In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents.

We used in ovo technology to protect chickens against multiple diseases by inoculating vaccines containing mixtures of live viral agents. A single in ovo injection of a vaccine containing serotypes 1, 2, and 3 of Marek's disease virus (MDV), a vaccine strain of serotype 1 infectious bursal disease virus (IBDV), and recombinant fowl pox vaccine with HN and F genes of Newcastle disease virus (rFP-NDV) induced protection against virulent MDV, IBDV, Newcastle disease virus, and fowl poxvirus. The multiple-agent vaccine induced specific antibodies against the viral agents present in the mixture and did not adversely affect the survival of hatched chickens. Inoculation of a vaccine containing serotypes 1, 2, and 3 of MDV and IBDV did not affect hatchability of eggs, although the addition of rFP-NDV to the mixture reduced hatchability by 23%-26%. In ovo vaccination with a vaccine containing MDV and IBDV vaccine viruses did not exacerbate the inhibitory effect of individual viral agents on humoral and cellular immune competence.     

The impact of in ovo injection of l-arginine on hatchability,immune system and caecum microflora of broiler chickens

The present article was conducted to evaluate the effect of in ovo injection of arginine on hatchability, immune system and caecum microflora of broiler chickens. For this reason, 300 fertile eggs were used in a completely randomized design with three experimental treatments. The experimental groups included: 1%–0.5% l -arginine (100 eggs), 2%–1% l -arginine (100 eggs), 3- control [included both sham control (injection of distilled water; 50 eggs) and control (no injection; 50 eggs)], which were injected on d 14 of incubation. After hatching, chicks of each experimental group (0.5% l -arginine, 1% l -arginine, and control groups) were randomly divided into four equal groups (as replicates) and reared for 30 days. Weight and feeding of chickens were recorded. Next, blood samples of chickens were collected on day 30 to evaluate antibody titre. Also, chickens were slaughtered on 24 and 30 days of the experiment to evaluate immune system organs and caecum microflora. Based on the results, in ovo injection of l -arginine had no significant effect on hatchability, body weight, antibody titre, spleen, bursa of Fabricius and thymus weight (p > .05). On the other hand, treatments significantly affected feed intake and feed conversion ratio (p < .05). As a novel finding, in ovo injection of l -arginine increased caecal Lactobacillus (p < .01), while decreasing Coliform and Escherichia Coli bacteria (p < .01). However, treatments did not influence caecal Enterococcus (p > .05). The overall results indicated that in ovo injection of 0.5% l -arginine had a better improving effect on caecal microflora and then considered as a recommended level of the present experiment.     

Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease.

The B1 strain of Newcastle disease virus (NDV-B1), which is nonpathogenic for newly hatched chickens, killed embryos when it was used to inoculate chicken eggs at embryonation day 18. Treatment of NDV-B1 with an alkylating agent, ethylmethane sulfonate (EMS) markedly reduced the pathogenicity of the virus for 18-day-old chicken embryos. Eggs inoculated with the modified virus (NDV-B1-EMS) hatched, and the virus was isolated from lungs and spleen of 1-day-old chickens. The hatched chickens developed antibody to NDV and were protected against challenge exposure (at 4 weeks of age) with a highly virulent GB-Texas strain of NDV. Presence of maternal antibody to NDV in embryonating eggs did not influence the protective ability of NDV-B1-EMS, which also induced protective immunity when administered to 4-week-old chickens. The 50% protective dose of NDV-B1-EMS in maternal antibody-negative and -positive embryos was calculated to be 10.77 and 17.70 embryo 50% lethal doses, respectively. Results of the study indicated that NDV-B1-EMS may be used as an embryo vaccine to protect chickens against Newcastle disease.     

Effect of in ovo administered reovirus vaccines on immune responses of specific-pathogen-free chickens

We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.     

Ileal MUC2 gene expression and microbial population,but not growth performance and immune response,are influenced by in ovo injection of probiotics in broiler chickens

1. The objective of present study was to evaluate the effects of intra-amniotic injection of different probiotic strains (Bacillus subtilis, Enterococcus faecium and Pediococcus acidilactici) on the intestinal MUC2 gene expression, microbial population, growth performance and immune response in broiler chicken.

2. In a completely randomised design, different probiotic strains were injected into the amniotic fluid of the 480 live embryos (d 18 of incubation), with 4 treatments and 5 replicates. Ileal MUC2 gene expression, microbial profile, growth performance and immune response were determined.

3. Injection of probiotic strains, especially B. subtilis, had significant effect on expression of the MUC2 on d 21 of incubation and d 3 post-hatch, but not on d 19 of incubation.

