Evaluation of the Antimicrobial Activity of a Formulation Containing Ascorbic Acid and Eudragit FS 30D Microparticles for the Controlled Release of a Curcumin-Boric Acid Solid Dispersion in Turkey Poults Infected with Salmonella enteritidis: A Therapeutic Model.

The selection of components within a formulation or for treatment must stop being arbitrary and must be focused on scientific evidence that supports the inclusion of each one. Therefore, the objective of the present study was to obtain a formulation based on ascorbic acid (AA) and Eudragit FS 30D microparticles containing curcumin-boric acid (CUR-BA) considering interaction studies between the active components carried out via Fourier transform infrared spectrometry (FTIR) and differential scanning calorimetry (DSC) to minimize antagonistic effects, and comprehensively and effectively treat turkey poults infected with Salmonella enteritidis (S. enteritidis). The DSC and FTIR studies clearly demonstrated the interactions between AA, BA, and CUR. Consequently, the combination of AA with CUR and/or BA should be avoided, but not CUR and BA. Furthermore, the Eudragit FS 30D microparticles containing CUR-BA (SD CUR-BA MP) showed a limited release of CUR-BA in an acidic medium, but they were released at a pH 6.8-7.0, which reduced the interactions between CUR-BA and AA. Finally, in the S. enteritidis infection model, turkey poults treated with the combination of AA and SD CUR-BA MP presented lower counts of S. enteritidis in cecal tonsils after 10 days of treatment. These results pointed out that the use of an adequate combination of AA and CUR-BA as an integral treatment of S. enteritidis infections could be a viable option to replace the indiscriminate use of antibiotics.

Orexin system is expressed in avian muscle cells and regulates mitochondrial dynamics.

Orexin A and B, orexigenic peptides produced primarily by the lateral hypothalamus that signal through two G protein-coupled receptors, orexin receptors 1/2, have been implicated in the regulation of several physiological processes in mammals. In avian (nonmammalian vertebrates) species; however, the physiological roles of orexin are not well defined. Here, we provide novel evidence that not only is orexin and its related receptors 1/2 (ORXR1/2) expressed in chicken muscle tissue and quail muscle (QM7) cell line, orexin appears to be a secretory protein in QM7 cells. In vitro administration of recombinant orexin A and B (rORX-A and B) differentially regulated prepro-orexin expression in a dose-dependent manner with up-regulation for rORX-A (P < 0.05) and downregulation for rORX-B (P < 0.05) in QM7 cells. While both peptides upregulated ORXR1 expression, only a high dose of rORX-B decreased the expression of ORXR2 (P < 0.05). The presence of orexin and its related receptors and the regulation of its own system in avian muscle cells indicate that orexin may have autocrine, paracrine, and/or endocrine roles. rORXs differentially regulated mitochondrial dynamics network. While rORX-A significantly induced the expression of mitochondrial fission-related genes (DNM1, MTFP1, MTFR1), rORX-B increased the expression of mitofusin 2, OPA1, and OMA1 genes that are involved in mitochondrial fusion. Concomitant with these changes, rORXs differentially regulated the expression of several mitochondrial metabolic genes (av-UCP, av-ANT, Ski, and NRF-1) and their related transcriptional regulators (PPARγ, PPARα, PGC-1α, PGC-1ß, and FoxO-1) without affecting ATP synthesis. Taken together, our data represent the first evidence of the presence and secretion of orexin system in the muscle of nonmammalian species and its role in mitochondrial fusion and fission, probably through mitochondrial-related genes and their related transcription factors.

An Impedance Aptasensor with Microfluidic Chips for Specific Detection of H5N1 Avian Influenza Virus.

In this research a DNA aptamer, which was selected through SELEX (systematic evolution of ligands by exponential enrichment) to be specific against the H5N1 subtype of the avian influenza virus (AIV), was used as an alternative reagent to monoclonal antibodies in an impedance biosensor utilizing a microfluidics flow cell and an interdigitated microelectrode for the specific detection of H5N1 AIV. The gold surface of the interdigitated microelectrode embedded in a microfluidics flow cell was modified using streptavidin. The biotinylated aptamer against H5N1 was then immobilized on the electrode surface using biotin-streptavidin binding. The target virus was captured on the microelectrode surface, causing an increase in impedance magnitude. The aptasensor had a detection time of 30 min with a detection limit of 0.0128 hemagglutinin units (HAU). Scanning electron microscopy confirmed the binding of the target virus onto the electrode surface. The DNA aptamer was specific to H5N1 and had no cross-reaction to other subtypes of AIV (e.g., H1N1, H2N2, H7N2). The newly developed aptasensor offers a portable, rapid, low-cost alternative to current methods with the same sensitivity and specificity.

