Chicken Fecal Samples Research Articles

Evaluation of the detection method by a flotation method using a wire loop for gastrointestinal parasites.

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples.

Contribution of veterinary sector to antimicrobial resistance in One Health compendium: an insight from available Indian evidence.

The application of antibiotics in the poultry and veterinary sectors is very common practice in India. Owing to the seriousness of antimicrobial resistance (AMR), the present study has illustrated the overall scenario of AMR in the poultry and veterinary sectors in India through an in-depth scoping review and key informant interview (KII). In the poultry sector, most of the studies reviewed have reported resistant bacteria isolated from chicken meat, eggs, cloacal swabs, and fecal samples, and only a few have reported the presence of resistant bacteria in and around the environment of poultry farms. The major resistant bacteria that have been reported are E. coli, Salmonella spp., S. aureus, Campylobacter jejuni, and K. pneumoniae. These bacterial isolates exhibited resistance to various antibiotics, such as azithromycin (21.43%), tetracycline (11.30-100%), chloramphenicol (4.76-100%), erythromycin (75-83.33%), ciprofloxacin (5.7-100%), gentamicin (17-100%), amikacin (4.76%), cotrimoxazole (42.2-60%), trimethoprim (89.4%), ceftriaxone (80%), and cefotaxime (14.29-70%). Like the poultry sector, different antibiotics are also used for treating clinical and subclinical bovine mastitis, which is one of the major problems plaguing the dairy sector. Several AMR bacterial strains, such as E. coli, Staphylococcus aureus, S. epidermidis, and Klebsiella pneumoniae, have been reported by many researchers and showed resistance against tetracycline (74%), oxytetracycline (47.37%), ciprofloxacin (51%), streptomycin (57.89%), cephalosporin (100%), and trimethoprim (70%). The KIIs have revealed several reasons behind these AMR scenarios, of which the growing need for the production of food animals and their products with inadequate infrastructure and a lack of proper knowledge on farm management among the farmers are the major ones. Though several government legislations and policies have been laid down, proper implementation of these policies, strict surveillance on antibiotic application in the poultry and veterinary sectors, awareness generation among farmers, and infrastructure development can help minimize the development and transmission of AMR bacteria within and from these sectors.

Genetic Characterization of Antibiotic-Resistant Staphylococcus spp. and Mammaliicoccus sciuri from Healthy Humans and Poultry in Nigeria.

Staphylococcus spp. poses a significant threat to human and animal health due to their capacity to cause a wide range of infections in both. In this study, resistance genes conferring antibiotic resistance in Staphylococcus spp. and Mammaliicoccus sciuri isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 61 Staphylococcus spp. isolates were obtained from urine, wounds, nasal and chicken fecal samples. Species identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed using the Kirby-Bauer method for 16 antibiotics. Whole-genome sequencing was used for characterization of the isolates. The 61 investigated isolates included Staphylococcus aureus, S. arlettae, M. sciuri, S. haemolyticus, and S. epidermidis. A total of 47 isolates (77%) belonged to human samples and 14 (23%) isolates were collected from poultry samples. All were phenotypically resistant to at least three antimicrobial(s). Multiple resistance determinants were detected in the human and poultry isolates analyzed. Phylogenetic analysis revealed close relatedness among the isolates within each species for S. arlettae, M. sciuri, and S. haemolyticus, respectively. This study delivered comprehensive genomic insights into antibiotic-resistant Staphylococcus species and M. sciuri isolates from human and poultry sources in Edo state, Nigeria, from a One Health perspective.

Antimicrobial Resistance and Molecular Characterization of Escherichia coli from Healthy Chickens in Shandong, China from 2009 to 2014.

