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.

Comparison of immunoadjuvant activities of four bursal peptides combined with H9N2 avian influenza virus vaccine

The bursa of Fabricius (BF) is a central humoral immune organ unique to birds. Four bursal peptides (BP-I, BP-II, BP-III, and BP-IV) have been isolated and identified from the BF. In this study, the immunoadjuvant activities of BPs I to IV were examined in mice immunized with H9N2 avian influenza virus (AIV) vaccine. The results suggested that BP-I effectively enhanced cell-mediated immune responses, increased the secretion of Th1 (interferon gamma)- and Th2 (interleukin-4)-type cytokines, and induced an improved cytotoxic T-lymphocyte (CTL) response to the H9N2 virus. BP-II mainly elevated specific antibody production, especially neutralizing antibodies, and increased Th1- and Th2-type cytokine secretion. BP-III had no significant effect on antibody production or cell-mediated immune responses compared to those in the control group. A strong immune response at both the humoral and cellular levels was induced by BP-IV. Furthermore, a virus challenge experiment followed by H&E staining revealed that BP-I and BP-II promoted removal of the virus and conferred protection in mouse lungs. BP-IV significantly reduced viral titers and histopathological changes and contributed to protection against H9N2 AIV challenge in mouse lungs. This study further elucidated the immunoadjuvant activities of BPs I to IV, providing a novel insight into immunoadjuvants for use in vaccine design.

Arterial vascularization of the cloacal bursa of the greater rhea (Rhea americana americana Linnaeus, 1758) / Vascularização arterial da bolsa cloacal de emas (Rhea americana americana Linnaeus, 1758)

The cloacal bursa is an organ in fowls, which triggers lymphocyte maturation and transference to other tissues. Since there is scanty information on its morphology in wild species in spite of its importance in the fowls´ immunological system, current analysis describes the arterial vascularization of the cloacal bursa in the greater rhea. Twenty juvenile rheas (male and female), which died of natural causes, were obtained from the Center for the Multiplication of Wild Animals (CEMAS/UFERSA). The animals were dissected and their thoracic aorta cannulated and red-stained Latex Neoprene 650 was introduced. The cloacal bursa was irrigated from the terminal branches of the abdominal aorta similar to the caudal mesenteric artery and to the right and left internal iliac arteries. The right and left internal pudenda arteries originated from the right and left internal iliac arteries which, in their turn, gave rise to cloacalbursa arteries and to the right and left cloacal arteries in all studied animals. The caudal mesenteric artery contributed to the arterial blood of the cloacal bursa in almost all studied animals through the anastomosis of right or left internal pudenda artery, with direct branches to the cloacal bursa. The cloacal bursa of the greater rhea is irrigated by cloacal-bursa arteries and cloacal right and left and also by the mesenteric caudal artery, with small variations with regard to the number and layout of the anastomoses and branches from the arteries. The latter are specific characteristics and differentiate them from standards perceived in other fowls.

A bolsa cloacal é o órgão das aves responsável pela maturação e transferência de linfócitos para outros tecidos. Apesar da importância deste órgão nos mecanismos imunológicos das aves, são escassas as informações a respeito de sua morfologia em espécies silvestres, fato que ensejou descrever a vascularização arterial do mesmo em emas. Foram utilizadas 20 emas jovens (machos e fêmeas) obtidas no Centro de Multiplicação de Animais Silvestres (CEMAS/UFERSA), as quais vieram a óbito por causas naturais. Os animais foram dissecados e a aorta torácica canulada e perfundida com Látex Neoprene 650 corado com pigmento vermelho. Verificou-se que a irrigação da bolsa cloacal é originada a partir de ramos terminais da artéria aorta abdominal, tais como, as artérias ilíacas internas, direita e esquerda e a artéria mesentérica caudal. Das artérias ilíacas internas, direita e esquerda, originaram-se as artérias pudendas internas, direita e esquerda, que por sua vez deram origem às artérias bursocloacais e cloacais, direita e esquerda em todos os animais estudados. A artéria mesentérica caudal contribuiu com o aporte sanguíneo arterial da bolsa cloacal em quase todos os animais estudados, por meio de anastomose com a artéria pudenda interna direita ou com a artéria pudenda interna esquerda, mas também emitiu ramos diretamente a bolsa cloacal. A bolsa cloacal de emas é irrigada pelas artérias bursocloacais e cloacais direita e esquerda e ainda, pela artéria mesentérica caudal, podendo apresentar pequenas variações quanto ao número e disposição de anastomoses e ramos emitidos, peculiares à espécie, o que as diferenciam do padrão comumente reportado para outras aves.