Food-producing animals and their health in relation to human health.

The fields of immunology, microbiology, and nutrition converge in an astonishing way. Dietary ingredients have a profound effect on the composition of the gut microflora, which in turn regulates the physiology of metazoans. As such, nutritional components of the diet are of critical importance not only for meeting the nutrient requirements of the host, but also for the microbiome. During their coevolution, bacterial microbiota has established multiple mechanisms to influence the eukaryotic host, generally in a beneficial fashion. The microbiome encrypts a variety of metabolic functions that complements the physiology of their hosts. Over a century ago Eli Metchnikoff proposed the revolutionary idea to consume viable bacteria to promote health by modulating the intestinal microflora. The idea is more applicable now than ever, since bacterial antimicrobial resistance has become a serious worldwide problem both in medical and agricultural fields. The impending ban of antibiotics in animal feed due to the current concern over the spread of antibiotic resistance genes makes a compelling case for the development of alternative prophylactics. Nutritional approaches to counteract the debilitating effects of stress and infection may provide producers with useful alternatives to antibiotics. Improving the disease resistance of animals grown without antibiotics will benefit the animals' health, welfare, and production efficiency, and is also a key strategy in the effort to improve the microbiological safe status of animal-derived food products (e.g. by poultry, rabbits, ruminants, or pigs). This review presents some of the alternatives currently used in food-producing animals to influence their health in relation to human health.

Bioenergetics in chicken embryo fibroblast cells: evidence of lower proton leak in spontaneously immortalized chicken embryo fibroblasts compared to young and senescent primary chicken embryo fibroblast cells.

A spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) is known to exhibit faster growth rate and greater sensitivity to oxidative stress compared to the primary parent CEF (pCEF1°) cells. Thus, major objectives of this study were to assess cell bioenergetics in pCEF1° and DF-1 cells under control conditions and in response to 4-hydroxy 2-nonenal (4-HNE) induced oxidative challenge. Cell bioenergetics were assessed by flux analysis of oxygen consumption rate (OCR). Under control conditions, DF-1 cells had higher OCR associated with ATP synthase activity and mitochondrial oxygen reserve capacity as well as lower OCR due to proton leak and non-mitochondrial cytochrome c oxidase activity. In response to 4-HNE (0 to 30 µM), DF-1 cells were more sensitive to oxidant challenge than both young (passage 8) and senescent (passage 19) pCEF1° cells. Both passages 8 and 19 pCEF1° cells exhibited higher proton leak in response to 4-HNE, but this was not observed in DF-1 cells. Inducible proton leak occurs by 4-HNE stimulated activation of uncoupling protein (UCP) and adenine nucleotide translocase (ANT). From mRNA expression data indicated that ANT and avian UCP were down-regulated and up-regulated, respectively, in DF-1 compared to pCEF1° cells. Thus, we hypothesize that DF-1 cells are unable to increase proton leak due to lower expression of ANT, but not avian UCP, and this inability to increase proton leak contributes to greater susceptibility to oxidative stress of DF-1 cells compared to pCEF1° cells.

Effect of chitosan on Salmonella Typhimurium in broiler chickens.

