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H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.
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Animais Geneticamente Modificados , Galinhas , Cadeias Pesadas de Imunoglobulinas , Anticorpos de Domínio Único , Animais , Galinhas/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , COVID-19/imunologia , Transgenes/genética , Linfócitos B/imunologia , Anticorpos Antivirais/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , HumanosRESUMO
Clostridial cellulitis or dermatitis affects commercial turkey flocks, primarily as they approach market age. In the field, this disease has been effectively controlled with antibiotics, but alternatives to antibiotics are needed. Bacterin-toxoid vaccination programs have been shown to prevent clostridial diseases in other species, including humans. Results from previous field studies indicate that vaccination with an experimental whole-cell Clostridium septicum (CS) bacterin-toxoid oil emulsion vaccine reduced clostridial dermatitis-associated mortality and antibiotic usage for some commercial turkey flocks, but vaccination was not always efficacious. To improve vaccine efficacy, studies were conducted to optimize the antigenic component of the experimental vaccine and to determine the appropriate antigen to adjuvant ratio, route, and volume for vaccine administration. It was determined that the phase of culture at time of formalin inactivation played a key role in serum antibody titer and larger volume vaccine doses produced higher serum antibody immune response regardless of antigen:adjuvant formulation ratio or route of injection. No significant differences (P > 0.05) were found between formulation ratios or between the subcutaneous and tail head injection sites. Based on these results, we propose to look further into the relationship between culture phase and antigenic components produced by CS under different culture conditions.
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Infecções por Clostridium , Clostridium septicum , Dermatite , Doenças das Aves Domésticas , Humanos , Animais , Perus , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Celulite (Flegmão)/veterinária , Imunidade Humoral , Doenças das Aves Domésticas/prevenção & controle , Galinhas , Clostridium , Vacinas Bacterianas , Toxoides , Dermatite/veterináriaRESUMO
Introduction: Coccidiosis caused by the Eimeria spp., an Apicomplexan protozoon, is a major intestinal disease that affects the poultry industry. Although most cases of coccidiosis are subclinical, Eimeria infections impair bird health and decrease overall performance, which can result in compromised welfare and major economic losses. Viable sporulated Eimeria oocysts are required for challenge studies and live coccidiosis vaccines. Potassium dichromate (PDC) is typically used as a preservative for these stocks during storage. Although effective and inexpensive, PDC is also toxic and carcinogenic. Chlorhexidine (CHX) salts may be a possible alternative, as this is a widely used disinfectant with less toxicity and no known carcinogenic associations. Methods: In vitro testing of CHX gluconate and CHX digluconate exhibited comparable oocyst integrity and viability maintenance with equivalent bacteriostatic and bactericidal activity to PDC. Subsequent use of CHX gluconate or digluconate-preserved Eimeria oocysts, cold-stored at 4°C for 5 months, as the inoculum also resulted in similar oocyst shedding and recovery rates when compared to PDC-preserved oocysts. Results and discussion: These data show that using 0.20% CHX gluconate could be a suitable replacement for PDC. Additionally, autofluorescence was used as a method to evaluate oocyst viability. Administration of artificially aged oocysts exhibiting >99% autofluorescence from each preserved treatment resulted in no oocyst output for CHX salt groups.
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Histomonas meleagridis, the etiological agent of histomonosis, is a poultry parasite primarily detrimental to turkeys. Characteristic lesions occur in the liver and ceca, with mortalities in turkey flocks often reaching 80-100%. Chickens and other gallinaceous birds can be susceptible but the disease was primarily considered sub-clinical until recent years. Treating and preventing H. meleagridis infection have become more difficult since 2015, when nitarsone was voluntarily removed from the market, leaving the poultry industry with no approved prophylactics, therapeutics, or vaccines to combat histomonosis. Phytogenic compounds evaluated for chemoprophylaxis of histomonosis have varied results with in vitro and in vivo experiments. Some recent research successes are encouraging for the pursuit of antihistomonal compounds derived from plants. Turkeys and chickens exhibit a level of resistance to re-infection when recovered from H. meleagridis infection, but no commercial vaccines are yet available, despite experimental successes. Safety and stability of live-attenuated isolates have been demonstrated; furthermore, highly efficacious protection has been conferred in experimental settings with administration of these isolates without harming performance. Taken together, these research advancements are encouraging for vaccine development, but further investigation is necessary to evaluate proper administration age, dose, and route. A summary of the published research is provided in this review.
