPLC analysis of the factor show that its polarity is relatively strong, and it has more positive charges.The works of purifying and looking for a new assay are still going on.

STATE OF ACTIN POLYMERIZATION IN B LYMPHOCYTES FROM DIFFERENT DEVELOPMENT STAGES OF BURSA OF FABRICIUS OF PEKING DUCK / 解剖学报

In this study we have examined the state of actin polymerization in B cells from different development stages in bursa of Fabricius of Peking duck by indirect immunofluorescence technique using antibody against actin. In B cells from bursa of Fabricius of 26th day embryo and 3th week after hatching, the polymeric actin was the main form in the actin pool. In B cells from bursa of Fabricius of 12th week after hatching, however, the monomeric actin was the main form, which might be a result of the shift from the polymeric actin pool to the monomeric pool. We concluded that the state of actin polymerization might be an important factor in the cellular functions of B cells of bursa of Fabricius.