Public concern with the incidence of antibiotic-resistant bacteria, particularly among foodborne pathogens such as Salmonella, has been challenging the poultry industry to find alternative means of control. The purposes of the present study were to evaluate in vitro and in vivo effects of chitosan on Salmonella enterica serovar Typhimurium (ST) infection in broiler chicks. For in vitro crop assay experiments, tubes containing feed, water, and ST were treated with either saline as a control or 0.2% chitosan. The entire assay was repeated in three trials. In two independent in vivo trials, 40 broiler chicks were assigned to an untreated control diet or dietary treatment with 0.2% chitosan for 7 days (20 broiler chicks/treatment). At day 4, chicks were challenged with 2×105 colony-forming units (CFU) ST/bird. In a third in vivo trial, 100 broiler chicks were assigned to untreated control diet or dietary treatment with 0.2% chitosan for 10 days (50 broiler chicks/treatment) to evaluate ST horizontal transmission. At day 3, 10 birds were challenged with 105 CFU ST/bird, and the remaining nonchallenged birds (n=40) were kept in the same floor pen. In all three in vitro trials, 0.2% chitosan significantly reduced total CFU of ST at 0.5 and 6 h postinoculation compared with control (p<0.05). In two in vivo trials, at 7 days, dietary 0.2% chitosan significantly reduced total CFU of recovered ST in the ceca in both experiments. Dietary 0.2% chitosan significantly reduced total ST CFU recovered in the ceca of horizontally challenged birds in the third in vivo trial. Chitosan at 0.2% significantly reduced the CFU of recovered ST in vitro and in vivo, proving to be an alternative tool to reduce crop, ceca, and consequently carcass ST contamination as well as decreasing the amount of ST shed to the environment.

Unraveling frontiers in poultry health (part 1) - Mitigating economically important viral and bacterial diseases in commercial Chicken and Turkey production.

This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.

Assessment of the Impact of Humic Acids on Intestinal Microbiota, Gut Integrity, Ileum Morphometry, and Cellular Immunity of Turkey Poults Fed an Aflatoxin B1-Contaminated Diet.

A recent study published data on the growth performance, relative weights of the organs of the gastrointestinal tract, liver histology, serum biochemistry, and hematological parameters for turkey poults fed an experimental diet contaminated with aflatoxin B1 (AFB1) and humic acids (HA) extracted from vermicompost. The negative effects of AFB1 (250 ng AFB1/g of feed) were significantly reduced by HA supplementation (0.25% w/w), suggesting that HA might be utilized to ameliorate the negative impact of AFB1 from contaminated diets. The present study shows the results of the remaining variables, as an extension of a previously published work which aimed to evaluate the impact of HA on the intestinal microbiota, gut integrity, ileum morphometry, and cellular immunity of turkey poults fed an AFB1-contaminated diet. For this objective, five equal groups of 1-day-old female Nicholas-700 turkey poults were randomly assigned to the following treatments: negative control (basal diet), positive control (basal diet + 250 ng AFB1/g), HA (basal diet + 0.25% HA), HA + AFB1 (basal diet + 0.25% HA + 250 ng AFB1/g), and Zeolite (basal diet + 0.25% zeolite + 250 ng AFB1/g). In the experiment, seven replicates of ten poults each were used per treatment (n = 70). In general, HA supplementation with or without the presence of AFB1 showed a significant increase (p < 0.05) in the number of beneficial butyric acid producers, ileum villi height, and ileum total area, and a significant reduction in serum levels of fluorescein isothiocyanate-dextran (FITC-d), a marker of intestinal integrity. In contrast, poults fed with AFB1 showed a significant increase in Proteobacteria and lower numbers of beneficial bacteria, clearly suggesting gut dysbacteriosis. Moreover, poults supplemented with AFB1 displayed the lowest morphometric parameters and the highest intestinal permeability. Furthermore, poults in the negative and positive control treatments had the lowest cutaneous basophil hypersensitivity response. These findings suggest that HA supplementation enhanced intestinal integrity (shape and permeability), cellular immune response, and healthier gut microbiota composition, even in the presence of dietary exposure to AFB1. These results complement those of the previously published study, suggesting that HA may be a viable dietary intervention to improve gut health and immunity in turkey poults during aflatoxicosis.

Using microsphere or fluorescein tracers and total oocyst output to measure ingestion of material following live-coccidiosis vaccinations.

One method of prevention of coccidiosis in broiler chickens raised without antibiotics relies on coccidiosis vaccination. Live-coccidiosis vaccines carry the risk for pathogenic effects if the Eimeria species overcycle. However, all chicks must receive an appropriate dose of Eimeria oocysts to induce immunity and reduce the risk of adverse effects. At the hatchery, coccidiosis vaccines are administered topically to boxes of chicks by spray or gel-droplet application. Determining the volume of vaccine ingested by individual chicks could provide a means of evaluating the success of different application methods. For each of 2 mass application methods (spray, gel-droplet), we used 3 quantification methodologies to determine the amount of vaccine material ingested by chicks: total oocyst counts from feces collected 5- to 8-days postvaccination; and counts of either microsphere or fluorescein tracers recovered from the gastrointestinal tract 30-min postvaccination. For each quantification methodology, chicks vaccinated via spray or gel-droplet application were compared to chicks vaccinated via oral gavage using the same concentration of oocysts per mL for all groups. Chicks vaccinated via gel-droplet application shed 10-fold more oocysts than those vaccinated by spray application. Individual chick consumption of vaccine material using tracers also revealed that chicks ingested more material following gel-droplet application than spray application, although the magnitude of the difference varied based on quantification methodology. The results of this study suggest that all 3 quantification methodologies can be used to help validate and improve mass vaccine application methods to ensure optimal ingestion, and therefore, coccidiosis vaccination success.

