Sunset Yellow FCF (SY), used frequently in ready-made foods, cosmetics, and the pharmaceutical industry, may cause many health problems. This study is intended to evaluate the morphological and cellular effects of SY on the embryonic chicken immune system throughout incubation. Babcock white leghorn eggs were randomly divided into four groups. Besides a control group, there were three treatment groups which received a single injection of 200, 1,000, or 2,000 ng of SY into the air sac just before incubation. The eggs were opened on the 10ᵗʰ, 13ᵗʰ, 16ᵗʰ, and 21ˢᵗ days of incubation. Samples of the bursa of Fabricius, thymus, and spleen were taken from embryos. Serial sections of 5 μm thickness were stained with histological methods and routine histological procedures were performed. An increase in the spleen volume was determined as the hatching time of the chicks approached. The highest eosinophil ratio was found in the SY₁,₀₀₀ and SY₂,₀₀₀ groups (P < 0.05), where the most significant change was developmental retardation in the thymus. In the bursa of Fabricius, there was less lymphocyte accumulation and eosinophilic cell infiltration with increasing doses. It was concluded that in ovo administered SY has undesired effects on embryonic development of the bursa of Fabricius, spleen, and thymus, and on spleen volume.

The innate immune system is a critical component directing the overall response of the immune system early in the inflammatory process. Evaluation of the innate immune system could offer a screening method for the selection of breeding stock from commercial chicken operations to improve flock health and prevent the loss of genes crucial to disease resistance. Three commercial broiler chicken lines (designated Lines A, B and C) were profiled for efficiency of their innate immunologic response. Oxidative burst and bactericidal functions of heterophils and monocytes, as well as heterophil degranulation, were analyzed. The birds were tested 1, 4, 8 and 15 days post-hatch. Individual lines differed in their ability to perform innate immunological responses during the first 15 days post-hatch. Although bactericidal capabilities were similar, oxidative burst responses by monocytes were low in comparison to that generated by heterophils. The fact that monocytes are not particularly adept at producing an oxidative burst at this age suggests that this is not a major avenue of innate defense by monocytes. Heterophil oxidative burst response was stronger in Line C than Line A during the first four days post-hatch. Line B showed no difference from Line C in heterophil oxidative burst response at 1 d, but produced a stronger response than Line C on 4 and 8 d post-hatch. Degranulation by heterophils showed significant differences in responses of Lines A and C depending on the day post-hatch, and stronger response in Line C vs Line B in the first four days post hatch. The first week post-hatch is an important time as chicks are particularly susceptible to infection as neonates. Mortality data of the commercial lines indicates that Line A is the most susceptible to demise, followed by Line C and then Line B. These results suggest that oxidative burst production efficiency is an important defensive function to monitor for immunoprofiling.

Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S. enteritidis PT 4 strain from the United Kingdom (UK). The two PT 4 strains were also compared in orally inoculated adult laying hens. In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids. In orally inoculated one-day old chickens, the UK S. enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain. The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain. The S. enteritidis PT 8 strain and one S. typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice. This strain of S. typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S. typhimurium strain isolated from a pig with septicemic 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.

OBJECTIVE To determine effects of in ovo administration of a probiotic on development of the intestinal microbiota of 2 genetic lineages (modern and heritage) of chickens. SAMPLE 10 newly hatched chicks and 40 fertile eggs to determine intestinal microbiota at hatch, 900 fertile eggs to determine effects of probiotic on hatchability, and 1,560 chicks from treated or control eggs. PROCEDURES A probiotic competitive-exclusion product derived from adult microbiota was administered in ovo to fertile eggs of both genetic lineages. Cecal contents and tissues were collected from embryos, newly hatched chicks, and chicks. A PCR assay was used to detect bacteria present within the cecum of newly hatched chicks. Fluorescence in situ hybridization and vitality staining were used to detect viable bacteria within intestines of embryos. The intestinal microbiota was assessed by use of 16S pyrosequencing. RESULTS Microscopic evaluation of embryonic cecal contents and tissues subjected to differential staining techniques revealed viable bacteria in low numbers. Development of the intestinal microbiota of broiler chicks of both genetic lineages was enhanced by in ovo administration of adult microbiota. Although the treatment increased diversity and affected composition of the microbiota of chicks, most bacterial species present in the probiotic were transient colonizers. However, the treatment decreased the abundance of undesirable bacterial species within heritage lineage chicks. CONCLUSIONS AND CLINICAL RELEVANCE In ovo inoculation of a probiotic competitive-exclusion product derived from adult microbiota may be a viable method of managing development of the microbiota and reducing the prevalence of pathogenic bacteria in chickens.

