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The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Objective: Probiotics have been used in poultry production to improve the performance and health of chickens raised without antibiotics. The combination of different probiotic strains has been used with the hope of conferring multiple benefits to the host. However, the inclusion of several strains does not necessarily boost benefits. There is a lack of studies that compare the efficacy of multi-strain probiotics to their individual components. In this study, the effects of a Bacillus-based probiotic product mix containing B. coagulans, B. licheniformis, B. pumilus, and B. subtilis against Clostridium perfringens were tested in vitro using a co-culture method. The individual strains and different combinations of the strains used in the product were also tested against C. perfringens., Results: The probiotic product mix tested in this study did not show effects against C. perfringens (P = 0.499). When tested individually, the strain of B. subtilis was the most efficient strain to decrease C. perfringens concentrations (P ≤ 0.01), and the addition of other Bacillus species strains significantly decreased its efficacy against C. perfringens. We concluded that the probiotic mix of Bacillus strains used in this study (B. coagulans, B. licheniformis, B. pumilus and B subtilis) was not effective in decreasing C. perfringens concentrations in vitro. However, when deconstructing the probiotic, the strain of B. subtilis alone or combined with the strain of B. licheniformis were effective against C. perfringens. This suggests that the anticlostridial properties of the particular strains of Bacillus used in this study were negatively affected when combined with other Bacillus spp. strains., (© 2023. The Author(s).)

A dust generator was developed to disperse and maintain a desired concentration of airborne dust in a controlled environment chamber to study poultry physiological response to sustained elevated levels of particulate matter. The goal was to maintain an indicated PM10 concentration of 50 µg/m 3 of airborne dust in a 3.7 m × 4.3 m × 2.4 m (12 ft × 14 ft × 8 ft) controlled environment chamber. The chamber had a 1.5 m 3 /s (3200 cfm) filtered recirculation air handling system that regulated indoor temperature levels and a 0.06 m 3 /s (130 cfm) exhaust fan that exchanged indoor air for fresh outdoor air. Dry powdered red oak wood dust that passed through an 80-mesh screen cloth was used for the experiment. The dust generator metered dust from a rectangular feed hopper with a flat bottom belt to a 0.02 m 3 /s (46 cfm) centrifugal blower. A vibratory motor attached to the hopper ran only when the belt was operated to prevent bridging of powdered materials and to provide an even material feed rate. A laser particle counter was used to measure the concentration of airborne dust and provided feedback to an Arduino-based control system that operated the dust generator. The dust generator was operated using a duty cycle of one second on for every five seconds off to allow time for dispersed dust to mix with chamber air and reach the laser particle counter. The control system maintained an airborne PM10 dust concentration of 54.92 ± 6.42 µg/m 3 in the controlled environment chamber during six weeks of continuous operation using red oak wood dust. An advantage of the automatically controlled dust generator was that it continued to operate to reach the setpoint concentration in response to changes in material flow due to humidity, partial blockages, and non-uniform composition of the material being dispersed. Challenges included dust being trapped by the recirculation filter and the exhaust fan removing airborne dust from the environmental chamber.

Avian necrotic enteritis (NE) is an infectious disease that impacts poultry worldwide causing economic losses. Discontinued use of antimicrobial growth promoters has been associated with high incidence of the disease, which has led to a necessity for finding new therapeutic alternatives. Flavonoids are polyphenolic compounds that have been studied for their health-promoting properties in animals and humans. This study presents a flavonoid-rich corn (PennHFD), as a potential alternative for ameliorating NE in broiler chickens. The effect of a diet formulated with PennHFD was compared to a diet based on commercially available corn in chickens subjected to a controlled challenge of NE based on a co-infection of Eimeria maxima and Clostridium perfringens. Birds fed on the PennHFD-based diet had lower incidence of intestinal lesions (P = 0.048), higher body weight gain (P < 0.01), lower feed conversion ratio (P < 0.01), and lower mortality rates (P = 0.023) compared to the control diet. Therefore, we concluded that the inclusion of the high-flavonoid PennHFD reduces the severity of an experimental challenge of NE in broiler chickens., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