Evaluation of Clostridium septicum hemolytic activity, administration route, and dosage volume of a clostridial dermatitis (cellulitis) bacterin-toxoid on humoral immune response in commercial turkeys.

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.

The Immuno-Suppressive Effects of Cyclic, Environmental Heat Stress in Broiler Chickens: Local and Systemic Inflammatory Responses to an Intradermal Injection of Lipopolysaccharide.

To assess effects of environmental heat stress (HS) on the local and systemic inflammatory responses to lipopolysaccharide (LPS), broilers were reared under thermoneutral (TN) or cyclic HS conditions. Thermoneutral temperatures followed commercial production settings, with HS broilers exposed to 35 °C for 14 h/day from 4 days onward. At 37 days, HS- and TN-broilers were assigned to either LPS (100 µg/mL) or endotoxin-free phosphate-buffered saline (PBS; vehicle) treatments, eight each to HS- and TN-LPS, four each to HS- and TN-PBS. Treatments were administered by intradermal injection of growing feather (GF) pulps; 10 µL/GF; 12 GF/broiler. Blood and GF were collected before and at 6 and 24 h post-injection to assess leukocyte population changes in GF-pulps and blood, reactive oxygen species (ROS) generation and cytokine expression in GF-pulps, and plasma concentrations of alpha-1 acid glycoprotein (AGP-1). HS-LPS broilers had lower (p ≤ 0.05) infiltration of heterophils and macrophages, ROS generation, and inflammatory cytokine expression in GF-pulps, and lacked the increases in heterophil, monocyte, and plasma AGP-1 concentrations observed in TN-LPS broilers. HS-broilers had similar or greater drops in blood lymphocytes 6 h post-LPS or -PBS injection, respectively, and lower baseline levels (p ≤ 0.05) of circulating T- and B-lymphocytes than TN-broilers. Results indicated that cyclic HS reduced the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection.

Characterizing the impact of Enterococcus cecorum infection during late embryogenesis on disease progression, cecal microbiome composition, and early performance in broiler chickens.

Enterococcus cecorum (EC) has been associated with septicemia and early mortality in broiler chickens. There is limited research investigating the pathogenicity of EC field strains obtained from affected birds. The purpose of this study was to evaluate the effect of in-ovo administration into the amnion with different EC field isolates at d 18 of embryogenesis (DOE18). In Exp 1, 7 EC field isolates alone or in combination (EC1-EC3, EC4-EC5, EC6, and EC7) were selected based on phenotypic characteristics and evaluated at different concentrations (1 × 102, 1 × 104, and 1 × 106 CFU/200 µL/embryo) to assess the impact on early performance and macroscopic lesions. Three isolates (n = 3; EC2, EC5, EC7) were selected for additional evaluation based on the significant (P < 0.05) BWG reduction (d 0-21) compared to the negative control (NC) and the presence of macroscopic lesions observed during posting sessions at d 14 and d 21. An additional isolate associated with enterococcal spondylitis was included in Exp 2 (EC11B). Treatment groups for Exp 2 include: 1) NC, 2) EC2, 3) EC5, 4) EC7, and 5) EC11B (n = 90-120/embryos/group). Groups 2 to 5 were challenged at 1 × 102 CFU/200 µL/embryo by in-ovo injection into the amnion at DOE18. Chicks were placed in battery cages for the duration of the study (21 d), and pen weights were recorded at d 0, d 7, d 14, and d 21 to calculate average BW and BWG. At d 14 and d 21 posthatch, liver, spleen, free thoracic vertebrae (FTV), and femoral head (FH) were aseptically collected to enumerate Enterococcus spp. using Chromagar Orientation as the selective media. Cecal contents were collected at d 21 to evaluate the effect of EC challenge on the cecal microbiome composition. There was a significant (P < 0.05) reduction in BW at d 21, and BWG from d 14 to 21 and d 0 to 21, for EC7 and EC11B. Enterococcus cecorum was recovered from the FTV of all challenged groups at d 14 and d 21. The most representative lesions were pericarditis, hydropericardium, focal heart necrosis, and FH osteomyelitis. However, lesions were not uniform across challenged groups or ages (d 14 and d 21). Alpha diversity of the cecal contents was markedly lower in EC5 and EC11B compared to all treatment groups suggesting that EC exposure during late embryogenesis affect the cecal microbiome up to 21 d posthatch. Additionally, these results highlight the differences in pathogenicity of EC strains isolated from field cases and suggest that hatchery exposure to EC during late embryogenesis is a potential route of introduction into a flock.

