A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge.

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (108 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 106 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds.

Morphological Assessment and Biomarkers of Low-Grade, Chronic Intestinal Inflammation in Production Animals.

The complex interaction between the intestinal mucosa, the gut microbiota, and the diet balances the host physiological homeostasis and is fundamental for the maximal genetic potential of production animals. However, factors such as chemical and physical characteristics of the diet and/or environmental stressors can continuously affect this balance, potentially inducing a state of chronic low-grade inflammation in the gut, where inflammatory parameters are present and demanding energy, but not in enough intensity to provoke clinical manifestations. It's vital to expand the understanding of inflammation dynamics and of how they compromise the function activity and microscopic morphology of the intestinal mucosa. These morphometric alterations are associated with the release of structural and functional cellular components into the feces and the blood stream creating measurable biomarkers to track this condition. Moreover, the identification of novel, immunometabolic biomarkers can provide dynamic and predictors of low-grade chronic inflammation, but also provide indicators of successful nutritional or feed additive intervention strategies. The objective of this paper is to review the mechanisms of low-grade inflammation, its effects on animal production and sustainability, and the biomarkers that could provide early diagnosis of this process and support studies of useful interventional strategies.

The use of histológical parameters to assess intestinal and liver health on broilers challenged isolatedly and simultaneously with cyclopiazonic acid and aflatoxin / B1 El uso de parámetros histológicos para evaluar la salud intestinal y hepática en pollos de engorde desafiados de forma aislada y simultánea con ácido ciclopiazónico y aflatoxina B1

Abstract Mycotoxins contaminate agricultural commodities, which contaminates animals. These toxins can damage vital organs, such as the liver, as well as the epithelial tissue. Among these mycotoxins are aflatoxin B1 (AFB1) and cyclopiazonic acid (CPA), which can occur simultaneously in food. In broilers, mycotoxicosis has an economic impact due to several factors, such as low feed conversion rate, incidence of other diseases, and interference with reproductive capacity, all of which may lead to a public health problem. The aim of the present study was to histologically assess, through the I See Inside (ISI) method, harmful effects on broiler liver, duodenum, jejunum, and ileum in the presence of AFB1 and CPA isolatedly and simultaneously. Groups challenged with mycotoxins showed significant damage to both gut and liver fragments. All challenged-groups in all fragments impaired the parameters analyzed for intestinal epithelium. In the liver, AFB1 was predominantly harmful when the parameters were analyzed separately, but when analyzing the total ISI score, CPA was also found to be harmful to this organ. The other point analyzed was the great variation between the weights of the birds contaminated by mycotoxin while the negative control group presents a lesser variation.

Resumen Las micotoxinas contaminan los productos agrícolas, que a su vez contaminan a los animales. Estas toxinas pueden dañar órganos vitales, como el hígado y el tejido epitelial. Entre estas micotoxinas se encuentran la aflatoxina B1 (AFB1) y el ácido ciclopiazónico (CPA), que pueden hallarse simultáneamente en los alimentos. En los pollos de engorde, la micotoxicosis tiene un impacto económico debido a varios factores, como la baja tasa de conversión alimenticia, la incidencia de otras enfermedades y la interferencia de la capacidad reproductiva, que pueden llevar a un problema de salud pública. El objetivo de la presente investigación es la de evaluar histológicamente, a través del método "I See Inside" (ISI), los efectos nocivos sobre el hígado, duodeno, yeyuno e íleon de pollos de engorde en presencia de AFB1 y CPA de forma aislada y simultánea. Los grupos desafiados con micotoxinas presentaron un daño significativo tanto en el intestino como en los fragmentos del hígado. Todos los grupos tratados tuvieron alteraciones en los parámetros analizados para el epitelio intestinal. En el hígado, AFB1 fue predominantemente dañino cuando los parámetros se analizaron por separado, pero al examinar la puntuación ISI total, también se encontró que el CPA era perjudicial para este órgano. Otra cuestión que fue investigada fue la gran variación entre los pesos de las aves contaminadas por micotoxinas mientras el grupo de control negativo presentó una variación menor.

Novel Models for Chronic Intestinal Inflammation in Chickens: Intestinal Inflammation Pattern and Biomarkers.

