Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies.

Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-α and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for IL-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella)-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.

Liquid Chromatography/Tandem Mass Spectrometry Analysis of Sophora flavescens Aiton and Protective Effects against Alcohol-Induced Liver Injury and Oxidative Stress in Mice.

In this study, we investigated the hepatoprotective effects of an ethanol extract of Sophora flavescens Aiton (ESF) on an alcohol-induced liver disease mouse model. Alcoholic liver disease (ALD) was caused by the administration of ethanol to male C57/BL6 mice who were given a Lieber-DeCarli liquid diet, including ethanol. The alcoholic fatty liver disease mice were orally administered ESF (100 and 200 mg/kg bw/day) or silymarin (50 mg/kg bw/day), which served as a positive control every day for 16 days. The findings suggest that ESF enhances hepatoprotective benefits by significantly decreasing serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), markers for liver injury. Furthermore, ESF alleviated the accumulation of triglyceride (TG) and total cholesterol (TC), increased serum levels of superoxide dismutase (SOD) and glutathione (GSH), and improved serum alcohol dehydrogenase (ADH) activity in the alcoholic fatty liver disease mice model. Cells and organisms rely on the Kelch-like ECH-associated protein 1- Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system as a critical defensive mechanism in response to oxidative stress. Therefore, Nrf2 plays an important role in ALD antioxidant responses, and its level is decreased by increased reactive oxidation stress (ROS) in the liver. ESF increased Nrf2, which was decreased in ethanol-damaged livers. Additionally, four polyphenol compounds were identified through a qualitative analysis of the ESF using LC-MS/MS. This study confirmed ESF's antioxidative and hangover-elimination effects and suggested the possibility of using Sophora flavescens Aiton (SF) to treat ALD.

Ziziphus jujuba Miller Ethanol Extract Restores Disrupted Intestinal Barrier Function via Tight Junction Recovery and Reduces Inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by the disruption of the intestinal barrier. The intestinal barrier is maintained by tight junctions (TJs), which sustain intestinal homeostasis and prevent pathogens from entering the microbiome and mucosal tissues. Ziziphus jujuba Miller (Z. jujuba) is a natural substance that has been used in traditional medicine as a therapy for a variety of diseases. However, in IBD, the efficacy of Z. jujuba is unknown. Therefore, we evaluated ZJB in Caco2 cells and a dextran sodium sulfate (DSS)-induced mouse model to demonstrate its efficacy in IBD. Z. jujuba extracts were prepared using 70% ethanol and were named ZJB. ZJB was found to be non-cytotoxic and to have excellent antioxidant effects. We confirmed its anti-inflammatory properties via the down-regulation of inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). To evaluate the effects of ZJB on intestinal barrier function and TJ improvement, the trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran 4 kDa (FITC-Dextran 4) permeability were assessed. The TEER value increased by 61.389% and permeability decreased by 27.348% in the 200 µg/mL ZJB group compared with the 50 ng/mL IL-6 group after 24 h. Additionally, ZJB alleviated body weight loss, reduced the disease activity index (DAI) score, and induced colon shortening in 5% DSS-induced mice; inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were down-regulated in the serum. TJ proteins, such as Zonula occludens (ZO)-1 and occludin, were up-regulated by ZJB in an impaired Caco2 mouse model. Additionally, according to the liquid chromatography results, in tandem with mass spectrometry (LC-MS/MS) analysis, seven active ingredients were detected in ZJB. In conclusion, ZJB down-regulated inflammatory factors, protected intestinal barrier function, and increased TJ proteins. It is thus a safe, natural substance with the potential to be used as a therapeutic agent in IBD treatment.

Identification of Critical Immune Regulators and Potential Interactions of IL-26 in Riemerella anatipestifer-Infected Ducks by Transcriptome Analysis and Profiling.

Riemerella anatipestifer (RA) is an economically important pathogen in the duck industry worldwide that causes high mortality and morbidity in infected birds. We previously found that upregulated IL-17A expression in ducks infected with RA participates in the pathogenesis of the disease, but this mechanism is not linked to IL-23, which primarily promotes Th17 cell differentiation and proliferation. RNA sequencing analysis was used in this study to investigate other mechanisms of IL-17A upregulation in RA infection. A possible interaction of IL-26 and IL-17 was discovered, highlighting the potential of IL-26 as a novel upstream cytokine that can regulate IL-17A during RA infection. Additionally, this process identified several important pathways and genes related to the complex networks and potential regulation of the host immune response in RA-infected ducks. Collectively, these findings not only serve as a roadmap for our understanding of RA infection and the development of new immunotherapeutic approaches for this disease, but they also provide an opportunity to understand the immune system of ducks.

