Chemical bursectomy of chickens with colchicine applied to the anal lips.

To induce chemical bursectomy, 30 microliter colchicine dissolved in saline solution (1 mg/ml) was applied on the anal lips of White Leghorn chickens once daily for four consecutive days after hatching. Histologic characteristics of the bursa of Fabricius, spleen, thymus, cecal tonsils, and rectal wall were studied 1-7 days after hatching. Total necrosis of the lymphoid cells and the follicle-associated epithelium in the bursa was observed during the four days of colchicine application. The bursal stroma remained unchanged, and only minor changes were found in the interfollicular surface epithelium. After colchicine application ceased, some regeneration of the epithelium, as evidenced by small epithelial buds, was found. At the end of the observation period the epithelial buds were often covered by the follicle-associated epithelium, which was capable of phagocytizing carbon. However, practically no lymphoid repopulation was seen in the buds. Since this method of colchicine application had no direct effect on other lymphoid organs or on the survival or weight of the chickens, this bursectomy model seems to be a new tool for use in studies of bursal function.     

Thymus-dependent immune functions in chickens bursectomized with colchicine applied to the anal lips

Thymus-dependent immune functions were investigated in chickens bursectomized neonatally with colchicine solution given $\text{per anum}$. Antibody responses to thymus-dependent antigens sheep red blood cells (SRBC) and human gammaglobulin (HGG) were delayed, reaching the normal level after the third antigen stimulation. Also the mitogenic responses of peripheral blood lymphocytes were preserved, and no changes in the thymic morphology were found. In contrast, antibody responses to bursa-dependent antigen $\text{Brucella abortus}$ were low and the switch of immunoglobulin isotypes from IgM to IgA and IgG was disturbed. It can be concluded that neonatal bursectomy with cloacal administration of colchicine does not significantly affect T celi functions, whereas B cell functions are partially deficient.     

The efficacy of chemical bursectomy in chickens with congenital leukosis-virus infection.

Since treatment with Freund's complete adjuvant in chickens with congenital avian leukosisvirus infection results in severe hypergammaglobulinaemia, it was of interest to determine how various means of chemical bursectomy affected such animals. Testosterone injected during the embryonic period or cyclophosphamide administered neonatally were, alone, relatively ineffectual in suppressing the response to influenza virus or production of natural rabbit haemagglutinins, nor did these treatments reliably depress IgG or IgM levels. However, the combination of both testosterone and cyclophosphamide completely suppressed antibody responses to the antigens and resulted in animals with serum immunoglobulin levels 10-(4)-10-(5) that of untreated hypergammaglobulinaemic controls.     

Effect of early bursectomy on germinal centre and immunoglobulin production in chickens.

Chickens were bursectomized in ovo on days 18, 19 or 20 of incubation or within 6 h of hatch and immunized at day 28 after hatch by an intravenous injection of sheep red blood cells (SRBC). The immune response in the blood was measured by haemagglutination tests on the serum and by immunocyto-adherence tests for enumeration of rosette-forming cells with SRBC. Total serum 19S and 7S immunoglobulins were determined by radial immunodiffusion and the number of germinal centres per mm2 of fixed and stained spleen tissue sections was determined. In ovo bursectomy produced undetectably low serum levels of haemagglutinins, a reduction in SRBC rosette-forming cells with normal or increased 19S and reduced 7S immunoglobulins together with a complete disappearance (absence) of germinal centres in the spleen. The role of 19S antibody in the generation of germinal centres is discussed. The finding that birds which are equipped with serum 19S but no 7S Ig lack splenic germinal centres casts doubt on the hypothesis that the switch 19S leads to 7S Ig is necessarily an intrabursal event.     

Effects of bursectomy and thymectomy on the development of resistance to Cryptosporidium baileyi in chickens

 Comparisons were made between the course of infection with Cryptosporidium baileyi in normal chickens and in chickens with functional deficiencies in either B-lymphocytes (bursectomized) or T-lymphocytes (thymectomized). Bursectomy did not influence the acquired immune response to C. baileyi infection as measured by the oocyst excretion. However, the total oocyst output of bursectomized birds was less than a quarter of that of the controls due to the reduced site for multiplication of the parasite. The total oocyst output of thymectomized chickens was more than 2 times higher and the patency was 2 times longer as compared with control animals. Moreover, thymectomized birds failed to acquire resistance to the challenge infection. These findings attest to the primary role of cell-mediated immunity in the expression of resistance to C. baileyi as opposed to the antibody-mediated mechanisms. Received: 24 March 1995 / Accepted: 16 June 1995     