Antimicrobial resistance (AMR) is a great threat to animal and public health. Here, we conducted a surveillance of Escherichia coli isolated from healthy chickens during 2009-2014 to identify the characteristics of AMR. A total of 351 (95.64%) E. coli isolates were obtained from 367 healthy chicken fecal samples collected from 6 farms located in Shandong Province, China. The susceptibility to 10 antimicrobials, the prevalence of antibiotic resistance genes (ARGs), phylogenetic clustering, and multilocus sequence typing were evaluated. The isolates exhibited high resistant rates (>95%) to ampicillin, cefotaxime, ciprofloxacin, ceftiofur, and enrofloxacin. The most prevalent ARGs were blaCTX-M (36.36%), aac(6')-Ib-cr (30.79%), qnrS (29.62%), oqxAB (27%), mcr-1 (15.83%), blaTEM (9.09%), qnrC (3.52%), qnrD (0.88%), and qepA (0.29%). Phylogenetic clustering analysis indicated that the most prevalent group was group D (37.89%), followed by group B1 (34.76%), A (24.22%), and B2 (3.13%). Fifty-seven sequence types (STs) were identified among the 124 blaCTX-M-positive strains, and the dominant STs were ST354 (13.71%), ST117 (5.65%), ST155, ST2309, and ST2505 (4.84% each). There was a significant association between 17 pairs of AMR phenotypes, 14 pairs of ARGs, and 11 pairs of AMR-ARGs. The strongest association was found between ST602 and qnrC (odds ratios: 22.2). This study implied that E. coli isolated from healthy chickens could potentially serve as a reservoir of AMR and ARGs, and significant associations exist among AMR, ARGs, phylogenetic groups, and STs. Our study highlighted the need for routine surveillance of AMR in healthy chickens, and promoting appropriate antibiotic use and implementing regular monitoring of resistance in broilers are crucial for fostering the development of the poultry industry and safeguarding public health.

Towards facilitated interpretation of shotgun metagenomics long-read sequencing data analyzed with KMA for the detection of bacterial pathogens and their antimicrobial resistance genes.

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k-mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU.

Identification of Digestive Tract Endoparasites of Laying Hens in Suruhwadang Village, Kademangan District, Blitar Regency

This study aims to determine the species, prevalence, and infection degree of endoparasite in the digestive tract of laying hens in Suruhwadang Village, Kademangan District, Blitar Regency. Ninety-six fecal samples were taken from four different farms. Twelve samples of chicken feces aged 20-50 weeks and twelve samples of chicken feces > 50 weeks were taken from each farm. The examinations taken in this study are fecal examinations using native, sediment, and floating methods. The results of identification of digestive tract endoparasites found consisting of Ascaridia galli (68.75%), Heterakis gallinarum (53.12%), Strongyloides avium (5.21%), Trichostrongylus tenuis (6.25%), Eimeria maxima (51,04%), and Eimeria acervulina (3,12%). The highest average degree of worm egg infection was found in layers >50 weeks of age of 373.96 ± 450.41 while the highest average degree of infection of protozoa was found in layers aged >50 weeks of 296,87 ± 600,92. The results of the Chi-Square test showed that there was no significant effect of the difference in the age of laying hens in the layer phase on the prevalence of digestive tract endoparasites, both worm egg and protozoan infections (P>0.05). The results of the Mann-Whitney test showed that there was a significant effect of the difference in the age of the laying hen in the layer phase on the degree of worm egg infection (P<0.05), but the difference in age of the laying hen in the layer phase did not significantly affect the degree of protozoa infection (P>0,05).

Antibiotic resistance and virulence profiles of Proteus mirabilis isolated from broiler chickens at abattoir in South Africa.

Proteus mirabilis has been identified as an important zoonotic pathogen, causing several illnesses such as diarrhoea, keratitis and urinary tract infections. This study assessed the prevalence of P. mirabilis in broiler chickens, its antibiotic resistance (AR) patterns, ESBL-producing P. mirabilis and the presence of virulence genes. A total of 26 isolates were confirmed as P. mirabilis from 480 pooled broiler chicken faecal samples by polymerase chain reaction (PCR). The disk diffusion method was used to evaluate the antibacterial susceptibility test, while nine virulence genes and 26 AR genes were also screened by PCR. All 26 P. mirabilis isolates harboured the ireA (siderophore receptors), ptA, and zapA (proteases), ucaA, pmfA, atfA, and mrpA (fimbriae), hlyA and hpmA (haemolysins) virulence genes. The P. mirabilis isolates were resistant to ciprofloxacin (62%) and levofloxacin (54%), while 8 (30.7%) of the isolates were classified as multidrug resistant (MDR). PCR analysis identified the blaCTX-M gene (62%), blaTEM (58%) and blaCTX-M-2 (38%). Further screening for AMR genes identified mcr-1, cat1, cat2, qnrA, qnrD and mecA, 12%, 19%, 12%, 54%, 27% and 8%, respectively for P. mirabilis isolates. The prevalence of the integron integrase intI1 and intI2 genes was 43% and 4%, respectively. The rise of ciprofloxacin and levofloxacin resistance, as well as MDR strains, is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Furthermore, because ESBL-producing P. mirabilis has the potential to spread to humans, the presence of blaCTX -M -producing P. mirabilis in broilers should be kept under control. This is the first study undertaken to isolate P. mirabilis from chicken faecal samples and investigate its antibiotic resistance status as well as virulence profiles in South Africa.

