Tissue cytokines in chickens from lines selected for high or low humoral antibody responses, given supplemental Limosilactobacillus reuteri and challenged with Histomonas meleagridis.

Histomonas meleagridis, a protozoan parasite, induces blackhead disease (histomoniasis) in poultry. During hatching, chicks from lines divergently selected for high (HAS) and low (LAS) antibody responses to sheep red blood cells were divided into two groups, each of HAS and LAS, and placed in pens with wood shavings as litter. Feed and water were allowed ad libitum. Half of the chicks from each line had Limosilactobacillus reuteri (L. reuteri) inoculated to their drinking water. On day 18, all chicks were given a transcloacal inoculation of 100,000 H. meleagridis cells. Then, 10 days later, they were euthanized, followed by collection of tissues from the brain, cecal tonsil, ceca, liver, thymus, and spleen for qPCR analyses of cytokines involved in immunological development. Changes in cytokine expressions were most numerous in the cecal tonsil, ceca, and liver. In the absence of a functional medication for control of histomoniasis, L. reuteri and/or its secretory product, reuterin, might serve, in some genetic populations, as a means to reduce the impact of histomoniasis in chickens. The data demonstrate that L. reuteri treatment had tissue specificity between the two genetic lines, in which the effects were targeted primarily toward the cecal tonsil, ceca, and liver, which are the primary tissue targets of the parasite (H. meleagridis), as well as the thymus and spleen. However, interactions among main effects reflect that responses to inflammatory markers observed in tissues for one genetic line may not be observed in another.

Sodium bisulfate feed additive aids broilers in growth and intestinal health during a coccidiosis challenge.

Sodium bisulfate (SB) was evaluated on its ability to improve broiler growth and intestinal structure with(out) a coccidia challenge. One thousand two hundred Cobb500 day-old males were randomly assigned within 4 experimental groups with a 2 × 2 factorial design, with (out) SB in the diet and with(out) a day 0 coccidia challenge using a 10× dose of a commercial vaccine. At day 7, oocysts per gram of feces were determined. At day 0, 14, 28, and 41, BW and feed consumption were measured. At day 21, 20 birds per treatment were subjectively scored for coccidia lesions, and jejunal histologic samples were collected for villi measurements. Twenty additional birds were given fluorescein isothiocyanate-dextran to determine gut permeability. At day 41, 10 birds per treatment had histologic samples collected. Statistical analysis was conducted in JMP Pro 14 using GLM procedure to compare disease state and diet. Means were separated using Dunnett's test (P ≤ 0.05) with the nonchallenged standard diet treatment that is considered the control. All parameters measured indicated an effect due to the coccidia inoculation. Therefore, effects of diet on (non)challenged treatments were determined using a Student t test (P ≤ 0.05). Limited differences due to diet were seen for the nonchallenged production data. Sodium bisulfate had a thinner villi base width (P = 0.04) on day 21 and greater villi height (P = 0.03), smaller base width (P = 0.04), thicker muscularis (P = 0.03), and lower crypt: height ratio (P = 0.01) on day 41. Challenged SB had similar gut permeability to the nonchallenged control (P = 0.94) on day 21. There was no difference in flock uniformity, feed intake, oocysts per gram of feces, or lesion scores between challenged treatments. Challenged SB had greater BW on day 14 (P < 0.0001), 28 (P < 0.0001), and 41 (P = 0.02). Feed conversion ratio from day 0 to 14 was also lower (P = 0.0002). Challenged SB had smaller crypts (P = 0.02) and therefore a smaller crypt: height ratio (P = 0.03) on day 21. Challenged control had a larger apical width (P = 0.03) and thicker muscularis (P = 0.04) on day 41. Overall, the addition of SB during coccidial enteropathy aided in BW, feed conversion ratio, and villi health with no observed effects on parasite cycling.

Early infection with Histomonas meleagridis has limited effects on broiler breeder hens' growth and egg production and quality.

