Microbiome analyses of poultry feeds: Part II. Comparison of different poultry feeds.

Within the realm of poultry feed mill operations, the persistent concern over microbial feed quality necessitates the establishment of a robust baseline for enhancing and sustaining the standards of commercial feeds. This dual-phase investigation, comprising Parts I, was previously published, and the current study presented here as Part II aimed to illuminate this baseline using 16S rRNA gene sequencing. In Part II, nine distinct commercial poultry feeds formulated as starters, growers, starter/growers, or supplements, the selected feeds underwent genomic DNA extraction, amplification with custom dual-indexed primers, and subsequent Illumina MiSeq sequencing. Through data analysis in QIIME2-2021.4 and R Studio, the study unveils alpha (Kruskal-Wallis) and beta (ANOSIM) diversity, taxonomic differences (ANCOM), and core microbiomes (core_members), deeming main and pairwise effects statistically significant at p < 0.05 and Q < 0.05. Notably, the investigation identified 30% common core microbial members across the nine feed types, shedding light on potential foodborne poultry pathogens such as Helicobacter and Campylobacter. Probiotic-associated feeds exhibited distinct microbial communities, emphasizing the need to explore their impact on the early poultry gastrointestinal tract (GIT) further.

Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods.

Given extensive variability in feed composition, the absence of a dedicated DNA extraction kit for poultry feed underscores the need for an optimized extraction technique for reliable downstream sequencing analyses. This study investigates the impact of five DNA extraction techniques: Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA amplification and Illumina MiSeq sequencing were conducted. QIIME2-2021.4 facilitated data analysis, revealing significant diversity and compositional differences influenced by extraction methods. Qiagen exhibited lower evenness and richness compared to other methods. 1-Day Direct and PEG enhanced bacterial diversities by employing bead beating and lysozyme. Despite similar taxonomic resolution, the Qiagen kit provides a rapid, consistent method for assessing poultry feed microbiomes. Modified techniques (MQ and MQC) improve DNA purification, reducing bias in commercial poultry feed samples. PEG and 1-Day Direct methods were effective but may require standardization. Overall, this study underscores the importance of optimized extraction techniques in poultry feed analysis, with potential implications for future standardization of effective methods.

Incidence of Salmonella serovars isolated from commercial animal feed mills in the United States and serovar identification using CRISPR analysis.

AIMS: In this study, we sought to determine the incidence and diversity of Salmonella in a broad collection of commercial animal feeds collected from animal feed mills across the United States over an 11-month period and utilize CRISPR analysis to identify individual serovars. METHODS AND RESULTS: Over two independent trials, 387 feed samples from 135 different animal feed mills in the United States were screened for Salmonella. A total of 6·2% (24/387) of samples were contaminated with Salmonella, which is concordant with similar studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-typing was used to serotype Salmonella isolates, and serovars Infantis and Tennessee were the most common. CONCLUSIONS: Serogroups O:4 and O:7 were enriched in the feed samples, suggesting that these serogroups are better adapted to surviving in low moisture animal feeds. The study supports the utility of CRISPR to determine serovar type since most of the serovars identified in this study have been also isolated and identified in earlier studies using more classical serotyping methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to a growing body of literature concerning the Salmonella prevalence in animal feeds and highlights the need to effectively mitigate pathogens in livestock and poultry feed.

Campylobacter jejuni and casein hydrolysate addition: Impact on poultry in vitro cecal microbiota and metabolome.

