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Animal manures contain a large and diverse reservoir of antimicrobial resistance (AMR) genes that could potentially spillover into the general population through transfer of AMR to antibiotic-susceptible pathogens. The ability of poultry litter microbiota to transmit AMR was examined in this study. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 μg/mL), chloramphenicol (Cm; 25 μg/mL), streptomycin (Sm; 100 μg/mL), and tetracycline (Tc; 25 μg/mL). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA1 and sul1, respectively, in the poultry litter community. AMR gene load was determined relative to total bacterial abundance using 16S rRNA qPCR. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (

The mature intestinal microbiome is a formidable barrier to pathogen colonization. Day-old chicks seeded with cecal contents of adult hens are resistant to colonization with Salmonella, the basis of competitive exclusion. Competitive exclusion products can include individual microbes but are commonly undefined intestinal communities taken from adult animals and in commercial production is amplified in fermentator and sold commercially in freeze dried lots. While superior to single and multiple species probiotics, reducing Salmonella colonization by multiple logs, undefined products have limited acceptance because of their uncharacterized status. In this study, the bacterial composition of the master stock, preproduction seed stocks and commercial lots of a poultry competitive exclusion product, was defined by 16S rRNA sequence analysis, targeting the 16S rRNA variable region (V1-V3). The samples contained a diversity of genera (22–52 distinct genera) however, the commercial lots displayed less diversity compared to the seeds and the master stock. Community composition varied between seeds and the master stock and was not a good predictor of potency, in terms of log10 reduction in Salmonella abundance. While there was significant correlation in composition between seeds and their commercial lots, this too was a not a good predictor of potency. There was linear correlation between unclassified Actinobacteria, Peptococcus, and unclassified Erysipelotrichaceae, and Salmonella abundance (r2 > .75) for commercial seeds. However, upon review of the literature, these three genera were not consistently observed across studies or between trials that examined the correlation between intestinal community composition and Salmonella prevalence or abundance.

