Production and characterization of monoclonal antibodies reactive with the chicken interleukin-15 receptor alpha chain

We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor.     

Analysis of cross-reactivity of five new chicken monoclonal antibodies which recognize the apical complex of Eimeria using confocal laser immunofluorescence assay

For Apicomplexa (members) the host cell invasion is realized with the help of the organelles located at the apical tip of parasites. In this research paper the characterization of five chicken monoclonal antibodies (mabs) produced against Eimeria acervulina sporozoites is described. All mabs reacted with molecules belonging to the apical complex of chicken Eimeria sporozoites. On immunofluorescence assay (IFA) one mab, 8E-1, recognized an apical tip molecule present on all chicken Eimeria sporozoites, two mabs (8D-2 and HE-4) recognized an antigen present on the apical tip of the same two Eimeria species (E. acervulina and E. brunetti), another mab (5D-11) recognized an antigen present on the apical tip of other two species (E. acervulina and E. maxima) while one mab (8C-3) identified antigens present on the sporozoites and sporocysts wall of only E. acervulina. Besides the apical tip antigens, two mabs (HE-4 and 8D-2) recognized some proteins located in the anterior half of the sporozoites. Collectively, these mabs proved that the apical complex of chicken Eimeria sporozoites share one or more antigens that are expected to play a role in host cell recognition and invasion.     

Intestinal immunomodulation by vitamin A deficiency and lactobacillus-based probiotic in Eimeria acervulina-infected broiler chickens

In a 2 X 2 factorial study, a broiler starter ration was amended for vitamin A (control, C; deficient, A) and probiotic status (-, P) to investigate their modulatory effects onthe host immune system. Birds were inoculated orally with Eimeria acervulina (EA) oocysts, and disease susceptibility was evaluated by assessment of fecal oocyst shedding. Humoral and local cellular mediated immunity were assessed by evaluation of antibody and cytokine (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) levels in sera and intestinal secretions on a 3-day interval after inoculation. Fecal oocyst shedding was highest (P < 0.05) in A- birds, followed by AP, C-, and CP birds. Feeding the probiotic reduced shed oocysts by 20% in A fed birds and by 26% in C fed birds. Intestinal IFN-gamma was relatively constant in all treatment groups except for A-, where it declined steadily and was lower (P < 0.05) from day 6 on. Serum IFN-gamma levels fluctuated within each treatment and over time were not revealing. Intestinal IL-2 was highest in CP birds at 3 and 9 days postinfection (DPI) and lowest in A- birds at 3, 9, and 12 DPI (P < 0.05); no difference between treatments was found at 6 DPI (P > 0.05). Eimeria-specific intestinal antibody (Ab) level was constant (P > 0.05) in C- birds but increased with time (P < 0.05) in A-, AP, and CP birds. Serum Ab levels were also constant in A- and CP birds but increased (P < 0.05) in C- and AP birds after 6 DPI. The data demonstrate for the first time a probiotic-enhanced immunity in vitamin A deficient birds. This study is also the first to demonstrate the probiotic effect on local cell-mediated immunity of chickens, best manifested by apparent lower intestinal invasion and development by EA, on the basis of higher IL-2 secretion and lower EA oocyst production.     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Influence of Apoptosis in Bovine Embryo's Development

The correlation between apoptosis and early bovine embryonic loss is still not fully elucidated. In the present study, the relationship between the arrest of bovine embryos at the different stages of development and apoptosis was evaluated. We used embryos 7 days after in vitro maturation and fertilization, and morphologic and biochemical apoptotic analyses were performed by using a phase contrast microscope and by the terminal transferase dUTP nick end‐labelling respectively. For the statistic, the apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. To evaluate the relation between ACR and fragmentation pattern, embryos were divided into five groups, groups I–V. To assess the relation between ACR and cytoplasmatic fragmentation, embryos were divided into three groups, according to the fragmentation percentage (<5%; 5–15% and >15%). Of the total 139 embryos included, 65 arrested at 2–8 cells; 14 arrested at 9–16 cells; 18 compacted morula and 42 were non‐arrested blastocysts. The average number of embryonic fragmentation at different stages of the development, 2–8 cells, 9–16 cells, compacted morula and blastocyst, was 16.0 ± 1.5, 28.7 ± 4.4, 4.4 ± 2.4 and 1 ± 0.3 respectively. The embryos at the stage of arrested 9–16 cells and compacted morula had higher ACR than those at the blastocyst stage, excluding the stage of 2–8 cells (the genome is not yet active). The correlation detected between embryonic development and ACR was 0.92 (p < 0.01). It was observed that embryos possessing high fragmentation showed the higher ACR value (r = 0.98, p < 0.05). Comparing the results between fragmentation percentage and ACR, it was observed that the embryos with higher percentage of fragmentation corresponded to higher ACR (r = 0.97, p < 0.01). These results clearly demonstrated that bovine embryonic arrest at different stages of development is correlated with the apoptotic mechanisms.     