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Outbreaks of histomonosis in turkeys are typically initiated by the ingestion of contaminated embryonated eggs of Heterakis gallinarum, potentially present in earthworms and mechanical vectors. Once an outbreak is started, infected turkeys can transmit the disease by horizontal transmission. Factors influencing horizontal transmission of histomonosis are poorly understood. Replication of horizontal transmission in experimental conditions has not been consistent, presenting an obstacle in searching for alternatives to prevent or treat the disease. Two pilot experiments and three validation experiments were conducted in the present study. In pilot experiment 1, one isolate of Histomonas meleagridis (named Buford) was used. Turkeys were fed a low-nutrient density diet corn-soy based (LOW-CS) and raised in floor pens. In pilot experiment 2, another isolate of H. meleagridis was used (named PHL). Turkeys were fed a low-nutrient density diet with the addition of wheat middlings (LOW-WM) and raised in floor pens. In experiment 3, conducted on floor pens, both isolates and diets were used in different groups. In experiment 4, turkeys were raised on battery cages and only the PHL isolate was used. Both diets (LOW-WM and LOW-CS) were used, in addition to a diet surpassing the nutritional needs of young poults (turkey starter, TS). In experiment 5, conducted in battery cages, only the PHL isolate was used, and the LOW-WM and TS diets were in different groups. The horizontal transmission was achieved only with the PHL isolate from all experiments. The transmission rate varied among experimental diets, with the TS diet having the lowest transmission rate in experiments 4 and 5. Variation was observed between experiments and within experimental groups.
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The goal of this research was to assess cyclic heat stress on gut permeability, bone mineralization, and meat quality in chickens. Two separate trials were directed. 320 day-of-hatch Cobb 500 male chicks were randomly assigned to four thermoneutral (TN) and four cyclic heat stress (HS) chambers with two pens each, providing eight replicates per treatment in each trial (n = 20 chicks/replicate). Environmental conditions in the TN group were established to simulate commercial production settings. Heat stress chickens were exposed to cyclic HS at 35 °C for 12 h/day from days 7−42. Performance parameters, intestinal permeability, bone parameters, meat quality, and leukocyte proportions were estimated. There was a significant (p < 0.05) reduction in body weight (BW), BW gain, and feed intake, but the feed conversion ratio increased in chickens under cyclic HS. Moreover, HS chickens had a significantly higher gut permeability, monocyte and basophil levels, but less bone mineralization than TN chickens. Nevertheless, the TN group had significant increases in breast yield, woody breast, and white striping in breast fillets compared to HS. These results present an alternative model to our previously published continuous HS model to better reflect commercial conditions to evaluate commercially available nutraceuticals or products with claims of reducing the severity of heat stress.
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Oral administration of fluorescein isothiocyanate dextran (FITC-d) has been used as an indicator for intestinal permeability in poultry research for several years. Under healthy conditions, tight junctions in the intestinal wall will not allow the 4-6kDa FITC-d to enter the bloodstream. Detection of FITC-d in serum (1-hour post-oral administration of FITC-d) has proven to be a reliable indicator of leaky gut syndrome (increased intestinal inflammation and disruption of tight junctions). Administration of supplementary phytobiotics in feed, particularly products with high beta-carotene levels or other pigments, has resulted in strong serum background fluorescence, which can render this assay unreliable. To account for this increase in background autofluorescence, the FITC-d assay procedure has been modified to accommodate these particular serum samples by including pre-administration serum collection from each treatment group to remove background fluorescence. The modified FITC-d procedure detailed will allow for analysis of intestinal permeability in pigmented serum.
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Galinhas , Aves Domésticas , Animais , Dextranos , Dieta/veterinária , Fluoresceína-5-Isotiocianato/análogos & derivados , Mucosa Intestinal , PermeabilidadeRESUMO
The aim of this study was to see how spray-dried plasma (SDP) supplementation affected broiler chicken performance, intestinal permeability, and bone strength during persistent heat stress. One-day-old chicks (n = 480) were randomly assigned into twelve environmental corrals; four thermoneutral (TN-negative control, maintained at 24 °C from d 21-42); four heat stress (HS, exposed to 35 °C from d 21-42); and four heat stress treated with 2% SDP in the feed until d 28 followed by 1% SDP until d 42 (HS-SDP). The performance and serum levels of fluorescein isothiocyanate-dextran (FITC-d) were evaluated at d 21, 28, 35, and 42. The tibias strength was evaluated on d 21 and 42. The increment in chicken temperature (p < 0.05) was observed two h following the increase in environmental temperature in both HS groups and was associated with decreased performance parameters compared with the TN group. At d 42 of age, the chickens exposed to HS had an impaired gut permeability and decreased tibia strength compared to the TN group (p < 0.05). However, partially feeding SDP mitigated these adverse effects significantly. These findings imply that using SDP strategically during stressful times, such as prolonged heat stress, may help mitigate its negative consequences.