4. Injection of the probiotic strains decreased significantly the Escherichia coli population and increased the lactic acid bacteria population during the first week post-hatch.

5. Inoculation of probiotics had no significant effect on antibody titres against Newcastle disease virus, antibody titres against sheep red blood cell and cell-mediated immune response of chickens compared to control.

6. In ovo injection of the probiotic strains had no significant effect on growth performance of broiler chickens.

7. It was concluded that injection of probiotic bacteria especially B. subtilis into the amniotic fluid has a beneficial effect on ileal MUC2 gene expression and bacteria population during the first week post-hatch, but has no effect on growth performance and immune response in broiler chickens.     

Antioxidant defence in the brain of 1-d-old chickens exposed in ovo to acrylamide

1. Acrylamide (ACR) is a potent neurotoxicant, although information on its toxic influence on the developing neural system is still limited. The effects of in-ovo-injected ACR on the antioxidant system activity in the brain of newly hatched chickens was examined. This model eliminated the mother’s contribution to embryonic development. It was also recognised as an adequate model for animal embryonic development.

2. ACR was injected on d 4 of embryogenesis – in doses of 1.25 and 2.50 mg/egg (n = 40 eggs/group/120 eggs). The doses corresponded well with ACR doses used in other animal studies and their concentrations in certain animal feeds.

3. Mortality and incidences of malformations were not found to increase significantly. Significant depletion of glutathione was detected in the cerebellum, cerebrum and medulla oblongata of specimens exposed to the highest doses of ACR. Enzymatic activity was affected by the highest ACR doses. Glutathione peroxidase (GPx) activity increased significantly in the cerebrum, medulla oblongata and the hypothalamus. Superoxide dismutase (SOD) activity increased significantly in hypothalamus and decreased in cerebellum and cerebrum. A significant depletion of catalase (CAT) activity was detected in cerebellum. In the hypothalamus, the increased SOD/GPx and SOD/CAT ratios suggest the risk of H₂O₂.

4. It was concluded that ACR significantly influences the antioxidative defence in the chicken brain at doses of 1.25 and 2.50 mg/egg.     

In ovo oral vaccination with cmpylobacter jejuni establishes early development of intestinal immunity in chickens

1. Chick embryos were orally immunised at day 16 of incubation by injection of heat‐killed Campylobacter jejuni organisms into the amniotic fluid. The response to vaccination was observed at 5 d after hatching or, in some birds which received a postnatal oral booster vaccination, at 7 d after hatching, and the response was observed at 14 d of age.

2. The titres of antibody in serum, bile and intestinal scrapings, the distribution of immunoglobulin‐containing cells in the spleen, duodenum and ileum and the expression on peripheral blood leukocytes (PBL) of the T cell surface markers CD3, CD4 and CD8 were determined.

3. Whereas low titres of anti‐flagellin antibody were detected in serum, bile and intestinal scrapings of unimmunised birds, high titres were observed in immunised birds.

4. An increase in antibody of all isotypes was detectable in serum but the elevation in IgA antibody in intestinal scrapings and bile was particularly striking. This response was reflected in a dramatic increase in immunoglobulin‐containing cells, detected by fluorescent histology, particularly diose associated with IgA and IgM isotypes in the spleen and intestine of immunised birds.

5. Secondary oral boosting after hatching resulted in a depression in serum anti‐flagellin antibody in immunised birds compared to pre‐boosting titres (although still significandy higher than in non‐immunised controls) but an increase in IgA antibody in intestinal scrapings and bile. The number of immunoglobulin‐containing cells was also increased after boosting.

6. Neither immunisation regimen caused a significant change in the numbers of circulating CD3, CD4 or CD8 T cells.

7. These results indicate that in ovo oral immunisation with C. jejuni antigens stimulates the precocious development of immunity in chicks.     

A modified surgical technique for penile amputation and preputial ablation in the horse

The aim of this study was to describe a modified surgical technique for treatment of severe penile pathology, and the long‐term outcome. The surgery consisted of subischial urethrostomy and penile amputation with preputial ablation, with the horse in dorsal recumbency. A redundant section of the penis root and body was left in situ, rather than being retroflexed as described elsewhere. Follow‐up was obtained using a structured owner telephone questionnaire. The 15 cases included: 11 squamous cell carcinomas (73.3%); 2 melanomas; one chronic preputial discharge with no associated neoplasia; and one paraphimosis following routine sedation. Length of survival ranged from 0.9 to 74.6 months (median 25.1 months). From the 14 horses with follow‐up, 9 survived >18 months (64.3%) [Correction added on 17 August 2015, after first online publication: The percentage in the preceding sentence was wrong and has been corrected to '64.3%' from '69.2%']. Four euthanasias were due to presenting or post operative complications, while 2 were unrelated to the procedure. This procedure presents a simplified, viable option for treatment of extensive mixed penile lesions; reducing surgical complexity and time in comparison to previously described techniques requiring retroversion.     

