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

IntroductionCoccidiosis 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 associationsMethodsIn 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 discussionThese 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

The Boron Advantage: The Evolution and Diversification of Boron’s Applications in Medicinal Chemistry

In this review, the history of boron’s early use in drugs, and the history of the use of boron functional groups in medicinal chemistry applications are discussed. This includes diazaborines, boronic acids, benzoxaboroles, boron clusters, and carboranes. Furthermore, critical developments from these functional groups are highlighted along with recent developments, which exemplify potential prospects. Lastly, the application of boron in the form of a prodrug, softdrug, and as a nanocarrier are discussed to showcase boron’s emergence into new and exciting fields. Overall, we emphasize the evolution of organoboron therapeutic agents as privileged structures in medicinal chemistry and outline the impact that boron has had on drug discovery and development

Value and Limitations of Formaldehyde for Hatch Cabinet Applications: The Search for Alternatives

Pioneer colonization by beneficial microorganisms promote a shift in the composition of the gut microbiota, excluding opportunistic pathogens. Commercially, the horizontal transmission of both apathogenic and pathogenic organisms is common during the hatching phase. The microbial bloom occurs as the humidity rises during hatch, exposing naïve chicks to a plethora of potentially harmful microbes. Horizontal transmission or introduction of pathogens may occur as infected chicks hatch or during handling after hatch pull. Moreover, contaminated infertile or non-viable embryonated eggs can serve as reservoirs for pathogenic organisms and even rupture during incubation. The organisms within the contents of these eggs can penetrate the shell of the embryonated eggs and subsequently contaminate the entire cabinet. Formaldehyde fumigation is commonly applied during the hatching phase to control the microbial bloom in the environment, but does not penetrate the eggshell prior to hatch. Additionally, this fumigation technique eliminates microbial organisms in the environment at hatch, including beneficial species. Furthermore, prolonged exposure to formaldehyde can damage the tracheal epithelia of neonatal chicks increasing susceptibility to infection by opportunistic microbes. Laboratory challenge models that mimic the microbial bloom that occurs in commercial hatch cabinets can be used to evaluate effective alternatives to control the microbial bloom and promote colonization by beneficial bacteria without the use of formaldehyde fumigation

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

ABSTRACT: 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

A Fast and Inexpensive Protocol for Empirical Verification of Neutralizing Epitopes in Microbial Toxins and Enzymes

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

Evaluación del desprendimiento de oocistos de Eimeria maxima y Eimeria acervulina en pollos de engorde

The purpose of the present study was to evaluate the day and the time of sample collection of an experimental challenge with Eimeria maxima (EM) and Eimeria acervulina (EA) in broiler chickens. Oneday old male Cobb-Vantress broiler chickens were randomly allocated to one of three groups with ten replicates (n=8 chickens/replicate). Chickens were placed in battery cages with a controlled ageappropriate environment: Group 1) Negative control (no challenge or treatment); 2) Challenge control (Eimeria challenge only); 3) Challenge + Salinomycin. Challenged chickens were orally gavaged with the mixed culture of EM/EA (10,000 sporulated EM containing 4% wild-type EA) at 14 days of age. Performance parameters were recorded at days 7, 14, 20, and 23. Lesions scores were recorded post-mortem on days 20 and 23. Oocyst per gram (OPG) was performed on days six, seven, and eight post-challenge, and samples were collected at 9:00 AM and 6:00 PM on each day, respectively. Oocyst counts were significantly different (P < 0.05) between morning and afternoon on day six post coccidia challenge. The results of this study show that the day and the time at which samples are collected can have a significant effect on the reliability and validity of dataEl propósito del presente estudio fue evaluar el día y la hora de recolección de la muestra de un desafío experimental con Eimeria maxima (EM) y Eimeria acervulina (EA) en pollos de engorde. Los pollos de engorde macho Cobb-Vantress de un día de edad se asignaron aleatoriamente a uno de tres grupos con diez réplicas (n = 8 pollos / réplica). Los pollos se colocaron en jaulas de batería con un entorno controlado apropiado para la edad: Grupo 1) Control negativo (sin desafío o tratamiento); 2) Control de desafío (solo desafío Eimeria); 3) Desafío + salinomicina. A los 14 días de edad, los pollos fueron desafiados por vía oral con el cultivo mixto de EM / EA (10,000 EM esporulados que contenían 4% de EA de tipo salvaje). Los parámetros de rendimiento se registraron en los días 7, 14, 20 y 23. Los puntajes de las lesiones se registraron post mortem en los días 20 y 23. El oocisto por gramo (OPG) se realizó en los días seis, siete y ocho después del desafío, y muestras se recolectaron a las 9:00 a.m. y a las 6:00 p.m. de cada día, respectivamente. Los recuentos de oocistos fueron significativamente diferentes (P < 0.05) entre la mañana y la tarde en el día seis después del desafío con coccidios. Los resultados de este estudio muestran que el día y la hora en que se recolectan las muestras pueden tener un efecto significativo en la confiabilidad y validez de los dato

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

ABSTRACT: 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

Assessment of Lippia origanoides Essential Oils in a Salmonella typhimurium, Eimeria maxima, and Clostridium perfringens Challenge Model to Induce Necrotic Enteritis in Broiler Chickens

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 &lt; 0.05) compared with Group 2. Digested feed supernatant, supplemented with LEO and inoculated with CP, reduced CP burden (p &lt; 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

Experimental Cyclic Heat Stress on Intestinal Permeability, Bone Mineralization, Leukocyte Proportions and Meat Quality in Broiler Chickens

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 &deg;C for 12 h/day from days 7&ndash;42. Performance parameters, intestinal permeability, bone parameters, meat quality, and leukocyte proportions were estimated. There was a significant (p &lt; 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