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The objective of the present research was to evaluate dietary supplementation of essential oils from Lippia origanoides (LEO) on necrotic enteritis (NE). Chickens were randomly assigned to three groups. Group 1: negative control; Group 2: positive control challenged with Salmonella typhimurium (day 1), Eimeria maxima (day 18), and C. perfringens (CP, days 22-23); Group 3: dietary supplementation LEO and challenged. On d 25 of age, serum samples were collected to evaluate fluorescein isothiocyanate-dextran (FITC-d), superoxide dismutase (SOD), gamma interferon (IFN-γ), Immunoglobulin A (IgA). Group 3 showed a significant reduction of the harmful effects of induced infection/dysbiosis and a significant reduction in NE lesion scores, morbidity and mortality compared with the positive challenge control group (p < 0.05) compared with Group 2. Digested feed supernatant, supplemented with LEO and inoculated with CP, reduced CP burden (p < 0.05). Group 3 also exhibited a significant reduction in FITC-d, IFN-γ and IgA compared with Group 2. However, a significant increase SOD was observed in Group 3 compared with both control groups. Further investigation to compare the effect of LEO and the standard treatment of clostridial NE is required.
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The objective of the present research was to assess the dietary supplementation of three formulations of essential oils (EO) in chickens under heat stress (HS). Day-of-hatch Cobb 500 chicks (n = 500) were randomly distributed into four groups: 1. HS control + control diets; 2. HS + control diets supplemented with 37 ppm EO of Lippia origanoides (LO); 3. HS + control diets supplemented with 45 ppm LO + 45 ppm EO of Rosmarinus officinalis (RO) + 300 ppm red beetroot; 4. HS + 45 ppm LO + 45 ppm RO + 300 ppm natural betaine. Chickens that received the EO showed significant (p < 0.05) improvement on BW, BWG, FI, and FCR compared to control HS chickens. Average body core temperature in group 3 and group 4 was significantly (p < 0.05) reduced compared with the HS control group and group 2. Experimental groups showed a significant reduction in FITC-d at 42 days, a significant increase in SOD at both days but a significant reduction of IFN-γ and IgA compared with HS control (p < 0.05). Bone mineralization was significantly improved by EO treatments (p < 0.05). Together these data suggest that supplemental dietary EO may reduce the harmful effects of HS.
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The purpose of this pilot study was to evaluate and determine the concentration of prostaglandin GF2α (PGF2α) and isoprostane 8-iso-PGF2α in plasma and intestine of specific pathogen-free (SPF) Leghorn chickens challenged with Eimeria maxima, with or without dietary supplementation of curcumin using solid-phase microextraction and ultra-performance liquid chromatography/tandem mass spectrometry. Eighty 1-day-old male SPF chickens were randomly allocated to one of four groups with four replicates (n = 5 chickens/replicate). Groups consisted of: (1) Control (no challenge), (2) Curcumin (no challenge), (3) Eimeria maxima (challenge), and (4) Eimeria maxima (challenge) + curcumin. At day 28 of age, all chickens in the challenge groups were orally gavaged with 40,000 sporulated E. maxima oocysts. No significant differences (P > 0.05) were observed in the groups regardless of the treatment or challenge with E. maxima. Enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days and 9 days post-challenge were significantly increased (P < 0.01) compared to the non-challenge control chickens. Interestingly, the enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days post-challenge were significantly reduced in chickens fed curcumin, compared to control chickens challenge with E. maxima. At 9 days post-challenge, only levels of isoprostane 8-iso-PGF2α in the enteric samples were significantly reduced in chickens challenged with E. maxima supplemented with curcumin, compared with E. maxima challenge chickens. No differences of isoprostane 8-iso-PGF2α or PGF2α were observed in plasma at both days of evaluation. Similarly, no significant differences were observed between the challenge control or chickens challenge with E. maxima and supplemented with curcumin at both times of evaluation. The results of this pilot study suggests that the antioxidant anti-inflammatory properties of curcumin reduced the oxidative damage and subsequent intestinal mucosal over-production of lipid oxidation products. Further studies to confirm and extend these results in broiler chickens are required.