A study was conducted to determine differences between Histomonas meleagridis-infected and control pullets based on disease signs, hen growth, and egg production and quality. Ross 708SF females were weighed and then placed in pens on the day of hatch (92 chicks/pen). At 25 D, 4 pens were infected with H. meleagridis in the cloaca, whereas 4 pens were control. At 5, 10, and 20 D after inoculation, 5 birds per pen (2 birds per pen at 20 D) were subjectively scored for blackhead disease. Birds were feed restricted based on BW and/or egg production. Individual BW were collected at 3, 5, 13, 15, 20, and 64 wk. Egg production was recorded at 24-63 wk. Egg quality was measured at 30, 34, 39, 42, and 56 wk and included shell and vitelline membrane strength, shell thickness, egg weight, and Haugh units. Hatchability was measured at 27, 37, and 60 wk and fertility at 27 and 37 wk. Treatment effects were determined by JMP Pro 14 using GLM with means separated using the Student t test (P ≤ 0.05). Cecal lesions were apparent on 5, 10, and 20 D and liver lesions on 10 and 20 D for the infected birds. The control had no histomoniasis lesions. Flock uniformity differed on wk 13 and 20 (P = 0.04; 0.04). Infected birds weighed less at 64 wk (P = 0.002). The onset of lay was not delayed. Infected birds produced more eggs during 1 period (P = 0.02). The infected birds produced heavier eggs at 30 wk (P = 0.04), eggs with a stronger and thicker shell at 42 wk (P = 0.05, 0.03), and eggs with a stronger vitelline membrane at 56 wk (P = 0.049). Hatchability and fertility did not differ (P > 0.05). H. meleagridis was observed in the infected birds' cecal samples at trial termination. This study indicates early infection with H. meleagridis has limited effects on pullet egg production and quality.

The effect of sodium bisulfate and coccidiostat on intestinal lesions and growth performance of Eimeria spp.-challenged broilers.

Coccidiosis is a high-prevalence disease that annually entails huge costs for the poultry industry. Control of coccidiosis in poultry production is based on the use of coccidiostats and vaccines. However, along with the problem of drug resistance, there is a concern about food safety and drug residues in poultry products. The objective of this study was to evaluate the effect of sodium bisulfate (SBS) in comparison with monensin (M) and their combination (SBSM) effects on controlling coccidiosis in broilers. In a randomized design, 300 chickens (Ross 308) were divided into 5 treatments and 4 replications (15 birds per replicate). All birds, except the negative control (NC), were orally inoculated with 4 Eimeria species on 14 D of age. Treatments included were as follows: NC, an unsupplemented basal diet, nonchallenged; positive control, a basal diet unsupplemented, challenged with Eimeria spp; a basal diet supplemented with 5 g/kg of SBS; a basal diet supplemented with 1 g/kg of M; and a basal diet supplemented with 5 g/kg SBS and 1 g/kg M (SBSM). Oocyst shedding per gram (OPG) of the faecal sample from each experimental unit was counted on 5 to 14 D after inoculation. Two chicks from each experimental unit were euthanized to investigate intestinal lesions on day 5 after inoculation. The NC birds showed the highest BW gain and the lowest feed conversion ratio. The birds in the SBSM group had improved feed consumption compared with the M group in the prechallenge period (P < 0.05). All supplemented treatments resulted in a significant decrease in OPG. The M and SBSM treatments showed more efficacy than the SBS group (P < 0.05) in reducing OPG. There was a significant reduction in cecal lesions owing to supplementation with SBS, but the effect of SBS in the upper part of the intestine was lower than the M and SBSM groups (P < 0.05). Based on the results of this study, SBS has protective effects against coccidiosis in ceca, and the combination of M and SBS (SBSM) did not show any further improvement effect compared with M alone on the control of coccidiosis.

Histomonas meleagridis isolates compared by virulence and gene expression.

Pathology and putative virulence factor expression of three Histomonas meleagridis isolates differing in geographic origin, cell passage number (56 or 100), or cell populations grown from a monoculture were compared. Turkey poults inoculated with the high cell passage number isolates or monoculture isolates varied in gross lesion severity and weight gain (P<0.0001). Screening of a published H. meleagridis cDNA library identified forty- eight cysteine proteinases (CP) and one superoxide dismutase (Fe-SOD) proposed to function in either tissue damage and/or invasion and oxidative defense. The Fe-SOD and eight CPs were analyzed using real time polymerase chain reaction. CP2, CP3, and CP8 showed significant differences in expression among the field isolates (P ≤ 0.05). The high passage isolates had decreased CP2, CP3 and CP4 expression when compared with their field isolate. CP7 did not differ between field isolates or the 56-passaged isolate. The Fe-SOD gene showed significant differences in expression among the various isolates. When exposing cultured H. meleagridis to air, Fe-SOD expression decreased rapidly during the first hour of air exposure but increased progressively through the next 3 h. This study provides information on gross pathology and virulence factors associated with various isolates of Histomonas meleagridis which can aid in determining the pathogenetic mechanisms used by this organism.