Analysis of gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae in rural residents of Weifang City, Shandong Province

Objective: To investigate the drug-resistant gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae (CR-ECL) in rural residents of Weifang City, Shandong Province. Methods: Fecal samples were collected from rural community residents in Weifang City, Shandong Province in 2017. Drug-resistant strains were screened using a carbapenem-resistant enterobacter chromogenic medium. CR-ECL positive strains were acquired via Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOFMS) analysis. The antibiotic resistance phenotype of CR-ECL was determined using a microbroth dilution assay. Whole genome sequencing (WGS) and analysis were conducted, along with an examination of the immediate vicinity of the blaNDM gene and phylogenetic analysis of the strains. Results: A total of 628 fecal samples were collected and tested, of which 6 were CR-ECL positive (detection rate 0.96%), all exhibiting multiple drug resistance (MDR) phenotypes. Six CR-ECL strains had four MLST genotypes (ST), all of which carried multiple drug resistance genes (blaNDM-1, blaNDM-5, etc.) and virulence genes (acrA, acrB, entB, fepC, etc.). There were mobile genetic elements ISAba125, TN3-IS3000, TN3 and IS5 in the genetic environment surrounding the blaNDM gene. The phylogenetic tree showed that the multi-locus sequence typing of the core genome (cgMLST) was consistent with the single nucleotide polymorphism (SNPs) results. The cgMLST results showed that the allele differences between strains 2BC0101B and 2BC0251B, 2BG0561B and 2BI0221B were 2 and 1, respectively. The SNPs results showed that the above two pairs of bacteria also clustered together. It was found that the strains of chicken fecal samples in the National Center for Biotechnology Information (NCBI) database were located in the center of the evolutionary tree, and the local sequences could be traced back to American human sequences. Conclusion: Multidrug-resistant CR-ECL is detected in rural community residents in Weifang City, Shandong Province.

Molecular Detection of Virulence-Associated Markers in Campylobacter coli and Campylobacter jejuni Isolates From Water, Cattle, and Chicken Faecal Samples From Kajiado County, Kenya.

Campylobacter is a zoonotic foodborne pathogen that is often linked with gastroenteritis and other extraintestinal infections in humans. This study is aimed at determining the genetic determinants of virulence-encoding genes responsible for flagellin motility protein A (flaA), Campylobacter adhesion to fibronectin F (cadF), Campylobacter invasion antigen B (ciaB) and cytolethal distending toxin (cdt) A (cdtA) in Campylobacter species. A total of 29 Campylobacter coli isolates (16 from cattle, 9 from chicken, and 4 from water samples) and 74 Campylobacter jejuni isolates (38 from cattle, 30 from chicken, and 6 from water samples) described in an earlier study in Kajiado County, Kenya, were examined for the occurrence of virulence-associated genes using polymerase chain reaction (PCR) and amplicon sequencing. The correlations among virulence genes were analyzed using Pearson's correlation coefficient (R) method. Among the 103 Campylobacter strains screened, 89 were found to harbour a single or multiple virulence gene(s), giving an overall prevalence of 86.4%. C. jejuni strains had the highest prevalence of multivirulence at 64.9% (48/74), compared to C. coli (58.6%, 17/29). The ciaB and flaA genes were the most common virulence genes detected in C. jejuni (81.1% [60/74] and 62.2% [46/74], respectively) and in C. coli (each at 62.1%; 18/29). Campylobacter isolates from chicken harboured the most virulence-encoding genes. C. jejuni strains from chicken and cattle harboured the highest proportions of the cdtA and ciaB genes, respectively. All the C. coli strains from water samples harboured the cadF and flaA genes. The results obtained further revealed a significant positive correlation between cadF and flaA (R = 0.733). C. jejuni and C. coli strains from cattle, chicken, and water harbour virulence markers responsible for motility/colonization, invasion, adherence, and toxin production, evoking their important role in campylobacteriosis development among humans and livestock. The identification of cattle, chicken, and water samples as reservoirs of virulent Campylobacter spp. highlights the possible risk to human health. These data on some virulence genes of Campylobacter will assist food safety and public health officials in formulating policy statements.