This study investigates the impact of casein hydrolysates on the poultry ceca inoculated with Campylobacter focusing on microbial molecular preferences for different protein sources in the presence of Campylobacter jejuni. Three casein sources (intact casein (IN), casein enzyme hydrolysate (EH), and casein acid hydrolysate (AH)) were introduced to cecal contents in combination with inoculated C. jejuni in an in vitro model system incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted and amplified using custom dual-indexed primers, followed by sequencing on an Illumina MiSeq platform. The obtained sequencing data were then analyzed via QIIME2-2021.11. Metabolite extracts were analyzed with ultra-high-performance liquid orbitrap chromatography-mass spectrometry (UHPLC-MS). Statistical analysis of metabolites was conducted using MetaboAnalyst 5.0, while functional analysis was performed using Mummichog 2.0 with a significance threshold set at P < 0.00001. DNA sequencing and metabolomic analyses revealed that C. jejuni was most abundant in the EH group. Microbial diversity and richness improved in casein supplemented groups, with core microbial differences observed, compared to non-supplemented groups. Vitamin B-associated metabolites significantly increased in the supplemented groups, displaying distinct patterns in vitamin B6 and B9 metabolism between EH and AH groups (P < 0.05). Faecalibacterium and Phascolarctobacterium were associated with AH and EH groups, respectively. These findings suggest microbial interactions in the presence of C. jejuni and casein supplementation are influenced by microbial community preferences for casein hydrolysates impacting B vitamin production and shaping competitive dynamics within the cecal microbial community. These findings underscore the potential of nutritional interventions to modulate the poultry GIT microbiota for improved health outcomes.

Research Note: Preliminary assessment of the impact of dietary yeast products on egg production and cecal microbial profiles of laying hens.

The objective of the current study was to conduct an initial comparison of commercial yeast products in layer hen diets on egg production parameters and the corresponding impact on the cecal microbiota. A short-term feeding study was conducted with 35 laying hens receiving either a control, or 1 of 4 different yeast fermentation products, Immunowall, Hilyses (both from ICC, São Paulo, Brazil), Citristim (ADM, Decatur, IL), and Maxi-Gen Plus (CBS Bio Platforms, Calgary, Canada) with 7 hens per treatment from 40 to 46 wk of age. At the end of the trial, hens were euthanized, the ceca removed and prepared for denatured gradient gel electrophoresis (DGGE) microbial compositional analyses. Although initial shell weight and shell thickness were similar among the treatment groups, hens fed Hilyses had lower shell weight and thickness at the end of the experiment. The most predominant DGGE bands with the strongest intensity were identified as Lactobacillus species and excised double bands were identified as Bacillus, Clostridium, or Lachnospiraceae. In this short-term feeding trial, the commercial yeast products tested had little effect on egg production and shell quality, and only moderately impacted the composition of mature layer hen cecal microbiota.

Reduction of Salmonella Infantis on skin-on, bone-in chicken thighs by cetylpyridinium chloride application and the impact on the skin microbiota.

Salmonella Infantis has been the etiological agent of numerous foodborne outbreaks of nontyphoidal Salmonella. Consequently, there is an emergent need to mitigate Salmonella Infantis among poultry. Thus, this study evaluated the efficacy of cetylpyridinium chloride (CPC) versus peroxyacetic acid (PAA), on bone-in, skin-on chicken thighs for the reduction of Salmonella and changes in the microbiota. Exactly 100 skin-on, bone-in chicken thighs (2 trials, 0 and 24 h, k = 5, n = 5, N = 50) were inoculated with 108 CFU/mL of a nalidixic acid resistant strain of S. Infantis for an attachment of 106 CFU/g. Thighs were treated with 20 s part dips (350 mL): a no inoculum, no treatment control (NINTC); no treatment control (NTC); tap water (TW); TW+CPC; TW+PAA. Following treatment, thighs were rinsed in 150 mL of nBPW, and rinsates were collected. Rinsates were spot plated for Salmonella and aerobic bacteria (APC). Log10 transformed counts were analyzed using a mixed-effects model (random effect = trial) with means separated using Tukey's HSD (P ≤ 0.05). The genomic DNA of rinsates was extracted, and the 16S rDNA was sequenced on an Illumina MiSeq. Microbiota data were analyzed using QIIME2, with data considered significant at P ≤ 0.05 (main effects) and Q≤0.05 (pairwise differences). Treatment × time interactions were observed for both Salmonella and APC (P < 0.05). The treatment of thighs with PAA and CPC reduced Salmonella and APC in respect to the controls. Numerically, thighs treated with CPC had less Salmonella (4.29 log10CFU/g) and less APC (4.56 log10CFU/g) at 24 h than all other treatments (P > 0.05). Differences in diversity metrics were not consistently observed between treatments; however, in trial 2, the NTC treated thighs were different than those treated with CPC (P < 0.05; Q < 0.05). In both trials, ANCOM, the analysis of microbiome compositional profiles, revealed shifts at both the phylum and order levels with thighs being different in the relative abundances of Proteobacteria (P < 0.05). In conclusion, treatment of skin-on poultry parts with CPC may reduce the risk of foodborne outbreaks caused by Salmonella Infantis.

