Evaluation of a receptor gene responsible for maternal blood IgY transfer into egg yolks using bursectomized IgY-depleted chickens.

In avian species, maternal immunoglobulin Y (IgY) is transferred from the blood to the yolks of maturing oocytes; however, the mechanism underlying this transfer is unknown. To gain insight into the mechanisms of maternal IgY transfer into egg yolks, IgY-depleted chickens were generated by removing the bursa of Fabricius (bursectomy) during egg incubation, and their egg production and IgY transport ability into egg yolks were determined. After hatching, blood IgY concentrations of the bursectomized chickens decreased gradually until sexual maturity, whereas those of IgA remained low from an early stage of growth (from at least 2 wk of age). Chickens identified as depleted in IgY through screening of blood IgY and IgA concentrations were raised to sexual maturity. At 20 wk of age, both blood and egg yolk IgY concentrations in the IgY-depleted group were 600-fold lower than those of the control group, whereas egg production did not differ between the groups. Intravenously injected, digoxigenin-labeled IgY uptake into the egg yolk was approximately 2-fold higher in the IgY-depleted chickens than in the controls, suggesting that IgY depletion may enhance IgY uptake in maturing oocytes. DNA microarray analysis of the germinal disc, including the oocyte nucleus, revealed that the expression levels of 73 genes were upregulated more than 1.5-fold in the IgY-depleted group, although we could not identify a convincing candidate gene for the IgY receptor. In conclusion, we successfully raised IgY-depleted chickens presenting a marked reduction in egg yolk IgY. The enhanced uptake of injected IgY into the egg yolks of the IgY-depleted chickens supports the existence of a selective IgY transport mechanism in maturing oocytes and ovarian follicles in avian species.

The enigma of the lower gut-associated lymphoid tissue (GALT).

Artiodactyls possess GALT that appears in fetal life and is located at the extreme end of the ileum. These IPP contain mostly B cells and involute early in postnatal life. Rabbits have a similarly located lymphoid organ, called the sacculus rotundus. Studies in sheep and rabbits have led to the concept that the lower hindgut GALT represents primary lymphoid tissue for B cells and is necessary for normal B cell development, analogous to the bursa of Fabricius. This review traces the history of the observations and theories that have led to the existing concept concerning the role of lower GALT. We then review recent data from piglets with resected IPP that challenges the concept that the IPP is primary B cell lymphoid tissue and that artiodactyls and rabbits are members of the GALT group in the same context as gallinaceous birds. Eliminating the IPP as the primary lymphoid tissue for B cells leads to the hypothesis that the IPP acts as first-responder mucosal lymphoid tissue.

Thymic development in surgically bursectomized embryonic chicken: expression of PCNA, CD3, CD4 and CD8 markers.

Little information is available on the functional relationship between bursa and thymus during chicken embryogenesis. We, therefore, investigated embryonic thymuses taken at 17 days in ovo from chickens bursectomized at 68-72 hours, with histological, histochemical (PAS, Alcian blue), and immunoreaction (anti-cytokeratin B, anti-PCNA/cyclin and anti-CD3, CD4 and CD8 antibodies) methods and compared these data with those from normal and sham-operated chickens of the same age. The bursectomized thymuses distinctly differed from normal and sham-operated thymuses: they were smaller, and the cortical zone was thinner and contained fewer epithelial cells and thymocytes. Only few cortical thymocytes were immunoreactive for PCNA, indicating low proliferative rate. More cortical thymocytes as compared with the normal, expressed CD3 on their cell membrane, whereas the thymocytes at the cortical-medullary border expressing anti- CD4 and anti-CD8 antidodies were less numerous than in normal thymus. The medullary zone contained few epithelial clusters made up of fewer cells than medullary clusters in normal chickens. Some cystic formations were enlarged and contained PAS- or Alcian-blue positive amorphous material. All these data suggest that early bursectomy affects both morphological and functional thymic development.

The effect of surgical immunomodulation on liver inflammation and clearance of DHBV infection.

The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration.

Effect of embryonic 19-nortestosterone treatment and surgical bursectomy on plasma concentrations of reproductive hormones, on inhibin content in adrenals and gonads and on the histological appearance of the gonads in the young chicken.

