Chicken intestinal organoids: a novel method to measure the mode of action of feed additives.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.

Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing.

Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.

miR-214-PTEN pathway is a potential mechanism for stress-induced immunosuppression affecting chicken immune response to avian influenza virus vaccine.

Stress-induced immunosuppression (SIIS) is one of common problems in the intensive poultry industry, affecting the effect of vaccine immunization and leading to high incidences of diseases. In this study, the expression characteristics and regulatory mechanisms of miR-214 in the processes of SIIS and its influence on the immune response to avian influenza virus (AIV) vaccine in chicken were explored. The qRT-PCR results showed that serum circulating miR-214 was significantly differentially expressed (especially on 2, 5, and 28 days post immunization (dpi)) in the processes, so had the potential as a molecular marker. MiR-214 expressions from multiple tissues were closely associated with the changes in circulating miR-214 expression levels. MiR-214-PTEN regulatory network was a potential key regulatory mechanism for the heart, bursa of Fabricius, and glandular stomach to participate in the process of SIIS affecting AIV immune response. This study can provide references for further understanding of stress affecting immune response.

Comprehensive analysis of differentially expressed mRNA profiles in chicken jejunum and cecum following Eimeria maxima infection.

Coccidiosis, a protozoan disease that substantially impacts poultry production, is characterized by an intracellular parasite. The study utilized 48 one-day-old Horro chickens, randomly divided into the infected (I) and control (C) groups. The challenge group of chickens were administered Eimeria maxima oocysts via oral gavage at 21-days-old, and each chicken received 2 mL containing 7×104 sporulated oocysts. The total RNAs of chicken jejunum and cecum tissues were isolated from three samples, each from I and C groups. Our study aimed to understand the host immune-parasite interactions and compare immune response mRNA profiles in chicken jejunum and cecum tissues at 4 and 7 days postinfection with Eimeria maxima. The results showed that 823 up- and 737 down-regulated differentially expressed mRNAs (DEmRNAs) in jejunum at 4 d infection and control (J4I vs. J4C), and 710 up- and 368 down-regulated DEmRNAs in jejunum at 7 days infection and control (J7I vs. J7C) were identified. In addition, DEmRNAs in cecum tissue, 1424 up- and 1930 down-regulated genes in cecum at 4 days infection and control (C4I vs. C4C), and 77 up- and 191 down-regulated genes in cecum at 7 days infection and control (C7I vs. C7C) were detected. The crucial DEmRNAs, including SLC7A5, IL1R2, GLDC, ITGB6, ADAMTS4, IL1RAP, TNFRSF11B, IMPG2, WNT9A, and FOXF1, played pivotal roles in the immune response during Eimeria maxima infection of chicken jejunum. In addition, the potential detection of FSTL3, RBP7, CCL20, DPP4, PRKG2, TFPI2, and CDKN1A in the cecum during the host immune response against Eimeria maxima infection is particularly noteworthy. Furthermore, our functional enrichment analysis revealed the primary involvement of DEmRNAs in small molecule metabolic process, immune response function, inflammatory response, and toll-like receptor 10 signaling pathway in the jejunum at 4 and 7 days postinfection. Similarly, in the cecum, DEmRNAs at 4 and 7 days postinfection were enriched in processes related to oxidative stress response and immune responses. Our findings provide new insights and contribute significantly to the field of poultry production and parasitology.

Mitigation of False Layer Syndrome Through Maternal Antibodies Against Infectious Bronchitis Virus.

The relationship between passive immunity and the development of false layer syndrome (FLS) and its associated lesions was investigated in this study by comparing the long-term reproductive effects of an infectious bronchitis virus (IBV) DMV/1639 wild-type strain and the GA08 vaccine in birds with and without maternal antibodies. There was a clear protective effect provided by maternal antibodies against both the early vaccination and challenge. It was also observed that vaccination at an early age, in the absence of maternal antibodies, can induce reproductive issues, such as reduced egg production and FLS-associated lesions (e.g., cystic oviduct and egg yolk coelomitis). This might indicate that maternal antibodies and the timing of IBV infection are more important in the generation of FLS than the IBV strain type.

