A method for recording chick embryo electrocardiogram using the IX-TA 220 electrocardiogram recording system.

1. In developmental embryology in chickens, the cardiovascular system is the first to become functional, the first heart muscular contraction (beat) happens as early as 33 h of incubation of a developmental journey that takes 21 d.2. An electrocardiogram (ECG) recording system (IX-TA 220) has been used to record the ECG of various species. The following trial describes the use of such a system for recording electrical tracing of the developing heart in chick embryos on d 19 of embryonic development with the electrodes piercing the eggshell in specific locations to a depth of about 2 mm. The recorded ECG offers an opportunity to measure or calculate ECG parameters like those measured/calculated in humans.3. The use of anaesthesia substantially reduced embryo motion, but may have a transient tachycardia effect on heart rate.4. This is the first time such a system has been successfully used for measuring heart electrical activities in chick embryos and provides a broader research opportunity in chicken embryo cardio-physiology.

The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease.

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV.

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.

Adaptation of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay.

The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] has been widely used for bioassays. Herein is described the use of the MTT dye with a virus-neutralization (VN) assay to titer infectious bursal disease virus (IBDV)-neutralizing antibodies. A standard VN assay using chicken embryo fibroblasts (CEFs) and IBDV was used for the assessment of IBDV-neutralizing antibodies. The percent of CEF killing due to IBDV was quantitated using MTT, and the absorbance (A) data were used to calculate the VN antibody titer. This method of calculation offers the expression of VN titer in terms of units of activity per unit of volume.

Evaluation of the humoral immune response to different antigens in Arkansas Regressor and Progressor chickens.

Arkansas Regressor and Progressor chickens were re-evaluated for their immune response to different antigens. Chickens received i.v. injection of either SRBC (10 birds per line) or Salmonella pullorum (SP; 10 birds per line) at 7 wk of age, and sera were collected at 6, 13, and 20 d postimmunization. A third group of birds (10 birds per line) received and i.m. injection of GAT emulsion at 7 and 12 wk of age, and sera were collected at 10 and 14 wk of age. There were significant differences between the two lines in their humoral immunity to SRBC, SP, and GAT. Such results suggest genetic control of humoral immunity to these antigens in these lines. It is unknown whether humoral immunity to these antigens is correlated to regression of tumors induced by Rous sarcoma virus.

Augmentation of natural cell-mediated cytotoxic activity by supernatant from in vitro mitogen-stimulated, in vivo hormone-treated lymphocytes in immature male (K strain) chickens.

Newly hatched White Leghorn male chicks were used in this study. Triiodothyronine (T3; 0.1 or 1 ppm) and Thyrotropin Releasing Hormone (TRH; 1 or 5 ppm) were added to the feed for an 8-week period starting at hatch. A fifth group received the unsupplemented diet and served as controls. Peripheral blood lymphocytes from each treatment group were cultured in vitro with or without different mitogens (PHA, Con-A, or LPS), and the culture supernatants were tested for the presence of lymphokines (LK). The natural cell-mediated cytotoxicity (NCMC) assay was carried out with or without supernatant using the standard chromium (Cr51) release assay. Control untreated chicks were used as donors for effector cells and the P815 mouse mastocytoma was used as a target. Supernatant from in vivo 1 ppm T3- or 5 ppm TRH-treated lymphocytes significantly suppressed NCMC (or cells mediating NCMC, e.g., NK cells). However, supernatant from 1 ppm T3-treated, PHA-stimulated lymphocytes significantly enhanced NK cells cytotoxicity, while supernatant from 5 ppm TRH-treated lymphocytes with PHA stimulation tended to suppress cytotoxicity. These results provide evidence supporting a regulatory role of the hypothalamo-pituitary thyroid axis on lymphokine (LK) production. The results also suggest that these hormones act on different subpopulation of lymphocytes, and therefore, the mediators released by them.

In vivo effects of TRH, T3 and cGH on antibody production and T- and B-lymphocytes proliferation in immature male chickens.