Two Blackhead Disease Outbreaks in Commercial Turkey Flocks Were Potentially Exacerbated by Poor Poult Quality and Coccidiosis.

Field visits at two different farms suggest a correlation between commercial turkey (Meleagridis gallopavo) flocks having increased mortality from blackhead disease (histomoniasis) if they suffer from poor poult quality at placement and coccidiosis (Eimeria spp.) before age 6 wk. In both cases, the flocks were all-in/all-out with curtain-sided houses and received a coccidiosis vaccine on day of hatch. At Farm I 2018, poults from different hatcheries were placed in two houses on the same farm (Houses 1 and 2). House 2 had poults considered poor quality and suffered from mortality associated with coccidiosis at 2 and 4 wk of age. At 8 wk, blackhead disease was diagnosed in both houses by postmortem examination. House 2 had mortality of >2000 poults, and the subpopulation of necropsied poults had gross lesions characteristic of histomoniasis. Gross lesions associated with blackhead disease were only found in eight poults in House 1, which was populated with good-quality poults and did not have a second spike in mortality due to coccidiosis. The Farm II 2020 poults were delivered from the same hatchery onto a three-house farm (Houses A, B, and C). House C had poults that were considered poor quality and had mortality associated with coccidiosis at 3 wk of age. At 8-9 wk, House C had mortality approaching 1000 birds, with all poults examined postmortem having clinical signs of blackhead disease. Houses A and B were populated with good-quality poults and had no diagnosed mortality from coccidiosis or blackhead disease. The similarity of these two cases suggest that poult quality at placement coupled with coccidiosis before 6 wk of age can influence the severity of blackhead disease in commercial turkey flocks.

PCR detection of Heterakis gallinarum in environmental samples.

Heterakis gallinarum is a widely distributed cecal nematode that parasitizes gallinaceous birds including chickens and turkeys. H. gallinarum infection poses a problem for the poultry industry as the nematode egg serves as a vector for the protozoan parasite, Histomonas meleagridis, the causative agent of histomonosis. The only means of detecting H. gallinarum in the environment is microscopic identification of the eggs in soil or feces; however, H. gallinarum eggs are often mistaken for those of Ascaridia galli. Three primer sets were designed from sequences cloned from the H. gallinarum genome to develop a diagnostic PCR. Each of these primer sets amplified a single product from H. gallinarum, but were unable to amplify DNA from H. meleagridis, Ascaridia galli, or Cestode sp. H. gallinarum DNA was amplified from Lumbricus sp. (earthworms) and Alphitobius diaperinus (darkling beetles), confirming that the earthworm acts as a paratenic host for H. gallinarum and suggesting that the darkling beetle may be a carrier for this nematode.

Heterakis gallinarum, the Cecal Nematode of Gallinaceous Birds: A Critical Review.

Heterakis gallinarum is a heavily prevalent poultry parasite that thrives in the ceca of various species of gallinaceous birds. It is a small roundworm, measuring between 4 and 15 mm long, in the family Heterakidae. Heterakis gallinarum has a direct life cycle not requiring an intermediate host to complete development, and it is generally believed that poultry raised at high density on litter are at greatest risk for accumulating large numbers of the nematode. This species typically only causes mild pathology that does not significantly affect bird performance. However, H. gallinarum is recognized as an economically important parasite by the poultry industry because its ovum serves as the vector for the protozoal parasite Histomonas meleagridis, the cause of histomonosis in poultry. Diagnosis of the nematode typically relies on fecal egg counts, which are prone to false negative diagnoses. Molecular tools are available for studying the nematode and diagnosing infected flocks. Treating and preventing H. gallinarum infection is made difficult due to the low efficacy of anthelmintics for eradicating H. gallinarum from infected birds and of disinfectants for destroying H. gallinarum ova on contaminated farms.

Estudio Recapitulativo- Heterakis gallinarum, el nematodo cecal de las aves gallináceas: una revisión crítica. Heterakis gallinarum es un parásito avícola muy prevalente que se desarrolla en el ciego de varias especies de aves gallináceas. Es un pequeño gusano redondo, que mide entre 4 y 15 mm de largo, de la familia Heterakidae. Heterakis gallinarum tiene un ciclo de vida directo que no requiere de un huésped intermedio para completar el desarrollo y en general se cree que las aves criadas en alta densidad en cama tienen un mayor riesgo de acumular grandes cantidades de nematodos. Esta especie generalmente solo causa una patología leve que no afecta significativamente el rendimiento de las aves. Sin embargo, H. gallinarum es reconocido como un parásito económicamente importante para la industria avícola debido a que sus huevos sirven como vector para el parásito protozoario Histomonas meleagridis, que es la causa de la histomoniasis en las aves. El diagnóstico del nematodo generalmente se basa en el recuento de huevos fecales, que puede establecer diagnósticos falsos negativos. Las herramientas moleculares están disponibles para estudiar el nematodo y diagnosticar las parvadas infectadas. El tratamiento y la prevención de la infección por H. gallinarum se dificultan debido a la baja eficacia de los antihelmínticos para erradicar H. gallinarum de las aves infectadas y de los desinfectantes para destruir los huevos de H. gallinarum en granjas infestadas.

