Paternal nicotine taking elicits heritable sex-specific phenotypes that are mediated by hippocampal Satb2.

Nicotine intake, whether through tobacco smoking or e-cigarettes, remains a global health concern. An emerging preclinical literature indicates that parental nicotine exposure produces behavioral, physiological, and molecular changes in subsequent generations. However, the heritable effects of voluntary parental nicotine taking are unknown. Here, we show increased acquisition of nicotine taking in male and female offspring of sires that self-administered nicotine. In contrast, self-administration of sucrose and cocaine were unaltered in male and female offspring suggesting that the intergenerational effects of paternal nicotine taking may be reinforcer specific. Further characterization revealed memory deficits and increased anxiety-like behaviors in drug-naive male, but not female, offspring of nicotine-experienced sires. Using an unbiased, genome-wide approach, we discovered that these phenotypes were associated with decreased expression of Satb2, a transcription factor known to play important roles in synaptic plasticity and memory formation, in the hippocampus of nicotine-sired male offspring. This effect was sex-specific as no changes in Satb2 expression were found in nicotine-sired female offspring. Finally, increasing Satb2 levels in the hippocampus prevented the escalation of nicotine intake and rescued the memory deficits associated with paternal nicotine taking in male offspring. Collectively, these findings indicate that paternal nicotine taking produces heritable sex-specific molecular changes that promote addiction-like phenotypes and memory impairments in male offspring.

rpoS-Regulated core genes involved in the competitive fitness of Salmonella enterica Serovar Kentucky in the intestines of chickens.

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.

Homeostatic regulation of rapid eye movement sleep by the preoptic area of the hypothalamus.

Rapid eye movement sleep (REMs) is characterized by activated electroencephalogram (EEG) and muscle atonia, accompanied by vivid dreams. REMs is homeostatically regulated, ensuring that any loss of REMs is compensated by a subsequent increase in its amount. However, the neural mechanisms underlying the homeostatic control of REMs are largely unknown. Here, we show that GABAergic neurons in the preoptic area of the hypothalamus projecting to the tuberomammillary nucleus (POAGAD2→TMN neurons) are crucial for the homeostatic regulation of REMs in mice. POAGAD2→TMN neurons are most active during REMs, and inhibiting them specifically decreases REMs. REMs restriction leads to an increased number and amplitude of calcium transients in POAGAD2→TMN neurons, reflecting the accumulation of REMs pressure. Inhibiting POAGAD2→TMN neurons during REMs restriction blocked the subsequent rebound of REMs. Our findings reveal a hypothalamic circuit whose activity mirrors the buildup of homeostatic REMs pressure during restriction and that is required for the ensuing rebound in REMs.

Early intestinal development of chickens divergently selected for high or low 8-wk body weight and a commercial broiler.

The early posthatch period is crucial to intestinal development, shaping long-term growth, metabolism, and health of the chick. The objective of this study was to determine the effect of genetic selection on morphological characteristics and gene expression during early intestinal development. Populations of White Plymouth Rocks have been selected for high weight (HWS) and low weight (LWS) for over 63 generations, and some LWS display symptoms of anorexia. Intestinal structure and function of these populations were compared to a commercial broiler Cobb 500 (Cobb) during the perihatch period. Egg weights, yolk-free embryo BW, yolk weights, and jejunal samples from HWS, LWS, and Cobb were collected on embryonic day (e) 17, e19, day of hatch, day (d) 3, d5, and d7 posthatch for histology and gene expression analysis. The RNAscope in-situ hybridization method was used to localize expression of the stem cell marker, olfactomedin 4 (Olfm4). Villus height (VH), crypt depth (CD), and VH/CD were measured from Olfm4 stained images using ImageJ. mRNA abundance for Olfm4, stem cell marker Lgr5, peptide transporter PepT1, goblet cell marker Muc2, marker of proliferation Ki67, and antimicrobial peptide LEAP2 were examined. Two-factor ANOVA was performed for measurements and Turkey's HSD was used for mean separation when appropriate. Cobb were heaviest and LWS the lightest (P < 0.01). at each timepoint. VH increased in Cobb and CD increased in HWS compared to LWS (P < 0.01). PepT1 mRNA was upregulated in LWS (P < 0.01), and Muc2 mRNA was decreased in both HWS and LWS compared to Cobb (P < 0.01). Selection for high or low 8-wk body weight has caused differences in intestinal gene expression and morphology when compared to a commercial broiler.