Application of Amplon in combination with peroxyacetic acid for the reduction of nalidixic acid-resistant Salmonella Typhimurium and Salmonella Reading on skin-on, bone-in tom turkey drumsticks.

Peroxyacetic acid (PAA) has become an important component of pathogen reduction in poultry processing, but there are potential concerns for continued exposure. The objective was to evaluate the effects of PAA and Amplon (AMP) used alone or in the combination. Bone-in tom turkey drumsticks (N = 100, n = 10, k = 5, 0 and 24 h) per study were obtained and inoculated with either nalidixic acid-resistant Salmonella Typhimurium or Salmonella Reading (64 µg/mL). The inocula were allowed to adhere to the drums at 4°C for 60 min for a final attachment of 108 and 107 cfu/g per S. Typhimurium and S. Reading, respectively. Drumsticks were treated with a no-treatment control; tap water, pH 8.5 (TW); TW+500 ppm PAA, pH 3.5 (PAA); TW+500 ppm AMP, pH 1.3 (AMP); TW + PAA + AMP (PAA + AMP). Treatments were applied as short duration dips (30 s) and allowed to drip for 2 min. After treatment, drums were stored at 4°C until microbial analyses at 0 and 24 h. Drums were rinsed in neutralizing buffered peptone water and spot plated for total aerobes and Salmonella. Bacterial counts were log10 transformed and analyzed using n-way ANOVA. All treatments reduced S. Reading on turkey legs at both 0 and 24 h (P < 0.0001; P < 0.0001). At 24 h, drums treated with PAA + AMP (3.92 log10 cfu/g) had less S. Reading than no-treatment control, TW, and AMP. Treatment by time interactions were observed for total aerobes among drums in both studies (P < 0.0001, P < 0.0001) and Salmonella among drums inoculated with S. Typhimurium (P < 0.0001). During the S. Reading and S. Typhimurium study, all treatments reduced Salmonella and total aerobes on drums. During the S. Typhimurium study, drums treated with PAA + AMP had the lowest numerical load of S. Typhimurium and total aerobes. The combination of AMP + PAA may exhibit a synergistic effect in reducing Salmonella on turkey drums, thus increasing the safety of turkey products for consumers.

Autoradiographic characterization of beta adrenergic receptors in coronary blood vessels and myocytes in normal and ischemic myocardium of the canine heart.

This light microscopic autoradiographic study was performed to test the hypotheses that (a) the density of beta adrenergic receptors (BAR) may differ in various components of the heart and (b) BAR in certain components of the heart may exhibit a selective response to pharmacologic and pathological stimuli. Blocks of canine left ventricle were frozen and tissue sections cut and incubated in (-)[3H]dihydroalprenolol (DHA) to label the BAR. For total and nonspecific binding, serial sections were incubated with and without 10(-5) M (+/-)propranolol. Scintillation spectrometry of sections demonstrated rapid binding, saturability, stereospecificity, a dissociation constant (KD) of 3.2 +/- 0.5 nM (SD) (n = 3), and a maximal binding of 31.3 +/- 3.1 fmol/mg of tissue protein. Isoproterenol was 12.5 times more effective than norepinephrine in displacing DHA. Sections incubated with 10(-5) - 10(-8) M metoprolol, a beta one selective antagonist, demonstrated a KD of 0.7 X 10(-6) M. For autoradiography, emulsion-coated coverslips were attached to the slides. After exposure, the slides were developed and stained, and grain density quantified. Specific BAR binding (n = 4 dogs) was 1,047 +/- 131 (SEM) grains/10(-2) mm2 for myocardial arterioles, 219 +/- 30 for myocardial arteries, 31 +/- 12 for the proximal left anterior descending coronary artery (LAD), and 231 +/- 34 for cardiac myocytes. Specific binding in the presence of 10(-5) M metoprolol was reduced approximately 75% for both arterioles and myocytes. However, at 10(-6) M metoprolol, the percent reduction in specific DHA binding was greater for myocytes (50%) than for arterioles (0%), and at 10(-7) M metoprolol, the percent reduction in specific DHA binding was 17% for myocytes with no reduction over arterioles. After 1 h of LAD occlusion, a selective increase (18%) in BAR density occurred over cardiac myocytes, but not over blood vessels in the ischemic myocardium. Thus, (a) specific BAR binding was five times greater in arterioles than in small arteries and myocardium and 34 times greater than in the proximal LAD; (b) BAR of myocytes were more sensitive than those of arterioles to displacement by the beta one selective antagonist, metoprolol; and (c) a selective increase in BAR occurs in cardiac myocytes but not in blood vessels after 1 h of ischemia in this experimental model.