An In Vitro Assay of Disinfectants on the Viability of Heterakis gallinarum Eggs.

Nematodes are widespread and common in poultry. Disinfectants are used to reduce infection rates in poultry houses, but there is little documentation of their effectiveness. An in vitro assay was developed to test the efficacy of products to damage Heterakis gallinarum eggs, and nine disinfectants and chemicals commonly used in the poultry industry were tested. Embryonated eggs of H. gallinarum were pipetted into wells of plastic cell culture plates (250-300 eggs/well in water). Measured amounts of test articles were added to the suspensions for 2, 4, 6, or 24 hr. After exposure, eggs were washed with water and treated with trypan blue (1 ml of 0.4% solution, added to each well) at room temperature for 2 min. Eggshell integrity was determined microscopically by counting the number of eggs that were clear (intact) or that contained blue dye (compromised). As a test of embryo viability, five eggs per well from treatments containing compromised eggs were transferred to a Petri dish and hatched manually, using forceps to open the eggshell. Released larvae were then observed for signs of controlled movement. In a test of Clorox bleach (NaOCl), Green Klean, Decon7, Kem San, PLT, Virkon S, NaCl, dry limestone (CaCO3), and diesel fuel, only NaOCl (bleach) and Green Klean damaged the eggshell, and only 20,625 ppm of NaOCl rendered the larvae nonviable.

Nota de Investigación- Un ensayo in vitro de desinfectantes sobre la viabilidad de los huevos de Heterakis gallinarum. Los nematodos son comunes y están generalizados en la avicultura. Los desinfectantes se usan para reducir las tasas de infección en las granjas avícolas, pero existe poca documentación de su efectividad. Se desarrolló un ensayo in vitro para probar la eficacia de los productos para afectar los huevos de Heterakis gallinarum, y se probaron nueve desinfectantes y productos químicos comúnmente utilizados en la industria avícola. Los huevos embrionados de H. gallinarum se pipetearon en pozos de placas plásticas de cultivo celular (250­300 huevos/pozo en agua). Se agregaron cantidades medidas de los compuestos a probar a las suspensiones durante dos, cuatro, seis o 24 horas. Después de la exposición, los huevos se lavaron con agua y se trataron con azul tripán (1 ml de solución al 0.4%, se agregaron a cada pozo) a temperatura ambiente durante dos minutos. La integridad de la cáscara del huevo se determinó microscópicamente al contar el número de huevos que estaban claros (intactos) o los que contenían colorante azul (afectado). Como prueba de la viabilidad de los embriones, se transfirieron cinco huevos por pozo de los tratamientos que contenían huevos afectados a una placa de Petri y se eclosionaron manualmente utilizando fórceps para abrir la cáscara del huevo. Las larvas liberadas fueron observadas para detectar signos de movimiento controlado. En una prueba con blanqueador Clorox (NaOCl), Green Klean, Decon7, Kem San, PLT, Virkon S, NaCl, caliza seca (CaCO3) y combustible diesel, solo NaOCl (lejía) y Green Klean dañaron la cáscara del huevo, y solo 625 ppm de NaOCl provocaron que las larvas no fueran viables.

Relationship Between Broiler Body Weights, Eimeria maxima Gross Lesion Scores, and Microscores in Three Anticoccidial Sensitivity Tests.