Impact of Eimeria meleagrimitis and intermittent amprolium treatment on performance and the gut microbiome composition of Turkey poults.

Introduction: Drug-sensitive live coccidiosis vaccines have been used to control coccidiosis and renew drug sensitivity in commercial chicken operations. However, only limited species coverage vaccines have been available for commercial turkey producers. This study aimed to assess the effect of an E. meleagrimitis vaccine candidate, with and without amprolium intervention, on performance and oocyst shedding. Additionally, the effect of vaccination, amprolium treatment, and E. meleagrimitis challenge on intestinal integrity and microbiome composition was evaluated. Methods: Experimental groups included: (1) NC (non-vaccinated, non-challenged control); (2) PC (non-vaccinated, challenged control); (3) VX + Amprol (E. meleagrimitis candidate vaccine + amprolium); and 4) VX (E. meleagrimitis candidate vaccine). For VX groups, 50% of the direct poults were orally vaccinated at DOH with 50 sporulated E. meleagrimitis oocysts and were comingled with contact or non-vaccinated poults for the duration of the study. From d10-14, VX + Amprol group received amprolium (0.024%) in the drinking water. All groups except NC were orally challenged with 95K E. meleagrimitis sporulated oocysts/mL/poult at d23. At d29, ileal and cecal contents were collected for 16S rRNA gene-based microbiome analysis. Results and Discussion: VX did not affect performance during the pre-challenge period. At d23-29 (post-challenge), VX groups had significantly (P < 0.05) higher BWG than the PC group. Contacts and directs of VX groups in LS had significantly reduced compared to PC. As anticipated, amprolium treatment markedly reduced fecal and litter OPG for the VX + Amprol group compared to the VX group which did not receive amprolium. The ileal and cecal content results showed that the PC group had different bacterial diversity and structure, including alpha and beta diversity, compared to NC. Linear discriminant analysis Effect Size (LEfSe) identified that Lactobacillus salivarius (ASV2) was enriched in PC's ileal and cecal content. Compared to NC and PC, the vaccinated groups showed no distinct clusters, but there were similarities in the ileal and cecal communities based on Bray-Curtis and Jaccard distances. In conclusion, these results indicate that vaccination with this strain of E. meleagrimitis, with or without amprolium intervention, caused a very mild infection that induced protective immunity and challenge markedly affected both the ileal and cecal microbiome.

Research Note: Isolation, speciation, and anticoccidial sensitivity of Eimeria spp. recovered from wild turkey feces in the United States.

Between 2018 and 2020, over 100 wild turkey fecal samples were collected from the Eastern and Central thirds of the United States, where commercial turkey production is uncommon. We hypothesized that anticoccidial-sensitive Eimeria spp. would be present in wild turkey fecal samples. Samples containing Eimeria spp. oocysts were amplified in vivo. If propagation was successful, the samples were PCR-speciated and subjected to anticoccidial sensitivity testing (AST) for key members of both ionophore and chemical categories of anticoccidial drugs. The purpose of this study was to isolate Eimeria spp. relevant to commercial turkey production that possessed sensitivity to monensin, zoalene, and amprolium. Future research would evaluate the efficacy of wild turkey Eimeria spp. as vaccine candidates for reducing coccidiosis in commercial turkey flocks utilizing single oocyst-derived stocks obtained in the present study.

An overview of the use of bacteriophages in the poultry industry: Successes, challenges, and possibilities for overcoming breakdowns.