Noninvasive evaluation of aortic stenosis severity utilizing Doppler ultrasound and electrical bioimpedance.

Aortic valve area was calculated noninvasively in 30 patients with aortic stenosis undergoing cardiac catheterization. Continuous wave Doppler ultrasound was employed to estimate the mean transvalvular pressure gradient. The mean left ventricular outflow tract flow velocity and cross-sectional area were determined from pulsed Doppler and two-dimensional ultrasound recordings. Electrical transthoracic bioimpedance cardiography performed simultaneously with the ultrasonic study and repeated at the time of catheterization measured heart rate, systolic ejection period and cardiac output. These noninvasive data permitted calculation of aortic valve area using the Gorlin equation (range 0.21 to 1.75 cm2) and the continuity equation (range 0.25 to 1.9 cm2). Subsequent cardiac catheterization showed valve area to range from 0.21 to 1.75 cm2. The mean Doppler pressure gradient estimate was highly predictive of the gradient measured at catheterization (r = +0.92, SEE = 10). Bioimpedance cardiac output measurements agreed with the average of Fick and indicator dye estimates (r = +0.90, SEE = 0.52). Valve area estimates utilizing continuous wave Doppler ultrasound and electrical bioimpedance were superior (r = +0.91, SEE = 0.12) to estimates obtained utilizing the continuity equation (r = +0.76, SEE = 0.29) and were more reliable in the detection of patients with severe aortic stenosis (9 of 11 versus 6 of 11). These data show that 1) electrical bioimpedance methods accurately estimate cardiac output in the presence of aortic stenosis; 2) the hybridized bioimpedance-Doppler ultrasound method yields accurate estimates of aortic stenosis area; and 3) the speed, accuracy and cost-effectiveness of aortic stenosis evaluation may be improved by this hybridized approach.

Quantification of aortic regurgitation utilizing continuous wave Doppler ultrasound.

Aortic regurgitation and mitral stenosis are hemodynamically similar, insofar as both result in passive ventricular filling across a narrow orifice driven by a declining pressure gradient. Because mitral stenosis is successfully characterized by Doppler ultrasound determination of the velocity half-time, or time constant, aortic regurgitation might be quantified in an analogous fashion. Eighty-six patients with diverse causes of aortic regurgitation underwent continuous wave Doppler examination before cardiac catheterization or urgent aortic valve replacement. The Doppler velocity half-time was defined as the time required for the diastolic aortic regurgitation velocity profile to decay by 29%, whereas catheterization pressure half-time was calculated as the time required for transvalvular pressure to decay by 50%. Doppler velocity and catheterization pressure half-times were linearly related (r = 0.91). Doppler velocity half-times were inversely related to regurgitant fraction (r = -0.88). Angiographic severity (1+ = mild to 4+ = severe) was also inversely related to pressure and velocity half-time; a Doppler half-time threshold of 400 ms separated mild (1+, 2+) from significant (3+, 4+) aortic regurgitation with high specificity (0.92) and predictive value (0.90). The Doppler velocity half-time was independent of pulse pressure, mean arterial pressure, ejection fraction and left ventricular end-diastolic pressure. Estimation of transvalvular aortic pressure half-time utilizing continuous wave Doppler ultrasound is a reliable and accurate method for the noninvasive evaluation of the severity of aortic regurgitation.

Effects of orthostatic postural changes on myocardial oxygen demands.