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Anti-Inflamatórios/farmacologia , Coccidiose/tratamento farmacológico , Curcumina/farmacologia , Dinoprosta/análogos & derivados , Dinoprosta/antagonistas & inibidores , Eimeria/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Ração Animal , Animais , Animais Recém-Nascidos , Galinhas/crescimento & desenvolvimento , Galinhas/parasitologia , Coccidiose/metabolismo , Coccidiose/parasitologia , Coccidiose/veterinária , Suplementos Nutricionais , Dinoprosta/metabolismo , Eimeria/crescimento & desenvolvimento , Eimeria/patogenicidade , Inflamação , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Masculino , Oocistos/efeitos dos fármacos , Oocistos/crescimento & desenvolvimento , Oocistos/patogenicidade , Estresse Oxidativo , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/parasitologia , Organismos Livres de Patógenos EspecíficosRESUMO
Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method - Golden Gate assembly with a bi-directional promoter (GBid) - for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to "scFv-like" simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert - two variable chains and one bi-directional promoter - are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.
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Técnicas de Visualização da Superfície Celular/métodos , Fragmentos Fab das Imunoglobulinas/metabolismo , Biblioteca de Peptídeos , Regiões Promotoras Genéticas , Animais , Especificidade de Anticorpos , Antígenos CD20/imunologia , Sequência de Bases , Galinhas , Humanos , Receptor ErbB-2/metabolismoRESUMO
Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) strains were isolated at 39.5 °C to rule out temperature-sensitive strains (ts+) and identified using random amplification of polymorphic DNA. Then, their minimum inhibitory concentrations (MIC100) were calculated in isolated strains from broiler breeders and laying hens vaccinated with ts+ MS-H and ts+ MG TS-11 vaccines in Mexico. We sampled 631 lots of hens. A total of 28 of the 123 MS isolates and 12 of the 23 MG isolates were analyzed using random amplification of polymorphic DNA, of which 24 and 3 matched the DNA banding patterns of the MS-H and MG-F strains, respectively. The isolated MS and MG strains were sensitive to tiamulin and tylosin and showed intermediate sensitivity or resistance to lincomycin, florfenicol, erythromycin, enrofloxacin, and curcumin. Although both the MS and MG strains were sensitive to the same antibiotics (MIC100 lower than 1 mg mL-1), the MG strains were 5 to 10 times more sensitive than the MS strains. MS is the most frequently isolated mycoplasma in Mexican poultry production. The MS vaccine used (ts+ MS-H) could reverse its thermosensitivity and therefore could regain its virulence. MS was less sensitive to tiamulin and tylosin compared to MG.
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Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.
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Aptâmeros de Nucleotídeos/imunologia , Antígenos CD40/imunologia , Imunoglobulina G/imunologia , Orthomyxoviridae/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos , Animais , Antígenos/imunologia , Aptâmeros de Nucleotídeos/genética , Linhagem Celular , Galinhas/imunologia , Galinhas/virologia , Epitopos/imunologia , Haptenos/imunologia , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Imunoglobulina G/farmacologia , Macrófagos/imunologia , Orthomyxoviridae/efeitos dos fármacos , Peptídeos/imunologia , Vacinas/imunologiaRESUMO
This study's objective was to evaluate the effect of the fermented soybean meal (FSBM) on Salmonella typhimurium (ST) to turkey poults using two models of infection. In the prophylactic model, one-day-old turkeys were randomly allocated to one of four different groups (n = 30 turkeys/group): (1) Control group, (2) FSBM group, (3) Control group challenged with ST (Control + ST), and (4) FSBM group challenged with ST (FSBM + ST). On day 9 of age, all poults were orally challenged with 106 colony forming units (CFU) ST and 24 h post-inoculation, intestinal samples were collected to determine ST recovery and morphometric analysis. Blood samples were collected to evaluate serum fluorescein isothiocyanate-dextran (FITC-d). In the therapeutic model, a similar experimental design was used, but turkeys were orally gavaged 104 CFU ST on day 1, and samples were collected at day 7. FSBM improved performance and reduced leaky gut in both experimental infective models. In the prophylactic model, FSBB induced morphology changes in the mucosa. Although the strains (Lactobacillus salivarius and Bacillus licheniformis) used for the fermentation process showed in vitro activity against ST, no significant effect was observed in vivo. The fermentation with different beneficial bacteria and different inclusion rates of FSBM requires further investigation.