Mitigación del síndrome de la falsa ponedora mediante anticuerpos maternos contra el virus de la bronquitis infecciosa. En este estudio se investigó la relación entre la inmunidad pasiva y el desarrollo del síndrome de la falsa ponedora (FLS) y sus lesiones asociadas comparando los efectos reproductivos a largo plazo de una cepa de tipo silvestre DMV/1639 del virus de la bronquitis infecciosa (IBV) y la cepa vacunal GA08, en aves con y sin anticuerpos maternos. Hubo un claro efecto protector proporcionado por los anticuerpos maternos tanto contra la vacunación temprana como contra el desafío. También se observó que la vacunación a una edad temprana, en ausencia de anticuerpos maternos, puede inducir problemas reproductivos, como una reducción de la producción de huevo y lesiones asociadas al síndrome de la falsa ponedora (p. ej., oviducto quístico y celomitis de yema de huevo). Esto podría indicar que los anticuerpos maternos y el momento de la infección por el virus de la bronquitis infecciosa son más importantes en la generación del síndrome de la falsa ponedora que el tipo de cepa del virus de la bronquitis infecciosa.

Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental Clostridial dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 µg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation.

The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

Host Immune Response Modulation in Avian Coronavirus Infection: Tracheal Transcriptome Profiling In Vitro and In Vivo.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

Immunogenicity and protective efficacy of a multivalent herpesvirus vectored vaccine against H9N2 low pathogenic avian influenza in chicken.

The application of recombinant herpesvirus of turkey, expressing the H9 hemagglutinin gene from low pathogenic avian influenza virus (LPAIV) H9N2 and the avian orthoavulavirus-1 (AOAV-1) (commonly known as Newcastle Disease virus (NDV)) fusion protein (F) as an rHVT-H9-F vaccine, is an alternative to currently used classical vaccines. This study investigated H9- and ND-specific humoral and mucosal responses, H9-specific cell-mediated immunity, and protection conferred by the rHVT-H9-F vaccine in specific pathogen-free (SPF) chickens. Vaccination elicited systemic NDV F- and AIV H9-specific antibody response but also local antibodies in eye wash fluid and oropharyngeal swabs. The ex vivo H9-specific stimulation of splenic and pulmonary T cells in the vaccinated group demonstrated the ability of vaccination to induce systemic and local cellular responses. The clinical protection against a challenge using a LPAIV H9N2 strain of the G1 lineage isolated in Morocco in 2016 was associated with a shorter duration of shedding along with reduced viral genome load in the upper respiratory tract and reduced cloacal shedding compared to unvaccinated controls.

Loop PDE of viral capsid protein is involved in immune escape of the emerging novel variant infectious bursal disease virus.

Infectious bursa disease (IBD) is an acute, highly contactable, lethal, immunosuppressive infectious disease caused by the Infectious bursa disease virus (IBDV). Currently, the emerged novel variant IBDV (nVarIBDV) and the sustainedly prevalent very virulent IBDV (vvIBDV) are the two most prevalent strains of IBDV in China. The antigenic properties of the two prevalent strains differed significantly, which led to the escape of nVarIBDV from the immune protection provided by the existing vvIBDV vaccine. However, the molecular basis of the nVarIBDV immune escape remains unclear. In this study, we demonstrated, for the first time, that residues 252, 254, and 256 in the PDE of VP2 are involved in the immune escape of the emerging nVarIBDV. Firstly, the IFA-mediated antigen-antibody affinity assay showed that PBC and PDE of VP2 could affect the affinity of vvIBDV antiserum to VP2, of which PDE was more significant. The key amino acids of PDE influencing the antigen-antibody affinity were also identified, with G254N being the most significant, followed by V252I and I256V. Then the mutated virus with point or combined mutations was rescued by reverse genetics. it was further demonstrated that mutations of V252I, G254N, and I256V in PDE could individually or collaboratively reduce antigen-antibody affinity and interfere with antiserum neutralization, with G254N being the most significant. This study revealed the reasons for the widespread prevalence of nVarIBDV in immunized chicken flocks and provided innovative ideas for designing novel vaccines that match the antigen of the epidemic strain.

Transcriptome analysis reveals that gga-miR-2954 inhibits the inflammatory response against Eimeria tenella infection.