The primary contaminants in poultry are Salmonella enterica, Campylobacter jejuni, Escherichia coli, and Staphylococcus aureus. Their pathogenicity together with the widespread of these bacteria, contributes to many economic losses and poses a threat to public health. With the increasing prevalence of bacterial pathogens being resistant to most conventional antibiotics, scientists have rekindled interest in using bacteriophages as antimicrobial agents. Bacteriophage treatments have also been investigated as an alternative to antibiotics in the poultry industry. Bacteriophages' high specificity may allow them only to target a specific bacterial pathogen in the infected animal. However, a tailor-made sophisticated cocktail of different bacteriophages could broaden their antibacterial activity in typical situations with multiple clinical strains infections. Bacteriophages may not only be used in terms of reducing bacterial contamination in animals but also, under industrial conditions, they can be used as safe disinfectants to reduce contamination on food-contact surfaces or poultry carcasses. Nevertheless, bacteriophage therapies have not been developed sufficiently for widespread use. Problems with resistance, safety, specificity, and long-term stability must be addressed in particular. This review highlights the benefits, challenges, and current limitations of bacteriophage applications in the poultry industry.

Humic Acids Preparation, Characterization, and Their Potential Adsorption Capacity for Aflatoxin B1 in an In Vitro Poultry Digestive Model.

Vermicompost was used for humic acid (HA) preparation, and the adsorption of aflatoxin B1 (AFB1) was investigated. Two forms of HA were evaluated, natural HA and sodium-free HA (SFHA). As a reference, a non-commercial zeolitic material was employed. The adsorbents were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), energy-dispersive X-ray spectroscopy (EDS), zeta potential (ζ-potential), scanning electron microscopy (SEM), and point of zero charge (pHpzc). The adsorbent capacity of the materials when added to an AFB1-contaminated diet (100 µg AFB1/kg) was evaluated using an in vitro model that simulates the digestive tract of chickens. Characterization results revealed the primary functional groups in HA and SFHA were carboxyl and phenol. Furthermore, adsorbents have a highly negative ζ-potential at the three simulated pH values. Therefore, it appears the main influencing factors for AFB1 adsorption are electrostatic interactions and hydrogen bonding. Moreover, the bioavailability of AFB1 in the intestinal section was dramatically decreased when sorbents were added to the diet (0.2%, w/w). The highest AFB1 adsorption percentages using HA and SFHA were 97.6% and 99.7%, respectively. The zeolitic material had a considerable adsorption (81.5%). From these results, it can be concluded that HA and SFHA from vermicompost could be used as potential adsorbents to remove AFB1 from contaminated feeds.

In vitro and in vivo evaluation of chlorhexidine salts as potential alternatives to potassium dichromate for Eimeria maxima M6 oocyst preservation.

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.

Assessing the effects of a mixed Eimeria spp. challenge on performance, intestinal integrity, and the gut microbiome of broiler chickens.

A mixed Eimeria spp. challenge model was designed to assess the effects of challenge on broiler chicken performance, intestinal integrity, and the gut microbiome for future use to evaluate alternative strategies for controlling coccidiosis in broiler chickens. The experimental design involved broiler chickens divided into two groups: a control group (uninfected) and a positive control group, infected with Eimeria acervulina (EA), Eimeria maxima (EM), and Eimeria tenella (ET). At day-of-hatch, 240 off-sex male broiler chicks were randomized and allocated to one of two treatment groups. The treatment groups included: (1) Non-challenged (NC, n = 5 replicate pens); and (2) challenged control (PC, n = 7 replicate pens) with 20 chickens/pen. Pen weights were recorded at d0, d16, d31, d42, and d52 to determine average body weight (BW) and (BWG). Feed intake was measured at d16, d31, d42, and d52 to calculate feed conversion ratio (FCR). Four diet phases included a starter d0-16, grower d16-31, finisher d31-42, and withdrawal d42-52 diet. At d18, chickens were orally challenged with 200 EA, 3,000 EM, and 500 ET sporulated oocysts/chicken. At d24 (6-day post-challenge) and d37 (19-day post-challenge), intestinal lesion scores were recorded. Additionally, at d24, FITC-d was used as a biomarker to evaluate intestinal permeability and ileal tissue sections were collected for histopathology and gene expression of tight junction proteins. Ileal and cecal contents were also collected to assess the impact of challenge on the microbiome. BWG and FCR from d16-31 was significantly (p < 0.05) reduced in PC compared to NC. At d24, intestinal lesion scores were markedly higher in the PC compared to the NC. Intestinal permeability was significantly increased in the PC group based on serum FITC-d levels. Cadherin 1 (CDH1), calprotectin (CALPR), and connexin 45 (Cx45) expression was also upregulated in the ileum of the PC group at d24 (6-day post-challenge) while villin 1 (VIL1) was downregulated in the ileum of the PC group. Additionally, Clostridium perfringens (ASV1) was enriched in the cecal content of the PC group. This model could be used to assess the effect of alternative coccidiosis control methods during the post-challenge with EA, EM, and ET.

