A Consistent and Predictable Commercial Broiler Chicken Bacterial Microbiota in Antibiotic-Free Production Displays Strong Correlations with Performance.

Defining the baseline bacterial microbiome is critical to understanding its relationship with health and disease. In broiler chickens, the core microbiome and its possible relationships with health and disease have been difficult to define, due to high variability between birds and flocks. Presented here are data from a large, comprehensive microbiota-based study in commercial broilers. The primary goals of this study included understanding what constitutes the core bacterial microbiota in the broiler gastrointestinal, respiratory, and barn environments; how these core players change across age, geography, and time; and which bacterial taxa correlate with enhanced bird performance in antibiotic-free flocks. Using 2,309 samples from 37 different commercial flocks within a vertically integrated broiler system and metadata from these and an additional 512 flocks within that system, the baseline bacterial microbiota was defined using 16S rRNA gene sequencing. The effects of age, sample type, flock, and successive flock cycles were compared, and results indicate a consistent, predictable, age-dependent bacterial microbiota, irrespective of flock. The tracheal bacterial microbiota of broilers was comprehensively defined, and Lactobacillus was the dominant bacterial taxon in the trachea. Numerous bacterial taxa were identified, which were strongly correlated with broiler chicken performance across multiple tissues. While many positively correlated taxa were identified, negatively associated potential pathogens were also identified in the absence of clinical disease, indicating that subclinical dynamics occur that impact performance. Overall, this work provides necessary baseline data for the development of effective antibiotic alternatives, such as probiotics, for sustainable poultry production.IMPORTANCE Multidrug-resistant bacterial pathogens are perhaps the greatest medical challenge we will face in the 21st century and beyond. Antibiotics are necessary in animal production to treat disease. As such, animal production is a contributor to the problem of antibiotic resistance. Efforts are underway to reduce antibiotic use in animal production. However, we are also challenged to feed the world's increasing population, and sustainable meat production is paramount to providing a safe and quality protein source for human consumption. In the absence of antibiotics, alternative approaches are needed to maintain health and prevent disease, and probiotics have great promise as one such approach. This work paves the way for the development of alternative approaches to raising poultry by increasing our understandings of what defines the poultry microbiome and of how it can potentially be modulated to improve animal health and performance.

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O'Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12-16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

Survival of turkey arthritis reovirus in poultry litter and drinking water.

Turkey reoviruses (TRVs) can cause arthritis, tenosynovitis, and enteric diseases in turkeys, leading to huge economic losses. The TRVs are tentatively divided into turkey arthritis reoviruses (TARVs) and turkey enteric reoviruses (TERVs) depending on the type of disease they produce. This study was conducted to determine the survival of these viruses in autoclaved and nonautoclaved poultry litter and drinking water at room temperature (approx. 25°C). Three isolates of TARV (TARV-O'Neil, TARV-MN2, and TARV-MN4) and one each of TERV (TERV-MN1) and chicken arthritis reovirus (CARV) were used in this study. The viruses were propagated and titrated on QT-35 cells. In autoclaved dechlorinated tap water, all 5 viruses were able to survive for 9 to 13 wk. In nonautoclaved water, all 5 viruses survived for at least 2 wk. In autoclaved litter, the viruses survived for 6 to 8 wk, and in nonautoclaved litter, they survived for 6 to 8 d only. The implications of these results are discussed below.

Effect of pimenta essential oil against Salmonella Agona and Salmonella Saintpaul in ground turkey meat and nonprocessed turkey breast meat.