Newly hatched White Leghorn male chicks were used in this study. Different doses of T3 (0.1 or 1 ppm) or TRH (1 or 5 ppm) were administered in the feed for an 8-week period. Chicken growth hormone (cGH) (10 micrograms/kg BW) was injected (i.v.) into a different group of chicks twice daily for 1 week starting at 7 weeks of age. A different group received both T3 (0.1 and 1 ppm) and cGH. Serum concentrations of T4, T3 and GH, antibody production against sheep red blood cells (SRBC) and Brucella Abortus (BA), and in vitro proliferative response of both T- and B-lymphocytes to mitogenic stimulation were measured. Supplementation of T3 (1 ppm) significantly lowered T4 and increased T3 concentrations. No effect of any hormone treatment on antibody production was observed. T3 supplementation and cGH injection alone or with T3 (0.1 ppm) significantly increased blastogenic response of lymphocytes to either Con-A or LPS mitogenic stimulation. It was concluded that T3 and GH are involved in lymphocyte activity of chickens.

Chicken growth hormone, triiodothyronine and thyrotropin releasing hormone modulation of the levels of chicken natural cell-mediated cytotoxicity.

Newly hatched White Leghorn male chicks received dietary supplements of either Triiodothyronine (T3) or Thyrotropin Releasing Hormone (TRH) until 8 weeks of age. Chicken growth hormone (cGH) (10 micrograms/kg BW) was injected into different chicks twice daily for 1 week starting at 7 weeks of age. Separate groups received both T3 and cGH. Natural cell-mediated cytotoxic (NCMC) activity against different target cells was tested. It was found that cytotoxic activity in cells involved in NCMC against P815 mouse mastocytoma was stimulated by cGH alone or in combination with T3 (1 ppm). These findings indicate that cGH and T3 stimulate NCMC activity; and that the cells responsible for this activity may be Natural Killer (NK) cells.

Effect of thyrotropin-releasing hormone, triiodothyronine, and chicken growth hormone on plasma concentrations of thyroxine, triiodothyronine, growth hormone, and growth of lymphoid organs and leukocyte populations in immature male chickens.

One-day-old White Leghorn male chicks were fed different levels of Thyrotropin Releasing Hormone (TRH) (1 and 5 ppm) or Triiodothyronine (T3) (.1 and 1 ppm) for an 8-wk period. In a second experiment, chicken growth hormone (cGH) (10 micrograms/kg of BW) was injected (iv) into different birds daily for 7 days starting at 7 wk of age. Different groups of birds received both T3 (.1 and 1 ppm) and cGH. Serum concentrations of thyroxine (T4), T3, and growth hormone (GH), lymphoid organ weights, total circulating white blood cells (WBC), and differential counts were measured following hormone treatments. It was found that T3, cGH, or a combination of both significantly lowered serum T4 concentrations. Triiodothyronine supplementation at 1 ppm, alone or with cGH significantly increased serum T3 concentrations. Chicken GH with T3 (.1 ppm) significantly increased serum GH concentrations. Thyrotropin releasing hormone supplementation did not affect serum concentrations of either T4, T3, or cGH. Relative bursa weights were greater in chicks that received T3 (1 ppm) or TRH (1 or 5 ppm) but not cGH. Relative spleen weights were enhanced in response to cGH alone or with T3 (1 ppm) but not TRH. Total WBC count was significantly increased in response to T3 (1 ppm). Supplementation of T3 (.1 or 1 ppm), TRH (1 ppm), and the combination of cGH and T3 (1 ppm) significantly increased the percentage of lymphocyte cell population. These results demonstrate the impact of feeding hormones on T3, T4, and cGH concentrations in the serum and suggest the involvement of the above hormones in the growth of lymphoid organs as well as the production of lymphocytes.

Effect of thyroidectomy of immature male chickens on circulating thyroid hormones and on response to thyroid-stimulating hormone and chronic cold exposure.

Previous studies in the author's laboratory established that thyroidectomized (Tx) immature male chickens had significant levels of circulating thyroid hormones, and it was proposed that extrathyroidal tissue might be present. Three experiments were conducted to further investigate this possibility. In all experiments, thyroid glands were removed surgically at 3 wk of age. In the first experiment, birds were kept until 20 wk of age. It was found that only triidothyronine levels were reduced significantly in the Tx birds. In the second experiment, Tx as well as sham-operated control groups received a single iv injection of bovine thyroid-stimulating hormone (TSH) to determine if extrathyroidal tissue in Tx birds would respond to exogenous TSH. It was found that circulating thyroxine (T4) concentrations in sham-operated control birds, but not Tx birds, were increased following TSH injection. In the third experiment, Tx and sham-operated birds were exposed chronically to cold (7 C), and only circulating T4 was found to be elevated in both groups. It was concluded that extrathyroidal tissue in Tx birds does not respond to TSH.