Coccidiosis is an important parasitic protozoan disease in poultry farming, causing huge economic losses in the global poultry industry every year. MicroRNAs (miRNAs) are a class of RNA macromolecules that play important roles in the immune response to pathogens. However, the expression profiles and functions of miRNAs during Eimeria tenella (E. tenella) infection in chickens remain mostly uncharacterized. In this study, high-throughput sequencing of cecal tissues of control (JC), resistant (JR), and susceptible (JS) chickens led to the identification of 35 differentially expressed miRNAs among the three groups. Functional enrichment analysis showed that the differentially expressed miRNAs were mainly associated with the TGF-beta, NF-kB, and Jak-STAT signaling pathways. Notably, gga-miR-2954 was found to be significantly upregulated after coccidial infection. Functional analysis showed that gga-miR-2954 inhibited the production of the inflammatory cytokines IL-6, IL-1ß, TNF-α, and IL-8 in sporozoite-stimulated DF-1 cells. Mechanistically, we found that gga-miR-2954 targeted the RORC gene and that RORC promoted the inflammatory response in sporozoite-stimulated DF-1 cells. In conclusion, our study was the first to identify differentially expressed miRNAs in chicken cecal tissue during E. tenella infection and found that gga-miR-2954 regulates the host immune response to coccidial infection in chickens by targeting the RORC gene.

Life cycle of chicken bursal secretory dendritic cell (BSDC).

The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Chitosan-nanoparticle-based oral Salmonella enteritidis subunit vaccine elicits cross-protection against Salmonella typhimurium in broilers.

Non-typhoidal Salmonella infection is a significant health and economic burden in poultry industry. Developing an oral vaccine to induce robust mucosal immunity in the intestines of birds, especially cross protection against different Salmonella serotypes is challenging. Therefore, a potent oral vaccine platform that can mitigate different serotypes of Salmonella is warranted for the poultry industry. We reported earlier that the Salmonella enteritidis (SE) immunogenic outer membrane proteins (OMPs) and flagellin (FLA) entrapped in mannose chitosan nanoparticles (OMPs-FLA-mCS NPs) administered prime-boost (d-3 and 3-wk later) by oral inoculation elicits mucosal immunity and reduces challenge SE colonization by over 1 log10 CFU in birds. In this study, we sought to evaluate whether the SE antigens containing OMPs-FLA-mCS NPs vaccine induces cross-protection against Salmonella typhimurium (ST) in broilers. Our data indicated that the OMPs-FLA-mCS NPs vaccine induced higher cross-protective antibody responses compared to commercial Poulvac ST vaccine (contains a modified-live ST bacterium). Particularly, OMPs-FLA-mCS-NP vaccine elicited OMPs and FLA antigens specific increased production of secretory IgA and IgY antibodies in samples collected at both post-vaccination and post-challenge timepoints compared to commercial vaccine group. Notably, the vaccine reduced the challenge ST bacterial load by 0.8 log10 CFU in the cecal content, which was comparable to the outcome of Poulvac ST vaccination. In conclusion, our data suggested that orally administered OMPs-FLA-mCS-NP SE vaccine elicited cross protective mucosal immune responses against ST colonization in broilers. Thus, this candidate vaccine could be a viable option replacing the existing both live and killed Salmonella vaccines for birds.

The effect of ghrelin on bursa and cecal tonsils of chickens infected with an attenuated virus strain of infectious bursal disease virus.