Focal duodenal necrosis (FDN) is an intestinal disease of egg-layer chickens characterized by multifocal necrosis of the duodenal loop and proximal jejunum. Affected flocks usually have decreased egg weights and drops in egg production. Previous studies have associated this condition with Clostridium perfringens infection. We tried to reproduce FDN by experimental infection of egg-laying chickens using different netB-positive and netB-negative C. perfringens strains, and duodenal homogenate obtained from FDN lesions. Chickens challenged with C. perfringens and/or duodenal homogenate developed duodenitis after challenge. Gross lesions included mucosal erosions, hyperemia, mucosal hemorrhages, and watery intestinal content. Microscopic lesions included mild enterocyte degeneration and necrosis, and mild-to-moderate hemorrhage and lymphoplasmacytic and heterophilic infiltration of the lamina propria. Two netB-positive C. perfringens strains closely related to necrotic enteritis pathogenic strains, by genomic composition, were re-isolated from lesions. Necrosis of intestinal crypts was observed in chickens challenged with duodenal homogenate with or without C. perfringens coinfection. Characteristic microscopic FDN lesions with significant necrosis and loss of villus enterocytes were not reproduced.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues.El síndrome del enanismo infeccioso en pollos de engorde se asocia con alteración de la morfología de las células madre intestinales y la expresión de genes. El síndrome del enanismo infeccioso (con las siglas en inglés RSS) en pollos de engorde se caracteriza por alteraciones en la morfología intestinal y en la expresión de genes, además de retraso en el crecimiento. El objetivo de este estudio fue realizar un estudio retrospectivo de la expresión genética en células madre y células diferenciadas en el intestino delgado de pollitos con el síndrome del enanismo infeccioso. Se analizaron dos modelos diferentes del síndrome del enanismo infeccioso: en pollos de engorde que fueron infectados experimentalmente y en pollos de engorde infectados naturalmente. Los pollitos infectados experimentalmente se expusieron a la cama de parvadas infectadas (RSS-litter chicks), o infectados con astrovirus (RSS-astrovirus chicks). Se adquirieron muestras intestinales de pollitos infectados naturalmente que mostraban signos clínicos del síndrome del enanismo infeccioso de granjas comerciales en Georgia y se llevaron a un laboratorio de diagnóstico avícola (RSS-Clinical-GA) y de granjas en Brasil que tenían antecedentes del síndrome del enanismo infeccioso (RSS-Clinical-BR). Los pollitos de granjas de Brasil (RSS-Clinical-BR) se separaron en aquellos que fueron positivos o negativos para gallivirus de acuerdo con la secuenciación del ADN. Se identificaron la morfología intestinal y el tipo de células intestinales en tejidos archivados fijados con formalina e incluidos en parafina. La hibridación in situ para ARNm específico de células se utilizó para identificar células madre intestinales que expresan olfactomedina 4 (Olfm4), células en proliferación que expresaban Ki67, células absorbentes que expresan el cotransportador 1 de glucosa y sodio (SGLT1) y el transportador de péptidos 1 (PepT1), y células caliciformes que expresan mucina 2 (Muc2). Los pollos expuestos a cama infectada (RSS-litter) y los infectados naturalmente de Georgia (RSS-clinical-GA) mostraron entre un 4% y un 7.5% de criptas quísticas, mientras que los pollos infectados de granjas de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un 11.7% de criptas quísticas. Los pollos infectados con astrovirus (RSS-astrovirus chicks) y los pollos de Brasil (RSS-clinical-BR) que eran negativos para gallivirus mostraron pocas criptas quísticas. Los pollos expuestos a cama infectada (RSS-litter chicks) y los pollos infectados de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un aumento en la profundidad de las criptas en comparación con los pollos control o negativos para el gallivirus, respectivamente. No se observó expresión de ARNm de Olfm4 en las células madre de pollitos expuestos a cama infectada (RSS-litter chicks) ni en pollos infectados de Georgia (RSS-clinical-GA), en contraste con la expresión normal de ARNm de Olfm4 en pollitos infectados con astrovirus (RSS-astrovirus chicks) y en pollitos infectados de Brasil (RSS-clinical-BR). Todos los pollos, independientemente del estado de infección, mostraron una expresión normal de ARNm para Ki67 en las células de la cripta, de ARNm para Muc2 en las células caliciformes y ARNm de SGLT1 o PepT1 en los enterocitos. Estos resultados demuestran que el síndrome del enanismo infeccioso, que puede ser inducido por diferentes etiologías, puede mostrar diferencias en la expresión del marcador para células madre Olfm4.

Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its micro biome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the symposium by leading international researchers in this field. The symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system were presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites, and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease, and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new-omics platforms and data management tools to more completely understand host microbiomes was stressed. Published version

The transfer of the intestinal microbiota from adult to juvenile animals reduces Salmonella prevalence and abundance. The mechanism behind this exclusion is unknown, however, certain member species may exclude or promote pathogen colonization and Salmonella abundance in chickens correlates with intestinal community composition. In this study, newly hatched chicks were colonized with Salmonella Typhimurium and 16S rRNA libraries were generated from the cecal bacterial community at 21, 28, 35, and 42 days of age. Salmonella was quantified by real-time PCR. Operational taxonomic units (OTUs) were assigned, and taxonomic assignments were made, using the Ribosomal Database Project. Bacterial diversity was inversely proportional to the Salmonella abundance in the chicken cecum (p < 0.01). In addition, cecal communities with no detectable Salmonella (exclusive community) displayed an increase in the abundance of OTUs related to specific clostridial families (Ruminococcaceae, Eubacteriaceae, and Oscillospiraceae), genera (Faecalibacterium and Turicibacter) and member species (Ethanoligenens harbinense, Oscillibacter ruminantium, and Faecalibacterium prausnitzii). For cecal communities with high Salmonella abundance (permissive community), there was a positive correlation with the presence of unclassified Lachnospiraceae, clostridial genera Blautia and clostridial species Roseburia hominis, Eubacterium biforme, and Robinsoniella peoriensis. These findings strongly support the link between the intestinal bacterial species diversity and the presence of specific member species with Salmonella abundance in the chicken ceca. Exclusive bacterial species could prove effective as direct-fed microbials for reducing Salmonella in poultry while permissive species could be used to predict which birds will be super-shedders.