Humoral immune responses in chickens and turkeys after infection with Toxoplasma gondii by using recombinant antigens

Toxoplasma gondii is a parasite which can be transmitted to humans via the consumption of contaminated meat products derived from different animal species, e.g., poultry. In Europe, the consumption rate of poultry meat is high and may pose a risk for humans. However, little is known about the prevalence and immune response against T. gondii in these animals. Based on these circumstances, we experimentally infected 18 turkeys and 16 chickens with the parasite. Turkeys were infected either with tachyzoites on different routes or with various amounts of oocysts. In contrast, chickens were only infected with different doses of oocysts. The immunoglobulin (Ig) Y humoral immune responses of these animals were investigated in a lineblot assay against the recombinant T. gondii antigens rGRA1, rGRA6, rGRA9, rSAG1, and rSUB1. By using the recombinant antigens rGRA6, rGRA9, and rSUB1 in the lineblot assay, we found a correlation between the humoral immune response and the parasite stage in turkeys. Thereby, an infection with oocysts induced a stronger, permanent long-lasting antibody response compared to tachyzoite-infected animals. Only a minor relation between the oocyst infection dose and the manifestation of the immune response in chickens was found 7 days post infection (dpi) by using rGRA1 and rGRA9. However, an inconstant detection of antigen-specific IgY antibodies in the lineblot assay seems not to be a sufficient method for the identification of a Toxoplasma infection in chickens. In contrast, the detection of anti-rGRA6, anti-rGRA9, and anti-rSUB1 IgY antibodies showed potential for the identification of an infection in turkeys.     

Redistribution of lipofuscin in aged neurons induced by colchicine

The effect of a single, 40 micrograms, intracerebroventricular injection of colchicine on the distribution of neuronal lysosomes and lipofuscin granules in aged mice was studied. At the light microscope level we observed that colchicine induced a redistribution of dipeptidyl aminopeptidase II (Dpp II), a lysosomal and lipofuscin granule marker enzyme, from the cell bodies of neurons to the dendrites; cell bodies became depleted of Dpp II while dendrites became enriched with this enzyme. Quantitation of this phenomenon at the electron microscope level demonstrated that colchicine induced a rapid and significant decrease in the density of lysosomes and lipofuscin granules from the somata of neurons whereas in dendrites we observed a significant increase in the density of these organelles.     

Humoral immunity in nasal mucosa of patients with common variable immunodeficiency

Humoral immunodeficiency, as reflected by the low serum immunoglobulin (Ig) concentrations in adult patients with common variable immunodeficiency (CVID), was even more severely expressed at the B-cell level in their nasal mucosa. No Ig-producing cells could be detected by immunohistochemistry in 11 of 19 mucosal specimens. The epithelial distribution of secretory component (SC) was normal in all specimens, but a sign of SC-dependent IgM transport was seen in only three. Epithelial IgA was completely lacking. All patients had had recurrent lower respiratory tract infections and 16 had recurrent or chronic infections of the upper respiratory tract. A previous report indicated that the intestinal mucosa is a privileged site for maturation of B cells in patients with CVID; the present study shows that this does not hold true for the nasal mucosa. This difference in B-cell maturation may in part explain the preferential susceptibility to infections in the respiratory tract of patients with CVID.     

Humoral and local antibodies in chickens with mixed infection with three Mycoplasma species.

Eight one-year-old commercial layer hens with strong humoral antibody response to Mycoplasma synoviae were examined for mycoplasmas. Cultures from the respiratory tract, the Harderian gland and the oviduct showed the group to be infected with M. synoviae, M. gallisepticum, M. gallinarum and Bordetella avium. The humoral antibody response to M. synoviae was strongly positive and supported by high titre specific IgG and IgM class antibody, while that against M. gallisepticum and M. gallinarum was weak or undetectable. However, an antibody response to M. gallisepticum and M. gallinarum was demonstrated in the Harderian gland, respiratory secretions and oviduct of some birds along with antibody to M. synoviae. In contrast with serum, antibodies of IgA and IgG classes against M. gallisepticum were observed in the extracts of spleen of all birds. These data indicate that adult chickens are capable of mounting an antibody response against at least three Mycoplasma species during a mixed infection but it may be necessary to examine tissues as well as serum to demonstrate them.     