Infectious bursal disease (IBD) significantly affects the poultry industry, causing substantial economic losses. This study aimed to investigate the effects of ghrelin on chicks infected with an attenuated virus strain of IBDV (aIBDV). Chicks were divided into 3 groups: a control group (group I), an aIBDV infection group (group II), and a ghrelin + aIBDV infection group (group III). Mice in groups II and III were fed until they reached 19 d of age and then inoculated with aIBDV to establish a subclinical infection model. Group III received an intraperitoneal injection of 0.5 nmol/100 g ghrelin from d 17 to 23. The present study utilized paraffin sectioning, H&E staining, and immunohistochemical staining to examine the effects of ghrelin on the bursa of fabricius and cecum tonsils in aIBDV-infected chicks. The results indicated that at 3 d postinfection (dpi), the average body weight of group III was significantly greater than that of group II (P < 0.05). At 3 and 7 dpi, the proportion of large lymphoid follicles in the bursa of fabricius in group III was notably greater than that in group II (P < 0.05). aIBDV infection resulted in bleeding, edema, and fibrosis in the cecal mucosal layer of chicks, but ghrelin administration mitigated these pathological changes. At 3 and 7 dpi, the thickness of the lamina propria in the cecal tonsils of group III was significantly lower than that in the cecal tonsils of group II (P < 0.05). Additionally, the percentage of large lymphoid follicles in the cecal tonsils of group III was significantly greater than that in group II at 3 and 5 dpi (P < 0.05). There were significantly fewer macrophages in the cecal tonsils of group III than in those of group II at 1, 3, and 5 dpi (P < 0.05). In conclusion, ghrelin supplementation improved performance and mitigated bursal atrophy in aIBDV-infected chicks. It also reduced histological lesions and immune responses in the cecum tonsil. Notably, the reduction in macrophages in the cecum tonsil following ghrelin administration may decrease the risk of aIBDV spread.

Intermittent mild cold acclimation ameliorates intestinal inflammation and immune dysfunction in acute cold-stressed broilers by regulating the TLR4/MyD88/NF-κB pathway.

To investigate the potential protective effect of prior cold stimulation on broiler intestine induced by acute cold stress (ACS). A total of 384 one-day-old broilers were divided into control (CON), ACS, cold stimulation Ⅰ (CS3+ACS), and cold stimulation Ⅱ (CS9+ACS) groups. Broilers in CON and ACS groups were reared normally, and birds in CS3+ACS and CS9+ACS groups were reared at 3℃ and 9℃ below CON group for 5 h, respectively, on alternate days from d 15 to 35. Broilers in ACS, CS3+ACS, and CS9+ACS groups were subjected to 10℃ for 24 h on d 43. Eventually, small intestine tissues were collected for histopathological observation and indexes detection. The results showed that intestinal tissues in all ACS-broilers exhibited inflammatory cell infiltrates, microvilli disruption, reduced villus length in jejunum and increased crypt depth in jejunum and ileum. Whereas these phenomena were relatively light in CS3+ACS group. Compared to CON group, mRNA expression of the TLR4/MyD88/NF-κB pathway-related genes (TLR4, MyD88, NF-κBp65, COX-2, iNOS, PTGEs, TNF-α), Th1/Th17-derived cytokines (IL-1ß, IL-2, IL-8, IL-12, IFN-γ, IL-17), and HSPs (HSP40, HSP60, HSP70, HSP90) was upregulated (P < 0.05), and that of Th2-deviated cytokines (IL-4, IL-6, IL-10, IL-13) and IκBα was downregulated (P < 0.05) in small intestine in almost all ACS-broilers. Compared to ACS group, mRNA expression of most of the TLR4/MyD88/NF-κB pathway-related genes, Th1/Th17-derived cytokines, and HSPs was downregulated and that of Th2-derived cytokines was upregulated in CS3+ACS group (P < 0.05). Protein expression levels of TLR4, MyD88, p-p65/p65, p-IκBα/IκBα, IKK, TNF-α, IL-1ß, IL-10, and HSPs were similar to their mRNA expression. The concentration of sIgA and activities of CAT, SOD, and GSH-px were decreased and MDA and H2O2 were increased in ACS and CS9+ACS groups compared to CON group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to gut immune dysfunction; while mild cold stimulation at 3℃ below normal rearing temperature alleviated cold stress-induced intestinal injure and dysfunction by modulating the TLR4/MyD88/NF-κB pathway in broilers.

Molecular and metabolic responses to immune stress in the jejunum of broiler chickens: transcriptomic and metabolomic analysis.