Vaccine-related fowl cholera must be considered when flock mortality increases after use of a live Pasteurella multocida vaccine product. All registered live vaccines serotype as Heddleston 3,4; however, in some regions this is also the most common serotype of outbreak isolates in broiler breeders and turkeys. Therefore, serotyping may not be useful for diagnosing vaccine-related fowl cholera. This project sought to apply a vaccine-specific test to differentiate vaccine-related disease from naturally occurring outbreaks. Results indicate that vaccine strains were commonly isolated from broiler breeders exhibiting signs of fowl cholera postvaccination, but some of these isolates exhibited only serotype 4 antigenicity. The isolates' lipopolysaccharides, the target antigen for serotyping, contained compositional changes that may explain the varying serotype results and virulence of the commercial preparations. These results suggest that vaccine-related disease may be common in broiler breeders, and live commercial vaccine preparations need to be assessed for serotype and titer prior to use in order to reduce vaccine-related fowl cholera.

Salmonella enterica cause significant illnesses worldwide. There has been a marked increase in resistance to fluoroquinolones and beta-lactams/cephalosporins, antibiotics commonly used to treat salmonellosis. However, S. enterica serovars vary in their resistance to these and other antibiotics. The systemic virulence of some Salmonella serovars is due to a low copy number, IncF plasmid (65-100 kb) that contains the ADP-ribosylating toxin, SpvB. This virulence plasmid is present in only nine Salmonella serovars. It is possible that the spvB-virulence plasmid excludes other plasmids and may explain why antibiotic resistance is slow to develop in certain Salmonella serovars such as S. Enteritidis. The distribution of plasmid entry exclusion genes traS/traT and traY/excA are variable in Salmonella IncF and IncI plasmids, respectively and may account for differences in emergent antimicrobial resistance for some Salmonella serovars. The goal of this study is to determine the contribution of the Salmonella spvB-virulence plasmid in F-plasmid exclusion. From conjugation experiments, S. Typhimurium exhibited lower conjugation frequency with incFI and incFII plasmids when the spvB-virulence plasmid is present. Furthermore, introduction of cloned incFI traS into a "plasmidless" S. Typhimurium LT2 strain and Escherichia coli DH5 alpha excluded incFI plasmid. However, deletion of the virulence plasmid traS did not affect plasmid exclusion significantly compared to a spvB control deletion. In addition, differences in F plasmid conjugation in natural Salmonella isolates did not correlate with IncF or SpvB-virulence plasmid genotype. There appear to be other plasmid or chromosomal genes at play in plasmid exclusion that may be responsible for the slow development of antibiotic resistance in certain serovars. United States Department of AgricultureUnited States Department of Agriculture (USDA) [2017-05586, VA-160130] This work was supported by grants from the United States Department of Agriculture (2017-05586; VA-160130).

The rise in Salmonella resistance to cephalosporins and fluoroquinolones has become a significant threat to public health. At issue, is whether agricultural use of antimicrobials is driving emergent antibiotic resistance in Salmonella and the degree to which large antimicrobial resistance gene reservoirs, present in animal manures, drive the emergence of resistance. Two in vivo studies were performed to address these questions. In the first study, chickens were administered Salmonella and commensals, including an Escherichia coli strain with a mobile, ceftiofur-resistance plasmid, in order to determine how antibiotic administration impacted resistance in E. coli and Salmonella. All antibiotic treatments increased streptomycin resistance in E. coli. However, only ceftiofur administration increased resistance in Salmonella and specifically to extended-spectrum -lactams and cephalosporins (ESBL). There was no significant increase in emergence of ESBL-resistant Salmonella in chickens administered a ceftiofur-resistance plasmid donor. In the second study, chickens were administered two different isolates of S. enterica Typhimurium and a resistant cecal microbiome to serve as a gene donor. Birds were subsequently administered chlortetracycline or streptomycin. Antimicrobial administration significantly altered aminoglycoside and tetracycline resistance in the Enterobacteriaceae population. However, there was no significant increase in antimicrobial resistant Salmonella. Administration of a resistant microbiome had no significant impact on prevalence of resistance in Enterobacteriaceae populations, including Salmonella. Evident, from both studies, was that these treatments had minimal effect on increasing the prevalence of resistance in Salmonella, suggesting that other factors such as vertical transmission may be more important in dissemination of antimicrobial resistant Salmonella in chickens.