Anticoccidial sensitivity tests (ASTs) serve to determine the efficacy of anticoccidial drugs against Eimeria field isolates in a controlled laboratory setting. The most commonly measured parameters are body weight gain, feed conversion ratio, gross intestinal lesion scores, and mortality. Due to the difficulty in reliably scoring gross lesion scores of Eimeria maxima , microscopic analysis of intestinal scrapings (microscores) can be used in the field to indicate the presence of this particular Eimeria. The goal of this study was to determine the relationship between E. maxima microscores and broiler body weights and gross E. maxima lesion scores in three ASTs. Day-old broiler chicks were raised for 12 days on a standard corn-soy diet. On Day 12, chicks were placed in Petersime batteries and treatment diets were provided. There were six birds per pen, four pens per treatment, and 12 treatments, for a total of 288 chicks per AST. The treatments were as follows: 1) nonmedicated, noninfected; 2) nonmedicated, infected; 3) lasalocid, infected; 4) salinomycin, infected; 5) diclazuril, infected; 6) monensin, infected; 7) decoquinate, infected; 8) narasin + nicarbazin, infected; 9) narasin, infected; 10) nicarbazin, infected; 11) robenidine, infected; and 12) zoalene, infected. On Day 14, chicks were challenged with an Eimeria field isolate by oral gavage. On Day 20, broilers were weighed, and gross lesion scores and microscores were classified from 0 to 4 depending on the severity of the gross lesion scores and E. maxima microscores. Data from three trials using different field isolates were statistically analyzed using a logarithmic regression model. There was no relationship (P = 0.1224) between microscores and body weight gain. There was a positive relationship between microscores and gross lesion scores (P = 0.004). However, there was also an interaction between isolate and treatment (P < 0.0001). Lastly, the interaction between isolate and gross lesion scores (P = 0.0041) demonstrates that the significance of the relationship between microscores and gross lesion scores may be dependent on pathogenicity of the challenge Eimeria or the amount of E. maxima in the inoculum.

Development of a Dry Medium for Isolation of Histomonas meleagridis in the Field.

Blackhead disease is caused by Histomonas meleagridis, an anaerobic protozoan parasite, and results in mortality rates of up to 100% in turkeys and 30% in chickens. Outbreaks of blackhead disease are unpredictable, and the harvesting of H. meleagridis strains from the field would be a great resource for researchers to study its epidemiology. Therefore, the objective of this study was to develop a dry medium that would allow storage at ambient temperatures until needed. Fifty milliliters of horse serum was dried and then mixed with dry medium M199 with Hanks balanced salts (10.6 g), sodium bicarbonate (0.35 g), and rice powder (0.8 g). To test the ability of reconstituted medium to support growth of H. meleagridis, groups of 10 flasks containing 0.2 g of dry medium were stored for 24 hr at 25 and 60 C before testing. Other groups of flasks containing dry medium were stored at 25, 37, and 42 C for 1, 3, or 6 mo. At each test period, the flasks were reconstituted with 10 ml of water, inoculated with 100 000 H. meleagridis cells, and incubated at 40 C for 48 hr. Fresh liquid medium was used as a control. There were no differences in cell counts in medium stored at 25 or 60 C for 24 hr. After 1 mo, cell counts in reconstituted medium were about half that of fresh liquid medium after 48 hr of incubation. But after 3 and 6 mo, the cell counts were not significantly different in all groups (P < 0.05) after 72 hr of incubation. These results show that dried Dwyer medium can be stored at ambient temperatures for extended times and would be an effective tool for obtaining isolates of H. meleagridis from the field.

Pathologic and molecular characterization of histomoniasis in peafowl ( Pavo cristatus).

Histomonas meleagridis is a flagellate protozoan organism that can cause severe necrotizing typhlitis and hepatitis in gallinaceous birds. Peafowl ( Pavo spp.) have been shown to be susceptible to histomoniasis in experimental settings, but there are few reports of natural histomoniasis in this species. A retrospective study of the archived cases at 2 veterinary diagnostic laboratories in the United States yielded 5 cases of peafowl with gross and histologic findings characteristic of histomoniasis. Lesions included bilateral, transmural fibrinonecrotic typhlitis and multifocal necrotizing hepatitis with associated trophozoites morphologically consistent with H. meleagridis. There was no evidence of Heterakis gallinarum infestation in the studied cases. DNA was extracted from formalin-fixed, paraffin-embedded liver and ceca from all 5 cases and was analyzed using multiple sets of primers with subsequent sequencing and genotyping. Four samples were positive for H. meleagridis, and 1 sample was positive for both H. meleagridis and Tetratrichomonas gallinarum. These results confirm that peafowl develop clinical disease similar to that described previously in other gallinaceous birds infected by H. meleagridis. The role of T. gallinarum remains unknown and further research is necessary to elucidate its role, if any, in the pathogenesis of the observed lesions.

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets.

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240-360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.

Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens.

The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.

Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species.