Ação trófica de aditivos fitogênicos, glutamina e ácido glutâmico sobre a Bursa de Fabrícius e intestino delgado de frangos de corte / Trophic action of phytogenic additives, glutamine and glutamic acid on Bursa Fabricii and small intestine of broilers

The aim of this study was to evaluate the effect of phytogenic additives and glutamine plus glutamic acid, associated or not, on histomorphometry of bursa of Fabricius and small intestine, oocyst count and lesion scores, and carbon turnover of duodenal mucosa of broiler chickens infected with Eimeria acervulina. A total of 450 male broiler chickens was distributed into a completely randomized design with six treatments and three replications. Treatments consisted of control diet (CD); CD + coccidiosis vaccine; CD + antibiotic performance enhancers and anticoccidial (APE/AC); CD + glutamine and glutamic acid (Gln/Glu); CD + phytogenic additives (PA); CD + Gln/Glu + PA. Birds on treatment CD + vaccine were vaccinated via drinking water at three days of age against coccidiosis. At 16 days of age all birds of all treatments were inoculated orally and individually with 500,000 oocysts of Eimeria acervulina. There was no treatment effect on lesion score in the intestinal epithelium of birds. The smaller number of excreted oocysts was observed in groups of birds fed diets containing APE/AC and PA. Were observed better results of villus height and crypt depth for duodenum and ileum of birds of treatments containing Gln/Glu at 7 days of age, and Gln/Glu and PA at 21 days of age. Higher percentage of cortical area from bursa follicles was observed in birds fed diets supplemented with Gln/Glu and PA at 7, 14 and 21 days of age. Increased turnover of intestinal mucosa was observed in treatments containing Gln/Glu, indicating acceleration in development and regeneration of damaged tissue. Glutamine plus glutamic acid and phytogenic additives can provide improvements to structure, and thus to intestinal function, as well as to better immune response against the infectious challenges. Phytogenic additives can be used for coccidiosis control of broiler chickens where the use of antibiotic performance enhancers and anticoccidials is prohibited...

O objetivo deste trabalho foi avaliar o efeito dos aditivos fitogênicos e da glutamina mais ácido glutâmico, associados ou não, sobre a histomorfometria da Bursa de Fabricius e intestino delgado, sobre contagem de oocistos e escores de lesão e sobre o turnover do carbono da mucosa intestinal de frangos de corte experimentalmente infectadas com Eimeria acervulina. Para isso foram utilizados 450 pintos de corte machos distribuídos em delineamento inteiramente casualisado, com seis tratamentos e três repetições. Os tratamentos consistiram de dieta controle (DC); DC + Vacina de coccidiose; DC + antibióticos melhoradores de desempenho e anticoccidiano (AMD/AC); DC + glutamina e ácido glutâmico (Gln/Glu); DC + sditivos fitogênicos (AFs); DC + Gln/Glu + AFs. As aves do tratamento DC + Vacina foram vacinadas via água de bebida, aos três dias de idade, contra coccidiose. Aos 16 dias de idade todas as aves de todos os tratamentos foram inoculadas oralmente e individualmente com 500.000 oocistos de Eimeria acervulina. Não houve efeito dos tratamentos para escore de lesão no epitélio intestinal das aves. O menor número de oocistos excretados foi observado nos grupos de aves alimentadas com dieta contendo AMD/AC e AFs. Foram observados melhores resultados para altura das vilosidades e profundidade das criptas do duodeno e ílio das aves dos tratamentos contendo Gln/Glu, aos 7 dias de idade e Gln/Glu e AFs aos 21 dias de idade. Maior porcentagem de área cortical dos folículos bursais foi observada em aves alimentadas com dieta suplementada com Gln/Glu e AFs aos 7, 14 e 21 dias de idade. Maior turnover da mucosa intestinal foi observada em aves dos tratamentos contendo Gln/Glu, indicando aceleração do desenvolvimento e regeneração do tecido lesado. Glutamina mais ácido glutâmico e aditivos fitogênicos podem oferecer melhorias à estrutura e, consequentemente, à função do intestino, bem como melhores condições para resposta imune frente à desafios infecciosos...

