Perspectives on chick embryo models in developmental and reproductive toxicity screening.

Teratology, the study of congenital anomalies and their causative factors intersects with developmental and reproductive toxicology, employing innovative methodologies. Evaluating the potential impacts of teratogens on fetal development and assessing human risk is an essential prerequisite in preclinical research. The chicken embryo model has emerged as a powerful tool for understanding human embryonic development due to its remarkable resemblance to humans. This model offers a unique platform for investigating the effects of substances on developing embryos, employing techniques such as ex ovo and in ovo assays, chorioallantoic membrane assays, and embryonic culture techniques. The advantages of chicken embryonic models include their accessibility, cost-effectiveness, and biological relevance to vertebrate development, enabling efficient screening of developmental toxicity. However, these models have limitations, such as the absence of a placenta and maternal metabolism, impacting the study of nutrient exchange and hormone regulation. Despite these limitations, understanding and mitigating the challenges posed by the absence of a placenta and maternal metabolism are critical for maximizing the utility of the chick embryo model in developmental toxicity testing. Indeed, the insights gained from utilizing these assays and their constraints can significantly contribute to our understanding of the developmental impacts of various agents. This review underscores the utilization of chicken embryonic models in developmental toxicity testing, highlighting their advantages and disadvantages by addressing the challenges posed by their physiological differences from mammalian systems.

Changes in Nucleolin Expression during Malignant Transformation Leading to Ovarian High-Grade Serous Carcinoma.

OBJECTIVE: Ovarian high-grade serous carcinoma (HGSC) is a fatal malignancy of women. Alterations in the expression of nuclear proteins are early steps in malignant transformation; nucleolin is one such protein. Changes in nucleolin expression and circulatory levels during ovarian HGSC development are unknown. The study goal was to determine if tissue and circulatory levels of nucleolin change in response to malignant transformation leading to ovarian HGSC. METHODS: Sera, ovaries, and BRCA+ fimbria from healthy subjects, and sera and tumor tissues from patients (n = 10 each), and healthy hens and hens with HGSC were examined in exploratory and prospective studies for nucleolin expression by immunohistochemistry, immunoblotting, gene expression, and immunoassay, and analyzed by analysis of variance (ANOVA). RESULTS: Compared with normal, nucleolin expression was higher in patients and hens with ovarian HGSC and in women with a risk of HGSC (P < 0.05). Compared with normal (1400 + 105 pg/mL, n = 8), serum nucleolin levels were 1.5 and 1.7-fold higher in patients with early- (n = 5) and late-stage (n = 5) HGSC, respectively. Additionally, serum nucleolin levels increased significantly (P < 0.05) prior to the formation of detectable masses. CONCLUSION: This pilot study concluded that tissue and serum levels of nucleolin increase in association with malignant changes in ovaries and fimbriae leading to ovarian HGSC.

Development of a novel trivalent invasive non-typhoidal Salmonella outer membrane vesicles based vaccine against salmonellosis and fowl typhoid in chickens.

Poultry animals act as natural reservoirs of invasive non-typhoidal Salmonella [iNTS] serovars and consumption of iNTS contaminated poultry meat and eggs is one of the major sources of iNTS infection in developed and developing countries. Irrational use of antibiotics in the poultry industry gives rise to the global emergence of multi drug resistant iNTS strains. Among different strategies to control iNTS infection in poultry farms, vaccination is now being widely used. There are several licensed vaccines available in the market for poultry animals to ameliorate iNTS infection but none of them have broad spectrum protective efficacy. In this study we have formulated a single novel trivalent iNTS outer membrane vesicles [OMVs] based immunogen which can confer long term broad spectrum protection against most prevalent iNTS serovars. We have isolated OMVs from Salmonella Typhimurium [ST], Salmonella Enteritidis [SE], and Salmonella Gallinarum [SG] and formulated the trivalent immunogen by mixing OMVs in a 1:1:1 ratio. One day old chicks were immunized thrice via oral route at two week intervals. Vaccination significantly induced serovar specific antibodies detected up to 180 days post immunization. Post challenge with both homologous and heterologous [S. Infantis] serovars, immunized birds showed reduced level of fecal shedding and organ invasion. A long term efficacy study also showed reduced levels of tissue invasion up to one year post immunization. These results demonstrate that our novel formulation of immunogen could be a broad spectrum potential vaccine for both layer and broiler breeds against iNTS mediated salmonellosis and fowl typhoid.