Detecting horizontal gene transfer among microbiota: an innovative pipeline for identifying co-shared genes within the mobilome through advanced comparative analysis.

Horizontal gene transfer (HGT) is a key driver in the evolution of bacterial genomes. The acquisition of genes mediated by HGT may enable bacteria to adapt to ever-changing environmental conditions. Long-term application of antibiotics in intensive agriculture is associated with the dissemination of antibiotic resistance genes among bacteria with the consequences causing public health concern. Commensal farm-animal-associated gut microbiota are considered the reservoir of the resistance genes. Therefore, in this study, we identified known and not-yet characterized mobilized genes originating from chicken and porcine fecal samples using our innovative pipeline followed by network analysis to provide appropriate visualization to support proper interpretation.

Uncovering changes in microbiome profiles across commercial and backyard poultry farming systems.

The microbiome profiles of poultry production systems significantly impact bird health, welfare, and the environment. This study investigated the influence of broiler-rearing systems on the microbiome composition of commercial and backyard chicken farms and their environment over time. Understanding these effects is vital for optimizing animal growth, enhancing welfare, and addressing human and environmental health implications. We collected and analyzed various samples from commercial and backyard farms, revealing significant differences in microbial diversity measurements between the two systems. Backyard farms exhibited higher alpha diversity measurements in soil and water samples, while commercial farms showed higher values for litter and feeder samples. The differences in microbial diversity were also reflected in the relative abundance of various microbial taxa. In backyard farms, Proteobacteria levels increased over time, while Firmicutes levels decreased. Campilobacterota, including the major poultry foodborne pathogen Campylobacter, increased over time in commercial farm environments. Furthermore, Bacteroides, associated with improved growth performance in chickens, were more abundant in backyard farms. Conversely, pathogenic Acinetobacter was significantly higher in backyard chicken fecal and feeder swab samples. The presence of Brevibacterium and Brachybacterium, associated with low-performing broiler flocks, was significantly higher in commercial farm samples. The observed differences in microbial composition and diversity suggest that farm management practices and environmental conditions significantly affect poultry health and welfare and have potential implications for human and environmental health. Understanding these relationships can inform targeted interventions to optimize poultry production, improve animal welfare, and mitigate foodborne pathogens and antimicrobial resistance risks. IMPORTANCE The microbiome of poultry production systems has garnered significant attention due to its implications on bird health, welfare, and overall performance. The present study investigates the impact of different broiler-rearing systems, namely, commercial (conventional) and backyard (non-conventional), on the microbiome profiles of chickens and their environment over time. Understanding the influence of these systems on microbiome composition is a critical aspect of the One-Health concept, which emphasizes the interconnectedness of animal, human, and environmental health. Our findings demonstrate that the type of broiler production system significantly affects both the birds and their environment, with distinct microbial communities associated with each system. This study reveals the presence of specific microbial taxa that differ in abundance between commercial and backyard poultry farms, providing valuable insights into the management practices that may alter the microbiome in these settings. Furthermore, the dynamic changes in microbial composition over time observed in our study highlight the complex interplay between the poultry gut microbiome, environmental factors, and production systems. By identifying the key microbial players and their fluctuations in commercial and backyard broiler production systems, this research offers a foundation for developing targeted strategies to optimize bird health and welfare while minimizing the potential risks to human and environmental health. The results contribute to a growing body of knowledge in the field of poultry microbiome research and have the potential to guide future improvements in poultry production practices that promote a sustainable and healthy balance between the birds, their environment, and the microbial communities they host.

Antimicrobial Effects of Aqueous Extract of Garcinia Kola Nuts on Salmonella Isolates from Chicken Dropping in Southern Taraba, North East, Nigeria