An ammonia gas (NH 3 ) generator was developed to maintain a set concentration of ammonia gas in a controlled environment chamber to study poultry physiological responses to sustained elevated levels of ammonia gas. The goal was to maintain 50 parts per million (ppm) of ammonia gas in a 3.7 m × 4.3 m × 2.4 m (12 ft × 14 ft × 8 ft) controlled environment chamber. The chamber had a 1.5 m 3 /s (3000 cfm) recirculation system that regulated indoor temperature and humidity levels and a 0.06 m 3 /s (130 cfm) exhaust fan that exchanged indoor air for fresh outdoor air. The ammonia generator was fabricated by coupling ultrasonic humidifiers with an Arduino-based microcontroller and a metallic oxide MQ-137 ammonia gas sensor. Preliminary evaluation under steady conditions showed the average MQ-137 gas sensor accuracy was within 1.4% of the 65.4 ppm concentration measured using a highly accurate infrared gas analyzer. Further evaluation was performed for a setpoint concentration of 50 ppm where ammonia generator reservoirs were filled with 2% or 10% ammonia liquid. For the system tested, it was found that two generators operating at the same time filled with 3.8 L (1.0 gallon) of 2% ammonia cleaning liquid each (7.6 L or 2.0 gallons total) could maintain a gas level of 49.45 ± 0.79 ppm in the chamber air for a duration of 30 h before refilling was required. One generator filled with 3.8 L of 10% ammonia cleaning liquid could maintain 51.24 ± 1.53 ppm for 195 h. Two ammonia generators were deployed for a six-week animal health experiment in two separate controlled environment chambers. The two ammonia generators maintained an average ammonia concentration of 46.42 ± 3.81 ppm and 45.63 ± 4.95 ppm for the duration of the experiment.

Bacterial chondronecrosis with osteomyelitis (BCO) is a septic necrosis of the skeletal system of unknown origin and an important cause of lameness in broiler chickens. Epithelial inflammation has been proposed as an avenue for bacterial translocation leading to BCO. We evaluated the effect of subclinical necrotic enteritis (SNE), an intestinal inflammatory event, with the development of BCO. In each of two experiments, chickens were divided into three treatments: 1) SNE challenge, including both dietary (wheat- and fish-based diet) and intestinal pathogenic challenges ( Eimeria maxima and Clostridium perfringens ), 2) dietary challenge only, and 3) control diet (corn- and soy-based diet). Floor ramps were introduced as part of an established method for increasing the frequency of BCO. The efficacy of the SNE challenge was corroborated by necropsy evaluation of a representative sample of the population. At the end of each experiment, all birds were evaluated for BCO. A high incidence of BCO was found, even in birds with no external signs of lameness. However, the incidence of BCO was not correlated with the intestinal challenge. Conclusions: under the conditions used in these studies, a treatment that is associated with severe damage to the intestinal mucosa does not change the incidence of BCO in broiler chickens., Competing Interests: The authors declare no conflict of interest.

Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/microL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leukocyte concentration in blood did not differ between groups, but the percentage of blood lymphocytes was lower in broilers from the Red#3+PG group compared with birds from the control group (52.4+/-2.9 and 56.9+/-2.9%, respectively). Cells recovered from the lavage fluid from both groups were primarily heterophils. The concentration of leukocytes was greater in the lavage fluid of broilers from the Red#3+PG group compared with broilers from the control group (763.2+/-158.7 and 402.9+/-62.6 white blood cells/microL, respectively), but the proportions among leukocytes were not different between the 2 groups. We propose that the increased concentration of leukocytes present within the airways was one of the components that enabled broilers pre-treated with aerosolized Red#3+PG to exhibit PH responses to intratracheal lipopolysaccharide.