The role of invasive Salmonella in contamination of ground poultry is poorly defined. Salmonella harborage sites were determined in experimentally infected chickens and turkeys. Bioluminescent-tagged Salmonella were used to follow their spread in bone, meat, and skin following infection. Immunohistochemistry and culture were used to localize Salmonella. Chicken neck skin was positive for S. Heidelberg and S. Typhimurium throughout the experimental period, and the bacteria were localized on the stratum corneum of the epidermis and feather follicles. S. Heidelberg and S. Typhimurium were intermittently detected in drumstick muscle of chickens, with Salmonella primarily localized in connective tissues and lymphatics. Twenty percent of the drumstick muscles were culture positive for Salmonella in chickens at 42 days of age. Blood and tibiotarsus bone were culture positive for S. Heidelberg and S. Typhimurium during the first 2 weeks of infection. Salmonella levels in neck skin and muscle were

Focal duodenal necrosis (FDN) is a poorly understood intestinal disease of egg layers, and has been associated with drops in egg production and decreased egg weights. The etiology of this disease is still unknown, but the condition has been associated with Clostridium colinum and Clostridium perfringens. In order to investigate the etiology, duodenal samples were taken from hens with FDN. The hens originated from table egg layer farms in three states. The samples were examined by histopathology, bacteriology, and immunohistochemistry. Macroscopically, all samples contained focal to multifocal, variably sized, reddened or brownish gray areas of mucosal erosion. Histopathology revealed mild to severe heterophilic and lymphoplasmacytic enteritis with loss of enterocytes at the villous tips, luminal fibrinonecrotic exudate, and variable numbers of Gram-positive and Gram-negative rod-shaped bacteria within the lesions in 16/30 samples. Clostridium perfringens was isolated by anaerobic bacteriology from 4/13 samples that had characteristic microscopic lesions of FDN. Polymerase chain reaction (PCR) revealed that all four isolates were Type A C. perfringens, positive for beta2 gene and negative for necrotic enteritis toxin B and enterotoxin genes. PCR for Clostridium colinum applied to DNA extracted from frozen intestinal samples yielded negative results in 14/14 duodenal samples. Immunohistochemistry (IHC) for 7C. perfringens, alpha and beta2 toxins stained a few to numerous long rod-shaped bacteria present in the lesions. IHC for alpha and beta2 toxins also stained enterocytes at the villous tips, inflammatory cells in the lamina propria, as well as degenerated and sloughed enterocytes present within the luminal exudate. These findings suggest that C. perfringens may play a role in the development of FDN. Experimental challenge studies with these isolates still need to be performed in order to reproduce the disease and fulfill Koch's postulates.

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S . Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA , sseI , sopE , and sodC1. S . Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S . Kentucky and S . Typhimurium grown microaerophilically in cecal contents, S . Kentucky persisted longer when chickens were coinfected with S . Typhimurium. The in vivo advantage that S . Kentucky has over S . Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS . Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S . Kentucky. The contributions of four of these operons ( mgl , prp , nar , and csg ) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S . Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella 's adaptation to its animal host and especially for S . Kentucky's emergence as the dominant serovar in poultry.