Four-day-old chick embryos were hormonally treated with 19-nortestosterone in order to inhibit bursa development. At days 1, 4, 8, 15, 22, 29, and 36 of age, plasma, adrenals, and gonads from intact and hormonal treated chicks were collected. In embryonic nortestosterone treated males the appearance of a left 'ovotestis-like' gonad was observed. The occurrence of this ovotestis-like left gonad in the 19-nortestosterone treated male is probably a secondary effect of the in ovo treatment since surgically bursectomised chicks did not show the testicular morphology and histological changes as observed in 19-nortestosterone treated chicks. Additionally, both male and female hormonally or surgically treated chicks showed relatively enlarged adrenal glands. Hormonal bursectomy affected organ inhibin contents and plasma inhibin, testosterone, and FSH levels in males. Male hormonal treated chicks showed lower levels of plasma inhibin (p=0.0001), testosterone (p=0.01), and FSH (p=0.004), and a lower total testes inhibin content (p=0.0003) compared to intact chicks. However, none of these were significantly different between female intact and hormonal treated chicks, again indicating that the observed hormonal changes in males are not the result of the disappearance of the bursa but of the hormonal 19-NT treatment. The total adrenal inhibin content as well as the adrenal inhibin concentration were significantly higher in hormonally treated chicks than in intact chicks (p=0.0001), regardless of the sex.

Investigation of the site of precursors for IgA-producing cells in the chicken intestine.

Bursectomized chicks received lymphocyte single cell suspensions harvested from the bursa of Fabricius (BF), ileal lymphoid aggregate (ILA), caecal tonsils (CT), spleen and peripheral blood. Four days after cell transfer, repopulation of the duodenal and CT lamina propria in age-matched recipient bursectomized chickens with IgA-secreting plasma cells was determined. The results indicate the highest level of reconstitution with cells derived from BF, but substantial numbers of IgA-secreting plasma cells were also observed in a number of birds that received lymphocytes originating from the ILA and CT.

Cyclosporin A, but not bursectomy, abolishes the protective immunity of chickens against Leucocytozoon caulleryi.

Chickens that survive primary infection with Leucocytozoon caulleryi show strong resistance to reinfection. Using bursectomized (BX) or cyclosporin A (CsA)-treated chickens, we performed experiments to determine which type of immunity, humoral or cellular immunity, plays an important role in the resistance of chickens against reinfection with L. caulleryi. BX chickens were inoculated with 2, 20 or 200 sporozoites of L. caulleryi intravenously at 3 weeks of age. Some BX chickens which were inoculated with 2 or 20 sporozoites survived the primary infection. These birds had no parasites in their peripheral blood after challenge infection with 5 x 10(3) sporozoites, even though they had no antibody to L. caulleryi. In contrast, CsA-treated chickens had parasitemia, serum-soluble antigen and antibodies, as did untreated chickens during primary infection. After secondary infection, CsA-treated chickens had parasitemia and serum-soluble antigen, even though they had specific antibodies to L. caulleryi whereas untreated chickens showed no parasitemia. The number of CD4(+), CD8(+), T cell receptor (TCR) alpha ss-bearing cells and TCRgamma delta-bearing cells decreased markedly in the peripheral blood of CsA-treated chickens compared to those of untreated chickens. Lymphocyte proliferation in response to concanavalin A, and T cell growth factor production, were also markedly suppressed in CsA-treated chickens. These results suggest that cell-mediated immunity plays an important role in the development of resistance of chickens against reinfection with L. caulleryi.

Development of maternal IgG-free chick obtained from surgically bursectomized hen.

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the development of the immune system. Surgical bursectomy on the 18th day of incubation deprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens are usually dead before sexual maturity under conventional conditions. When surgically bursectomized chickens were housed in an isolated clean room and antibiotics were administered to them, they could survive to sexual maturity. Finally, we succeeded in obtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgically bursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicks reached six times higher than that of normal chicks 5 days after hatching.

A role for bursa fabricii and bursin in the ontogeny of the pineal biosynthetic activity in the chicken.

The tripeptide bursin (Lys-His-Gly-NH2) is a B cell differentiation hormone derived from the bursa fabricii. The latter is a cloacal diverticulum and the site of B lymphocyte differentiation and selection in aves; also the bursa fabricii is involved in endocrine functions. Herein we demonstrate that in the chicken, the bursa fabricii and bursin are crucial to the ontogeny of both the pineal response to antigenic challenge and pineal circadian synthetic activity. In early embryonically bursectomized chickens, the plasma melatonin response to immunization by porcine thyroglobulin (Tg) was abolished. Also, the amplitudes of both plasma melatonin and pineal N-acetyltransferase (NAT) circadian rhythms were reduced by 50%, whereas the activity of hydroxyindole-O-methyltransferase (HIOMT) remained unchanged. Conversely, administration of either minute amounts (100 pg, 100 fg) or highly dilute (5 x 10(-27) g) bursin, with the exception of a highest dose (100 micrograms), to bursaless embryos induced recovery of normal antigen-induced melatonin response and normal amplitudes of melatonin and NAT rhythms. These findings establish that early in embryonic life, the bursa fabricii and its derived signal (bursin) are essential for normal development of pineal synthetic activity and underline the efficacy of very dilute bursin as an informative signal.