The objective of this study was to compare the effects of 3 diet formulations containing different protein sources (animal, plant, and a combination of animal and plant) on the colonization of Campylobacter jejuni in the gastrointestinal tract of broiler chickens. A freshly isolated strain of C. jejuni (biotype IV, serotype HS O:21, O:29, HL untypable) from a broiler chicken was used to infect 3-day-old chicks that had been free of C. jejuni; 0.5 mL of an inoculum containing 108 colony-forming units was administered orally. Shedding of the organism was studied, and C. jejuni in the ceca, jejuni, and crop were enumerated by quantitative culture. The isolates recovered from the birds during the study period of 35 d were characterized and confirmed as C. jejuni by the use of standard methods and underwent biotyping, serotyping, antimicrobial susceptibility testing by disk diffusion and the E-test, and flagellin gene typing. A cyclical pattern of shedding of C. jejuni was observed in all the birds. Colonization was highest in the ceca. The ceca of birds receiving plant-protein-based feed had significantly less colonization then the ceca of birds receiving the other types of feed, whereas the differences in colonization of the jejuni and crops were not significant. Characterization by biotyping, serotyping, and flagellin gene typing showed that 95% of the recovered isolates were identical to the strain used for infecting the chicks. However, with the Lior-HL typing scheme, 74% of the recovered isolates were HL untypable. Antimicrobial resistance testing did not reveal significant differences between the infecting strain and the recovered isolates among the different feed groups.

The left ovary of chicken embryos was removed and incubated in culture medium with a thymidine analogue, bromodeoxyuridine (BrdU), in vitro. In addition, fertile chicken eggs were injected with BrdU via the extraembryonic vessels and incubated for 24 hours. The ovaries were then processed for immunohistochemical localization of calbindin-D28K (a 28-kd vitamin D-dependent calcium-binding protein) and BrdU. Calbindin-D28K was detected in the germinal epithelium and in cells surrounding the oogonia and oocytes (future granulosa cells) of the embryonic chicken ovary. However, Brdu was observed in the nucleus of the oogonia and oocytes of the chicken embryonic ovaries. Comparison of the 2 adjacent sections, immunostained for calbindin-D28K and BrdU consecutively, indicated that BrdU, the marker for cell proliferation was not detected in calbindin-D28K-containing cells, namely, germinal epithelium and future granulosa cells, in the ovary of chicken embryos. These results suggested that calbindin-D28K-containing cells in the ovary were not in the process of cell division during the 24-hour incubation of chicken embryos.

Campylobacter jejuni A74/O and A74/C are congenic strains. An oral dose of 10(5) organisms of strain A74/C colonizes chicken intestines. Strain A74/O, from which A74/C is derived, does not colonize the chicken intestines with an oral dose of 10(5) organisms. In this study, the congenic bacteria were compared to identify possible colonization mechanisms. Differences were not observed in plasmid content or by HindIII, Pst I, Acc I, HincII, Ava I, Ava II, Xba I, and BamHI restriction enzyme digestion of total DNA. Transmission electron microscopy of negatively stained samples revealed no differences between the strains. Sections of cecal tissue from nonfed day-of-hatch chicks were cultured with each strain for 2 hours and then examined by light and electron microscopy. Both strains caused necrosis of villus epithelial cells. Immunofluorescent or silver staining revealed strain A74/C located deep in numerous epithelial crypts, but strain A74/O only was present in one sample mixed with sloughed necrotic cells. Similarly, organisms were detected by transmission electron microscopy deep in crypts in tissues cultured with A74/C, but not A74/O. Cells of A74/C detected in crypts did not appear to associate with epithelial cells. The strains did not differ in chemotactic behavior to mucin or fucose.