Effect of in ovo bursectomy on the course of an infectious bronchitis virus infection in line C White Leghorn chickens

Summary White Leghorn line C chicks were surgically bursectomised (Bx) in ovo to eliminate antibody production. After inoculation with infectious bronchitis virus (IBV) at 14 days after hatching, Bx chicks experienced a more severe and longer lasting infection than intact chicks. The severity and duration of clinical infection in the Bx chicks resembled that previously observed in the highly susceptible line 15 I chicks, however no increase in mortality was observed, in contrast to the high levels of mortality recorded in IBV-inoculated line 15 I chicks. After secondary challenge the degree of damage to the ciliated epithelium of the trachea was greater in the Bx chicks than in the intact chicks.The results indicate that, although antibodies play an important role in recovery from IBV infection, other immunological factor(s) may also be involved.     

B cell activation in chickens induced by Staphylococcusaureus

Conventional mammalian polyclonal B cell activators were evaluated for activity in chicken spleen and peripheral blood lymphocyte (PBL) cultures. Although lipopolysaccharide was found to have a marginal influence on proliferation, two strains of the bacterium $\text{Staphylococcus}$$\text{aureus}$ (Cowan I and Wood 46 strains) induced moderate proliferation in both spleen and PBL cultures. In spleen cell cultures the proliferating cell population was identified as the B cell. The mitogenic response required the presence of adherent cells since their removal eliminated the response. Evidence of $\text{in vitro}$ polyclonal immunoglobulin synthesis could not be obtained. However, when administered intravenously, $\text{S.}$$\text{aureus}$ induced polyclonal immunoglobulin synthesis.     

Paratyphoid infection induced by Eimeria tenella in the broiler-type chickens

Two experiments were conducted to examine the effects of the Eimeria tenella infection on mortality, weight gain and lesions of paratyphoid infection in broilers. When 4-day-old birds were exposed to approximately 106 colony forming units (CFU) of S. typhimurium for 5 consecutive days after the coccidial inoculation, the effect on mortality of concurrent infection, 17.7%, was about the sum of the E. tenella-inoculated group and the S. typhimurium-inoculated group (15.5%). When 11-day-old birds were similarly exposed to approximately 107 CFU of S. typhimurium, the gross lesions of paratyphoid infection characterised by visible foci over the entire liver were observed only in the concurrently infected birds killed 10 and 14 days after coccidial inoculation. The S. typhimurium counts in the liver and spleen of birds killed 7 days after coccidial inoculation were significantly greater than that of birds inoculated with S. typhimurium alone. In both experiments, the S. typhimurium counts in the caecal contents of birds inoculated with both organisms were significantly greater than those of birds infected with S. typhimurium alone.     

应用小肽融合蛋白免疫诱导抗HER-2体液免疫反应

目的应用HER-2 B细胞表位模拟小肽Mut与Hsc70的融合蛋白诱导抗小肽和抗HER-2的体液免疫反应。方法将Hsc70和Mut小肽序列定向插入pQE40载体中,构建pQE40-Hsc70-Mut原核表达质粒,并诱导表达、纯化融合蛋白Hsc70-Mut。常规免疫Balb/c小鼠,应用ELISA、Western blot检测小鼠血清中抗小肽Mut和抗HER-2的体液免疫反应。结果成功构建了pQE40-Hsc70-Mut原核表达质粒,Hsc70-Mut融合蛋白在大肠杆菌SG13009能够实现可溶性表达,应用Ni-NTA纯化可得到纯度>90%的融合蛋白;Hsc70-Mut免疫小鼠可产生抗Mut小肽的免疫反应,部分小鼠同时可产生抗HER-2的体液免疫反应。结论 Hsc70-Mut融合蛋白免疫小鼠可产生抗小肽和抗HER-2的体液免疫反应,为HER-2疫苗的研制奠定了一定基础,同时应用小肽融合蛋白免疫也为小肽免疫提供了一种新的免疫策略。     

Re-excretion of infectious bronchitis virus in chickens induced by cyclosporin

Following inoculation of day-old chicks with Moroccan strain 'G' of infectious bronchitis virus (IBV) which has a predilection for the gut, virus was recovered from the trachea up to day 7 and from the cloaca up to day 35. After this no virus could be detected, even following the natural stress of re-housing with unfamiliar birds at 9 weeks. When the birds were 12.5 weeks old, they were injected with cyclosporin, a selective T-cell suppressor. Four of the five birds re-excreted virus very erratically, as did two of five contacts. This was accompanied by the appearance of IBV-specific IgM in the sera of both groups. The results suggest that in long-term infections with IBV, virus persistence is controlled by T lymphocytes.     