The effects of orthostatic tilting were studied in 12 mongrel dogs anesthetized with sodium pentobarbital. Orthostasis produced significant decreases in cardiac output and left ventricular dimensions. Secondary decreases in coronary flow and myocardial oxygen consumption were also documented. These changes were attenuated when the animals were pretreated with propranolol. The upright posture may be of value in treatment of angina pectoris.

Mucocutaneous lymph node syndrome.

In 1967, Kawasaki reported an acute, febrile, mucocutaneous condition accompanied by swelling of cervical lymph nodes that afflicted infants and young children in Japan. He called the entity mucocutaneous lymph node syndrome. In 1974, the condition was reported in Hawaii, and several cases have been reported subsequently from the continental United States and other countries. One percent to two % of patients had died suddenly of cardiac failure. Rickettsia-like bodies have been demonstrated from skin and lymph node biopsy specimens, and the pathologic features suggest a relationship to infantile periarteritis nodosa.

How in the world is WHO doing?

The World Health Organization has chalked up some impressive victories in its 43 years but is nowhere near reaching its ambitious goal of "Health for All by the Year 2000." Uphill battles against some intractable health conundrums and shrinking financial resources face the Geneva, Switzerland-based organization, threatening its stated goal of guaranteeing universal health care within the decade.

N-dimethylisopropyl propranolol. Effects on myocardial oxygen demands.

N-Dimethylisopropyl propranolol (DMP) is a quaternary derivative which lacks significant beta-adrenergic blocking and local anesthetic effects. It has been reported, nonetheless, to be effective in treating experimental arrhythmias and in limiting the extent of ST-segment elevations following experimental coronary occlusion. The present study examined the effects of DMP on the hemodynamics and myocardial oxygen demands of anesthetized dogs. After a single dose of 3 mg/kg, heart rate fell from 146 +/- 8 to 124 +/- 6 beats/min (P less than 0.0025), and aortic systolic pressure fell from 151 +/- 11 to 141 +/- 9 mm Hg (0.05 less than P less than 0.10), resulting in a 16.8% reduction in the tension-time index. Stroke volume was reduced by 10% despite a 54% increase in left ventricular end-diastolic pressure, suggesting a negative inotropic effect. This was supported by a decrease in maximum extrapolated contractile element velocity from 9.10 +/- 1.05 to 6.61 +/- 65 units/sec (P less than 0.0025). Myocardial oxygen consumption was reduced from 12.0 +/- 1.4 to 9.9 +/- 1.5 ml/min/100 g tissue (P less than 0.05). Myocardial oxygen extraction was unchanged, indicating that the decrease in oxygen consumption resulted from a reduction in myocardial oxygen demand. When heart rate and systolic pressure were artificially restored to control levels, after the administration of DMP, myocardial oxygen consumption remained significantly below the control level. DMP, therefore, appeared to reduce myocardial oxygen demands primarily by its negative inotropic effect. This drug may have application in the treatment of ischemic heart disease.

Direct endocardial recording from an accessory atrioventricular pathway: localization of the site of block, effect of antiarrhythmic drugs, and attempt at nonsurgical ablation.

We recorded a discrete 0.95 mV potential consistent with accessory atrioventricular pathway (AP) activation during serial electrophysiologic studies in a patient with Ebstein's anomaly and Wolff-Parkinson-White syndrome. Bipolar pacing from the catheter electrode in which the AP potential was recorded resulted in a stimulus-ventricle interval identical to the AP-ventricle interval during antegrade conduction, and a stimulus-atrium interval identical to the AP-atrium interval during retrograde conduction. With the patient in the drug-free state, antegrade AP block during atrial pacing and retrograde AP block during ventricular pacing occurred distal to the AP potential (AP-ventricle junction and AP-atrium junction, respectively), supporting the "impedance mismatch" hypothesis. Procainamide and disopyramide each lengthened the antegrade AP effective refractory period by affecting the AP-ventricle junction (possibly by decreasing the current generated by the AP). Both drugs also lengthened the retrograde AP effective refractory period but produced a greater effect on the ventricle-AP junction than on the AP-atrium junction, suggesting marginal geometry of the former. R wave synchronous shocks of 160 and 320 W-sec delivered between the catheter electrode recording the largest unipolar AP potential and a skin electrode produced transient, complete, antegrade block over the AP, suggesting the feasibility of this new nonsurgical technique for AP ablation.