In the large poultry industry, where farmed chickens are fed at high density, the prevalence of pathogens and repeated vaccinations induce immune stress, which can significantly decrease the production performance and increase the mortality. This study was designed to shed light on the molecular mechanisms and metabolic pathways involved in immune stress through an in-depth analysis of transcriptomic and metabolomic changes in jejunum samples from the broilers. Two groups were established for the experiment: a control group and an LPS group. LPS group received an intraperitoneal injection of LPS solution at a dose of 250 µg per kg at 12, 14, 33, and 35 d of age, whereas the control group received a sterile saline injection. The severity of immune stress was assessed using the Disease Activity Index. A jejunal section was collected to measure the intestinal villus structure (villus length and crypt depth). RNA sequencing and metabolomics data analysis were conducted to reveal differentially expressed genes and metabolites. The results showed that the DAI index was increased and jejunal villus height/crypt depth was decreased in the LPS group. A total of 96 differentially expressed genes and 672 differentially accumulating metabolites were detected in the jejunum by LPS group compared to the control group. The comprehensive analysis of metabolomic and transcriptomic data showed that 23 pathways were enriched in the jejunum and that appetite, nutrient absorption, energy and substance metabolism disorders and ferroptosis play an important role in immune stress in broilers. Our findings provide a deeper understanding of the molecular and metabolic responses in broilers to LPS-induced immune stress, suggesting potential targets for therapeutic strategies to improve the production performance of broiler chickens.

Host genetics drives differences in cecal microbiota composition and immune traits of laying hens raised in the same environment.

Vaccination is one of the most effective strategies for preventing infectious diseases but individual vaccine responses are highly heterogeneous. Host genetics and gut microbiota composition are 2 likely drivers of this heterogeneity. We studied 94 animals belonging to 4 lines of laying hens: a White Leghorn experimental line genetically selected for a high antibody response against the Newcastle Disease Virus (NDV) vaccine (ND3) and its unselected control line (CTR), and 2 commercial lines (White Leghorn [LEG] and Rhode Island Red [RIR]). Animals were reared in the same conditions from hatching to 42 d of age, and animals from different genetic lines were mixed. Animals were vaccinated at 22 d of age and their humoral vaccine response against NDV was assessed by hemagglutination inhibition assay and ELISA from blood samples collected at 15, 19, and 21 d after vaccination. The immune parameters studied were the 3 immunoglobulins subtypes A, M, and Y and the blood cell composition was assessed by flow cytometry. The composition of the cecal microbiota was assessed at the end of the experiment by analyzing amplified 16S rRNA gene sequences to obtain amplicon sequence variants (ASV). The 4 lines showed significantly different levels of NDV vaccine response at the 3 measured points, with, logically, a higher response of the genetically selected ND3 line, and intermediate and low responses for the unselected CTR control line and for the 2 commercial lines, respectively. The ND3 line displayed also a higher proportion of immunoglobulins (IgA, IgM, and IgY). The RIR line showed the most different blood cell composition. The 4 lines showed significantly different microbiota characteristics: composition, abundances at all taxonomic levels, and correlations between genera and vaccine response. The tested genetic lines differ for immune parameters and gut microbiota composition and functions. These phenotypic differences can be attributed to genetic differences between lines. Causal relationships between both types of parameters are discussed and will be investigated in further studies.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Unmasking the dynamics of Mycoplasma gallisepticum: deciphering HD11 macrophage polarization for innovative infection control strategies.

Mycoplasma gallisepticum (MG) is a highly contagious avian respiratory pathogen characterized by rapid spread, widespread distribution, and long-term persistence of infection. Previous studies have shown that chicken macrophage HD11 cells play a critical role in the replication and immunomodulation of MG. Macrophages are multifunctional immunomodulatory cells that polarize into different functions and morphologies in response to exogenous stimuli. However, the effect of MG infection on HD11 polarization is not well understood. In this study, we observed a time-dependent increase in both the expression of the MG-related virulence protein pMGA1.2 and the copy number of MG upon MG infection. Polarization studies revealed an upregulation of M1-type marker genes in MG-infected HD11 cells, suggesting that MG mainly induces HD11 macrophages towards M1-type polarization. Furthermore, MG activated the inflammatory vesicle NLRP3 signaling pathway, and NLRP3 inhibitors affected the expression of M1 and M2 marker genes, indicating the crucial regulatory role of the NLRP3 signaling pathway in MG-induced polarization of HD11 macrophages. Our findings reveal a novel mechanism of MG infection, namely the polarization of MG-infected HD11 macrophages. This discovery suggests that altering the macrophage phenotype to inhibit MG infection may be an effective control strategy. These findings provide new perspectives on the pathogenic mechanism and control measures of MG.