Persistent Mullerian Duct Syndrome in a Miniature Schnauzer Dog with Signs of Feminization and a Sertoli Cell Tumour

A 5‐year‐old male Miniature Schnauzer was presented with unilateral cryptorchidism and signs of feminization. Abdominal ultrasonography revealed an enlarged right testis and a large, fluid‐filled cavity that appeared to arise from the prostate. Computed tomography revealed the cavity to be consistent with an enlarged uterine body, arising from the prostate, and showed two structures resembling uterine horns that terminated close to the adjacent testes. The dog had a normal male karyotype, 78 XY. Gonadohysterectomy was performed and both the surgical and the histological findings confirmed the presence of a uterus in this male animal, resulting in a diagnosis of persistent Mullerian duct syndrome (PMDS). The enlarged intra‐abdominal testis contained a Sertoli cell tumour. Computed tomography proved to be an excellent diagnostic tool for PMDS.     

RESEARCH PAPER: Segmental dorsolumbar epidural analgesia via the caudal approach using multiple port catheters with ketamine or lidocaine or in combination in cattle

ObjectiveTo determine the analgesic and systemic effects of epidural administration of ketamine, lidocaine or a combination of ketamine/lidocaine in standing cattle.Study designProspective, randomized, experimental trial.AnimalsSix healthy male cattle weighing between 335 and 373 kg.MethodsThe animals received 0.5 mg kg^?1 of ketamine (K), 0.2 mg kg^?1 of 2% lidocaine (L) or 0.25 mg kg^?1 ketamine plus 0.1 mg kg^?1 lidocaine (KL). All the drugs were injected into the dorsolumbar epidural space via a caudal approach through a non‐styletted multiple‐port catheter. Each animal received each treatment at random. Evaluations of analgesia, sedation, ataxia, heart rate, arterial pressure, respiratory rate, skin temperature and rectal temperature were obtained at 0 (basal), 5, 10, 15, 30, 45, 60, 75, 90 minutes after epidural injection, and then at 30‐minute intervals until loss of analgesia occurred. Skin temperature was taken at these intervals up to 60 minutes. All the animals received a standard noxious stimulus; a 4‐point scale was used to score the response. A second scale was used to score ataxia and a third for sedation.ResultsThe duration of analgesia in the upper and lower flanks in cattle was 140 ± 15, 50 ± 14 and 80 ± 22 minutes (mean ± SD) after dorsolumbar epidural KL, K or L, respectively. The cardiovascular changes were within acceptable limits in these clinically healthy cattle.ConclusionsDorsolumbar epidural administration of KL to cattle resulted in longer duration of analgesia of the upper and lower flanks in standing conscious cattle, than the administration of K or L alone.Clinical relevanceFurther research is necessary to determine whether this combination using this technique provides sufficient analgesia for flank surgery in standing cattle.     

Effects of corticosteroids on lymphocyte subpopulations and lymphokine secretion in chickens.

Various effects of glucocorticosteroids on the avian immune system were examined in chickens treated intramuscularly with 0.1 to 2.5 mg dexamethasone or prednisolone. Kinetic changes in body weight gain, percentages of lymphocyte subpopulations, and T-cell functions were examined following treatment with dexamethasone or prednisolone every other day. Chickens treated with dexamethasone or prednisolone showed a decrease in body-weight gain compared with age-matched, untreated chickens. In general, the total number of splenic lymphocytes of chickens treated with the two drugs was significantly lower than in controls in a dose-dependent manner. Flow cytometric analysis of splenic lymphocyte subpopulations revealed that the percentages of lymphocytes expressing CD8, gamma delta T-cell receptor, Ia, or IgM antigens and natural killer cells were lower in dexamethasone-treated chickens than in the controls, whereas the percentages of T lymphocytes bearing CD3, CD4, or alpha beta TCR antigens were higher. Furthermore, splenic T cells obtained from dexamethasone-treated chickens showed a significant depression in concanavalin A-induced lymphoproliferation and interleukin 2 and gamma-interferon production. The results characterize a variety of immunosuppressive effects of glucocorticoids on the avian immune system.     

Comparison of disease susceptibility and subclass-specific antibody response in SC and FP chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima

Susceptibility to disease and the subclass-specific antibody response to Eimeria tenella, E. acervulina, and E. maxima were compared in two inbred strains of chickens, FP (B15B21) and SC (B2B2). FP strain was more susceptible to coccidiosis than SC chickens based on oocyst production, lesion score, and clinical signs. FP chickens infected with E. tenella had more severe cecal lesions and a significantly lower hematocrit level than SC chickens. FP chickens infected with E. acervulina excreted five times as many oocysts at 6 days postinfection as SC and showed a 71% reduction in plasma carotenoid level compared with controls (56% reduction in SC chickens). Body-weight change did not correlate with other signs of disease. Both SC and FP chickens produced high levels of serum IgM and IgG and biliary IgA. Although SC chickens had a slightly higher antibody response than FP chickens at 7 days postinoculation, both strains maintained high levels of IgM, IgG, and IgA for a prolonged period post primary inoculation. Although SC and FP chickens show different disease susceptibility to coccidiosis, they demonstrate similar antibody response.