Pathogenesis of highly virulent infectious bursal disease virus infection in intact and bursectomized chickens.

The pathogenesis of highly virulent infectious bursal disease (IBD) virus (IBDV) infection was studied using 6-week-old intact and 5-week-old bursectomized chickens inoculated with highly virulent strain 90-11 or reference strain I. Chickens inoculated with 10(0.7) EID50 of strain 90-11 showed neither clinical signs nor lesions during the 4-day observation period. In contrast, birds inoculated with 10(2.7) or 10(4.7) EID50 developed severe clinical IBD, as well as gross and histologic lesions, typical of IBD, and produced IBDV antigen demonstrable by immunostaining in the bursa of Fabricius (BF), thymus, spleen and bone marrow from day 2 post-inoculation (PI) onwards. The antigen was also detected by the agar-gel precipitation and latex microsphere agglutination tests in a bursal suspension of these birds from day 2 or day 3 PI on. Birds inoculated with 10(6.1) EID50 of strain I developed only slight clinical signs at day 4 PI. Their lesion- and antigen-scores in the BF were almost the same as those in virulent strain-infected chickens, but lesion- and antigen-scores in the other organs were negligible. Bursectomized chickens inoculated with strain 90-11 did not develop clinical IBD despite the presence of infection that was evidenced by histologic lesions in the thymus and spleen as well as IBDV antigen demonstrable by immunostaining in these organs.

Chick gonadogenesis following early surgical bursectomy. II. Ultrastructural data on the embryonic left and right female gonads.

We examined the ultrastructural characteristics of the medullary steroidogenic cells in left and right female gonads of surgically bursectomized chick embryos killed on the 17th day of incubation. The steroidogenic cells of the bursectomized embryos have a more developed system of cisternae in the smooth endoplasmic reticulum than controls, and their mitochondria show some alterations in the density of the matrix and in the shape of the cristae. On the basis of these results, an enhancement of the steroidogenic activity in both gonads is suggested.

Bursectomy and in vitro response of adrenal gland to adrenocorticotropic hormone and testis to human chorionic gonadotropin in immature male chickens.

This investigation was carried out to determine the effects of bursectomy on in vitro response of adrenal glands to adrenocorticotropic hormone (ACTH) and testes to human chorionic gonadotropin (HCG) in immature male chickens. One-day-old Single Comb White Leghorn male chicks were surgically bursectomized (BSX). Sham-operated (sham) chicks were used as controls. At 5 wk of age, adrenal cortical cells (ACC) from BSX and sham birds were incubated in vitro with various concentrations (0, 10(-7), 10(-6), and 10(-5) M) of ACTH for 2 h, and then corticosterone was measured in all supernatants. At 15 wk of age, Leydig cells from testes of both groups were incubated in vitro with HCG (3,000 IU/mL) for 3 h, and then testosterone was measured in all supernatants. Corticosterone concentrations in response to both 10(-6) and 10(-5) ACTH were significantly lower for BSX than for sham ACC. Leydig cells from sham birds produced significantly more testosterone than those of BSX birds whether or not the cells were stimulated with HCG. Furthermore, Leydig cells from sham birds, but not from BSX birds, produced significantly more testosterone when stimulated with HCG than when saline was added. These results indicated that the bursa of Fabricius or its products might have a stimulatory effect on the response of ACC and Leydig cells to ACTH and HCG, respectively.

The effect of anti-bursa serum (ABS) on the intensity of acid phosphatase reaction in bursa-dependent structures of the chicken spleen and on the level of antibodies in the blood serum.

It has been found that administering rabbit anti-bursa serum (ABS) to chickens, like neonatal bursectomy, brings about a decreased acid phosphatase (APh) reaction in germinal centres type I and II, and in the periellipsoidal lymphatic tissue (PEL) of the spleen. It has turned out, that following a stimulation with sheep erythrocytes (SRBC) in the blood serum of the ABS-treated chickens appear traces of specific anti-SRBC agglutinins, while in bursectomized chickens there is only a slight decrease in the titre of those antibodies. The results may serve as a base for further studies on the role and participation of the spleen structures in chicken immunological response.

The effect of bursectomy on natural xenoreactive antibodies and vascularized rat cardiac xenograft rejection in the chicken.