T-cell immunodeficiency induced by T-cell mitogens combined with cyclophosphamide injection.

In the murine system in vivo administration of several T-cell mitogens (LcA, Con A, anti-Thy 1.2 mAb) followed by cyclophosphamide (CP) inhibited the functional activity of T-cell subsets (Th1, Th2, Ts) but not that of B cells. T-cell counts in the spleen of treated mice proved to be significantly decreased. Conversely, T mitogens or CP alone produced a negligible effect if any. Adoptively transferred thymocytes from intact donors restored T-dependent splenocyte responses in experimental mice. In addition, it did not suppress the normal response to sheep red blood cells (SRBC). Our results indicated that the acquired immune deficiency under study is caused by polyclonal elimination (deletion) of mitogen-stimulated T cells, and could be regarded as a model of CP-induced tolerance.     

Effects of adenosine on neutrophil polarization induced by N-formyl-methionyl-leucyl-phenylalanine,sodium propionate and colchicine

Polarization of human neutrophils (a characteristic bipolar shape change) can be induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP); sodium propionate, which causes a rapid acidification of the cytosol; or colchicine, which disrupts microtubules. We have previously reported that adenosine, endogenously produced in human neutrophil suspensions, inhibits FMLP-induced polarization. We report here that endogenously produced adenosine also inhibits sodium propionate-induced polarization but has no effect on colchicine-induced polariaation. These results suggest that neutrophil polarization may be a multistep process inducible by compounds that trigger different biochemical events.     

Influence of maturation on ulcer-development and immunodeficiency induced by activity-stress in rats

Young adult rats housed in the activity-wheel cages and fed only 1 hr daily, have ulcers in the glandular stomach and reveal immunodefeciency. The present study was attempted to investigate how to separate the ulceration and immunodeficiency in order to utilize the activity-stress (A-S) model as an animal model of the human stress ulcer. Six and 10 weeks old rats were used as subjects in this study. They were stressed for 3, 5, or 7 days. In the younger rats, the longer stress exposure caused to deteriorate more both the ulcer and immunodeficiency. The ulceration was found in all of the three stress periods, while the immunodeficiency was recognized in the 5- and 7-day stressed rats. In the elder rats, the ulceration was found in the 5- and 7-day stressed rats, whereas the immunodeficiency was seen only in the 7-day stressed rats. In the free-feeding control rats, relative weights of the thymus and spleen in the elder rats were smaller than those of the younger rats. In the adrenal weight, there was no difference between two age rats. The thymus and spleen of rats exposed to the stress revealed atrophy in both ages. These results suggest that immature rats are more susceptible for the stress than young mature rats, and application of mature rats to the A-S experiment appears to be available in the case of utilizing A-S rats as the stress ulcer model.     

Three types of chemical modification-effects induced by various chemical reagents on the glutamate receptors in molluscan neurons

The effects of specific protein modifying reagents on the dose (Glu)-response (delta G) relationship of the glutamate-hyperpolarizing (Glu-H) receptors in molluscan neurons (Onchidium verruculatum) were analyzed. The effects could be classified into three types. Type I, parallel shift of the dose-response curve towards higher concentration by modification of COO- groups by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The double reciprocal plots of the dose-response curves before and after the treatment indicated competitive inhibition type modification. Type II, a decrease in the slope of the dose-response curve through modification of -SH by N-ethylmaleimide and -NH3 groups by trinitrobenzenesulfonic acid. The double reciprocal plots indicated non-competitive inhibition type modification. The combined effects of Type I and Type II, after modification of arginyl residues with diacetyl trimer. The modification was most effective when applied during an activated state of receptors and channels by Glu. EEDQ had an irreversible after-effect on Glu-H type Onchidium neurons to activate an additional Na+ permeability increase in Glu induced hyperpolarizing response. Pretreatment of N-acetylimidazole (NAI), Glu-H (including weak D) receptors in Helix aspersa produced no significant difference in Glu response. However, simultaneous application of NAI and Glu induced an additional Na+ permeability increase, probably by modification of tyrosyl residues. This indicates the greatest effectiveness of the NAI modification during the activated state of the receptors and the channels by Glu. It is suggested that the Glu-H receptor protein possesses both negatively and positively charged residues, containing COO- and arginyl+ in the receptive site, and that several amino acid resides (SH, -NH, arginyl, and tyrosyl) act in the Glu-activated receptors and channels as their subsites. The role of the subsites in the receptors and channels is discussed.