Aim: The onerous threat of antimicrobial resistance to public health highlights the need for continuous research that will discover more potential medicinal plants that possess inhibitory potentials on bacteria especially those with multidrug-resistant qualities. Hence, this study investigated the antibacterial effect of hot and cold aqueous extracts of Garcinia Kola on Salmonella isolates.
 Study Design: This is experimental research involving fecal sample of chicken.
 Place and Duration of Study: This research was carried out in Wukari metropolis.
 Study Design: Sterile universal containers were used to collect one gram each of chicken fecal samples from domestic chicken coops and dissolved in Buffered Peptone Water to recover injured cell. A loop-full of the sample was streaked on prepared Salmonella-Shigella Agar plates. Suspected isolates were confirmed molecularly using PCR to identify the invA virulent gene from the isolate. Hot and cold water served as a menstruum for extracting bioactive contents from Garcinia Kola. Following evaporation of the crude hot and cold-water extracts, 0.25 mg, 0.5 mg, 1.0 mg, and 2.0 mg of the extract were respectively dissolved into 10 ml of distilled water. The Agar-well diffusion method was used in conducting the antimicrobial susceptibility tests.
 Results: Significant zone of inhibition (ZIB) of 9 mm, 12 mm and 17 mm was observed for isolates subjected to 0.50 mg-10 ml, 1.00mg-10 ml, and 2.00 mg-10 ml concentrations of cold-water extracts respectively while No zone of Inhibition (NZI) was observed at 0.25 g-10 ml concentrations. For hot water extracts, growth inhibition ranging from 9 mm, 11 mm, 15 mm, and 23 mm was respectively noticed for concentrates of 0.25 mg-10 ml, 0.50 mg-10, 1.00 mg-10 ml, and 2.00 mg-10 ml. Heavy growth persisted for the negative control plate which contained distilled water without extracts. Augmentin 30 µg was used as a positive control (≥31 mm).
 Conclusion: This research simply has amplified the medicinal importance of the consumption of Garcinia Kola particularly as it relates to the management of gastroenteritis caused by Zoonotic Non-enteric Salmonella. However, the clinical toxicity and safety of the plant need more understanding.

Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria.

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated 'One Health' surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.

The Spread of Heat-Labile Enterotoxin-Harboring Escherichia fergusonii in Broiler Chickens and Pigs in Okinawa, Japan.

In 2012, Escherichia fergusonii harboring a heat-labile enterotoxin (LT) was isolated from healthy chickens in South Korea. However, little is known regarding the prevalence, spread, and pathogenicity of these strains in humans and animals. This study aimed to understand the public health threats, such as the distribution, antimicrobial resistance, and genetic diversity of E. fergusonii carrying LTs. E. fergusonii containing LT was isolated from 15.0% (52/346) of chicken fecal samples from all three tested chicken farms but not from 360 pig fecal samples. The antimicrobial susceptibility testing revealed that over 75% of strains were resistant to ampicillin, kanamycin, nalidixic acid, streptomycin, or tetracycline; additionally, 71.2% (37/52) of strains were resistant to all five of these antimicrobials. The 52 strains were clustered into eight pulsed-field gel electrophoresis (PFGE) types, with types V and type VI accounting for 84.6% (44/52). In the present study, multiple chicken farms harbored E. fergusonii with similar antimicrobial resistance patterns and genetic clonality. Since the pathogenicity of LT-bearing E. fergusonii in humans and animals, such as food poisoning and sporadic diarrhea via meat, the transmission of the strains, and the dissemination of antimicrobial resistance genes are unknown, additional research is required.

Wind Speed and Landscape Context Mediate Campylobacter Risk among Poultry Reared in Open Environments.

Foodborne pathogens cause over 9 million illnesses in the United States each year, and Campylobacter from chickens is the largest contributor. Rearing poultry outdoors without the use of antibiotics is becoming an increasingly popular style of farming; however, little is understood about how environmental factors and farm management alter pathogen prevalence. Our survey of 27 farms in California, Oregon, Washington, and Idaho, USA, revealed a diversity of management practices used to rear poultry in the open environment. Here, we assess environmental and management factors that impact Campylobacter spp. prevalence in 962 individual chicken fecal samples from 62 flocks over a three-year period. We detected Campylobacter spp. in 250/962 (26.0%) of fecal samples screened, in 69.4% (43/62) of flocks, and on 85.2% (23/27) of farms. We found that Campylobacter spp. prevalence was predicted to increase in poultry on farms with higher average wind speeds in the seven days preceding sampling; on farms embedded in more agricultural landscapes; and in flocks typified by younger birds, more rotations, higher flock densities, and the production of broilers. Collectively, our results suggest that farms in areas with higher wind speeds and more surrounding agriculture face greater risk of Campylobacter spp. introduction into their flocks.