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The purpose of this study was to evaluate heat stress as a model to induce gastrointestinal leakage in broiler chickens. On the day of hatch, 320 chicks were allocated into 8 environmental chambers, 4 thermoneutral (TN) and 4 continuous heat stress (HS). Each chamber was divided into 2 pens containing separate feeders and water jugs (8 replicates per treatment, 20 birds/pen). The environment was established to simulate production setting as best possible for the first 21 D. A gradual reduction of temperature from 32°C to 24°C with relative humidity at 55 ± 5% was adopted for the first 21 D. At the time of HS, the HS groups were exposed to 35°C from Day 21 to 42, while thermoneutral ones were maintained at 24°C from Day 21 to 42. Chickens were equipped with a Thermochron temperature logger for continuous monitoring of core body temperature. The environmental temperature and relative humidity were continuously recorded. Fluorescein isothiocyanate-dextran (FITC-d) was orally gavaged to 2 chickens/replicate (n = 16) randomly selected on days 21, 28, 35, and 42. After 1 h of oral gavage, blood samples were collected to determine the passage of FITC-d. Tibias were removed from all chickens to evaluate break strength only on 21 D and 42 D (before HS and at the end of the trial). Performance parameters were evaluated weekly from 21 D to the end of the trial. Body temperature was significantly (P < 0.05) increased after 2 h of starting HS and remained that way until the end of the study. Chronic HS caused an increase in core body temperature which decreased feed intake, body weight, and feed efficiency (28, 35, and 42 D) when compared with control TN chickens. Similarly, serum FITC-d was significantly increased in HS chickens at all points of evaluation. Chronic HS also caused a significant reduction of bone strength at 42 D when compared with the control chickens. The results from the present study suggest that HS can be a robust model to induce gut leakage in broiler chickens.
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Trato Gastrointestinal/fisiopatologia , Transtornos de Estresse por Calor/veterinária , Permeabilidade , Animais , Animais Recém-Nascidos , Temperatura Corporal , Densidade Óssea , Galinhas , Dextranos/farmacocinética , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Temperatura Alta/efeitos adversosRESUMO
The effects of in ovo administration of a defined lactic acid microbiota (LAM), previously isolated from adult hens, in the cecae microbiota structure and Enterobacteriaceae colonization after exposure to virulent Escherichia coli during the hatching phase of broiler chickens were evaluated. Embryos inoculated with LAM showed a significant (P < 0.05) reduction of Enterobacteriaceae colonization at day-of-hatch (DOH) and day (d) 7. Furthermore, there was a significant increase in total lactic acid bacteria on DOH, body weight (BW) DOH, BW d7, and d0-d7 BW gain and reduced mortality d0-d7 was observed in the LAM group compared with that in phosphate-buffered saline (PBS) control. The bacterial composition at the family level revealed that the Enterobacteriaceae was numerically reduced, whereas the Ruminococcaceae was significantly increased in the LAM group when compared with that in the PBS control. Moreover, the bacterial genera Proteus and Butyricicoccus and unidentified bacterial genera of family Lachnospiraceae and Erysipelotrichaceae were significantly enriched in the LAM group. In contrast, the Clostridium of the family Peptostreptococcaceae and unidentified genus of family Enterobacteriaceae were significantly abundant in the PBS control group. In summary, in ovo administration of a defined LAM isolated from adult hens did not affect hatchability, improved body weight gain and reduced mortality at d7, induced variations in the cecae microbiota structure and reduced Enterobacteriaceae colonization on a virulent E. coli horizontal infection model in broiler chickens.
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Dendritic cells (DC) are important antigen-presenting cells and are among the least characterized immune cells in the chicken. In order to obtain chicken DC, current protocols require isolation of bone marrow myeloid progenitor cells and induction of DC differentiation with supplemental cytokines or negative selection of splenic cell preparations. Chicken peritoneal exudate cells (PEC) have traditionally been a source of various immune cells for ex vivo studies, primarily to investigate heterophils and macrophages. In this study, we observe the presence of CD205+ PEC populations, a marker of DC, as an additional resource to isolate and study chicken primary DCs. A panel of monoclonal antibodies was developed against the chicken CD205 DC marker and used to isolate CD205+ DC from the PEC population using magnetic bead cell sorting. This study reports the development of new anti-CD205 monoclonal antibodies as a reagent for chicken DC research, as well as PEC as a potential source of CD205+ DC for ex vivo studies in the chicken.
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Antígenos CD/metabolismo , Separação Celular/veterinária , Galinhas/imunologia , Células Dendríticas/citologia , Lectinas Tipo C/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Células Dendríticas/imunologia , Sefarose/imunologiaRESUMO
In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.
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Anticorpos Biespecíficos/administração & dosagem , Antígenos Virais/imunologia , Antígenos CD40/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/prevenção & controle , Animais , Galinhas , Influenza Aviária/imunologiaRESUMO
In vivo targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, Clostridium perfringens alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC) enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected in silico using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for in vitro toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin's hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and de facto identification of neutralizing epitopes in microbial toxins and enzymes within 2 weeks from in silico analysis of a putative target sequence.