Salmonella enterica Agona (S. Agona) and Salmonella enterica Saintpaul (S. Saintpaul) are among the emerging drug-resistant Salmonella in turkey production and processing. Rapid solutions to control emerging and uncommon serotypes such as S. Agona and S. Saintpaul are needed. This study tested pimenta essential oil (PEO) as a processing antibacterial against S. Agona and S. Saintpaul in experiments representative of different stages of turkey processing. The compound effectively reduced S. Agona and S. Saintpaul in nutrient broth studies and with mature biofilm assays. PEO was tested against a combination of S. Agona and S. Saintpaul in ground turkey meat and nonprocessed breast meat. In the first experiment with ground turkey, samples were inoculated with a mixture of S. Agona and S. Saintpaul (∼3 log10 CFU/g) and treated with PEO at different concentrations (0% PEO, 0.25% PEO, 0.5% PEO, 1% PEO, 2% PEO, and 2.5% PEO). In the second experiment with turkey breast, samples inoculated with ∼3 log10 CFU/g (SA+SP) were dipped in different concentrations of PEO with chitosan (CN) for 2 min. In both these experiments, samples were stored at 4°C, and Salmonella recovery was carried out at 0, 1, 3, 5, and 7 d. All experiments followed a completely randomized design and were repeated 6 times (n = 6). Statistical analysis was done using the PROC-ANOVA procedure of SAS. In the ground turkey meat, PEO at or above 2% reduced 2 log10 CFU/g of Salmonella by day 1. PEO at 2.5% in ground turkey meat resulted in enrichment-negative samples by 1 min, indicative of the rapid killing effect of the compound at a high concentration of PEO (P ≤ 0.05). A maximum reduction of 1.7 log10 CFU Salmonella/g of turkey breast meat was obtained after 2 min of dip treatment containing CN and 2.5% PEO. Results indicate that PEO could be used as a plant-based processing antibacterial against S. Agona and S. Saintpaul in turkey processing. Upscaling to plant-level studies is necessary before recommending its usage.

Combination of autochthonous Lactobacillus strains and trans-Cinnamaldehyde in water reduces Salmonella Heidelberg in turkey poults.

Reducing the colonization of Salmonella in turkeys is critical to mitigating the risk of its contamination at later stages of production. Given the increased susceptibility of newly hatched poults to Salmonella colonization, it is crucial to implement interventions that target potential transmission routes, including drinking water. As no individual intervention explored to date is known to eliminate Salmonella, the United States Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) recommends employing multiple hurdles to achieve a more meaningful reduction and minimize the potential emergence of resistance. Probiotics and plant-derived antimicrobials (PDAs) have demonstrated efficacy as interventions against Salmonella in poultry. Therefore, this study aimed to investigate the use of turkey-derived Lactobacillus probiotics (LB; a mixture of Lactobacillus salivarius UMNPBX2 and L. ingluviei UMNPBX19 isolated from turkey ileum) and a PDA, trans-cinnamaldehyde (TC), alone and in combination (CO), against S. Heidelberg in turkey drinking water and poults. The presence of 5% nutrient broth or cecal contents as contaminants in water resulted in S. Heidelberg growth. TC eliminated S. Heidelberg, regardless of the contaminants present. In contrast, the cecal contents led to increased survival of Lactobacillus in the CO group. Unlike TC, LB was most effective against S. Heidelberg when the nutrient broth was present, suggesting the role of secondary metabolites in its mechanism of action. In the experiments with poults, individual TC and LB supplementation reduced cecal S. Heidelberg in challenged poults by 1.2- and 1.7-log10 colony-forming units (CFU)/g cecal contents, respectively. Their combination yielded an additive effect, reducing S. Heidelberg by 2.7 log10 CFU/g of cecal contents compared to the control (p ≤ 0.05). However, the impact of TC and LB on the translocation of S. Heidelberg to the liver was more significant than CO. TC and LB are effective preharvest interventions against S. Heidelberg in poultry production. Nonetheless, further investigations are needed to determine the optimum application method and its efficacy in adult turkeys.

Amino acid and energy digestibility of conventional corn dried distillers grains with solubles, high protein distillers dried grains, and corn fermented protein, and impacts on turkey poult growth performance and intestinal characteristics.