Ultrasonography in soft-tissue foreign-body detection: a phantom study.

PURPOSE: Foreign body implantation into the soft tissues, either in the early period or late period, is a common cause of emergency department admissions. Direct X-rays are preferred in the diagnosis of soft-tissue foreign-bodies. Herein, we aimed to analyse the detection rates of foreign bodies of various sizes placed in phantoms implanted into soft tissue by ultrasonography. MATERIAL AND METHODS: A total of 740 pieces of chicken fillet were prepared as phantoms. No objects were implanted into 100 phantoms. We inserted glass, porcelain, plastic, wood, pencil tip, chicken bone, iron, walnut shell, and fishbone with a length of < 1 cm, 1-3 cm, and 3-5 cm into 20 pieces of chicken phantom in each size of foreign body (FB). In addition, 1-3 cm long peanut shell, < 1 cm rose spikes, < 1 cm cactus thorns, < 1 cm pellets, and < 1 cm staples were inserted into 20 pieces of chicken for each object. Each of the chicken pieces was placed inside a latex glove and examined by ultrasonography. RESULTS: The sensitivity of ultrasonography in the detection of the cactus thorn was 5%, whereas it ranged between 82.5 and 100% for other objects. For glass, plastic, wood, iron, and fishbone, we found that when the size exceeded 1 cm, the sensitivity increased. CONCLUSIONS: In the evaluation of soft tissue FBs, as the size of the FBs increases, the diagnostic value of ultrasonography increases.

A novel chicken model of fatty liver disease induced by high cholesterol and low choline diets.

Fatty liver diseases, common metabolic diseases in chickens, can lead to a decrease in egg production and sudden death of chickens. To solve problems caused by the diseases, reliable chicken models of fatty liver disease are required. To generate chicken models of fatty liver, 7-week-old ISA female chickens were fed with a control diet (17% protein, 5.3% fat, and 1,300 mg/kg choline), a low protein and high fat diet (LPHF, 13% protein, 9.1% fat, and 1,300 mg/kg choline), a high cholesterol with low choline diet (CLC, 17% protein, 7.6% fat with additional 2% cholesterol, and 800 mg/kg choline), a low protein, high fat, high cholesterol, and low choline diet (LPHFCLC, 13% protein, 12.6% fat with additional 2% cholesterol, and 800 mg/kg choline) for 4 wk. Our data showed that the CLC and LPHFCLC diets induced hyperlipidemia. Histological examination and the content of hepatic lipids indicated that the CLC and LPHFCLC diets induced hepatic steatosis. Plasma dipeptidyl peptidase 4, a biomarker of fatty liver diseases in laying hens, increased in chickens fed with the CLC or LPHFCLC diets. Hepatic ballooning and immune infiltration were observed in these livers accompanied by elevated interleukin 1 beta and lipopolysaccharide induced tumor necrosis factor mRNAs suggesting that the CLC and LPHFCLC diets also caused steatohepatitis in these livers. These diets also induced hepatic steatosis in Plymouth Rock chickens. Thus, the CLC and LPHFCLC diets can be used to generate models for fatty liver diseases in different strains of chickens. In ISA chickens fed with the CLC diet, peroxisome proliferator-activated receptor γ, sterol regulatory element binding transcription factor 1, and fatty acid synthase mRNAs increased in the livers, suggesting that lipogenesis was enhanced by the CLC treatment. Our data show that treatment with CLC or LPHFCLC for 4 wk induces fatty liver disease in chickens. These diets can be utilized to rapidly generate chicken models for fatty liver research.