For poultry producers, chronic low-grade intestinal inflammation has a negative impact on productivity by impairing nutrient absorption and allocation of nutrients for growth. Understanding the triggers of chronic intestinal inflammation and developing a non-invasive measurement is crucial to managing gut health in poultry. In this study, we developed two novel models of low-grade chronic intestinal inflammation in broiler chickens: a chemical model using dextran sodium sulfate (DSS) and a dietary model using a high non-starch polysaccharide diet (NSP). Further, we evaluated the potential of several proteins as biomarkers of gut inflammation. For these experiments, the chemical induction of inflammation consisted of two 5-day cycles of oral gavage of either 0.25mg DSS/ml or 0.35mg DSS/ml; whereas the NSP diet (30% rice bran) was fed throughout the experiment. At four times (14, 22, 28 and 36-d post-hatch), necropsies were performed to collect intestinal samples for histology, and feces and serum for biomarkers quantification. Neither DSS nor NSP treatments affected feed intake or livability. NSP-fed birds exhibited intestinal inflammation through 14-d, which stabilized by 36-d. On the other hand, the cyclic DSS-treatment produced inflammation throughout the entire experimental period. Histological examination of the intestine revealed that the inflammation induced by both models exhibited similar spatial and temporal patterns with the duodenum and jejunum affected early (at 14-d) whereas the ileum was compromised by 28-d. Calprotectin (CALP) was the only serum protein found to be increased due to inflammation. However, fecal CALP and Lipocalin-2 (LCN-2) concentrations were significantly greater in the induced inflammation groups at 28-d. This experiment demonstrated for the first time, two in vivo models of chronic gut inflammation in chickens, a DSS and a nutritional NSP protocols. Based on these models we observed that intestinal inflammation begins in the upper segments of small intestine and moved to the lower region over time. In the searching for a fecal biomarker for intestinal inflammation, LCN-2 showed promising results. More importantly, calprotectin has a great potential as a novel biomarker for poultry measured both in serum and feces.

Field evaluation of feeding spray-dried plasma in the starter period on final performance and overall health of broilers.

The effect of feeding spray-dried plasma (SDP) during the starter period was evaluated with a commercial broiler integrator on performance and overall health of broilers. The I See Inside (ISI) methodology assessing gut health in broilers was used as a tool to evaluate the impact of dietary interventions under commercial conditions. One hundred farms with approximately 1.1 million broilers were used at a Brazilian broiler integrator. Two groups of farms were fed either a control or an SDP diet containing 1% SDP, from 0 to 10 d of age. Diets were formulated to have similar nutritional density, containing zinc bacitracin and CuSO4 from 0 to 28 d. After 10 d, both groups were fed common commercial diets. Performance data were analyzed together or by type of ventilation system: positive pressure or negative pressure. Birds were sent to market as they reached 3.05 kg; therefore, age at slaughter (AS) was evaluated as a dependent variable along with other performance measures. From the 100 farms in the trial, 35 (16 control and 19 SDP farms) were selected for the assessment of broilers health, biosecurity, and local management. For that, 6 broilers per farm at 14 ± 2 d of age were necropsied and ileum sampled for the ISI methodology evaluation. Biosecurity and management were also evaluated to obtain the influence of those parameters on animal health. SDP-fed birds demonstrated improved feed conversion ratio, reduced mortality, and 1 d less for AS (P < 0.05) vs. control group (P < 0.05) regardless of the type of ventilation. During necropsy, birds fed SDP showed lower coccidiosis and locomotor system lesions as the overall ISI score compared to controls. Histologic intestinal alterations were also lower in SDP-fed broilers (P < 0.05). In conclusion, feeding 1% SDP in the starter period to broilers resulted in improved performance and health under both good and bad management and biosecurity standards independent of the type of ventilation. Overall, there was good agreement between the ISI method and performance improvements observed.

Comparing performance and resistance of two broilers breeds challenged by Eimeria acervulina.

The proposal on the ban of anticoccidials drugs from poultry feed highlights the importance of broiler breeders resistance to Eimeria as a criteria for animal genetic selection. The aim of this study was to compare the resistance between two commercial broiler breeds to Eimeria acervulina. Eight hundred male chicks from two commercial breeds (400 animals each) were housed from 1-42 days of age and randomly divided into 4 treatments with 8 replicates of 25 birds each. Following a factorial design, the treatments were AU, BU (breed A and B, unchallenged), AC and BC (breed A and B, challenged at 14 days of age by gavage with 1 × 106 oocysts of a field strain of E. acervulina). From 1-42 days, feed intake, body weight gain (BWG), feed conversion ratio (FCR), feed efficiency and mean weight (MW) were weekly measured. For the clinical evaluation, feces were sampled from 18 to 24 days for the counting of excreted oocysts and twenty birds per treatment were euthanized at 20 days of age for the scoring of macroscopic lesions in the gut. The breeds did not show statistical differences in performance after the Eimeria challenge in all phases. Breed A presented a higher increase in FCR from 21 to 45 days in comparison to the breed B, which presented a deeper reduction in FI, BWG and MW at all periods post-infection when compared to the breed A. Despite of the challenge, breed A presented better performance indexes (P < 0.05) up to the 28 days of age in comparison to the breed B, however, no significant difference in performance was detected between the breeds at the final age of 42 days. Regardless of the breed, the challenged birds presented significant worsens in all performance parameters from 14 to 42 days of age. The breed B presented a higher (P < 0.05) fecal count of oocysts from 19 to 21 days and higher (P < 0.05) score of macroscopic lesions in the gut at 21 days in comparison to the breed A. The clinical evaluation pointed out a lower resistance of the breed B to the E. acervulina challenge, which could result in significant impacts on the performance in field production conditions.