Peeling back the many layers of competitive exclusion.

Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities' response to Salmonella in chickens with low (≤5.85 Log10 genomes/g) or high (≥6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella's SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community's stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.

Sleep disturbances in autism spectrum disorder: Animal models, neural mechanisms, and therapeutics.

Sleep is crucial for brain development. Sleep disturbances are prevalent in children with autism spectrum disorder (ASD). Strikingly, these sleep problems are positively correlated with the severity of ASD core symptoms such as deficits in social skills and stereotypic behavior, indicating that sleep problems and the behavioral characteristics of ASD may be related. In this review, we will discuss sleep disturbances in children with ASD and highlight mouse models to study sleep disturbances and behavioral phenotypes in ASD. In addition, we will review neuromodulators controlling sleep and wakefulness and how these neuromodulatory systems are disrupted in animal models and patients with ASD. Lastly, we will address how the therapeutic interventions for patients with ASD improve various aspects of sleep. Together, gaining mechanistic insights into the neural mechanisms underlying sleep disturbances in children with ASD will help us to develop better therapeutic interventions.

Pioneer colonizers: Bacteria that alter the chicken intestinal morphology and development of the microbiota.

Microbes commonly administered to chickens facilitate development of a beneficial microbiome that improves gut function, feed conversion and reduces pathogen colonization. Competitive exclusion products, derived from the cecal contents of hens and shown to reduce Salmonella colonization in chicks, possess important pioneer-colonizing bacteria needed for proper intestinal development and animal growth. We hypothesized that inoculation of these pioneer-colonizing bacteria to day of hatch chicks would enhance the development of their intestinal anatomy and microbiome. A competitive exclusion product was administered to broiler chickens, in their drinking water, at day of hatch, and its impact on intestinal morphometrics, intestinal microbiome, and production parameters, was assessed relative to a control, no treatment group. 16S rRNA gene, terminal restriction fragment length polymorphism (T-RFLP) was used to assess ileal community composition. The competitive exclusion product, administered on day of hatch, increased villus height, villus height/width ratio and goblet cell production ∼1.25-fold and expression of enterocyte sugar transporters 1.25 to 1.5-fold in chickens at 3 days of age, compared to the control group. As a next step, chicks were inoculated with a defined formulation, containing Bacteroidia and Clostridia representing pioneer-colonizing bacteria of the two major bacterial phyla present in the competitive exclusion product. The defined formulation, containing both groups of bacteria, were shown, dependent on age, to improve villus height (jejunum: 1.14 to 1.46-fold; ileum: 1.17-fold), goblet cell numbers (ileum 1.32 to 2.51-fold), and feed efficiency (1.18-fold, day 1) while decreasing Lactobacillus ileal abundance by one-third to half in birds at 16 and 42 days of age, respectively; compared to the phosphate buffered saline treatment group. Therefore, specific probiotic formulations containing pioneer colonizing species can provide benefits in intestinal development, feed efficiency and body weight gain.

Conditions Necessary for the Transfer of Antimicrobial Resistance in Poultry Litter.

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 (<104 CFU/g). There was high abundance of resistance to Sm (106 to 107 CFU/g) and Tc (106 to 107 CFU/g) and a sizeable antimicrobial-resistance gene load in regards to gene copies per bacterial genome (aadA1: 0.0001-0.0060 and sul1: 0.0355-0.2455). While plasmid transfer was observed from Escherichia coli R100, as an F-plasmid donor control, to the Salmonella recipient in vitro, no AMR Salmonella were detected in a poultry litter microcosm with the inclusion of E. coli R100. Confirmatory experiments showed that isolated poultry litter bacteria were not interfering with plasmid transfer in filter matings. As no R100 transfer was observed at 25 °C, conjugative plasmid pRSA was chosen for its high plasmid transfer frequency (10-4 to 10-5) at 25 °C. While E. coli strain background influenced the persistence of pRSA in poultry litter, no plasmid transfer to Salmonella was ever observed. Although poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low under conditions commonly used to assess plasmid conjugation.

Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

Epidemiology of a Salmonella enterica subsp. enterica serovar Typhimurium strain associated with a songbird outbreak.

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.

Bacterial composition of a competitive exclusion product and its correlation with product efficacy at reducing Salmonella in poultry.

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 (r 2 > .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.

Runting Stunting Syndrome in Broiler Chickens Is Associated with Altered Intestinal Stem Cell Morphology and Gene Expression.

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. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

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.

Variation in Salmonella enteritidis RAPD-PCR patterns may not be due to genetic differences.

Salmonella Enteritidis is a leading cause of gastroenteritis associated with consumption of contaminated poultry meat and eggs. Because pulsed-field gel electrophoresis (PFGE) has limited utility in distinguishing between clonal Salmonella Enteritidis isolates, random amplified polymorphic DNA (RAPD) PCR has been recommended as an alternative molecular fingerprinting tool. This study's objective was to determine whether increasing PCR stringency would improve the repeatability of RAPD DNA patterns based on assessment of target sites within the genome. An in silico PCR was performed to predict amplification products from an Salmonella Enteritidis genome sequence for three different RAPD primers (1247, 1283, and OPA4) and to determine whether any primer would be more likely to amplify variable regions within the genome. A comparison of within- and between-isolate similarities in RAPD patterns was performed using primer 1247, which was predicted by in silico analysis to yield a variable size range of amplicons. In order to reduce artifactual variability associated with the method, three different methods for template preparation were evaluated. All were found to provide comparable results with respect to the similarities observed with repeated analyses of the same Salmonella Enteritidis isolates (n = 18, P = 0.91). Although the median within-isolate similarity (76.0%) was significantly greater than the median between-isolate similarity (66.7%; P = 0.001), duplicate RAPD-PCR runs of the same Salmonella Enteritidis isolates produced DNA patterns that ranged in similarity between 61.5 and 100%. These results indicate that the repeatability of RAPD-PCR is insufficient to distinguish genetic differences among related and unrelated Salmonella Enteritidis isolates.

Strength Lies in Diversity: How Community Diversity Limits Salmonella Abundance in the Chicken Intestine.

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.

Free-Living Aquatic Turtles as Sentinels of Salmonella spp. for Water Bodies.

Reptile-associated human salmonellosis cases have increased recently in the United States. It is not uncommon to find healthy chelonians shedding Salmonella enterica. The rate and frequency of bacterial shedding are not fully understood, and most studies have focused on captive vs. free-living chelonians and often in relation to an outbreak. Their ecology and significance as sentinels are important to understanding Salmonella transmission. In 2012-2013, Salmonella prevalence was determined for free-living aquatic turtles in man-made ponds in Clarke and Oconee Counties, in northern Georgia (USA) and the correlation between species, basking ecology, demographics (age/sex), season, or landcover with prevalence was assessed. The genetic relatedness between turtle and archived, human isolates, as well as, other archived animal and water isolates reported from this study area was examined. Salmonella was isolated from 45 of 194 turtles (23.2%, range 14-100%) across six species. Prevalence was higher in juveniles (36%) than adults (20%), higher in females (33%) than males (18%), and higher in bottom-dwelling species (31%; common and loggerhead musk turtles, common snapping turtles) than basking species (15%; sliders, painted turtles). Salmonella prevalence decreased as forest cover, canopy cover, and distance from roads increased. Prevalence was also higher in low-density, residential areas that have 20-49% impervious surface. A total of 9 different serovars of two subspecies were isolated including 3 S. enterica subsp. arizonae and 44 S. enterica subsp. enterica (two turtles had two serotypes isolated from each). Among the S. enterica serovars, Montevideo (n = 13) and Rubislaw (n = 11) were predominant. Salmonella serovars Muenchen, Newport, Mississippi, Inverness, Brazil, and Paratyphi B. var L(+) tartrate positive (Java) were also isolated. Importantly, 85% of the turtle isolates matched pulsed-field gel electrophoresis patterns of human isolates, including those reported from Georgia. Collectively, these results suggest that turtles accumulate Salmonella present in water bodies, and they may be effective sentinels of environmental contamination. Ultimately, the Salmonella prevalence rates in wild aquatic turtles, especially those strains shared with humans, highlight a significant public health concern.