Evaluation of Baloxavir Marboxil and Peramivir for the Treatment of High Pathogenicity Avian Influenza in Chickens.

Control measures in the case of high pathogenicity avian influenza (HPAI) outbreaks in poultry include culling, surveillance, and biosecurity; wild birds in captivity may also be culled, although some rare bird species should be rescued for conservation. In this study, two anti-influenza drugs, baloxavir marboxil (BXM) and peramivir (PR), used in humans, were examined in treating HPAI in birds, using chickens as a model. Chickens were infected with H5N6 HPAI virus and were treated immediately or 24 h from challenge with 20 mg/kg BXM or PR twice a day for five days. As per our findings, BXM significantly reduced virus replication in organs and provided full protection to chickens compared with that induced by PR. In the 24-h-delayed treatment, neither drug completely inhibited virus replication nor ensured the survival of infected chickens. A single administration of 2.5 mg/kg of BXM was determined as the minimum dose required to fully protect chickens from HPAI virus; the concentration of baloxavir acid, the active form of BXM, in chicken blood at this dose was sufficient for a 48 h antiviral effect post-administration. Thus, these data can be a starting point for the use of BXM and PR in treating captive wild birds infected with HPAI virus.

Suppression of GABAergic transmission in the spinal dorsal horn induces pain-related behaviour in a chicken model of spina bifida.

Spina bifida aperta (SBA), one of the most common congenital malformations, causes various neurological disorders. Pain is a common complaint of patients with SBA. However, little is known about the neuropathology of SBA-related pain. Because loss of g-aminobutyric acid GABAergic neurons in the spinal cord dorsal horn is associated with pain, we hypothesised the existence of crosstalk between SBA-related pain and alterations in GABAergic transmission in the spinal cord. Therefore, we investigated the kinetics of GABAergic transmission in the spinal cord dorsal horn in a chicken model of SBA. Neonatal chicks with SBA exhibited various pain-like behaviours, such as an increased number of vocalisations with elevated intensity (loudness) and frequency (pitch), reduced mobility, difficulty with locomotion, and escape reactions. Furthermore, the chicks with SBA did not respond to standard toe-pinching, indicating disruption of the spinal cord sensorimotor networks. These behavioural observations were concomitant with loss of GABAergic transmission in the spinal cord dorsal horn. We also found apoptosis of GABAergic neurons in the superficial dorsal horn in the early neonatal period, although cellular abnormalisation and propagation of neuro-degenerative signals were evident at middle to advanced gestational stages. In conclusion, ablation of GABAergic neurons induced alterations in spinal cord neuronal networks, providing novel insights into the pathophysiology of SBA-related pain-like complications.

Characterization of ascites-derived aldehyde dehydrogenase-positive ovarian cancer stem cells isolated from Leghorn chickens.

Leghorn chickens are used as a preclinical model of ovarian cancer as they develop epithelial ovarian adenocarcinoma spontaneously at a very high frequency. Ovarian cancer is the most lethal disease among all gynecological malignancies in women. A small proportion of ovarian cancer stem cells are responsible for drug resistance and relapse of ovarian cancer. The objectives of this study are to isolate ovarian cancer stem cells from ascites of Leghorn chickens that spontaneously developed ovarian cancer and to determine their invasiveness, spheroid formation in three-dimensional culture devoid of extracellular matrix over several months. Ovarian cancer cells obtained from ascites were subjected to ALDEFLOUR assay that measures aldehyde dehydrogenase (ALDH) activity to separate ALDH1+ and ALDH1- cells by fluorescence-activated cell sorting. The cells were cultured using serum-free media for up to 6 mo in ultra-low attachment plates. Invasiveness of ALDH1+ and ALDH1- cells was determined by Matrigel invasion assay. Cellular uptake of acetylated low-density lipoprotein was evaluated. A small proportion (<4.75%) of ovarian cancer cells isolated from ascites were found to be ALDH1+ cells. ALDH1+ cells formed a greater number of spheroids and were also highly invasive in extracellular matrix compared to ALDH1- cells. Several spheroids developed 0.1- to 1-mm-long capillary-like tubules connecting other spheroids, thus forming a complex network that underwent remodeling over several months. Cells in the spheroids incorporated acetylated low-density lipoprotein suggestive of scavenger receptor activity. In summary, ALDH1+ ovarian cancer stem cells isolated from ascites of chickens appear to be invasive and form spheroids with complex networks of tubules reminiscent of vascular mimicry. Understanding the structure and function of spheroids and tubular network would provide valuable insight into the biology of ovarian cancer and improve poultry health.