Comparative Analysis of Antibiotic Resistance and Biofilm Characteristics of Two Major Enterococcus Species from Poultry Slaughterhouses in South Korea.

The spread of antibiotic-resistant Enterococcus in the poultry industry poses significant public health challenges due to multidrug resistance and biofilm formation. We investigated the antibiotic resistance profiles and biofilm characteristics of E. faecalis and E. faecium isolates from chicken meat in poultry slaughterhouses in South Korea. Ninety-six isolates (forty-eight each of E. faecalis and E. faecium) were collected between March and September 2022. Both species were analyzed using MALDI-TOF, PCR, antibiotic susceptibility testing, and biofilm assays. A high level of multidrug resistance was observed in E. faecalis (95.8%) and E. faecium (93.8%), with E. faecium exhibiting a broader range of resistance, particularly to linezolid (52.1%) and rifampicin (47.9%). All E. faecalis isolates formed biofilm in vitro, showing stronger biofilm formation than E. faecium with a significant difference (p < 0.001) in biofilm strength. Specific genes (cob, ccf, and sprE) were found to be correlated with biofilm strength. In E. faecium isolates, biofilm strength was correlated with resistance to linezolid and rifampicin, while a general correlation between antibiotic resistance and biofilm strength was not established. Through analysis, correlations were noted between antibiotics within the same class, while no general trends were evident in other analyzed factors. This study highlights the public health risks posed by multidrug-resistant enterococci collected from poultry slaughterhouses, emphasizing the complexity of the biofilm-resistance relationship and the need for enhanced control measures.

Effects of Growth Hormone-Releasing Hormone (GHRH) Plasmid Treatment on the Reproductive Productivity of Sows in Primiparous and Multiparous Sow Breeds.

The effect of growth hormone-releasing hormone (GHRH) plasmid treatment on sow reproductive performance was examined. Forty pregnant sows (three-way crossbreed: Landrace × Yorkshire × Duroc) at 85 days of gestation were included in the study and consisted of twenty primiparous and twenty multiparous sows (third parity). Sows were randomly assigned to the control and treatment groups. The treatment group received 5 mg dose of GHRH plasmid injection via electroporation, whereas the control group received a phosphate buffer solution. Reproductive indicators, including serum insulin-like growth factor-1 (IGF-1) concentration and weaned piglet data, were assessed. In the GHRH plasmid-treated group, serum IGF-1 concentration significantly increased compared with that in the control group, a trend observed in primiparous and multiparous sows. The key indicator of reproductive performance, litter size, showed that for control primiparous sows (C-PS), it was 10.90 ± 0.99 kg, while for control multiparous sows (C-MS), it was 14.00 ± 0.67 kg. Furthermore, for primiparous sows treated with GHRH plasmid (G-PS), the litter size was 11.60 ± 0.97 kg, and for multiparous sows treated with GHRH plasmid (G-MS), it was 14.00 ± 0.82 kg. The GHRH plasmid-treated group also exhibited a higher number of total births and surviving piglet numbers, along with a decrease in stillborn piglets; however, there was no significant difference in birth weight. The results suggest that GHRH plasmid treatment can enhance the reproductive performance of sows.

Development of a Parturition Detection System for Korean Native Black Goats.

Korean Native Black Goats deliver mainly during the cold season. However, in winter, there is a high risk of stunted growth and mortality for their newborns. Therefore, we conducted this study to develop a KNBG parturition detection system that detects and provides managers with early notification of the signs of parturition. The KNBG parturition detection system consists of triaxial accelerometers, gateways, a server, and parturition detection alarm terminals. Then, two different data, the labor and non-labor data, were acquired and a Decision Tree algorithm was used to classify them. After classifying the labor and non-labor states, the sum of the labor status data was multiplied by the activity count value to enhance the classification accuracy. Finally, the Labor Pain Index (LPI) was derived. Based on the LPI, the optimal processing time window was determined to be 10 min, and the threshold value for labor classification was determined to be 14 240.92. The parturition detection rate was 82.4%, with 14 out of 17 parturitions successfully detected, and the average parturition detection time was 90.6 min before the actual parturition time of the first kid. The KNBG parturition detection system is expected to reduce the risk of stunted growth and mortality due to hypothermia in KNBG kids by detecting parturition 90.6 min before the parturition of the first kid, with a success rate of 82.4%, enabling parturition nursing.