Amino acids (AA) are an expensive nutritional components of poultry diets. Distillers dried grains with solubles (DDGS) is the primary co-product produced by the dry grind bioethanol industry, although new technologies are being implemented to produce high protein distillers dried grains (HP-DDG) and corn fermented protein (CFP), but data on their nutritive value in poultry are lacking. Two experiments (EXP) were conducted to determine the energy and AA digestibility of DDGS, HP-DDG, and CFP in poults in addition to a feeding trial to evaluate increasing dietary levels of HP-DDG and CFP on growth performance and intestinal characteristics. In EXP 1, 6 different DDGS sources were evaluated using poults to determine their nitrogen-corrected apparent metabolizable energy (AMEn) concentrations, and cecectomized roosters were used to determine their standardized ileal (SID) AA digestibility (SID-AA). In EXP 2, AMEn and SID-AA for HP-DDG and CFP were determined in young poults, and a feeding trial was conducted to evaluate growth performance and intestinal morphology and permeability of poults fed diets containing 7.5 and 15% HP-DDG or CFP from 1 to 32 d of age. In EXP 1, the AMEn concentration among the DDGS samples ranged from 2,530 to 3,573 kcal/kg DM but was not different (P = 0.57) among the samples, with an average SID for LYS of 66.6%. In EXP 2, different (P = 0.001) AMEn concentrations for HP-DDG and CFP were observed (3,114 and 3,760 kcal/kg DM, respectively), with the SID for LYS being 66.55 and 77.00% for HP-DDG and CFP, respectively. Including HP-DDG or CFP into the diet at 7.5 and 15% had no effect (P > 0.05) on growth, feed intake, or feed conversion. Neither co-product nor its inclusion rate affected intestinal morphology and permeability (P > 0.05). Overall, DDGS, HP-DDG, and CFP are excellent sources of AMEn and digestible AA, with dietary inclusion rates of up to 15% of HP-DDG or CFP having no impact on growth or intestinal characteristics.

Prolonged repeated inseminations trigger a local immune response and accelerate aging of the uterovaginal junction in turkey hens.

Artificial insemination is a standard practice in the turkey breeder industry to ensure the production of fertile eggs. Even though hens are inseminated on a weekly basis, their fertility tends to decline after a few weeks of production. Avian species have a specialized structures called sperm storage tubules (SSTs), located in the uterovaginal junction (UVJ) of the oviduct. The ability of SSTs to store sperm is directly correlated with the fertility of the hen. The objective of the study was to examine changes in the transcriptome of the turkey hen's UVJ in response to the presence of sperm at three key stages of production. We hypothesized that repeated and prolonged exposure to sperm would alter the transcriptome of the UVJ. Samples were collected from virgin hens prior to the onset of lay, as well as from sham-inseminated (extender only) and semen-inseminated hens at early lay, peak lay, and late lay. Gene expression profiling of the UVJ was examined, and a differential expression analysis was conducted through pairwise comparisons between semen- and sham-inseminated groups at each production stage and across production stages. In the early laying stage, no significant gene expression changes were found between semen- and sham-inseminated groups. However, at peak lay, genes related to lipid biosynthesis, Wnt signaling, cell proliferation, and O-glycan biosynthesis were upregulated in the semen group, while the immune response and cytokine-cytokine receptor interaction were downregulated. In the late lay stage, the transcription pathway was upregulated in the semen group, whereas the translation pathway was downregulated. The local immune response that was suppressed during peak lay was increased at the late laying stage. In the semen-inseminated group, the UVJ exhibited advanced aging at the late laying stage, evidenced by reduced telomere maintenance and translation processes. The results from this study provide valuable insights into the alteration of the UVJ function in response to the presence of sperm at different stages of production and throughout the production cycle. Targeting the modulation of local immune response and addressing aging processes after peak production could potentially prevent or delay the decline in fertility of turkey breeder hens.

Evidence of host specificity in Lactobacillus johnsonii genomes and its influence on probiotic potential in poultry.

To date, the selection of candidate strains for probiotic development in production animals has been largely based upon screens for desired phenotypic traits. However, increasing evidence indicates that the use of host-specific strains may be important, because coevolution with the animal host better prepares a bacterial strain to colonize and succeed in its respective host animal species. This concept was applied to Lactobacillus johnsonii in commercial poultry production because of its previous correlation with enhanced bird performance. Using 204 naturally isolated chicken- and turkey-source L. johnsonii, we demonstrate that there is a strong phylogenetic signal for coevolution with the animal host. These isolates differ phenotypically, even within host source, and these differences can be correlated with certain L. johnsonii phylogenetic clades. In commercial turkey poults, turkey-specific strains with strong in vitro phenotypes performed better early in life than strains lacking those phenotypes. A follow-up performance trial in broiler chickens demonstrated that chicken-specific strains result in better overall bird performance than nonchicken-specific strains. Collectively, this work provides evidence for the impact of host adaptation on a probiotic strain's potential. Furthermore, this top-down approach is useful for screening larger numbers of isolates for probiotic candidates.