Effect of Salmonella vaccination of breeder chickens on contamination of broiler chicken carcasses in integrated poultry operations.

While measures to control carcass contamination with Salmonella at the processing plant have been implemented with some success, on-farm interventions that reduce Salmonella prevalence in meat birds entering the processing plant have not translated well on a commercial scale. We determined the impact of Salmonella vaccination on commercial poultry operations by monitoring four vaccinated and four nonvaccinated breeder (parental) chicken flocks and comparing Salmonella prevalences in these flocks and their broiler, meat bird progeny. For one poultry company, their young breeders were vaccinated by using a live-attenuated Salmonella enterica serovar Typhimurium vaccine (Megan VAC-1) followed by a killed Salmonella bacterin consisting of S. enterica serovar Berta and S. enterica serovar Kentucky. The other participating poultry company did not vaccinate their breeders or broilers. The analysis revealed that vaccinated hens had a lower prevalence of Salmonella in the ceca (38.3% versus 64.2%; P < 0.001) and the reproductive tracts (14.22% versus 51.7%; P < 0.001). We also observed a lower Salmonella prevalence in broiler chicks (18.1% versus 33.5%; P < 0.001), acquired from vaccinated breeders, when placed at the broiler farms contracted with the poultry company. Broiler chicken farms populated with chicks from vaccinated breeders also tended to have fewer environmental samples containing Salmonella (14.4% versus 30.1%; P < 0.001). There was a lower Salmonella prevalence in broilers entering the processing plants (23.4% versus 33.5%; P < 0.001) for the poultry company that utilized this Salmonella vaccination program for its breeders. Investigation of other company-associated factors did not indicate that the difference between companies could be attributed to measures other than the vaccination program.

Differentiating Vaccine-Related Fowl Cholera from Naturally Occurring Disease.

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.

The Role of the Salmonella spvB IncF Plasmid and Its Resident Entry Exclusion Gene traS on Plasmid Exclusion.

Salmonella enterica cause significant illnesses worldwide. There has been a marked increase in resistance to fluoroquinolones and ß-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 DH5α 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.

The Contribution of Eimeria Coinfection and Intestinal Inflammation to Cecal Colonization and Systemic Spread of Salmonella Typhimurium Deficient in Tetrathionate Reductase or Type III Secretion Systems Salmonella Pathogenicity Island 1 or 2.

Intestinal inflammation may provide a growth advantage for Salmonella and enhance its systemic spread in chickens. Salmonella triggers intestinal inflammation in the host by using type III secretion systems (T3SS) and produces the inflammatory end product tetrathionate. In mice, tetrathionate respiration confers a growth advantage for Salmonella Typhimurium over the competitive microbiome in the inflamed intestine. Coccidia also promote intestinal inflammation and enhance Salmonella intestinal growth and systemic spread in chickens. The objective of this study was to evaluate the contribution of inflammation, induced by Eimeria spp. or Salmonella Typhimurium, to Salmonella colonization and dissemination in chickens. In addition, the fitness costs associated with defects in tetrathionate reductase and T3SS associated with Salmonella Pathogenicity Island 1 or 2 (SPI-1 or SPI-2) were evaluated in in vivo competition experiments with wild-type Salmonella strain, with or without Eimeria coinfection. One-day-old specific-pathogen-free chickens were orally inoculated with a sham inoculum or with 4 × 102Eimeria oocysts cocktail of Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria mitis. At 6 days of age, birds were orally administered a 1:1 ratio of Salmonella Typhimurium wild-type and mutant deficient in tetrathionate reductase, SPI-1, or SPI-2 (108 colony forming units/bird). Ceca, livers, and drumsticks were collected at 3, 7, 14, and 42 days after Salmonella infection, for bacteriology. Intestinal inflammation was scored by histology. Significantly higher intestinal inflammation was observed in challenge groups compared with the control. However, there were no significant differences in intestinal inflammation scores between groups coinfected with both Eimeria spp. and Salmonella Typhimurium and birds infected with Salmonella alone, and Eimeria coinfection did not increase Salmonella prevalence or abundance. Contrary to mouse studies, tetrathionate reductase did not enhance Salmonella Typhimurium cecal colonization or systemic spread in chickens. SPI-1 and SPI-2 played a significant role in Salmonella dissemination and cecal colonization in chickens, respectively.