Genetic Characterization and Phylogeographic Analysis of the First H13N6 Avian Influenza Virus Isolated from Vega Gull in South Korea.

Avian influenza virus (AIV) is a pathogen with zoonotic and pandemic potential. Migratory birds are natural reservoirs of all known subtypes of AIVs, except for H17N10 and H18N11, and they have been implicated in previous highly pathogenic avian influenza outbreaks worldwide. This study identified and characterized the first isolate of the H13N6 subtype from a Vega gull (Larus vegae mongolicus) in South Korea. The amino acid sequence of hemagglutinin gene showed a low pathogenic AIV subtype and various amino acid substitutions were found in the sequence compared to the reference sequence and known H13 isolates. High sequence homology with other H13N6 isolates was found in HA, NA, PB1, and PA genes, but not for PB2, NP, M, and NS genes. Interestingly, various point amino acid mutations were found on all gene segments, and some are linked to an increased binding to human-type receptors, resistance to antivirals, and virulence. Evolutionary and phylogenetic analyses showed that all gene segments are gull-adapted, with a phylogeographic origin of mostly Eurasian, except for PB2, PA, and M. Findings from this study support the evidence that reassortment of AIVs continuously occurs in nature, and migratory birds are vital in the intercontinental spread of avian influenza viruses.

Promotion of Th1 and Th2 responses over Th17 in Riemerella anatipestifer stimulation in chicken splenocytes: Correlation of gga-miR-456-3p and gga-miR-16-5p with NOS2 and CCL5 expression.

Riemerella (R.) anatipestifer poses a significant threat to ducks, resulting in mortality rates ranging from 5-75%. This disease is highly infectious and economically consequential for domestic ducks. Although other avian species, such as chickens, also display susceptibility, the impact is comparatively less severe than in ducks. IL-17A has a pronounced correlation with R. anatipestifer infection in ducks, which is less in chickens. This study performed an in vitro transcriptome analysis using chicken splenic lymphocytes collected at 4-, 8-, and 24-hour intervals following R. anatipestifer stimulation. The primary objective was to discern the differentially expressed genes, with a specific focus on IL-17A and IL-17F expression. Moreover, an association between specific miRNAs with NOS2 and CCL5 was identified. The manifestation of riemerellosis in chickens was linked to heightened expression of Th1- and Th2-associated cells, while Th17 cells exhibited minimal involvement. This study elucidated the mechanism behind the absence of a Th17 immune response, shedding light on its role throughout disease progression. Additionally, through small RNA sequencing, we identified a connection between miRNAs, specifically miR-456-3p and miR-16-5p, and their respective target genes NOS2 and CCL5. These miRNAs are potential regulators of the inflammatory process during riemerellosis in chickens.

Demonstration of SARS-CoV-2 Exposure in Korean Native Cattle and Korean Native Black Goats in Korea.

The COVID-19 pandemic is caused by the zoonotic SARS-CoV-2 virus. A wide range of animals that interact with humans have been investigated to identify potential infections. As the extent of infection became more apparent, extensive animal monitoring became necessary to assess their susceptibility. This study analyzed nasal swabs and blood samples collected from randomly selected Korean native cattle and Korean native black goats. The tests conducted included real-time qPCR to detect SARS-CoV-2 antigens, an ELISA to detect antibodies, and a plaque reduction neutralization test (PRNT) to determine the presence of neutralizing antibodies. Among the 1798 animals tested (consisting of 1174 Korean native cattle and 624 Korean native black goats), SARS-CoV-2 viral RNA was detected in one Korean native cattle and one Korean native black goat. ELISA testing revealed positive results for antibodies in 54 Korean native cattle (4.60%) and 16 Korean native black goats (2.56%), while PRNTs yielded positive results in 51 Korean native cattle (4.34%) and 14 Korean native black goats (2.24%). The presence of SARS-CoV-2 antigens and/or antibodies was identified in animals on farms where farmworkers were already infected. It is challenging to completely rule out the possibility of reverse zoonotic transmission from humans to livestock in Korea, although the transmission is not to the same extent as it is in highly susceptible animal species like minks, cats, and dogs. This is due to the limited geographical area and the dense, intensive farming practices implemented in these regions. In conclusion, continuous viral circulation between humans and animals is inevitable, necessitating ongoing animal monitoring to ensure public health and safety.