Studies were undertaken to assess the chicken embryo and newly hatched chicken as models for studying the effects of bone-active agents. Initially, 1,25-dihydroxycholecaliferol (1,25[OH]2D3), sodium fluoride (NaF), parathyroid extract, epidermal growth factor, and prostaglandin E2, were tested for lethality over a broad dose range. One or 3 injections of 1,25(OH)2D3 into the yolk sac of chicken embryos resulted in death of embryos given greater than 0.1 ng/injection, whereas 0.01 ng was tolerated by the embryos. Administering 1,25(OH)2D3 intraperitoneally to newly hatched chickens as a single injection or weekly for 3 weeks resulted in no deaths at doses up to 50 ng. One or 3 IV injections of less than 400 micrograms were tolerated by the embryo. Giving chickens feed and water containing 2.4 g of NaF/kg was lethal but no deaths occurred when chickens were given feed containing less than 1.2 g of NaF/kg. Mortality associated with the administration of epidermal growth factor to embryos was inconsistent, in that death occurred in embryos given a single injection of greater than 250 ng, but no deaths occurred in embryos given 3 injections at similar doses. Parathyroid extract and prostaglandin F2 were not lethal when administered to embryos and chickens in a single-injection or multiple-injection regimen. Overall, lethality in chicken embryos given a particular agent reflected the dose of bone-active agent injected, rather than the number of injections. Three of the bone-active agents were selected to characterize their microscopic bone effects in chicken embryos and chickens. Administration of 1,25(OH)2D3 to embryos on day 14 at doses of 100, 10, 1, and 0.1 ng led to subperiosteal hyperosteoidosis in all 5 of the tibiotarsi examined from the high-dose (100 ng) group necropsied on day 18 of incubation. Three of 5 of the tibiotarsi from the 10-ng treatment group were similarly affected. Bone effects were noticed in chickens hatched from the aforementioned treatment groups or in chickens given 1,25(OH)2D3 intraperitoneally and examined at 3 and 6 weeks of age. Administration of NaF to chicken embryos on the 10, 12th, and 14th days of incubation via the IV route at doses of 160, 80, 40 and 20 micrograms/embryo led to subperiosteal hyperosteoidosis in tibiotarsi from 3 of 10 embryos (examined at 18 days of incubation) from the 2 high-dose groups. Tibiotarsi of chickens from this treatment group were microscopically normal at 3 weeks after hatching. When newly hatched chickens were given a diet containing NaF at dosages of 1.2 g/kg, 0.6 g/kg, and 0.3 g/kg, a dose-dependent increase in osteoid was seen at 3 and 6 weeks. In addition, cortical thinning and expansion of the medullary canal were observed only at 3 weeks. In contrast to the effects observed with 1,25(OH)2D3 and NaF, parathyroid extract caused no microscopic bone alterations when given to embryos or chickens. Overall, the bone alterations in the embryo were attributed to increased subperiosteal osteoid formation and defective mineralization. These findings were consistent with known effects of NaF and 1,25(OH)2D3 on bone, and they establish the chicken embryo as a sensitive model for studying bone-active agents.

Immunoreactivity for 28 kd vitamin D-dependent calcium-binding protein (calbindin-D28k) has been localized in the germinal epithelium and cells surrounding oogonia and oocytes (future granulosa cells) of developing and growing ovaries of chicken embryos. The protein first appeared prominently in the germinal epithelium of the developing left ovary in 8-day embryos. At the twelfth day of incubation, cells surrounding oogonia and oocytes reacted intensely for calbindin-D28k. The number and intensity of calbindin-D28k-containing cells increased in both types of cells as the embryos further developed. Calbindin-D28k remained in the germinal epithelium throughout the study period observed (up to 10 weeks). However, the protein was present transiently in the future granulosa cells. It gradually decreased after hatching, and was virtually absent from granulosa cells in a 10-week old chicken. Compared with the known process of onset of sexual development, these results indicated possible involvement of calbindin-D28k in the early phases of oogenesis in chicken ovaries.