Broilers reared under commercial conditions inhale irritant gases and aerosolized particulates contaminated with gram-negative bacteria and bacterial lipopolysaccharide (LPS). Previous studies demonstrated that i.v. injections of LPS can trigger an increase in the pulmonary arterial pressure (PAP); however, the pulmonary hemodynamic response to aerosolized LPS entering via the most common route, the respiratory tract, had not been evaluated in broilers. In experiment 1, broilers reared on new wood shavings litter in clean environmental chambers either were not pretreated (control group) or were pretreated via aerosol inhalation of substances (food color dyes and propylene glycol) known to sensitize the airways. One day later, the broilers were anesthetized, catheterized to record the PAP, and an intratracheal aerosol spray of LPS (1 mL of 2 mg/mL of LPS) was administered. Broilers in the control group as well as broilers pretreated with aerosolized distilled water or yellow and blue food color dyes did not develop pulmonary hypertension (PH; an increase in PAP) after the intratracheal spray of LPS, whereas broilers that had been pretreated with red food color did develop PH in response to intratracheal LPS. In experiment 2, birds raised under commercial conditions on used wood shavings litter developed PH in response to intratracheal LPS regardless of whether they had been pretreated with aerosolized red food color dye. In experiment 3, broilers reared in clean environmental chambers on new wood shavings litter were used to demonstrate that Red Dye #3 and propylene glycol are capable of priming the responsiveness of the airways to a subsequent intratracheal LPS challenge. Common air contaminants such as LPS can result in PH leading to pulmonary hypertension syndrome (ascites) in broilers with appropriately primed airways.

Previous hemodynamic evaluations demonstrated that pulmonary arterial pressure (PAP) is higher in broilers that are susceptible to pulmonary hypertension syndrome (PHS, ascites) than in broilers that are resistant to PHS. We compared key pulmonary hemodynamic parameters in broilers from PHS-susceptible and PHS-resistant lines (selected for 12 generations under hypobaric hypoxia) and in broilers from a relaxed (control) line. In experiment 1 the PAP was measured in male broilers in which a flow probe positioned on one pulmonary artery permitted the determination of cardiac output and pulmonary vascular resistance (PVR). The PAP and relative PVR were higher in susceptible broilers than in relaxed and resistant broilers, whereas absolute and relative cardiac output did not differ between lines. In experiment 2 male and female broilers from the 3 lines were catheterized to measure pressures in the wing vein, right atrium, right ventricle, pulmonary artery, and pulmonary veins (WP, wedge pressure). The transpulmonary pressure gradient (TPG) was calculated as (PAP-WP), with PAP quantifying precapillary pressure and WP approximating postcapillary pulmonary venous pressure. When compared with resistant and relaxed broilers, PAP values in susceptible broilers were > or =10 mmHg higher, TPG values were > or =8 mmHg higher, and WP values were < or =2 mmHg higher, regardless of sex. The combined hemodynamic criteria (elevated PAP and PVR combined with a proportionally elevated TPG) demonstrate that susceptibility to PHS can be attributed primarily to pulmonary arterial hypertension associated with increased precapillary (arteriole) resistance rather than to pulmonary venous hypertension caused by elevated postcapillary (venous and left atrial) resistance.

The pathogenesis of pulmonary hypertension syndrome (PHS, ascites) includes the development of systemic arterial hypoxemia (reduction in the saturation of hemoglobin with O(2), HbO(2)), which can be mimicked in clinically healthy broilers by i.v. injections of microparticles (MP). In experiment 1, arterial blood samples were collected from clinically healthy broilers before and after i.v. MP injections, and during a subsequent 100% O(2) inhalation period. The arterial samples were analyzed for HbO(2), partial pressure of O(2) and CO(2), and pH using a blood gas analyzer. In experiment 2, broilers that initially averaged > or =75% HbO(2) were assigned to a "high O(2)" group, whereas those that initially averaged < 75% HbO(2) were assigned to a "low O(2)" group. The HbO(2) and heart rate (HR) were measured using a pulse oximeter before, during, and after broilers in both groups inhaled 100% O(2). In experiment 3, HbO(2) and HR were measured using a pulse oximeter before, during, and after broilers inhaled 100% O(2), after i.v. MP injections, and during a second period of 100% O(2) inhalation. The HbO(2) rapidly decreased after i.v. MP injections, and subsequently providing 100% O(2) to breathe increased the HbO(2) above preinjection control levels in experiments 1 and 3. In experiment 2, inhaling 100% oxygen eliminated the initial spontaneous differences in HbO(2) between the high O(2) and low O(2) groups, whereas the return to breathing ambient air restored the initial group differences in HbO(2). These experiments indicate that MP-induced and spontaneous hypoxemia can be attributed to a diffusion limitation rather than to arterial-venous shunts, because the hypoxemia resulting from arterial-venous shunts cannot be wholly eliminated by providing 100% O(2) to inhale.