Epidemiological investigation and drug resistance of Eimeria species in Korean chicken farms.

BACKGROUND: Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. RESULTS: An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. CONCLUSION: The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.

Role of Physiology, Immunity, Microbiota, and Infectious Diseases in the Gut Health of Poultry.

"Gut health" refers to the physical state and physiological function of the gastrointestinal tract and in the livestock system; this topic is often focused on the complex interacting components of the intestinal system that influence animal growth performance and host-microbial homeostasis. Regardless, there is an increasing need to better understand the complexity of the intestinal system and the various factors that influence gut health, since the intestine is the largest immune and neuroendocrine organ that interacts with the most complex microbiome population. As we face the post-antibiotic growth promoters (AGP) era in many countries of the world, livestock need more options to deal with food security, food safety, and antibiotic resilience to maintain agricultural sustainability to feed the increasing human population. Furthermore, developing novel antibiotic alternative strategies needs a comprehensive understanding of how this complex system maintains homeostasis as we face unpredictable changes in external factors like antibiotic-resistant microbes, farming practices, climate changes, and consumers' preferences for food. In this review, we attempt to assemble and summarize all the relevant information on chicken gut health to provide deeper insights into various aspects of gut health. Due to the broad and complex nature of the concept of "gut health", we have highlighted the most pertinent factors related to the field performance of broiler chickens.

Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection.

Riemerella anatipestifer is one of the most devastating pathogens affecting the global duck farms. Infection is involved in secretion of proinflammatory cytokines, including interleukin- (IL-) 17A. During the immune response to infection, IL-22 and IL-17A are often produced concurrently and at high levels in inflamed tissues. Little is known about duck IL-22 (duIL-22) during R. anatipestifer infection. We describe the characterization of duIL-22 and its mRNA expression analysis in splenic lymphocytes and macrophages treated with heat-killed R. anatipestifer and in the spleens and livers of R. anatipestifer-infected ducks. Full-length cDNA of duIL-22 encoded 197 amino acids. The deduced amino acid sequence of duIL-22 shared a 30.4-40.5% similarity with piscine counterparts, 57.4-60.1% with mammalian homologs, and 93.4% similarity to the chicken. Duck IL-22 mRNA expression level was relatively high in the skin of normal ducks. It was increased in mitogen-stimulated splenic lymphocytes and in killed R. anatipestifer-activated splenic lymphocytes and macrophages. Compared with healthy ducks, IL-22 transcript expression was significantly upregulated in the livers and spleens on days 1 and 4 postinfection, but not on day 7. IL-17A was significantly increased in the spleens only on day 4 postinfection and in the livers at all time points. When splenic lymphocytes were stimulated with heat-killed R. anatipestifer, CD4+ cells predominantly produced IL-22 while IL-17A was expressed both by CD4+ and CD4- cells. These results suggested that IL-22 and IL-17A are likely expressed in different cell types during R. anatipestifer infection.

Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens.

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries cNK-2 to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were randomly allocated into eight treatment groups: two control groups (NC: E. acervulina infected non-B. subtilis control; CON: non-infected control); three B. subtilis-empty vector (EV) groups (EV6: 106 cfu/day/bird; EV8: 108 cfu/day/bird; EV10: 1010 cfu/day/bird), and three B. subtilis-cNK-2 groups (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens, except those in the CON group, were challenged with 5,000 freshly sporulated E. acervulina oocysts through oral gavage on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16. Body weight, weight gains, and fecal oocyst shedding were measured. To investigate the efficacy of oral B. subtilis-cNK-2 against coccidiosis, gene expression of gut health-related biomarkers was measured using RT-PCR. Markers included SOD1, CAT, and HMOX1 for oxidative stress in the spleen and intestinal mucosa, OCLN, ZO-1, and JAM2 for tight junction proteins, and MUC2 for mucin gene expression in the gut. The results showed that oral treatment of young chickens with B. subtilis-cNK-2 improved growth performance, enhanced gut integrity, and reduced fecal oocyst shedding. Altogether, these results confirm B. subtilis-cNK-2 treatment as a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce coccidiosis-induced body weight loss, and to decrease gut damage based on the enhanced expression of proteins associated with gut integrity and intestinal health.