Isolation and immunomodulatory activity of bursal peptide, a novel bursal peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF), which is unique to birds, serves as the central humoral immune organ and plays a significant role in B lymphocyte differentiation. In this study, a new bursal peptide (BP-IV) was isolated from BF, which promoted colony-forming unit pre-B formation and regulated B cell differentiation. BP-IV also exerted immunomodulatory effects on antigen-specific immune responses via both humoral and cellular immunity in chicken and mice that had been immunized with inactivated avian influenza virus (AIV; H9N2 subtype), including enhancing AIV-specific antibody and cytokine production. The results of this study provided novel insights into the use of a potential candidate reagent for B cell development and future immuno-pharmacological use.

The potential molecular effects of bursal septpeptide II on immune induction and antitumor activity

The bursa of Fabricius (BF) is the acknowledged central humoral immune organ in birds. Bursal septpeptide II (BSP-II) is an immunomodulatory bioactive peptide isolated from BF. To understand the effects of BSP-II on immune induction, gene expression profiles of hybridoma cells treated with BSP-II were evaluated. Pathway analysis showed that regulated genes were involved in cytokine-cytokine receptor interactions, T cell receptor signaling pathway, and pathway in cancer. It was observed that BSP-II reduced tumor cells proliferation and stimulated p53 expression. These results indicate potential mechanisms underlying the effects of the humoral immune system on immune induction, including antitumor activities. Our study has provided a novel insight into immunotherapeutic strategies for treating human tumors.

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.

Evaluation of bursal lymphoid depletion: comparison between the conventional histology method and digital lymphocyte depletion evaluation system / Avaliação da depleção linfóide bursal: comparação entre a histologia convencional e o sistema de avaliação digital da depleção linfocitária

Fifty-five bursa of Fabricius (BF) were evaluated by optical microscopy for three different avian histopathologists (H1, H3 and H4) to determine the degree of lymphoid depletion. One histologist evaluated the same slides at two different times (H1 and H2) with four-months interval between the observations. The same BFs were evaluated using the system of Digital Lymphocyte Depletion Evaluation (ADDL), being performed by three differents operators of the system, not histopathologists. The results showed was a significant difference between the histopathologists and between the scores established by the same expert (H1 and H2). However, there were not significant differences between the scores with the ADDL system, obtained using ADDL. The results make clear the fragility of the subjective lymphocyte depletion score classification by the traditional histologic method, while the ADDL system proves to be more appropriated for the assessment of the lymphoid loss in the BF.(AU)

Cinquenta e cinco bursas de Fabricius (BF) foram avaliadas através da microscopia óptica por três diferentes histopatologistas aviários (H1, H3 e H4) para determinar o grau de depleção linfóide. Um histopatologista avaliou as amostras em dois momentos distintos (H1 e H2) com quatro meses de intervalo entre as observações. As mesmas BF foram avaliadas utilizando-se o sistema de Avaliação Digital da Depleção Linfocitária (ADDL), sendo realizadas por três diferentes operadores do sistema, não histopatologistas. Os resultados mostraram diferenças significativas entre os histopatologistas e entre um mesmo histopatologista (H1 e H2). Contudo, não houve diferenças significativas entre os escores obtidos utilizando-se ADDL. Estes resultados caracterizam a fragilidade da classificação subjetiva em escores de depleção linfóide, enquanto o sistema ADDL prova ser um sistema robusto de avaliação da perda linfocitária na BF.(AU)

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Vascularização da bolsa cloacal em Gallus gallus domesticus (Linhagem Master Gris Cou Plumé) / Vascularization of the cloacal bursa in Gallus gallus domesticus (Master Gris Cou Plumé Lineage)