Salmonella remains the leading cause of foodborne illness in the United States, and the dissemination of drug-resistant Salmonellae through the food chain has important implications for treatment failure of salmonellosis. We investigated the ecology of Salmonella in integrated broiler production in order to understand the flow of antibiotic susceptible and resistant strains within this system. Data were analyzed from a retrospective study focused on antimicrobial resistant Salmonella recovered from commercial broiler chicken farms conducted during the initial years of the U.S. FDA’s foray into retail meat surveillance by the National Antimicrobial Resistance Monitoring System (NARMS). Sixty-three percent of Salmonella were pan-susceptible to a panel of nineteen antimicrobials used by the NARMS program. Twenty-five antimicrobial resistance phenotypes were observed in Salmonella isolated from two broiler chicken farms. However, Salmonella displaying resistance to streptomycin, alone, and in combination with other antibiotics was the most prevalent (36.3%) antimicrobial resistance phenotype observed. Resistance to streptomycin and sulfadimethoxine appeared to be linked to the transposon, Tn21. Combinations of resistance against streptomycin, gentamicin, sulfadimethoxine, trimethoprim, and tetracycline, were observed for a variety of S. enterica serovars and genetic types as defined by pulsed-field gel electrophoresis. There were within and between farms differences in the antibiotic susceptibilities of Salmonella and some of these differences were linked to specific serovars. However, farm differences were not linked to antibiotic usage. Analysis of the temporal and spatial distribution of the endemic Salmonella serovars on these farms, suggests that preventing vertical transmission of antibiotic-resistant Salmonella would reduce carcass contamination with antibiotic-resistant Salmonella and subsequently human risk exposure.

OBJECTIVE To determine effects of in ovo administration of a probiotic on development of the intestinal microbiota of 2 genetic lineages (modern and heritage) of chickens. SAMPLE 10 newly hatched chicks and 40 fertile eggs to determine intestinal microbiota at hatch, 900 fertile eggs to determine effects of probiotic on hatchability, and 1,560 chicks from treated or control eggs. PROCEDURES A probiotic competitive-exclusion product derived from adult microbiota was administered in ovo to fertile eggs of both genetic lineages. Cecal contents and tissues were collected from embryos, newly hatched chicks, and chicks. A PCR assay was used to detect bacteria present within the cecum of newly hatched chicks. Fluorescence in situ hybridization and vitality staining were used to detect viable bacteria within intestines of embryos. The intestinal microbiota was assessed by use of 16S pyrosequencing. RESULTS Microscopic evaluation of embryonic cecal contents and tissues subjected to differential staining techniques revealed viable bacteria in low numbers. Development of the intestinal microbiota of broiler chicks of both genetic lineages was enhanced by in ovo administration of adult microbiota. Although the treatment increased diversity and affected composition of the microbiota of chicks, most bacterial species present in the probiotic were transient colonizers. However, the treatment decreased the abundance of undesirable bacterial species within heritage lineage chicks. CONCLUSIONS AND CLINICAL RELEVANCE In ovo inoculation of a probiotic competitive-exclusion product derived from adult microbiota may be a viable method of managing development of the microbiota and reducing the prevalence of pathogenic bacteria in chickens.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the "sweating" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

SUMMARY the intestinal microbiota are composed of a large diversity of microorganisms. some have been cultured and characterized, but most remain uncultivated and thus uncharacterized. the intestinal microbiota play a significant role in host health, nutrition, physiology, and performance. In recent studies, researchers have focused on the complex mechanisms involved in host-microbiota symbiosis. Host physiological homeostasis contributes to the stability and composition of the microbiota. Very little is known about microbial competition or metabolism within the intestinal environment, and even less is known about how microbial interactions influence intestinal health and pathogen exclusion. Here we explore factors such as the challenges of studying the intestinal microbiota, how the composition of the microbiota is affected by intestinal substrates and the host corporeal composition, and whether it is possible to explain the effects of the intestinal microbiota modified by growth promoters. Finally, we discuss the desirable effects of growth promoters on the microbiota with regard to intestinal health and pathogen exclusion. Understanding how feed additives affect the intestinal community and the gut environment is the first step in designing strategies to manipulate performance and gut health.

Impacts • Salmonella was detected in 88% of the broiler breeder houses that were sampled in four south-eastern states and was identified on all 49 farms enrolled in the study. • Management characteristics associated with increased environmental Salmonella prevalence included treatment of the flock for any disease, dusty conditions in the house, dry conditions under the slats and picking-up dead birds more than one time per day. • The intraclass correlations for samples collected from the same house (0.27) and from different houses on the same farm (0.24) were relatively high, indicating that farm-level clustering is an important consideration when evaluating Salmonella prevalences.