Effect of plant-derived antimicrobials against multidrug-resistant Salmonella Heidelberg in ground Turkey.

Salmonella Heidelberg (SH) is a highly invasive human pathogen for which turkeys can serve as reservoir hosts. Colonization of turkeys with SH may result in potential contamination and is a greater challenge to prevent in comminuted products. Antimicrobial efficacy of 3 GRAS-status plant-derived antimicrobials (PDAs), lemongrass essential oil (LG), citral (CIT), and trans-cinnamaldehyde (TC), against SH in ground turkey, a comminuted product implicated in several outbreaks, was evaluated in this study. Ground turkey samples inoculated with ∼3.50 log10 CFU/g of a three-strain SH cocktail were treated with either LG, CIT, or TC at either 0.5, 1, or 2% (vol/wt). Samples were stored at 4°C, and bacterial enumeration was performed on d 0, 1, 3, and 5. Appropriate controls were included alongside all treatments. Fluorescence microscopy was performed to evaluate the direct impact of the PDAs against SH in vitro. Appearance and aroma difference testing of raw patties was also performed for select treatments with trained sensory panelists. Treatment with 2% TC yielded a 2.5 log10 CFU/g reduction by d 1 and complete reduction by d 5 (P < 0.05). By d 3, 2% CIT and 2% LG resulted in SH reduction of at least 1.7 log10 CFU/g (P < 0.05). Addition of 1% TC resulted in reduction of at least 1.8 log10 CFU/g by d 3 (P < 0.05). Participants could distinguish PDA-treated raw patties by aroma. Most participants (7/11) could not distinguish patties treated with 0.5% TC based on appearance. Microscopic images indicate that all PDAs resulted in disruption of the SH membrane. Results of the present study indicate that the three tested PDAs, LG, CIT, and TC are effective against SH in ground turkey, indicating their potential use as interventions to mitigate Salmonella contamination in comminuted turkey products.

Effect of caprylic acid alone or in combination with peracetic acid against multidrug-resistant Salmonella Heidelberg on chicken drumsticks in a soft scalding temperature-time setup.

The antimicrobial efficacy of caprylic acid (CA), a medium-chain fatty acid, against multidrug-resistant Salmonella Heidelberg (MDR SH) on chicken drumsticks in a soft-scalding temperature-time setup was investigated. Based on the standardization experiments in nutrient media and on chicken breast fillet portions, intact chicken drumsticks were spot inoculated with MDR SH and immersed in water with or without antimicrobial treatments at 54°C for 2 min. The treatments included 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA. Additionally, the efficacy of the potential scald treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4°C was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. Appropriate controls were included for statistical comparisons. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum (∼2.5 log10 CFU/g) experiments, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA resulted in 0.7-, 1.0-, 2.5-, 1.4-, and 1.5- log10 CFU/g reduction of MDR SH on drumsticks, respectively (P < 0.05). The same treatments resulted in 0.9-, 1.3-, 2.5-, 2.2-, and 2.6- log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with a higher inoculum (∼4.5 log10 CFU/g) level (P < 0.05). Moreover, the antimicrobial treatments inactivated MDR SH in the treatment water to undetectable levels, whereas 2.0- to 4.0- log10 CFU/mL MDR SH survived in the positive controls (P < 0.05). Also, the treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4°C; however, the magnitude of reduction remained the same as observed during the treatment (P < 0.05). Additionally, none of the treatments affected the color of the drumsticks (P > 0.05). Results indicate that CA could be an effective natural processing aid against MDR SH on chicken products.

Effect of lemongrass essential oil against multidrug-resistant Salmonella Heidelberg and its attachment to chicken skin and meat.