A rapid high-precision flow cytometry based technique for total white blood cell counting in chickens

The automated analysis of total white blood cell count and white blood cell differentials is routine in research and clinical diagnosis in mammalian species. In contrast, in avian haematology these parameters are still estimated by conventional microscopic procedures due to technical difficulties associated with the morphological peculiarities of avian erythrocytes and thrombocytes. Both cell types are nucleated and fairly resistant to cell lysis, a prerequisite for automated leukocyte quantification and differentiation by commercial instruments. By using an anti-CD45 monoclonal antibody in combination with selected subset specific markers we have established a simple (no-lyse no-wash single-step one-tube) flow cytometry based technique for high precision chicken blood cell quantification. EDTA-blood samples are diluted, spiked with fluorescence beads and incubated with a mixture of fluorochrome conjugated chicken leukocyte specific antibodies. We demonstrate that total leukocyte numbers as well as thrombocyte, monocyte, T-cell, B-cell and heterophilic granulocyte numbers can be determined by flow cytometry in a single step without prior cell lysis, cell separation or cell washing steps. Importantly, we also show that blood samples can be fixed prior to cell staining which enables shipping of samples making the technology widely available. Comparison of this technique with conventional microscopy revealed superior precision. By comparing leukocyte differentials of two chicken populations and during immune system development after hatch we demonstrate that large sample numbers can be analysed within hours. This technique will help to overcome previous restrictions in immune status analysis in chickens in experimental systems, during vaccine testing and health status monitoring in chicken flocks. Advances in avian genomics should facilitate the development of appropriate tools for other avian species in the future which will make this technique broadly applicable.     

Effects of cyclophosphamide in newly hatched chickens after inoculation with avian nephritis virus

Effects of immunosuppression were compared in newly hatched chickens given cyclophosphamide (CY) after inoculation with avian nephritis virus (ANV). All CY-treated infected chickens died within 13 days after inoculation of the virus and had heavy urate deposits throughout the body. However, non-CY-treated infected, CY-treated noninfected, and non-CY-treated noninfected control chickens survived through the observation period. In a chronologic study, the value of serum uric acid in CY-treated infected chickens was more than 3 times higher than that in non-CY-treated infected chickens, and more than 9 times higher than in noninfected chickens. Serum uric acid values were coincident with the positive degree of ANV antigen in the tubular epithelial cells in the kidneys and with the severity of renal degeneration. Serologic and immunohistologic examinations did not reveal detectable antibody and IgG- and IgM-containing cells in the spleen and kidneys of CY-treated infected chickens. However, non-CY-treated infected chickens had an increased number of IgM- and IgG-containing cells and antibody against ANV on postinoculation day 6. These findings demonstrated that CY treatment enhanced the susceptibility of chickens to ANV infection.     

Effect of in ovo injection of threonine on immunoglobulin A gene expression in the intestine of Japanese quail at hatch

The objective of this study was to investigate the effect of in ovo injection of threonine (THR) on immunoglobulin A (IgA) gene expression of Japanese quail on hatch day. A total of 540 Japanese quail eggs were assigned into nine groups of 60 each and were set in a single‐stage incubator. Treatments were as follows: non‐injected (control), two diluent levels (0.05 or 0.1 ml saline), two sites of injection (in or under the air sac) and with or without nutrients (0.5 mg/ml THR). Eggs were injected on d 11 of incubation. On hatch day, after euthanizing hatched quail chicks, the intestine was removed and the jejunum was separated. The relative mRNA expression of jejunal IgA increased (p < 0.05) by the injection of 0.05 ml THR under the air sac when compared to the control group or other treatments of injection. Compared to the control group, no differences were imputable to treatments of 0.1‐ml injections on IgA gene expression. Differences with other injected groups were not significant. It was concluded that injection of 0.05 ml saline containing 0.5 mg THR/ml under the air sac can improve jejunal IgA mRNA expression in newly hatched Japanese quail chicks.     

Hatchability rate and embryonic growth of broiler chicks following in ovo injection royal jelly

1. The objectives of this study were to compare the hatchability, chick body and internal organs weights and plasma testosterone concentration of hatchlings after in ovo administration of royal jelly (RJ) on d 7 of incubation.