Estudou-se a vascularização da bolsa cloacal em aves da linhagem Master Gris Cou Plumé, quanto a sua origem, número e distribuição dos vasos. Procedeu-se a dissecação dos vasos por meio de duas incisões paramedianas na base do pigóstilo, em 20 exemplares de seis semanas de idade após fixação em solução aquosa de formol a 10%. Anteriormente à fixação, a artéria isquiádica direita foi canulada para perfusão do sistema arterial com solução aquosa a 50% de Neoprene Látex "450", corado com pigmento vermelho. A bolsa cloacal foi irrigada pelas artérias: bursocloacal direita, presente em 19 exemplares (95%) apresentando de um a cinco ramos arteriais; bursocloacal esquerda, presente em todos os casos (100%), apresentando de um a cinco ramos arteriais; cloacal direita, observada em 13 aves (65%), exibindo de um a quatro ramos arteriais; cloacal esquerda, presente em 13 aves (65%), emitindo de dois a três ramos arteriais; e ilíacas internas direita e esquerda, observadas uma única vez (5%), que enviaram dois ramos arteriais. Quanto à distribuição, pode-se afirmar que os quadrantes caudais da bolsa cloacal, de ambos antímeros, foram os que mais receberam ramos arteriais, sendo que o esquerdo recebeu ramos da A. bursocloacal, A. cloacal e A. ilíaca interna em 19 casos (95%), 13 (65%) e um (5%), respectivamente. Os quadrantes craniais direito e esquerdo foram irrigados por ramos da A. bursocloacal em cinco (25%) e quatro casos (20%), respectivamente. Foram observadas grandes variações no aporte sanguíneo da bolsa cloacal da linhagem estudada em relação às demais da espécie Gallus gallus domesticus, concluindo-se que estas diferenças podem estar associadas à maior ou menor funcionalidade do órgão, ou mesmo das características morfofuncionais da linhagem empregada. Estatisticamente não foram observadas diferenças em relação às artérias responsáveis pelo suprimento sanguíneo, quando considerado o antímero e o número de ramos arteriais enviados à bolsa cloacal.

The vascularization of the cloacal bursa in fowls from Master Gris Cou Plumé lineage was studied in relation to origin, number and distribution of vessels. After cannulation of the right sciatic artery in 20 specimens of six-week-old, the arterial system was perfused with a 50% aqueous solution of Neoprene Latex "450" colored with red pigment. Subsequently the specimens were fixated in aqueous 10% formaldehyde. Vessels were dissected through two paramedian incisions at the base of the pygostyle. The cloacal bursa showed to be irrigated by the arteries: right bursocloacal, present in 19 specimens (95%) with one to five arterial branches; left bursocloacal, present in all cases (100%) and showing one to five arterial branches; right cloacal, observed in 13 birds (65%) with one to four arterial branches; left cloacal, also present in 13 birds, sending two to three arterial branches; and right and left internal iliac arteries, observed only in a single individual (5%), which sent two arterial branches. As for distribution, it can be stated that the caudal quadrants of both sides of cloacal bursa were the ones that most received arterial branches. The left one received branches from the bursocloacal, cloacal and internal iliac arteries in 19 (95%), 13 (65%) and one (5%) cases, respectively. The right and the left cranial quadrants were irrigated, respectively, by branches of the bursocloacal artery in five cases (25%) and in four cases (20%). Great variations in blood supply of cloacal bursa were observed in the lineage studied in relation to others lineages of the species Gallus gallus domesticus, concluding that these differences may be associated with greater or lesser functionality of this organ, or even the morphofunctional characteristics of the tested lineage. Statistically were not found significant differences in relation to the arteries responsible by blood supply when considered antimere and number of arterial branches sent to the cloacal bursa.

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.

Distribution and Quantification of Lymphocytes in the Major Lymphoid Organs of Naturally Gumboro Infected Broilers / Distribución y Cuantificación de Linfocitos en los Órganos Linfoides Principales de Pollos de Engorda Infectados Naturalmente con Gumboro

Gumboro disease is caused by the infectious bursal disease virus (IBDV) which rapidly destroys immature B-lymphocytes of bursa of Fabricious, and causes immune suppression and high mortality in commercial broiler farms in Bangladesh. To investigate the possible effect of IBDV on lymphocytes and its distribution in the major lymphoid organs, bursa of Fabricious including spleen and thymus of naturally Gumboro-infected broilers, a research was conducted in the Department of Anatomy and Histology, collaboration with the Department of Pathology, Bangladesh Agricultural University, Bangladesh. Bursa of Fabricious, spleen and thymus of 21-days-old Gumboro-infected and non-infected broilers of same age (control) were routinely processed and stained by hematoxylin and eosin to examine the distribution of lymphocytes in the major lymphatic organs as well as quantified the number of lymphocytes under high power magnification field and compared with those of control. The number of lymphocytes in bursa of Fabricious, spleen and thymus of Gumboro-infected broilers were 27.20 ± 1.53, 66.50 ± 2.70 and 79.30 ± 3.92 whereas 121 ± 3.82, 89.90 ± 2.09 and 106.30 ± 4.07 were in non-infected control respectively. The numbers of lymphocytes were significantly (p < 0.05) lower in all lymphatic organs of Gumboro-infected broilers than those of non-infected control. The significant numbers of lymphocytes decrease in spleen and thymus suggest that IBVD not only destroy lymphocytes in bursa of Fabricious, but also in spleen and thymus and thus may severely suppress the immune response of IBVD affected broilers.