Salmonella continues to cause significant foodborne outbreaks, best illustrated with recent outbreaks associated with peanut butter, raw tomatoes, and serrano peppers. To ascertain the likely source of the outbreak, bacterial typing is essential to this process. While PCR has become an important detection tool for pathogens in foods, PCR can also identify strain differences by targeting gene(s) or sequences exhibiting polymorphisms or variability in its distribution within the bacterial population. Over 2,500 Salmonella enterica serovars identified based on antigenic differences in lipopolysaccharide and flagellin have been identified to date. We developed an allelotyping PCR scheme that identifies the O and H alleles associated with S. enterica serovars Enteritidis, Hadar, Heidelberg, Typhimurium, and others, with the same antigen alleles but in different O- and H-allele combinations (e.g., S. enterica Kentucky), and validated it as a screen to identify samples contaminated with these Salmonella serovars. We correctly identified poultry samples containing S. enterica serovars Enteritidis, Kentucky, and Typhimurium from our multiplex screen of primary enrichments of environmental drag swabs. PCR agreed well (kappa values = 0.81 to 1.0) with conventional serotyping methods used to type salmonellae isolated from primary enrichment. Coupled with Salmonella-specific PCR, such as invA, this allelotyping PCR could prove useful in the identification of Salmonella and specific S. enterica serovars present in foods or the environment and could decrease the time and cost associated with conventional serotyping methods.

Intensifying concerns about the use of antimicrobials in meat and poultry production has enhanced interest in the application of prebiotics, probiotics and enzymes to enhance growth and prevent disease in food animals. Growth-promoting antibiotics enhance growth of animals by reducing the load of bacteria in the intestine, by reducing colonization by intestinal pathogens or by enhancing the growth and/or metabolism of beneficial bacteria in the intestine. Recently, molecular ecology, utilizing DNA-sequence heterogeneity of the 16S rRNA gene, has revealed a surprising diversity of uncharacterized bacteria inhabiting this ecosystem. We used this approach to determine the effect of growth-promoting antibiotics on the development and composition of the ileal bacterial community. Pairwise comparisons, correspondence analysis and community diversity indices revealed significant differences among the treatments (bacitracin/virginiamycin or monensin) and controls. Antibiotics reduced the diversity of the ileal bacterial community and induced communities rich in Clostridia throughout the life of the broiler chicken. These results indicate that some bacterial species, such as lactobacilli, were suppressed and also suggest that many intestinal Clostridia may be non-pathogenic. Future studies should focus on characterizing the important bacterial species needed to stabilize the intestinal microbiota and identifying those commensals that stimulate and enhance development of intestinal function.

Runting-stunting syndrome (RSS) in poultry has been known for more than 40 years, but the precise etiology remains unknown and a licensed vaccine is consequently not currently available. In order to mitigate the symptoms associated with RSS, a series of experiments was performed to investigate whether a combined bacteriotherapeutic treatment consisting of probiotics, prebiotics, and organic acids could influence the outcome of this disease. Initially two groups of commercial broiler chickens were either left uninoculated or inoculated with filtrate from homogenized intestines of RSS-affected broiler chickens. One group from each of these two challenge groups was treated, with a bacteriotherapeutic regimen. After 12 days chickens were euthanatized, the body weight was measured, and duodenal lesions were enumerated. Five consecutive broiler chicken flocks were then raised either on litter from RSS-affected birds or on fresh wood shavings. Treatment had no beneficial effect on the number and severity of intestinal lesions. There appeared to be a significant build-up of RSS agent(s) in poultry litter, with each consecutive flock placement, independent of bacteriotherapeutic treatment, as more individuals exhibited intestinal lesions on built-up litter in RSS-affected houses (28.9% vs. 44%). While treatment did not appear to consistently reduce intestinal lesions, it did significantly improve the mean body weights (P0.05) and uniformity of 12-day-old chickens placed on reused litter in houses in which RSS-infected birds were previously raised. A combination of litter management and bacteriotherapy may be needed to ameliorate the adverse effects of RSS on intestinal health and body weight in broiler chickens.