2. Fertile eggs (n = 150) were injected into the air sac or yolk sac with 0.5 ml normal saline solution or normal saline and pure RJ. The eggs were randomly divided into 5 groups of 30 eggs each: NC, control eggs receiving no injection; ASA, air sac–injected eggs given normal saline solution; ARJ, air sac–injected eggs injected with pure RJ; YSA, yolk sac–injected eggs receiving normal saline solution and YRJ, yolk sac–injected eggs given pure RJ.

3. Injection of RJ significantly decreased hatchability (46.7%) compared with injection of saline solution (68.3%). Hatchability was lower in ARJ (33.3 %) and YRJ (60.0%) groups than in the NC group (90.0%). Hatchability in ASA (70.0%) and YSA (66.66%) groups were comparable to the NC group.

4. In ovo injection of RJ into both sacs increased chicks’ absolute and relative body, heart, liver and testes weights compared to the control group whereas plasma testosterone concentration was similar among the different groups.

5. It was concluded that in ovo injection of RJ may be an effective method to increase the body weight of hatched chicks as an absolute value (CWT) and chicks' internal organ weights.     

应用PCR/RFLP技术对广西禽白血病病毒的检测与分型研究

对2000～2003年间从广西的南宁、玉林、桂林、钦州、柳州、梧州等地采集的有肿瘤/可疑肿瘤的250份鸡病例进行肿瘤病的检测.结果发现,外源性禽白血病阳性的有32份,阳性率为12.8%.其中,同时为马立克氏病阳性的有30份,两者共感染率高达93.75%(30/32);根据禽白血病病毒囊膜糖蛋白gp85基因的限制性片段长度多态性分析,对其中20份外源性禽白血病病毒进行分型鉴定,结果证实全部属于A亚群.     

The kinetics of hemopexin and alpha1-acid glycoprotein levels induced by injection of inflammatory agents in chickens.

The acute phase response to inflammation induces changes in the secretion of hepatic proteins. To examine the time course of an acute phase protein response in broiler chickens, the plasma levels of hemopexin (HX) and alpha1-acid glycoprotein (AGP) and liver HX mRNA were measured at various time points from 3 hr to 336 hr after an intraabdominal injection of either lipopolysaccharide (LPS), complete Freund's adjuvant, incomplete Freund's adjuvant, phytohemagglutin, or mineral oil. Uninjected chicks served as controls. The accumulation of liver HX mRNA began within 3 hr of stimulation and peaked at 12 hr. Relative to control levels, plasma HX and AGP levels increased by 6-12 hr postchallenge and peaked at 24 hr. Complete Freund's adjuvant and LPS treatments induced the greatest increase in plasma HX (threefold; P < 0.05). Plasma levels of HX and AGP returned to control levels at 336 and 168 hr postinjection, respectively. A second experiment demonstrated that turpentine induced a similar AGP response as LPS and that albumin is a negative acute phase protein. The results suggest that plasma levels of HX or AGP could be used as an indicator of the systemic component of a local inflammatory response in chickens.     

Midline scrotal ablation technique for unilateral cryptorchid castration in horses

Thirty-nine unilateral cryptorchid horses were castrated, using a midline scrotal ablation technique. This approach was satisfactory to access both inguinal rings, to eliminate the need for 2 incisions, and to allow for completion of the surgery by primary closure. These horses recovered from surgery with few complications, returned to work promptly, and had excellent cosmetic results.     

Effect of in ovo threonine supplementation on early growth,immunological responses and digestive enzyme activities in broiler chickens

1.?The effects of injecting threonine in ovo on early growth, some immunological responses and the activity of digestive enzymes of broiler chicks were investigated. Fertile eggs were distributed into 6 groups, each of 60. These were: untreated control, sham control, 10, 20, 30 or 40 mg threonine. Threonine was dissolved in 0·5 ml sterile saline and inoculated into the yolk sac of the 14-d-old embryo through the narrow end of the egg.

2.?The ratio of chick to egg weight was 1·6% higher in the group given 30 mg threonine and at 28 d of age chicks receiving threonine were 29 to 79 g heavier than untreated controls.

3.?Food conversion ratio until 7 d after hatching was improved in those chicks receiving 10, 20 or 40 mg threonine but there was no significant effect on the activities of amylase, pepsin or trypsin.

4.?The humoral response to sheep red blood cells was significantly greater in those groups receiving 10, 20 or 30 mg threonine supplementation than in untreated controls.

5.?The response to phytohaemagglutinin-P, a measure of the cell-mediated immune response, was not affected, however.

6.?It is concluded that injections of 20 to 30 mg threonine into yolk sac can improve post-hatching growth and humoral responses of broiler chicks.