Salmonella Heidelberg (S. Heidelberg) is a major pathogen implicated in foodborne outbreaks for which poultry products can serve as an epidemiological source. This study determined the efficacy of GRAS-status lemongrass essential oil (LGEO) against S. Heidelberg in vitro and on the pathogen's attachment to skin and meat. At first, employing in vitro assays, the effect of LGEO on multidrug-resistant S. Heidelberg multiplication and motility was examined. Biofilm inhibition and inactivation assays were also performed. The quorum-sensing modulating effect of LGEO was determined. In follow-up experiments, chicken skin or meat samples inoculated with S. Heidelberg were treated with various concentrations of LGEO at different time points at simulated scalding (54°C) and chilling (4°C) temperatures. The samples were incubated, and the surviving populations of S. Heidelberg were enumerated to determine if LGEO could be a potential processing aid in poultry operations. Duplicate samples were included in each treatment, and the experiments were repeated at least 3 times. Significant reductions of S. Heidelberg of at least 4.0 log10 CFU/mL after 24 h in nutrient broth and poultry cecal contents was observed with 0.5% LGEO. Complete inhibition of motility, biofilm formation, and inactivation of pre-formed biofilms was observed with 0.15% LGEO (P ≤ 0.05). Concentrations of LGEO at 0.5% and 1% affected violacein production (P ≤ 0.05). On skin samples, all concentrations significantly reduced S. Heidelberg by 1.2 to 3.9 log10 CFU/sample after 2 min at 54°C. We obtained a significant reduction of the pathogen in meat samples at 54°C and skin samples at 4°C with 2% LGEO. All concentrations significantly reduced S. Heidelberg from the treatment water kept at 4°C and 54°C (P ≤ 0.05). In conclusion, LGEO could potentially serve as a natural antimicrobial strategy in scalding and chilling waters to reduce S. Heidelberg during processing. However, additional studies are warranted before recommending its commercial use.

Draft Genome Sequence of "Candidatus Arthromitus" UMNCA01, a Suspected Commensal Isolated from the Gut Microbiome of Commercial Turkey.

"Candidatus Arthromitus" UMNCA01 was recovered from ileal samples of commercial turkey poults and may have probiotic capabilities. The complete genome was determined using the Illumina MiSeq and HiSeq sequencing platforms. The complete genome consists of 1,631,326 bp and has a G+C content of 26.14%, 1,540 coding sequences (CDS), and 37 RNA coding genes.

Effect of Turkey-Derived Beneficial Bacteria Lactobacillus salivarius and Lactobacillus ingluviei on a Multidrug-Resistant Salmonella Heidelberg Strain in Turkey Poults.

Effects of turkey-derived beneficial bacteria Lactobacillus ingluviei UMNPBX19 and Lactobacillus salivarius UMNPBX2 on Salmonella Heidelberg (SH) in turkey poults was investigated. Using in vitro studies, we determined each strain's resistance to pH 2.5 and 0.3% bile salts and their ß-hemolysis activity. We also tested each strain's adherence to avian epithelial cells and exhibition of antimicrobial activity against major poultry-associated Salmonella. Moreover, using three in vivo experiments, we determined the effect of the strains in combination (LBIS) against SH in turkey poults. The treatment groups were negative control (-SH, -LBIS), SH control (+SH, -LBIS), and LBIS group (+SH, +LBIS). Supplementation of LBIS was done in drinking water throughout the study at a dose of 8 log CFU/gal. On day 7, poults were challenged with a 2011 ground turkey outbreak strain of SH at 5 × 105 CFU/mL, and the surviving pathogens were determined on day 7 postinoculation from the cecum, spleen, and liver. Both Lactobacillus strains exerted resistance to low pH and bile salts ( P < 0.05), showed adhesion to epithelial cells ( P < 0.05), but did not exhibit ß-hemolysis. Cell-free culture supernatants of strains showed antimicrobial activity against Salmonella ( P < 0.05). Results from the in vivo studies revealed that LBIS significantly reduced dissemination of SH to the liver and spleen in all experiments, and colonization in the cecum in two of the three experiments (1.9- and 3.9-log CFU/g reductions), compared with the control. The results indicate that turkey-derived L. ingluviei UMNPBX19 and L. salivarius UMNPBX2 have potential beneficial effects against SH in turkeys. However, more studies to this effect are warranted.

Antibiotics and Host-Tailored Probiotics Similarly Modulate Effects on the Developing Avian Microbiome, Mycobiome, and Host Gene Expression.