Birds display remarkable diversity in the distribution and morphology of scales and feathers on their feet, yet the genetic and developmental mechanisms governing this diversity remain unknown. Domestic pigeons have striking variation in foot feathering within a single species, providing a tractable model to investigate the molecular basis of skin appendage differences. We found that feathered feet in pigeons result from a partial transformation from hindlimb to forelimb identity mediated by cis-regulatory changes in the genes encoding the hindlimb-specific transcription factor Pitx1 and forelimb-specific transcription factor Tbx5. We also found that ectopic expression of Tbx5 is associated with foot feathers in chickens, suggesting similar molecular pathways underlie phenotypic convergence between these two species. These results show how changes in expression of regional patterning genes can generate localized changes in organ fate and morphology, and provide viable molecular mechanisms for diversity in hindlimb scale and feather distribution.

Generation of Avian Induced Pluripotent Stem Cells.

Avian species are among the most diverse vertebrates on our planet and significantly contribute to the balance of the ecology. They are also important food source and serve as a central animal model to decipher developmental biology and disease principles. Derivation of induced pluripotent stem cells (iPSCs) from avian species would enable conservation of genetic diversity as well as offer a valuable cell source that facilitates the use of avian models in many areas of basic and applied research. In this chapter, we describe methods used to successfully reprogram quail fibroblasts into iPSCs by using human transcription factors and the techniques critical to the characterization of their pluripotency.

Amino acid sequence of Japanese quail (Coturnix japonica) and northern bobwhite (Colinus virginianus) myoglobin.

Myoglobin has an important physiological role in vertebrates, and as the primary sarcoplasmic pigment in meat, influences quality perception and consumer acceptability. In this study, the amino acid sequences of Japanese quail and northern bobwhite myoglobin were deduced by cDNA cloning of the coding sequence from mRNA. Japanese quail myoglobin was isolated from quail cardiac muscles, purified using ammonium sulphate precipitation and gel-filtration, and subjected to multiple enzymatic digestions. Mass spectrometry corroborated the deduced protein amino acid sequence at the protein level. Sequence analysis revealed both species' myoglobin structures consist of 153 amino acids, differing at only three positions. When compared with chicken myoglobin, Japanese quail showed 98% sequence identity, and northern bobwhite 97% sequence identity. The myoglobin in both quail species contained eight histidine residues instead of the nine present in chicken and turkey.

Nonviral minicircle generation of induced pluripotent stem cells compatible with production of chimeric chickens.

Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such as meat and egg production or even disease resistance. However, these cell types have significant limitations because they are hard to culture long term while maintaining developmental plasticity. Induced pluripotent stem cells (iPSCs) are a novel class of stem cells that have proven to be robust, leading to the successful development of transgenic mice, rats, quail, and pigs and may potentially overcome the limitations of previous pluripotent stem cell systems in chickens. In this study we generated chicken (c) iPSCs from fibroblast cells for the first time using a nonviral minicircle reprogramming approach. ciPSCs demonstrated stem cell morphology and expressed key stem cell markers, including alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1. These cells were capable of rapid growth and expressed high levels of telomerase. Late-passage ciPSCs transplanted into stage X embryos were successfully incorporated into tissues of all three germ layers, and the gonads demonstrated significant cellular plasticity. These cells provide an exciting new tool to create transgenic chickens with broad implications for agricultural and transgenic animal fields at large.

Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism.

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. Here, we show that mutation or inhibition of Valosin-Containing Protein (VCP), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, we find that VCP inhibition causes an altered splicing pattern of the large pruning gene molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). Our data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, our results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism.

Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate.

Germ cells (GCs) are critically important as the vehicle that passes genetic information from one generation to the next. Correct development of these cells is essential and perturbation in their development often leads to reproductive failure and disease. Despite the importance of GCs, little is known about the mechanisms underlying the acquisition and maintenance of the GC character. Using a reprogramming strategy, we demonstrate that overexpression of ectopic transcription factors in embryonic fibroblasts can lead to the generation of chicken induced primordial germ cells (ciPGCs). These ciPGCs express pluripotent markers POU5F1, SSEA1, and the GC defining proteins, CVH and DAZL, closely resembling in vivo sourced PGCs instead of embryonic stem cells. Moreover, CXCR4 expressing ciPGCs were capable of migrating to the embryonic gonad after injection into the vasculature of stage 15 embryos, indicating the acquisition of a GC fate in these cells. Direct availability of ciPGCs in vitro would facilitate the study of GC development as well as provide a potential strategy for the conservation of important genetics of agricultural and endangered birds using somatic cells.