While characterizing the intestinal bacterial community of broiler chickens, we detected ε-proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69: 6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter -specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis . The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli . These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.

Epidemiological studies indicate that Campylobacter species may be responsible for the majority of cases of sporadic gastroenteritis in humans. These studies also suggest that poultry may be one of the most common sources of the bacteria for humans. Campylobacter and related genera in the family Campylobacteraceae are oral and intestinal commensals of vertebrates and some nonvertebrates, a characteristic that complicates rational approaches to controlling Campylobacter contamination of poultry. This review will discuss the phylogeny, genomics, and physiology of campylobacters with the intention of revealing how these organisms have evolved to fill their intestinal ecological niche in poultry and how their physiology must be understood in order to enact effective control strategies.

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli , and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 μg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 μg/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus , with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae -related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10 9 CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens , Corynebacterium ammoniagenes , Corynebacterium urealyticum , Clostridium aminovalericum , Arthrobacter sp., and Denitrobacter permanens , that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella , pathogenic Escherichia coli , Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.

Using PCR, we screened 105 isolates of poultry-associated Campylobacter jejuni for the presence of class 1 integrons. Of those isolates, 21% (22 of 105) possessed the integrase gene, but only 5 isolates produced an amplicon in a 5′-3′ conserved sequence PCR directed toward amplification of the resistance cassettes. DNA sequencing demonstrated that all five isolates possessed the aminoglycoside resistance gene, aacA4 .

The domestic chicken is a common model organism for human biological research and of course also forms the basis of a global protein industry. Recent methodological advances have spurred the recognition of microbiomes as complex communities with important influences on the health and disease status of the host. In this minireview, we provide an overview of the current state of knowledge of the chicken gastrointestinal microbiome focusing on spatial and temporal variability, the presence and importance of human pathogens, the influence of the microbiota on the immune system, and the importance of the microbiome for poultry nutrition. Review and meta-analysis of public data showed cecal communities dominated by Firmicutes and Bacteroides at the phylum level, while at finer levels of taxonomic resolution, a phylogenetically diverse assemblage of microorganisms appears to have similar metabolic functions that provide important benefits to the host as inferred from metagenomic data. This observation of functional redundancy may have important implications for management of the microbiome. We foresee advances in strategies to improve gut health in commercial operations through management of the intestinal microbiota as an alternative to in-feed subtherapeutic antibiotics, improvements in pre- and probiotics, improved management of polymicrobial poultry diseases, and better control of human pathogens via colonization reduction or competitive exclusion strategies.

Alternative growth promoters such as probiotics, prebiotics and competitive exclusion products have been used with some success in animal production. A compelling application of these products is their potential to reduce pathogen populations by altering the environmental bacterial community of a built up litter system over multiple grow-outs. The objectives of this study were determine the bacterial composition of the litter over multiple production cycles and describe the impact of alternative growth promoters on composition and abundance of bacteria viewed as beneficial or pathogenic. In this study, probiotics were found to have the ability to positively alter the litter bacterial community. Bacterial diversity and the proportion of the community consisting of beneficial bacteria were increased in the litter when probiotics were administered. As the litter aged, diversity decreased and the composition shifted from Firmicute to Actinobacteria. Detection of pathogenic Clostridium species increased in litter with time regardless of the type of product used. However, detection of Enterococcus cecorum decreased over time and treatments produced less E. cecorum in reused litter after 1 flock cycle. These findings suggest that administering alternatives to antibiotics can change the litter bacterial composition, increase the abundance of beneficial bacteria and reduce the prevalence of some pathogens.