Establishment of new assessment method for the synergistic effect between umami peptides and monosodium glutamate using electronic tongue.

In order to investigate the synergistic effect between umami peptides and monosodium glutamate (MSG), a new assessment method was established using electronic tongue. After 36 kinds of umami peptides synthesized by peptide solid phase synthesis, their taste characteristics were preliminarily explored by electronic tongue technology, and then the umami intensity was ranked before and after addition of MSG, using a concentration of 0.35% of MSG as control. In addition, the sensory evaluation was utilized to verify the results of the electronic tongue. Finally, the umami intensity and the synergistic effect of umami peptides and MSG were also investigated by the aroma chicken model (ACM). Results showed that peptide Lys-Gly-Ser-Leu-Ala-Asp-Glu-Glu (KE-8) and Arg-Leu (RL) have the strongest umami taste, Asp-Asp-Asp (DDD) and Glu-Ser-Val (ESV) have the strongest synergistic effect with MSG, which could increase the umami intensity. The ESV and Glu-Asp-Asp (EDD) showed the strongest synergistic effect with ACM. The evaluation method could provide the objective data for further investigating for the synergistic theory.

Ultrasound Evaluation of Soft-Tissue Foreign Bodies by US Army Medics.

OBJECTIVE: The study's primary objective was to determine army medics' accuracy performing bedside ultrasound (US) to detect radiolucent foreign bodies (FBs) in a soft-tissue hand model. Secondary objectives included the assessment of US stand-off pad effects on soft-tissue FB detection rates and assess established FB detectable lower limit size of 2 mm. METHODS: Prospective, single blinded, observational study of US-naïve Army medics' abilities utilizing bedside US to detect wooden FBs in a chicken thigh model with or without an US stand-off pad. After a 2 h training period, medics' abilities to detect 1-3 mm FB utilizing a SonoSite® M-Turbo US and 13-6 MHz linear probe were assessed. RESULTS: After a 2 h training period, 28 medics had a sensitivity and specificity of 73% and 78% detecting 1-3 mm FBs utilizing standard US equipment. The medics' sensitivity and specificity were both 78% in detecting radiolucent FBs 2 mm and larger without a stand-off pad. The sensitivity and specificity decreased to 48%, 62%, and 67% when utilizing a stand-off pad to detect 1, 2, and 3 mm soft-tissue FBs. Sub 2 mm detection rates decreased from 82% for 2 mm FB to 64% for 1 mm FBs without utilizing a stand-off pad. CONCLUSION: Army medics with minimal US experience successfully identified FBs embedded in hand models with accuracies similar to radiologists and emergency medicine physicians. However, radiolucent FB detection sensitivity and specificity decreased in US-naïve Army medics utilizing stand-off pads. In addition, this study reconfirmed the lower limit of FB detection rates at 2 mm. These results support Army medics' utilization of US to evaluate for superficial radiolucent FBs of the hand.

Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors.

Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.

Early neonatal loss of inhibitory synaptic input to the spinal motor neurons confers spina bifida-like leg dysfunction in a chicken model.