Contribución de la coinfección por Eimeria y de la inflamación intestinal a la colonización cecal y a la propagación sistémica de Salmonella Typhimurium deficiente en tetrationato reductasa o de sistemas de secreción de tipo III de islas de patogenicidad 1 o 2 de Salmonella. La inflamación intestinal puede proporcionar una ventaja para el crecimiento de Salmonella y aumentar su propagación sistémica en pollos. Salmonella desencadena la inflamación intestinal en el huésped mediante el uso de sistemas de secreción tipo III (T3SS) y produce el producto final inflamatorio, tetrationato. En ratones, la respiración con tetrationato confiere una ventaja de crecimiento para Salmonella Typhimurium sobre el microbioma competitivo en el intestino inflamado. Coccidia también promueve la inflamación intestinal y mejora el crecimiento intestinal de Salmonella y la propagación sistémica en pollos. El objetivo de este estudio fue evaluar la contribución de la inflamación, inducida por Eimeria spp. o Salmonella Typhimurium, en la colonización y diseminación de Salmonella en pollos. Además, se evaluaron los costos de aptitud asociados con defectos en la tetrationato reductasa y T3SS asociados con las islas de patogenicidad 1 o 2 de Salmonella (SPI-1 o SPI-2) mediante experimentos de competencia in vivo con cepas de Salmonella de tipo silvestre, con o sin coinfección con Eimeria. Pollos libres de patógenos específicos de un día de edad se inocularon por vía oral con un inóculo falso o con 4 × 102 de un coctel de ooquistes de Eimeria que incluyó Eimeria tenella, Eimeria acervulina, Eimeria maxima y Eimeria mitis. A los seis días de edad, se les administró a las aves administró por vía oral una proporción 1: 1 de Salmonella Typhimurium de tipo silvestre o tipo mutante que es deficiente de tetrationato reductasa, SPI-1 o SPI-2 (108 unidades formadoras de colonias/ave). Se recolectaron ciegos, hígados y pernas a los tres, siete, catorce y 42 días después de la infección por Salmonella, para bacteriología. La inflamación intestinal se calificó por histología. Se observó inflamación intestinal significativamente mayor en los grupos de desafío en comparación con el control. Sin embargo, no hubo diferencias significativas en las puntuaciones de inflamación intestinal entre los grupos coinfectados con Eimeria spp. y Salmonella Typhimurium y las aves infectadas con Salmonella por si sola y la coinfección con Eimeria no aumentó la prevalencia o abundancia de Salmonella. A diferencia de los estudios en ratones, la tetrationato reductasa no mejoró la colonización cecal de Salmonella Typhimurium o la diseminación sistémica en pollos. Las islas de patogenicidad SPI-1 y SPI-2 jugaron un papel importante en la diseminación de Salmonella y en la colonización cecal en pollos, respectivamente.

Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.

BACKGROUND: Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. RESULTS: By analyzing the nucleotide sequences of the genes for O-antigen biosynthesis including wba operon and the central variable regions of the H1 and H2 flagellin genes in Salmonella, designated PCR primers for four multiplex PCR reactions were used to detect and differentiate Salmonella serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z10; and H2 antigen complexes, I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z15. Through the detection of these antigen gene allele combinations, we were able to distinguish among S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. The assays were useful in identifying Salmonella with O and H antigen gene alleles representing 43 distinct serovars. While the H2 multiplex could discriminate between unrelated H2 antigens, the PCR could not discern differences within the antigen complexes, 1,2; 1,5; 1,6; 1,7 or e,n,x; e,n,z15, requiring a final confirmatory PCR test in the final serovar reporting of S. enterica. CONCLUSION: Multiplex PCR assays for detecting specific O and H antigen gene alleles can be a rapid and cost-effective alternative approach to classical serotyping for presumptive identification of S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.