La enfermedad de Gumboro es causada por el virus de la bursitis infecciosa (VBI), que destruye rápidamente los linfocitos B inmaduros de la bolsa de Fabricio, y causa supresión inmune y la elevada mortalidad en las granjas comerciales de pollos de engorde en Bangladesh. Para investigar el posible efecto del VBI en los linfocitos y su distribución en los órganos linfoides principales, la bolsa de Fabricio, incluyendo el bazo y el timo de pollos de engorde naturalmente infectados con Gumboro, se realizó una investigación en el Departamento de Anatomía e Histología, y el Departamento de Patología, Universidad Agrícola de Bangladesh, Bangladesh. Tanto la bolsa de Fabricio, bazo y el timo de pollos de engorde con 21 días de edad infectados con Gumboro y no infectados de la misma edad (control) se procesaron de forma rutinaria y se tiñeron con H & E para examinar la distribución de los linfocitos en los órganos linfáticos principales, así cuantificar el número de linfocitos bajo campo de alta magnificación y compararlos con los de control. El número de linfocitos en la bolsa de Fabricio, bazo y timo de pollos infectados con Gumboro fue 27,20 ± 1,53, 66,50 ± 2,70 y 79,30 ± 3,92, respectivamente, mientras que en los controles no infectados fue 121 ± 3,82, 89,90 ± 2,09 y 106,30 ± 4,07 respectivamente. El número de linfocitos fue significativamente (p < 0,05) más bajo en todos los órganos linfáticos de pollos de engorde infectados con Gumboro que los no infectados. La disminuición significativa de linfocitos en el bazo y timo, sugiere que el VBI no sólo destruye linfocitos en la bolsa de Fabricio, sino también en el bazo y el timo y, por tanto, puede suprimir severamente la respuesta inmune de pollos de engorde afectados por VBI.

[ effect of different levels of cadmium on the histopathological changes and the rate of lymphoid cells apoptosis of bursa of fabricius in broiler chickens]

Cadmium toxicity can cause kidney failure, liver damage and a weakened immune system in experimental and naturally occurring toxicities. This study was designed to investigate the effects of cadmium [Cd] on the histology and the rate of lymphoid apoptosis in the bursa of fabricius of chicken. One-hundred 20-day-old male Ross broilers were purchased and randomly divided into four groups. The control group [C] received no Cd, whereas groups 1, 2, and 3 had rations administered containing 25, 50 and 100 ppm cadmium as CdCl, respectively. At days 14, 28 and 42, seven chicks from each group were randomly selected and sacrificed. The bursa of Fabricius of each chick was removed, weighed, fixed in 10% buffered formalin and processed for histopathology and assessment of the rate of lymphoid cells apoptosis. The apoptotic cells were demonstrated in paraffin embedded tissue sections using the TUNEL[terminal oxynucleotidyl transferase-mediated dUTP nick end labeling] method. The concentration of Cd in the liver samples was measured by atomic absorption. Areverse correlation between the levels of Cd in the rations and the body weight of the chickens [p < 0.01] was found. The concentration of Cd in the liver showed a positive correlation with the levels of Cd in the rations [p < 0.01]. The number of apoptotic lymphoid cells was significantly increased in those groups receiving higher levels of Cd [especially groups 2 and 3] [p<0.01]. Morphologically, plicas and lymphoid follicles of groups 2 and 3 were smaller than of the control group. In the histological analysis they were found to be hypocellular and some of them were edematous. Compared to the control group, there was an increase in the number of intraepithelial cysts in groups 2 and 3 at days 28 and 42. In addition, atrophic changes of bursal paranchyma were observed in group 3 after 42 days. It can be concluded that under experimental conditions the higher concentrations of Cd in the rations [50 and 100 ppm]has detrimental effects on the bursa of Fabricius of chickens

Screening proteins interacting with infectious bursa disease virus Gt VP2 from cDNA library of B lymphoid cells of the bursa of Fabricius / 生物工程学报