The effects of essential oil from Lippia origanoides and herbal betaine on performance, intestinal integrity, bone mineralization and meat quality in broiler chickens subjected to cyclic heat stress.

Essential oils (EO) affect performance, intestinal integrity, bone mineralization, and meat quality in broiler chickens subjected to cyclic heat stress (HS). Day-of-hatch Cobb 500 male broiler chicks (n = 475) were randomly divided into four groups. Group 1: No heat stress (Thermoneutral) + control diets with no antibiotics; Group 2: heat stress control + control diets; Group 3: heat stress + control diets supplemented with thymol chemotype (45 ppm) and herbal betaine (150 ppm) formulation EO1; Group 4: heat stress + control diets supplemented with phellandrene (45 ppm) and herbal betaine (150 ppm) formulation EO2. From day 10-42, the heat stress groups were exposed to cyclic HS at 35°C for 12 h (8:00-20:00). BW, BWG, FI, and FCRc were measured at d 0, 10, 28, and 42. Chickens were orally gavaged with FITC-d on days 10 (before heat stress) and 42. Morphometric analysis of duodenum and ileum samples and bone mineralization of tibias were done. Meat quality was assessed on day 43 with ten chickens per pen per treatment. Heat stress reduced BW by day 28 (p < 0.05) compared to thermoneutral chickens. At the end of the trial, chickens that received both formulations of EO1 and EO2 had significantly higher BW than HS control chickens. A similar trend was observed for BWG. FCRc was impaired by EO2 supplementation. There was a significant increase in total mortality in EO2 compared with EO1 EO1 chickens had lower FITC-d concentrations at day 42 than the HS control. In addition, EO1 treatment is not statistically different if compared to EO2 and thermoneutral. Control HS broilers had significantly lower tibia breaking strength and total ash at day 42 than heat-stressed chickens supplemented with EO1 and EO2. Heat stress affected intestinal morphology more than thermoneutral chickens. EO1 and EO2 improved intestinal morphology in heat-stressed chickens. Woody breast and white striping were more common in thermoneutral chickens than heat stress chickens. In conclusion, the EO-containing diet could improve broiler chicken growth during cyclic heat stress, becoming increasingly relevant in antibiotic-free production in harsh climates.

Evaluation of the efficacy of humic acids to counteract the toxic effects of aflatoxin B1 in turkey poults.

This study aims to evaluate the efficacy of humic acid (HA) from worm compost as an adsorbent for aflatoxin B1 (AFB1) in turkey poults. The experiment involved the inclusion of 0.25% (w/w) HA in the diet of turkey poults consuming aflatoxin-contaminated feed (250 ng AFB1/g). A total of 350 1-day-old female Nicholas-700 turkey poults were randomly allocated to five equal groups: negative control (basal diet); positive control (basal diet + 250 ng AFB1/g; HA (basal diet + 0.25% HA); HA + AFB1 (basal diet + HA + 250 ng AFB1/g); and zeolite + AFB1 (basal diet + 0.25% zeolite + 250 ng AFB1/g). Each group had seven replicates of 10 poults (n = 70). The impact of HA addition was evaluated in terms of performance parameters, relative organ weights, liver histological lesions, and serum biochemical and hematological constituents. In general, the addition of HA improved body weight (BW), body weight gain (BWG), and feed conversion rate (FCR). Furthermore, HA effectively mitigated the toxic effects caused by AFB1 in the majority of the analyzed variables. The results indicated that HA effectively counteracted the AFB1-induced toxic effects in turkey poults. Based on these findings, it can be concluded that HA is capable of removing AFB1 from the contaminated diet.