The microbiome is important to all animals, including poultry, playing a critical role in health and performance. Low-dose antibiotics have historically been used to modulate food production animals and their microbiome. Identifying alternatives to antibiotics conferring similar modulatory properties has been elusive. The purpose of this study was to determine if a host-tailored probiotic could recapitulate effects of a low-dose antibiotic on host response and the developing microbiome. Over 13 days of life, turkey poults were supplemented continuously with a low-dose antibiotic or oral supplementation of a prebiotic with or without two different probiotics (8 cage units, n = 80 per group). Gastrointestinal bacterial and fungal communities of poults were characterized by 16S rRNA gene and ITS2 amplicon sequencing. Localized and systemic host gene expression was assessed using transcriptome sequencing (RNA-Seq), kinase activity was assessed by avian-specific kinome peptide arrays, and performance parameters were assessed. We found that development of the early-life microbiome of turkey poults was tightly ordered in a tissue- and time-specific manner. Low-dose antibiotic and turkey-tailored probiotic supplementation, but not nontailored probiotic supplementation, elicited similar shifts in overall microbiome composition during development compared to controls. Treatment-induced bacterial changes were accompanied by parallel shifts in the fungal community and host gene expression and enhanced performance metrics. These results were validated in pen trials that identified further additive effects of the turkey-tailored probiotic combined with different prebiotics. Alternative approaches to low-dose antibiotic use in poultry are feasible and can be optimized utilizing the indigenous poultry microbiome. Similar approaches may also be beneficial for humans.IMPORTANCE Alternative approaches are greatly needed to reduce the need for antibiotic use in food animal production. This study utilized a pipeline for the development of a host-tailored probiotic to enhance performance in commercial turkeys and modulate their microbiota, similar to the effects of low-dose antibiotic administration. We determined that a host-tailored probiotic, developed in the context of the commercial turkey gut microbiome, was more effective at modulating these parameters than a nontailored probiotic cocktail. Furthermore, the host-tailored probiotic mimicked many of the effects of a low-dose antibiotic growth promoter. Surprisingly, the effects of the antibiotic growth promoter and host-tailored probiotic were observed across kingdoms, illustrating the coordinated interkingdom effects of these approaches. This work suggests that tailored approaches to probiotic development hold promise for modulating the avian host and its microbiota.

Antibiotic degradation during manure composting.

On-farm manure management practices, such as composting, may provide a practical and economical option for reducing antibiotic concentrations in manure before land application, thereby minimizing the potential for environmental contamination. The objective of this study was to quantify degradation of chlortetracycline, monensin, sulfamethazine, and tylosin in spiked turkey (Meleagris gallopavo) litter during composting. Three manure composting treatments were evaluated: a control treatment (manure pile with no disturbance or adjustments after initial mixing), a managed compost pile (weekly mixing and moisture content adjustments), and vessel composting. Despite significant differences in temperature, mass, and nutrient losses between the composting treatments and the control, there was no difference in antibiotic degradation among the treatments. Chlortetracycline concentrations declined rapidly during composting, whereas monensin and tylosin concentrations declined gradually in all three treatments. There was no degradation of sulfamethazine in any of treatments. At the conclusion of the composting period (22-35 d), there was >99% reduction in chlortetracycline, whereas monensin and tylosin reduction ranged from 54 to 76% in all three treatments. Assuming first-order decay, the half-lives for chlortetracycline, monensin, and tylosin were 1, 17, and 19 d, respectively. These data suggest that managed compositing in a manure pile or in a vessel is not better than the control treatment in degrading certain antibiotics in manure. Therefore, low-level manure management, such as stockpiling, after an initial adjustment of water content may be a practical and economical option for livestock producers in reducing antibiotic levels in manure before land application.

Comparative pathogenicity of early and recent isolates of avian metapneumovirus subtype C in turkeys.

The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.