Basal and Infectious Enteritis in Broilers Under the I See Inside Methodology: A Chronological Evaluation.

Recently, the inflammation of the intestinal mucosa has been related to many diseases in humans and animals. The concept of Microscopic Enteritis (ME) used in human pathology through the Marsh classification system has no counter-part in veterinary medicine. In poultry science, the I See Inside (ISI) methodology, unlike the current linear measures of villi and crypts, generates possibilities to describe and understand the avian ME. Through specific parameters, graded from 0 to 3, the model links proliferative and/or inflammatory reactions in the intestinal layers to some loss in performance. Herein, two trials were conducted in order to describe the development of ME through the ISI methodology in chickens challenged or not with Eimeria spp. and Clostridium perfringens. In each trial, a total of 64 birds were divided in 2 treatments with 4 replicates containing 8 birds each: non-challenged (NCH) and challenged (CH) through gavage with an Eimeria spp. vaccine at 1 day of age and 108 CFU/mL of Clostridium perfringens administered at 10, 11, and 12 days of age. At 7, 14, 21, and 28 days of age birds were euthanized and samples of ileum and liver were collected for ISI evaluation, cytokines and presence of macrophages, CD4+ and CD8+ cell. The results allowed the description of the avian Microscopic Enteritis and of its two basic components: a basal enteritis (BE) in NCH broilers, over which the infectious enteritis is developed in CH birds. In addition, the chronology of ME translated by the ISI methodology parameters were associated to losses in zootechnical performance.

Applying I see inside histological methodology to evaluate gut health in broilers challenged with Eimeria.

The present study evaluated the effects of coccidiosis on histological parameters and performance of broilers submitted to a mild challenge with Eimeria sp. A total of 132 broilers were randomly divided into two groups with 6 replicates of 11 birds each: negative control (NC) - birds uninfected and challenge (CH) - birds infected by gavage at day-one with 10x the manufacturer recommended dose of Bio-Coccivet R® live vaccine by Biovet, with strains of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. praecox, E. tenella and E. mitis. From 1 to 28 d of age, weekly the zootechnical performance was evaluated and samples of the liver, duodenum, jejunum and ileum were collected and submitted to histological analysis by the I See Inside methodology (ISI). The ISI methodology is a metric evaluation of histological alterations in the intestine and liver. Which translates macroscopic and microscopic alterations into numbers and allows their correlation with the animal zootechnical performance. Pearson's correlation was used to demonstrates how the damages to the gut, evaluated by ISI method, could affect negatively the performance of broilers. The presence of oocysts in histological analysis of the jejunum and ileum at 7d indicate a negative effect on performance over the next 2 weeks (14d and 21d). When the ISI total score was measured in the ileum at 14d, we observed a strong negative effect on zootechnical performance from that period on 14d, 21d and 28d. The obtained data demonstrates that the higher the ISI scores, the worse are the zootechnical performance results of broilers challenged with Eimeria sp. and the I See Inside scores could be applied to evaluate coccidiosis effect on performance in future.

Applying I see inside histological methodology to evaluate gut health in broilers challenged with Eimeria.

The present study evaluated the effects of coccidiosis on histological parameters and performance of broilers submitted to a mild challenge with Eimeria sp. A total of 132 broilers were randomly divided into two groups with 6 replicates of 11 birds each: negative control (NC) - birds uninfected and challenge (CH) - birds infected by gavage at day-one with 10x the manufacturer recommended dose of Bio-Coccivet R® live vaccine by Biovet, with strains of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. praecox, E. tenella and E. mitis. From 1 to 28 d of age, weekly the zootechnical performance was evaluated and samples of the liver, duodenum, jejunum and ileum were collected and submitted to histological analysis by the I See Inside methodology (ISI). The ISI methodology is a metric evaluation of histological alterations in the intestine and liver. Which translates macroscopic and microscopic alterations into numbers and allows their correlation with the animal zootechnical performance. Pearson's correlation was used to demonstrates how the damages to the gut, evaluated by ISI method, could affect negatively the performance of broilers. The presence of oocysts in histological analysis of the jejunum and ileum at 7d indicate a negative effect on performance over the next 2 weeks (14d and 21d). When the ISI total score was measured in the ileum at 14d, we observed a strong negative effect on zootechnical performance from that period on 14d, 21d and 28d. The obtained data demonstrates that the higher the ISI scores, the worse are the zootechnical performance results of broilers challenged with Eimeria sp. and the I See Inside scores could be applied to evaluate coccidiosis effect on performance in future.