To screen the interactive proteins with IBDV Gt VP2 protein from cDNA library of B Lymphoid cells of the bursa of Fabricius. The expression cDNA library plasmids was transformed to the yeast competent cells, which have the bait plasmid-Gt VP2. After testing for growth in synthetic complete medium lacking histidine and uracil and for production of beta-galactosidase (X-gal), we obtained 16 positive clones. We searched the gene sequences of positive clones in the NCBI website. The blast results showed that five positive clones were the gallus sequences. They were Gallus gallus breed mitochondrial DNA, O_G1cNAc transferase, Tumor protein p53 binding protein, Stathmin and Chondroitin sulfate Ga1NAcT-2, respectively. This study is helpful for the further identifying the receptors of IBDV in B Lymphoid cells of the bursa of Fabricius.

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.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease of chicken. Agar gel immunodiffusion using IBDV antigen extracted from bursa of Fabricius of infected chicken has been used officially for diagnosis of IBDV in Korea. In this study, in order to replace the IBDV whole virus antigen with non-infectious antigen, recombinant VP2 protein (rVP2) of IBDV was produced using recombinant baculovirus expression system. Purified baculovirus-expressed rVP2 was used as an antigen in an agar gel immunodiffusion (AGID). rVP2 antigen precipitated specifically IBDV antibodies. AGID using rVP2 antigen detected anti-IBDV antibodies from 6 dpi to 28 dpi (termination of the experiment) when specific pathogen free chickens were experimentally infected with IBDV 52/70 strain. This was consistent with result by AGID using IBDV antigen, virus neutralization test (VNT) and a commercial ELISA kit (except for one serum). The sensitivity of rVP2 was the same with that of IBDV antigen when field sera (n=324) were tested by AGID. However, AGID using rVP2 antigen detected maternal antibodies from broiler chickens (n=20) on a broiler farm up to 15 days old, although the detection rate of the AGID was relatively low compared to a commercial ELISA kit. Our results indicate that IBDV whole virus antigen from IBDV infected chickens would be replaced with recombinant VP2 protein as an antigen for AGID.

Celulite associada às lesões na bolsa de Fabrício de frangos de corte ao abate, sob inspeção sanitária / Cellulitis associated with lesions of bursa of Fabricius from broilers under sanitary inspection

Foi realizado um estudo epidemiológico de caso controle, onde foram avaliados 128 frangos de corte, entre 40 e 48 dias deidade, sob inspeção sanitária, vacinados contra a Doença Infecciosa da Bolsa de Fabrício (DIB) no 14o dia de vida (via água debebida). O grupo caso foi constituído por 68 aves portadoras de celulite e o grupo controle, por 60 aves sem a doença. Asbolsas de Fabrício foram medidas em bursômetro e registradas de acordo com sua escala. Após o exame macroscópicoforam coletados fragmentos de pele, lesada e íntegra, e de bolsa de Fabrício, fixados em formol a 10%, para a realização dahistopatologia. Foi criado o seguinte escore para análise histológica das lesões: escore 0 – sem lesão; escore 1 – discretashiperplasia e rarefação linfóide de folículos; 2 – edema, infiltrado inflamatório, necrose de folículos, rarefação linfóide severa eatrofia discreta de folículos; escore 3 – presença de cistos foliculares e epiteliais, atrofia severa de folículos e fibrose. A análiseestatística constou dos testes de qui-quadrado Kruskall-Wallys e Regressão Linear. A mensuração do bursômetro mostrou,no grupo caso 28 frangos com bolsas de diâmetro 10mm (tamanho 3); 37, com diâmetro 13mm (tamanho 4); três, comdiâmetro 16mm (tamanho 5); e no grupo controle 26 frangos com bolsas de diâmetro 10mm (tamanho 3); 31, com diâmetro13mm (tamanho 4); três, de diâmetro 16mm (tamanho 5). Macroscopicamente não foram observadas alterações.Microscopicamente foram registrados uma bolsa com escore 0; três, com escore 1; 25, com escore 2; e 99, com escore 3.Houve diferença estatística (p< 0,05) entre os grupos caso e controle em relação ao diâmetro e aos escores de lesão de bolsade Fabrício obtidos. Conclui-se que lesões amareladas em placas no subcutâneo de frangos de corte devem ser consideradascomo celulite, e critério para julgamento da carcaça, e são favorecidas pelas lesões mais graves na bolsa de Fabrício