Effect of Various Inoculum Levels of Multidrug-Resistant Salmonella enterica Serovar Heidelberg (2011 Ground Turkey Outbreak Isolate) on Cecal Colonization, Dissemination to Internal Organs, and Deposition in Skeletal Muscles of Commercial Turkeys after Experimental Oral Challenge.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is a major foodborne pathogen colonizing poultry. The pathogen is associated with a significant number of foodborne outbreaks through contaminated poultry meat, including turkeys. Recently, multidrug-resistant (MDR) strains of S. Heidelberg have emerged as a threat to human public health in the United States. The objective of this study was to determine the cecal colonization, dissemination to internal organs, and the potential for skeletal muscle deposition of an MDR S. Heidelberg isolate from the 2011 ground turkey outbreak in the United States after the experimental oral challenge of poults (young turkeys) and adult turkey hens. In the poult study, two separate experiments using day-old, straight-run, commercial hybrid converter poults were randomly assigned to five challenge groups (0, 10∧2, 10∧4, 10∧6, 10∧8 CFU groups; 12 poults/group; N = 60/experiment) and a week after, treatment groups were challenged separately with 0-, 2-, 4-, 6-, and 8- log10 CFU of S. Heidelberg orally. After 14 days post-challenge, the poults were euthanized, and samples were collected to determine MDR S. Heidelberg colonization in the cecum, dissemination to liver and spleen, and deposition in the thigh, drumstick, and breast muscles. A similar experimental design was followed for the adult turkey hens. In two separate experiments, 11-week-old commercial Hybrid Converter turkey hens (4 hens/group; N = 20/experiment) were challenged with MDR S. Heidelberg and on day 16 post-challenge, birds were euthanized and samples were collected to determine Salmonella populations in the samples. The results indicated that, in turkey poults, the recovery of MDR S. Heidelberg was highest in the cecum followed by spleen, liver, thigh, drumstick, and breast. All tested inoculum levels resulted in more than 3.5 log10 CFU/g colonization in the poult cecum. The cecal colonization, dissemination to internal organs, and tissue deposition of MDR S. Heidelberg were high in poults. The pathogen recovery from the cecum of adult turkey hens ranged from 37.5 to 62.5% in the challenge groups. The results signify the importance of controlling MDR S. Heidelberg in turkeys at the farm level to improve the safety of turkey products.

Rapid detection of avian pneumovirus in tissue culture by microindirect immunofluorescence test.

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.

Avian pneumovirus and its survival in poultry litter.

The survival of avian pneumovirus (APV) in turkey litter was studied at different temperature (room temperature, [approximately 22-25 C], 8 C, and -12 C) conditions. Built-up turkey litter from a turkey breeder farm known to be free of APV was obtained and was divided into two portions. One portion was sterilized by autoclaving and the other portion was kept nonautoclaved. Both samples were inoculated with a Vero cell-propagated Minnesota isolate of APV subtype C (APV/MN2A) with a titer of 10(5) 50% tissue culture infective dose at 1% level. These samples were then stored at three different temperatures: -12 C, 8 C, and room temperature (20-25 C). The samples were tested for the presence of viral RNA by reverse transcriptase-polymerase chain reaction and for the presence of live virus by virus isolation in Vero cells at the intervals of 1, 2, 3, 7, 14, 30, 60, and 90 days. Our studies revealed the presence of APV RNA even after 90 days in the autoclaved litter samples kept at -12 C and at 8 C. The virus was isolated from the autoclaved litter kept at -12 C up to 60 days. From the nonautoclaved litter, viral RNA was detected up to 60 days and virus was isolated up to 14days. The present study indicated that APV could survive in built-up turkey litter up to 60 days postinoculation at a temperature of-12 C.

Effects of bacterial coinfection on the pathogenesis of avian pneumovirus infection in turkeys.

Four- and nine-week-old poults were inoculated with cell culture propagated avian pneumovirus (APV) into each conjunctival space and nostril, followed by inoculation 3 days later with Escherichia coli, Bordetella avium (BA), or Ornithobacterium rhinotracheale or a mixture of all three (EBO). Clinical signs were evaluated on days 3, 5, 7, 9, 11, and 14 postinoculation (PI) of APV. The poults were euthanatized on days 2, 4, 6, 10, and 14 PI, and blood and tissues were collected. The poults that received APV followed by EBO or BA alone developed more severe clinical signs related to nasal discharge and swelling of intraorbital sinuses than did poults inoculated with APV alone or bacteria alone. More severe pathologic changes were found in poults inoculated with APV+BA that extended to the air sacs and lungs, particularly in 9-wk-old poults. Bordetella avium was recovered from tracheas and lungs of birds that were inoculated with APV followed by EBO or BA alone. APV was detected by immunohistochemical staining in the upper respiratory tract longer in the groups of poults inoculated with APV and pathogenic bacteria than in those that received only APV, particularly when BA was involved. Viral antigen was also detected in the lungs of poults that were inoculated with APV followed by administration of EBO or BA alone. Loss of cilia on the epithelial surface of the upper respiratory tract was associated with BA infection and may enhance infection with APV, allowing deeper penetration of the virus into the respiratory tract.