Many pathogenic and commensal organisms are multidrug resistant due to exposure to various antibiotics. Often, this antimicrobial resistance is encoded by integrons that occur on plasmids or that are integrated into the bacterial chromosome. Integrons are commonly associated with bacterial genera in the family Enterobacteriaceae . We determined that class 1 integrases were present in approximately 46% of the isolates from the family Enterobacteriaceae ; class 2 integrases were present only among Escherichia coli and Salmonella isolates. Seven percent of veterinary isolates were positive for class 3 integrase by DNA-DNA hybridization but could not be confirmed to be positive by PCR. None of the veterinary isolates possessed the class 4 integrase gene. The distribution of these integrase genes was variable within the members of the family Enterobacteriaceae when some or all integrase classes were absent from a particular genus. There was also considerable variability in the distribution of these integrases within a species, depending on the animal host. Unlike the class 1 integrases, the other integrase class, intI2 , appears to be more restricted in its distribution among the members of the family Enterobacteriaceae . There is also considerable variability in the distribution of the class 1 integrases within E. coli strains isolated from different food animals. The class 1 integrases are the most widely disseminated of the four classes among the members of the family Enterobacteriaceae from both the clinical and normal flora of animals. This is the first report to closely examine the distribution of class 2 integrases in members of the family Enterobacteriaceae isolated in the United States.

Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH , from a fowl cholera isolate of P. multocida . Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3′ sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3′ and 2-6′ sialyl lactose, while NanH demonstrated activity only on 2-3′ sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8′ sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.

Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces.

Salmonella infections have been implicated in large-scale die-offs of wild birds in the United States. Although we know quite a bit about the epidemiology of Salmonella infection among domestic fowl, we know little about the incidence, epidemiology, and genetic relatedness of salmonellae in nondomestic birds. To gain further insight into salmonellae in these hosts, 22 Salmonella isolates from diseased nondomestic birds were screened for the presence of virulence and antibiotic resistance-associated genes and compared genetically using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA analysis. Of the 22 Salmonella isolates examined, 15 were positive for the invasion gene invA and the virulence plasmid-associated genes spvC and pef . Most (15 of 22) were generally sensitive to antibiotics. However, two Salmonella isolates from pet birds were identified as Salmonella enterica serovar Typhimurium DT104. Despite the general susceptibility of these Salmonella isolates to most antimicrobial agents, antibiotic resistance-associated genes intI1 , merA , and aadA1 were identified in a number of these isolates. Five distinct Xba I and nine distinct Bln I DNA patterns were observed for the 22 Salmonella isolates typed by PFGE. PFGE analysis determined that Salmonella isolates from passerines in Georgia and Wyoming were genetically related.

Florfenicol is an antibiotic approved for veterinary use in cattle in the United States in 1996. Although this drug is not used in poultry, we have detected resistance to florfenicol in clinical isolates of avian Escherichia coli . Molecular typing demonstrated that the florfenicol resistance gene, flo , was independently acquired and is plasmid encoded.

Salmonellae are gastrointestinal pathogens of man and animals. However, strains that are host-specific avian pathogens are often avirulent in mammals, and those which are nonspecific are commensal in poultry. The objective of this study was to determine whether host specificity was exhibited by bacterial abilities to invade epithelial cells or resist leukocyte killing. In this study, leukocytes isolated from humans and chickens were used to kill Salmonella in vitro. Both Salmonella pullorum , an avian-specific serotype, and Salmonella typhimurium , a broad-host-range serotype, were sensitive to killing by polymorphonuclear leukocytes isolated from both species. Both serotypes replicated in cells of the MQ-NCSU avian-macrophage cell line. In contrast, S. pullorum was noninvasive for cultured epithelial Henle 407, chick kidney, chick ovary, and budgerigar abdominal tumor cells. In the bird challenge, however, S. typhimurium rapidly caused inflammation of the intestinal mucosa, but S. pullorum preferentially targeted the bursa of Fabricius prior to eliciting intestinal inflammation. Salmonella serotypes which cause typhoid fever in mice have been shown to target the gut-associated lymphoid tissue. Observations from this study show that S. pullorum initiated a route of infection in chicks comparable to the route it takes in cases of enteric fever.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.

Salmonella enterica serotype typhimurium ( S. typhimurium ) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes flo S. typhimurium ( flo St ), int , invA , and spvC were produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene flo St , reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the flo St -positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene flo St , described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the flo St gene or its resulting phenotype.