This work is an epidemiological study of case control, evaluating 128 broiler chickens, between 40 and 48 days old, undersanitary inspection, vaccinated against the DIB in the 14th day of age (through drinking water). The case group consists of 68broiler chickens with celulitis and the control group consists of 60 healthy animals. The bursals, when collected, were measuredin bursometer and registered in accordance with its scale. After the macroscospic examination were collected skin fragments,both injured and not injured, as well as bursal fragments, fixed with 10% phormol, in order to finish the histopathology, and alsofor the and processing by the established techniques, as well as included in paraffin and stained with hematoxilin and eosin, atthe Pathological Anatomy Service Professor Jéfferson Andrade dos Santos (UFF). A score has been created for the histophatologicalanalysis of the injuries with the following criteria: score 0 - without injury; score 1 - discrete hyperplasia and lymphoid folliclesrarefaction; score 2 - edema, inflammatory infiltrate, follicle necrosis, severe lymphoid rarefaction and follicle discrete atrophy;3 - presence of epithelial and follicle cysts, severe follicle atrophy and fibrosis. The statistical analysis was made with the Qui-square Kruskall-Wallys tests and Linear Regression. The bursometer analysis has demonstrated that: in case group- 28broiler chickens with burse the diameter 10 mm (size 3); 37, with diameter 13 mm (size 4); three, with diameter 16 mm (size 5);and in control group – 26 broiler chickens with burse the diameter 10 mm (size 3); 31, with diameter 13 mm (size 4); três, threewith diameter 16 mm (size 5). Through microscopical analysis we registered 1 bursal with score 0; 3 with score 1; 25 with score2; and 99 with score 3. There was statistical difference (p£ 0.05) between the case and control groups in relation to the diameterand to the scores of bursal injuries that...

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF / Apoptosis and expression of VP2 and GADPH in an experimental infectious bursal disease in SPF chicks

Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB) para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2) e da enzima gliceraldeído fosfato dehidrogenase (GAPDH). Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo) foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.

Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV) to evaluate early apoptosis and the expression of viral protein 2 (VP2) and glyceraldehyde-3-phosphate dehydrogenease (GAPDH). Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling) technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR). A SYBR GREEN PCR kit was used and the reaction was carried out in an ABI Prism 7000 SDS. Apoptotic indexes progressively increased indicating a role of IBDV in inducing hypotrophy of the BF. Also, it was showed that as long as apoptosis increased, viral protein expression decreased, which suggests that apoptosis plays a role as a defense mechanism against viral replication.

Determinación del catabolismo de los anticuerpos maternos y su interacción con diferentes planes vacunales para la enfermedad de gumboro en pollos de engorde / Evaluation of maternal antibodies catabolism and its interaction with vaccination programs against infectious bursal disease in broilers

Con el fin de evaluar el catabolismo de los anticuerpos maternos y su interferencia con tres planes vacunales diferentes contra la enfermedad de Gumboro se realizó un estudio utilizando pollos de la estirpe Ross 308, divididos en cuatro grupos: grupo 1: control (sinvacuna); grupo 2: vacunado los días 1, 7 y 15; grupo 3: vacunados los días 1 y 12; y grupo 4: vacunado el día 12. En el primer experimento el biológico se suministró en el agua de bebida, y en el segundo se hizo en el pico. Se sacrificaron 15 aves de cada grupo a los 1, 12, 21 y 42 días de edad y se evaluó peso corporal, tamaño y peso de la bolsa de Fabricio, relación peso bolsa/peso corporal (PB/PC), grado de depleción linfoide, presencia de cepas estándar y/o variantes del virus y los títulos de anticuerpos para la enfermedad de Gumboro.El catabolismo de los anticuerpos maternos ocurrió entre los 21 y 28 días de edad. En los grupos vacunados no se evidenció una respuesta inmune activa frente a ningún plan vacunal.La relación PB/PC no indicó atrofia de la bolsa en el experimento 1, aunque las lesiones histopatológicas en el último muestreo fueron grado 3; en el segundo experimento solamente el grupo 3 presentó una disminución en la relación PB/PC; las lesiones histopatológicas enel tercer y cuarto muestreos, en todos los grupos, fueron clasificadas como grado 3 y 4. En los dos experimentos se detectó la presencia de cepas tanto clásicas como variantes y una baja respuesta humoral. Ninguno de los tres planes vacunales confirió adecuada proteccióna las aves...