Host specific adaptations of Ligilactobacillus aviarius to poultry

The genus Ligilactobacillus encompasses species adapted to vertebrate hosts and fermented food. Their genomes encode adaptations to the host lifestyle. Reports of gut microbiota from chicken and turkey gastrointestinal tract have shown a high persistence of Ligilactobacillus aviarius along the digestive system compared to other species found in the same host. However, its adaptations to poultry as a host has not yet been described. In this work, the pan-genome of Ligilactobacillus aviarius was explored to describe the functional adaptability to the gastrointestinal environment. The core genome is composed of 1179 gene clusters that are present at least in one copy that codifies to structural, ribosomal and biogenesis proteins. The rest of the identified regions were classified into three different functional clusters of orthologous groups (clusters) that codify carbohydrate metabolism, envelope biogenesis, viral defence mechanisms, and mobilome inclusions. The pan-genome of Ligilactobacillus aviarius is a closed pan-genome, frequently found in poultry and highly prevalent across chicken faecal samples. The genome of L. aviarius codifies different clusters of glycoside hydrolases and glycosyltransferases that mediate interactions with the host cells. Accessory features, such as antiviral mechanisms and prophage inclusions, variate amongst strains from different GIT sections. This information provides hints about the interaction of this species with viral particles and other bacterial species. This work highlights functional adaptability traits present in L. aviarius that make it a dominant key member of the poultry gut microbiota and enlightens the convergent ecological relation of this species to the poultry gut environment.

Evaluation of antibiotic sensitivity of potential probiotic Bacillus strains isolated from chicken and pig feces

Nowadays, the misuse and overuse of antibiotics in livestock production leads to the appearance of antibiotic resistant strains that promotes the use of probiotics in feed to minimise/replace antibiotic consumption. Among them, Bacilli are common supplements for feed effectiveness and livestock health benefits. However, there is a growing concern about the development of antibiotic resistance and transfer of antibiotic resistance genes among bacteria. Thus, the European Food Safety Authority (EFSA) has suggested that products containing Bacillus strains intended to use as feed additive must be examined for susceptibility to antimicrobials of human and veterinary importance. In this study, the antibiotic susceptibility of potential probiotic Bacillus strains was investigated. A total of 59 Bacillus species were isolated from 10 samples of chicken and pig feces. Then, the in vitro probiotic characteristics including low pH tolerance, catalase activity, protease, amylase and cellulase enzymes production were tested to select appropriate isolates for further studies. Thirty-three promising probiotic strains were assessed for their antibiotic susceptibility against 13 antibiotics by disc diffusion method. In most cases, isolated Bacillus strains were susceptible to tested antibiotics. The obtained results demonstrated the potential application of Bacillus spp. as feed supplements in animal production.

Impact of Freeze Storage on the Estimation of Phenotypic Antimicrobial Resistance Prevalence in Escherichia coli Collected from Faecal Samples from Healthy Humans and Chickens.

Many studies on phenotypic antimicrobial resistance (AMR) of bacteria from healthy populations are conducted on freeze-stored samples. However, the impact of this practice on phenotypic AMR is not known. We investigated the prevalence of phenotypic AMR in Escherichia coli from chicken (n = 10) and human (n = 11) faecal samples collected from healthy subjects, subject to freeze storage (-20 °C and -80 °C) for 1, 2, 3, and 6 months. We compared counts of E. coli and prevalence of phenotypic resistance against five antimicrobials commonly used in chicken farming (ciprofloxacin, enrofloxacin, doxycycline, gentamicin, and florfenicol) with samples processed within 24 h of collection. Prevalence of phenotypic AMR was estimated by performing differential counts on agar media with and without antimicrobials. At -20 °C, there was a considerable reduction in E. coli counts over time, and this reduction was greater for human samples (-0.630 log10 units per 100 days) compared with chicken samples (-0.178 log10 units per 100 days). For most antimicrobials, AMR prevalence estimates decreased in freeze-stored samples both in humans and chickens over time. Based on these results, we conclude that results on the prevalence of phenotypic AMR on samples from freeze-stored samples are unreliable, and only fresh samples should be used in such studies.

RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples

Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA–CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA–CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.

Storage media and RNA extraction approaches substantially influence the recovery and integrity of livestock fecal microbial RNA.

BackgroundThere is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples.MethodsFecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination.ResultsThe quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or −80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples.ConclusionOur findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.