Broilers are susceptible to pulmonary hypertension syndrome (PHS; ascites syndrome) when their pulmonary vascular capacity is anatomically or functionally inadequate to accommodate the requisite cardiac output without an excessive elevation in pulmonary arterial pressure. The consequences of an inadequate pulmonary vascular capacity have been demonstrated experimentally and include elevated pulmonary vascular resistance (PVR) attributable to noncompliant, fully engorged vascular channels; sustained pulmonary arterial hypertension (PAH); systemic hypoxemia and hypercapnia; specific right ventricular hypertrophy, and right atrioventricular valve failure (regurgitation), leading to central venous hypertension and hepatic cirrhosis. Pulmonary vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and effective volume of blood vessels, as well as functional limitations related to the tone (degree of constriction) maintained by the primary resistance vessels (arterioles) within the lungs. Surgical occlusion of 1 pulmonary artery halves the anatomical pulmonary vascular capacity, doubles the PVR, triggers PAH, eliminates PHS-susceptible broilers, and reveals PHS-resistant survivors whose lungs are innately capable of handling sustained increases in pulmonary arterial pressure and cardiac output. We currently are using i.v. microparticle injections to increase the PVR and trigger PAH sufficient in magnitude to eliminate PHS-susceptible individuals while allowing PHS-resistant individuals to survive as progenitors of robust broiler lines. The microparticles obstruct pulmonary arterioles and cause local tissues and responding leukocytes to release vasoactive substances, including the vasodilator NO and the highly effective vasoconstrictors thromboxane A(2) and serotonin [5-hydroxytryptamine (5-HT)]. Nitric oxide is the principal vasodilator responsible for modulating (attenuating) the PAH response and ensuing mortality triggered by i.v. microparticle injections, whereas microparticle-induced increases in PVR can be attributed principally to 5-HT. Our observations support the hypothesis that susceptibility to PHS is a consequence of anatomically inadequate pulmonary vascular capacity combined with the functional predominance of the vasoconstrictor 5-HT over the vasodilator NO. The contribution of TxA(2) remains to be determined. Selecting broiler lines for resistance to PHS depends upon improving both anatomical and functional components of pulmonary vascular capacity.

Two experiments were conducted to evaluate the effects of arginine (Arg) and vitamin E (VE) on ascites (pulmonary hypertension syndrome) parameters, nitric oxide synthase (NOS) activity, and cardiopulmonary performance after an acute challenge with epinephrine (Epi). One-day-old male broilers (n = 100) were fed a commercial corn-soybean meal-based diet meeting NRC (1994) requirements, including 1.2% Arg and 40 IU of VE/kg. In experiment 1, birds were provided tap water (control), water with 0.3% Arg (HArg), water with 400 IU of VE/L (HVE), or a combination of both compounds (Arg-VE). In experiment 2, the treatment groups were similar but the VE was incorporated in the diet (400 IU/ kg of feed). At d 18, temperature was reduced to amplify the incidence of pulmonary hypertension. Body weight and hematocrit were recorded weekly. From d 28 to 42, cardiopulmonary performance was evaluated in clinically healthy, anesthetized birds (n = 7 to 8/treatment). A pulmonary artery and a systemic artery were cannulated, the birds were allowed to stabilize for 10 min (basal), an i.v. injection of Epi was applied (1 or 0.5 mg/kg of BW, experiment 1 and 2, respectively), and a second dose was applied 20 min later. Pulmonary arterial pressure (PAP), mean arterial pressure (MAP), and heart rate (HR) were recorded continuously and data were analyzed by repeated measures ANOVA. The NOS activity was estimated through the conversion of 14C-Arginine to 14C-citrulline in isolated pulmonary arteries. Right/total ventricular weight ratio (RV/TV) was recorded at the end of the experiment. Body weight, RV/TV, and hematocrit values were not significantly affected by the dietary treatments. The PAP increased (P < 0.01) within 30 s after Epi in all treatments, except the HArg treatment in experiment 2. Overall, the time taken for PAP to return to basal levels was longer in the Arg-VE birds and shorter in the HArg birds, particularly after the second challenge. However, although NOS activity was highly variable, birds fed HArg tended to have the lowest NOS activity of all groups. The levels of VE supplementation used in these experiments did not improve cardiopulmonary performance or NOS activity in isolated pulmonary arteries.