The clinical application of xenotransplantation between distantly related species is currently prevented by the occurrence of hyperacute rejection (HAR). Controversy exists over the importance of natural xenoreactive antibody (NAb)-mediated activation of the classical complement pathway vs. direct activation of the alternative C pathway in this process. In order to evaluate HAR of xenografts (Xgs) in the absence of NAb, this study utilized K strain leghorn chickens that were bursectomized (Bx) on day 17 in ovo (n = 18) to prevent B cell development and production of NAb. Aged-matched untreated siblings served as controls (n = 13). Based on pretransplant antibody levels, the Bx chickens were divided into two groups: totally Bx (Total Bx, n = 9) and partially Bx (Part Bx, n = 9). Chickens then underwent heterotopic cardiac xenotransplantation using PVG rats as donors, where the Xg was connected to the circulation of the chicken recipient utilizing cannulae. For the control group, Xg survival was 28 +/- 3 min (mean +/- SEM), while Part Bx prolonged survival to 80 +/- 15 min. Total Bx extended rat Xg survival to 102 +/- 11 min, with 5 of 9 Xgs functioning well at the time of termination of the study (90-120 min). Three chickens in the Total Bx group with rat cardiac Xgs that were functioning at 120 min were given a 1 ml i.v. injection of heat inactivated control chicken serum. This led to loss of Xg function within 10 min, confirming the important role for NAb in HAR in this species combination. Histologic examination of the Xgs following perfusion revealed significant arterial endothelial injury in the control and Part Bx groups but not in the Total Bx group. Conversely, Xgs from the Total Bx group showed marked venous congestion, which was not seen in the other two groups. This study demonstrates that: (1) Bx effectively eliminates NAb; (2) Xg survival is significantly prolonged in the absence of NAb in this rat-to-chicken xenogeneic combination; (3) the presence of NAb is associated with arterial endothelial injury; and (4) in the absence of NAb, marked venous congestion and injury occurs, which is possibly mediated by alternative C pathway activation or other humoral mechanisms.

Abrogation of age-related resistance to chicken infectious anemia by embryonal bursectomy.

Embryonally bursectomized (Ebx) chickens developed signs and lesions typical of chicken infectious anemia (CIA) when infected with CIA-1 isolate of chicken infectious anemia virus (CIAV) at 21 or 38 days of age. In both cases, the chickens had low hematocrit values after the 14th day of inoculation, and the percentage of CD4+ and CD8+ cells in the thymus was markedly reduced at 21 days postinoculation. Even though intact chickens became infected, they never developed low hematocrit values. The data support the hypothesis that age-related resistance to CIA is antibody-mediated and is not due to disappearance of the CIAV target cell; the data also suggest that CD4+/CD8+ cells are the target for infection.

Pathogenesis of marble spleen disease in bursectomized and non-bursectomized ring-necked pheasants following oral inoculation with cell-culture-propagated virus.

Seventy-two 13-week-old ring-necked pheasants were inoculated orally with 5.0 x 10(2) tissue-culture infective dose (TCID) of cell-culture-propagated marble spleen disease virus. Inoculated birds exhibited neither mortality nor clinical disease. Gross and histologic lesions were typical of marble spleen disease. The mean splenic weight was significantly (P less than 0.02) higher in inoculated birds than in controls between 6 and 10 days postinoculation (PI). The histologic splenic lesions, which consisted of reticuloendothelial cell hyperplasia, intranuclear inclusions within reticuloendothelial cells, and lymphoid depletion, were most prominent between 6 and 10 days PI. In a second experiment, 1-day-old pheasants were chemically bursectomized by dosing birds with 1.2 mg cyclophosphamide on 3 consecutive days. At 7 weeks of age, 54 bursectomized birds were inoculated orally with 5.0 x 10(2) TCID of marble spleen disease virus. Gross and histologic lesions were detected in one of the inoculated pheasants, but the mean splenic weight was not significantly different from control birds at any time PI. These results are evidence of the role of the bursa of Fabricius in the pathogenesis of marble spleen disease.

Effect of surgical and chemical in ovo bursectomy on lymphocyte density scores and histological evaluations of primary and secondary lymphoid organs in hypertensive and hypotensive turkeys.

The effects of surgical and chemical in ovo bursectomy on the histology of primary and secondary lymphoid organs were studied in hypertensive and hypotensive lines of turkeys. The effects of bursectomy were measured by determining the presence of lymphatic nodules and the presence of lymphocytes in secondary lymphoid organs: spleen, cecal tonsil, Meckel's diverticulum, and Peyer's patches; as well as in the thymus and bursa of Fabricius. No treatment effect on splenic nodule formation occurred, but a significantly lower lymphocyte density score was present in the surgically bursectomized group. Both nodule formation and lymphocyte density scores for the cecal tonsil and Peyer's patches were significantly reduced in the surgically bursectomized and testosterone-treated groups as compared with the control and sham groups. The most consistent depression in lymphatic scores in both the surgically and chemically bursectomized groups occurred in the Peyer's patches followed by cecal tonsils and Meckel's diverticulum. As expected, there were no differences in thymocyte density scores. There was no consistent effect on lymphatic scores due to blood pressure selection; however, the hypertensive line showed significantly lower scores than the hypotensive line in the bursa, cecal tonsils, and Peyer's patches. No differences between the sexes were noted in any organ.