Research Note: Characterization of monoclonal antibodies and development of sandwich ELISA for detecting chicken IL7.

IL7 is a hematopoietic growth factor required for development and maintenance of lymphocytes including T cells, B cells, and natural killer cells. Recently, chicken IL7 (chIL7) has been cloned and studied in viral and parasite infection models. However, no monoclonal antibodies (mAb) that specifically detect chIL7 have been developed so far. In this study, recombinant chIL7 that expressed for immunization and mAb against chIL7 were developed and characterized to assess their immunologic properties. Five mAb exhibiting specific binding to chIL7 were generated and investigated for their applicability by Western blot, ELISA, and neutralization assays. A sandwich ELISA mAb pair that enables the measurement of chIL7 protein levels in biological samples from Eimeria-infected chickens was identified and several mAb neutralized chicken primary thymocyte proliferation mediated by chIL7. The mAb developed in this study will be valuable reagents for fundamental and applied immunological studies in poultry.

Characterization of immunological properties of chicken chemokine CC motif ligand 5 using new monoclonal antibodies.

CCL5 (formerly RANTES) belongs to the CC (or ß) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. CCL5 is mainly produced and secreted by T cells, macrophages, epithelial cells, and fibroblasts and acts as a chemoattractant to recruit effector cells to the inflammation sites. Chicken CCL5 (chCCL5) protein is closely related to avian CCL5 orthologs but distinct from mammalian orthologs, and its modulatory roles in the immune response are largely unknown. The present work was undertaken to characterize the immunological properties of chCCL5 using the new sets of anti-chCCL5 mouse monoclonal antibodies (mAbs). Eight different mAbs (6E11, 6H1, 8H11, 11G1, 11G11, 12H1, 13D1, and 13G3) were characterized for their specificity and binding ability toward chCCL5. Two (13G3 and 6E11) of them were selected to detect native chCCL5 in chCCL5-specific antigen-capture ELISA. Using 13G3 and 6E11 as capture and detection antibodies, respectively, the ELISA system detected serum chCCL5 secretions in Clostridium perfringens- and Eimeria-infected chickens. The intracellular expressions of chCCL5 in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 13G3 and 6E11 mAbs. Furthermore, 6E11, but not 13G3, neutralized chCCL5-induced chemotaxis in vitro using chicken PBMCs. These molecular characteristics of chCCL5 demonstrate the potential application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species.

Detection of Necrotic Enteritis B-like Toxin Secreted by Clostridium perfringens Using Capture Enzyme-Linked Immunosorbent Assay.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G, which affects global poultry industry by compromising the performance, health, and welfare of chickens. The causative main virulent factor responsible for NE pathogenesis has been shifted from a phospholipase C portion of an α-toxin, to an NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, in NE development. Therefore, the ability to detect NetB toxin will enable early diagnosis of field NE. Because the NetB protein can only be detected by western blot analysis with polyclonal anti-NetB antiserum, we developed a NetB-specific monoclonal antibody (mAb)-based capture enzyme-linked immunosorbent assay (ELISA). Twenty mAbs reacting with Escherichia coli-expressed NetB protein were selected, isotyped, and conjugated with horseradish peroxidase for antibody pair tests. Multiple mAb pairs were found to detect E. coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates. The developed capture (sandwich) ELISA could be useful to identify in vitro production of native NetB protein secreted from netB-positive field C. perfringens isolates and to conduct a large field test of commercial chickens undergoing NE infection. Here, we first report that native NetB toxin can be detected in C. perfringens NetB-specific mAb-based capture ELISA.

Anti-inflammatory activity of diindolylmethane alleviates Riemerella anatipestifer infection in ducks.

3,3'-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1ß) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer-infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken.

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α. The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4.

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.