Spina bifida aperta (SBA), one of the most common congenital malformations, causes lifelong neurological complications, particularly in terms of motor dysfunction. Fetuses with SBA exhibit voluntary leg movements in utero and during early neonatal life, but these disappear within the first few weeks after birth. However, the pathophysiological sequence underlying such motor dysfunction remains unclear. Additionally, because important insights have yet to be obtained from human cases, an appropriate animal model is essential. Here, we investigated the neuropathological mechanisms of progression of SBA-like motor dysfunctions in a neural tube surgery-induced chicken model of SBA at different pathogenesis points ranging from embryonic to posthatch ages. We found that chicks with SBA-like features lose voluntary leg movements and subsequently exhibit lower-limb paralysis within the first 2 weeks after hatching, coinciding with the synaptic change-induced disruption of spinal motor networks at the site of the SBA lesion in the lumbosacral region. Such synaptic changes reduced the ratio of inhibitory-to-excitatory inputs to motor neurons and were associated with a drastic loss of γ-aminobutyric acid (GABA)ergic inputs and upregulation of the cholinergic activities of motor neurons. Furthermore, most of the neurons in ventral horns, which appeared to be suffering from excitotoxicity during the early postnatal days, underwent apoptosis. However, the triggers of cellular abnormalization and neurodegenerative signaling were evident in the middle- to late-gestational stages, probably attributable to the amniotic fluid-induced in ovo milieu. In conclusion, we found that early neonatal loss of neurons in the ventral horn of exposed spinal cord affords novel insights into the pathophysiology of SBA-like leg dysfunction.

Turkey model for flexor tendon research: in vitro comparison of human, canine, turkey, and chicken tendons.

BACKGROUND: Flexor tendon injuries are one of the most common hand injuries and remain clinically challenging for functional restoration. Canine and chicken have been the most commonly used animal models for flexor tendon-related research but possess several disadvantages. The purpose of this study was to explore a potential turkey model for flexor tendon research. METHODS: The third digit from human cadaveric hands, canine forepaws, turkey foot, and chicken foot were used for this study. Six digits in each of four species were studied in detail, comparing anatomy of the flexor apparatus, joint range of motion tendon excursion, tendon cross-sectional area, work of flexion, gliding resistance at the level of the A2 pulley, modulus of elasticity, suture retention strength, and histology across species. RESULTS: Anatomically, the third digit in the four species displayed structural similarities; however, the tendon cross-sectional area of the turkey and human were similar and larger than canine and chicken. Furthermore, the turkey digit resembles the human's finger with the lack of webbing between digits, similar vascularization, tendon excursion, work of flexion, gliding resistance, mechanical properties, and suture holding strength. More importantly, human and turkey tendons were most similar in histological appearance. CONCLUSIONS: Turkey flexor tendons have many properties that are comparable to human flexor tendons which would provide a clinically relevant, economical, nonhuman companion large animal model for flexor tendon research.

Regenerative capacity of the enteric nervous system: is immaturity defining the point of no return?

BACKGROUND: Intestinal obstruction in newborns is associated with intestinal motility disorders after surgery. Alterations in the enteric nervous system (ENS) might cause abnormal peristalsis, which may then result in intestinal motility disorders. We aimed to quantify alterations in the myenteric plexus after a ligation and to test if these alterations were reversible. METHODS: Small intestines of chicken embryos were ligated in ovo at embryonic day (ED) 11 for either 4 d (ED 11-15) or 8 d (ED 11-19). Both treated groups and control group were sacrificed and intestinal segments examined by means of both light and electron microscopy. RESULTS: The number of proximal myenteric ganglia increased (ED 19, 30.7 ± 3.16 versus 23.1 ± 2.03; P < 0.001) in the 8-d ligature group but had values similar to the control group in the 4-d ligature group. The size distribution was skewed toward small ganglia in the 8-d ligature group (ED 19, 83.71 ± 11.60% versus 3.88 ± 4.74% in the control group; P < 0.001) but comparable with the control group in the 4-d ligature group. Subcellular alterations in the 4-d ligature group were reversible. CONCLUSIONS: The pathologic alterations in the ENS were fully reversible in the 4-d ligature group. This reversibility might be linked to the degree of immaturity of the ENS.

Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens.

To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections.

Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine.

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts.