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Background Excessive and irrational use of antibiotics as growth promoters in poultry has been one of key factors contributing to increased emergence of antibiotics resistant bacteria. Several alternatives for antibiotic growth promoters are being sought, and the search for effective probiotics to be used as feed additives is amongst the promising ones. Our study aimed to isolate and test potential probiotics bacteria from cloacal swabs of various indigenous chicken (Gallus domesticus) breeds from rural outskirts of the Kathmandu valley (Nepal). Methods Selective isolation of probiotics was conducted by micro-aerophilic enrichment of sample in MRS Broth at 37°C, followed by culturing on MRS agar supplemented with 5 g/L of CaCO3. Isolated bacterial colonies producing transparent halo were selected as potential lactic acid bacteria (LAB), and tested for their antibacterial activity, phenotypic and biochemical characteristics, acidic yield, and tolerance to acid and bile. Results A total of 90 potential LAB were isolated from cloacal samples collected from 41 free-ranging chickens of indigenous breeds. Of these, 52 LAB isolates (57%) showed variable antibacterial activity to at least one bacterial pathogen. Of 52 LAB, 46 isolates fulfilled phenotypic and biochemical criteria of Lactobacillus spp. Of these, 37 isolates produced varying percentage yields of lactic acid, 27 isolates showed survival at pH 3.0, and 17 isolates showed survival tolerances in the presence of 0.3% and 0.5% bile salts for 24 hours. Phylogenetic analysis of 16S rDNA sequencing of LAB isolates fulfilling in vitro probiotics properties showed that 3 isolates had genetic identity of 99.38% with Lactobacillus plantarum, while one isolate was genetically similar (99.85%) with the clade of L. reuteri, L. antri and L. panis. Conclusion Our study identified four Lactobacillus spp. strains having potential probiotics properties. Further investigations are needed to evaluate these isolates to be used as poultry probiotics feed supplement.

This study addresses the variability of the mitochondrial cytochrome oxidase subunit I (COI) and 16S rDNA (16S), and nuclear internal transcriber spacer ITS2 (ITS2) genes in a set of purposely collected samples of the cattle tick, Rhipicephalus microplus (Canestrini, 1888) introduced in the Nearctic-Neotropical range (Mexico to Argentina), and in geo-referenced sequences from GenBank. The main aims of the study are (i) to provide evidence of the phylogeny of the tick in the region, as consequence of a single or several introductions, (b) to explore a possible impact of environmental traits, and (c) to check for the effect of geographical distance on genetic variability. We included published sequences of Rhipicephalus annulatus (Nearctic, Afrotropical and Mediterranean), R. microplus (Afrotropical, Asia), to fully characterize the Neotropical populations (total: 74 16S, 44 COI, and 49 ITS-2 sequences included in the analysis). Only clade A of R. microplus spread in the Nearctic-Neotropics. The K statistic, a measure of phylogenetic signal, supports low divergence rates of every tested gene in populations of R. microplus in the target region even under diverging environmental conditions. This test demonstrates that spatial distance and genetic variability are negatively correlated. The low variability of these genes may be due to (i) the recent introduction of the tick in the Neotropics, (ii) a high degree of panmixia because exchange of populations, and (iii) low environmental pressures, promoting a lack of genetic drift. These results have implications for the ecology and control of cattle tick infestations.

This article belongs to the Special Issue Zebrafish as a Powerful Tool for Drug Discovery 2021., Mycobacteriosis affects wild fish and aquaculture worldwide, and alternatives to antibiotics are needed for an effective and environmentally sound control of infectious diseases. Probiotics have shown beneficial effects on fish growth, nutrient metabolism, immune responses, disease prevention and control, and gut microbiota with higher water quality. However, the identification and characterization of the molecules and mechanisms associated with probiotics is a challenge that requires investigation. To address this challenge, herein we used the zebrafish model for the study of the efficacy and mechanisms of probiotic interventions against tuberculosis. First, bacteria from fish gut microbiota were identified with high content of the surface glycotope Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) that has been shown to induce protective immune responses. The results showed that probiotics of selected bacteria with high α-Gal content, namely Aeromonas veronii and Pseudomonas entomophila, were biosafe and effective for the control of Mycobacterium marinum. Protective mechanisms regulating immunity and metabolism activated in response to α-Gal and probiotics with high α-Gal content included modification of gut microbiota composition, B-cell maturation, anti-α-Gal antibodies-mediated control of mycobacteria, induced innate immune responses, beneficial effects on nutrient metabolism and reduced oxidative stress. These results support the potential of probiotics with high α-Gal content for the control of fish mycobacteriosis and suggested the possibility of exploring the development of combined probiotic treatments alone and in combination with α-Gal for the control of infectious diseases., This research was funded by Junta de Comunidades de Castilla-La Mancha (JCCM), Spain, and EU-FEDER (grant GALINFEC SBPLY/17/180501/000185). MC was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain (grant FJC-2018-038277-I).

Coronavirus-like organisms have been previously identified in Arthropod ectoparasites (such as ticks and unfed cat flea). Yet, the question regarding the possible role of these arthropods as SARS-CoV-2 passive/biological transmission vectors is still poorly explored. In this study, we performed in silico structural and binding energy calculations to assess the risks associated with possible ectoparasite transmission. We found sufficient similarity between ectoparasite ACE and human ACE2 protein sequences to build good quality 3D-models of the SARS-CoV-2 Spike:ACE complex to assess the impacts of ectoparasite mutations on complex stability. For several species (e.g., water flea, deer tick, body louse), our analyses showed no significant destabilisation of the SARS-CoV-2 Spike:ACE complex, suggesting these species would bind the viral Spike protein. Our structural analyses also provide structural rationale for interactions between the viral Spike and the ectoparasite ACE proteins. Although we do not have experimental evidence of infection in these ectoparasites, the predicted stability of the complex suggests this is possible, raising concerns of a possible role in passive transmission of the virus to their human hosts, S.D.L. is funded by a Fundamental Research Grant Scheme from the Ministry of Higher Education Malaysia [FRGS/1/2020/STG01/UKM/02/3]. P.A. is funded by the Wellcome Trust [Wellcome Trust 221327/Z/20/Z]. S.D.-S. is funded by Junta de Comunidades de Castilla-La Mancha (JCCM), Spain and EU-FEDER (grant GALINFEC SBPLY/17/180501/000185). The APC was funded by the Wellcome Trust.

Exploring tick associations with complex microbial communities and single-microbial partners, especially intracellular symbionts, has become crucial to understand tick biology. Of particular interest are the underlying interactions with biological consequences i.e. tick fitness, vector competence. In this study, we first sequenced the 16S rRNA bacterial phylogenetic marker in adult male ticks of Hyalomma lusitanicum collected from 5 locations in the province of Cáceres to explore the composition of its microbial community. Overall, 16S rRNA sequencing results demonstrated that the microbial community of H. lusitanicum is mostly dominated by Francisella-like endosymbionts (FLEs) (ranging from 52% to 99% of relative abundance) suggesting it is a key taxon within the microbial community and likely a primary endosymbiont. However, further research is required to explore the mechanisms underlying the interaction between FLEs and H. lusitanicum., This study and S.D-S were financially supported by the Consejería de Educación, Cultura y Deportes, JCCM, Spain co-financed by European Regional Development Fund (FEDER), project IMpROVE (SBPLY/17/180501/000342).

Biting midges of the genus Culicoides are known vectors of arboviruses affecting human and animal health. However, little is known about Culicoides imicola microbiota and its influence on this insect's biology. In this study, the impact of biotic and abiotic factors on C. imicola microbiota was characterized using shotgun-metagenomic sequencing of whole-body DNA samples. Wild-caught C. imicola adult nulliparous females were sampled in two locations from Sicily, Italy. The climatic variables of temperature and soil moisture from both localities were recorded together with potential host bloodmeal sources. Shared core microbiome among C. imicola populations included Pseudomonas, Escherichia, Halomonas, Candidatus Zinderia, Propionibacterium, and Schizosaccharomyces. Specific and unique taxa were also found in C. imicola from each location, highlighting similarities and differences in microbiome composition between the two populations. DNA and protein identification showed differences in host preferences between the two populations, with Homo sapiens and Canis lupus familiaris L. being the preferred bloodmeal source in both locations. A principal component analysis showed that the combined effect of host preferences (H. sapiens) and local soil moisture factors shape the microbiome composition of wild-caught populations of C. imicola. These results contribute to characterizing the role of the microbiome in insect adaptation and its utility in predicting geographic expansion of Culicoides species with potential implications for the control of vector-borne diseases.

Recently, our knowledge of the composition and complexity of tick microbial communities has increased and supports microbial impact on tick biology. Results support a phylogenetic association between ticks and their microbiota across evolution; this is known as phylosymbiosis. Herein, using published datasets, we confirm the existence of phylosymbiosis between Ixodes ticks and their microbial communities. The strong phylosymbiotic signal and the phylogenetic structure of microbial communities associated with Ixodid ticks revealed that phylosymbiosis may be a widespread phenomenon in tick–microbiota evolution. This finding supports the existence of a species-specific tick hologenome with a largely unexplored influence on tick biology and pathogen transmission. These results may provide potential targets for the construction of paratransgenic ticks to control tick infestations and tick-borne diseases., This investigation was conducted within the H2020 Collaborative Management Platform for detection and Analyses of (Re-)emerging and foodborne outbreaks in Europe (COMPARE) Grant 643476. We acknowledge support provided by the project IMPROVE (SBPLY/17/180501/000342) funded by Consejería de Educación y Deportes (JCCM, Spain) and cofounded by FEDER that supports S.D.S.

The poultry red mite (PRM), Dermanyssus gallinae (De Geer, 1778), is a worldwide distributed ectoparasite and considered a major pest affecting the laying hen industry in Europe. Based on available information in other ectoparasites, the mite microbiome might participate in several biological processes and the acquisition, maintenance and transmission of pathogens. However, little is known about the role of PRM as a mechanical carrier or a biological vector in the transmission of pathogenic bacteria. Herein, we used a metaproteomics approach to characterize the alphaproteobacteria in the microbiota of PRM, and variations in its profile with ectoparasite development (nymphs vs. adults) and feeding (unfed vs. fed). The results showed that the bacterial community associated with D. gallinae was mainly composed of environmental and commensal bacteria. Putative symbiotic bacteria of the genera Wolbachia, C. Tokpelaia and Sphingomonas were identified, together with potential pathogenic bacteria of the genera Inquilinus, Neorickettsia and Roseomonas. Significant differences in the composition of alphaproteobacterial microbiota were associated with mite development and feeding, suggesting that bacteria have functional implications in metabolic pathways associated with blood feeding. These results support the use of metaproteomics for the characterization of alphaproteobacteria associated with the D. gallinae microbiota that could provide relevant information for the understanding of mite-host interactions and the development of potential control interventions., This work was partially supported by the European Cooperation in Science and Technology (COST), Improving Current Understanding and Research for Sustainable Control of the Poultry Red Mite Dermanyssus Gallinae–COREMI Project (Action FA 1404) (https://www.coremi.eu/home.html). JFLB was supported by Doctorado Industrial contract (DI-14-06917) from Ministerio de Ciencia, Innovación y Universidades (Spain) and Sabiotec S. A. MV was supported by the University of Castilla-La Mancha (Spain).

Much work has been dedicated to identifying members of the microbial gut community that have potential to augment the growth rate of agricultural animals including chickens. Here, we assessed any correlations between the fecal microbiome, a proxy for the gut microbiome, and feed efficiency or weight gain at the pedigree chicken level, the highest tier of the production process. Because selective breeding is conducted at the pedigree level, our aim was to determine if microbiome profiles could be used to predict feed conversion or weight gain in order to improve selective breeding. Using 16s rRNA amplicon sequencing, we profiled the microbiomes of high and low weight gain (WG) birds and good and poor feed efficient (FE) birds in two pedigree lineages of broiler chickens. We also aimed to understand the dynamics of the microbiome with respect to maturation. A time series experiment was conducted, where fecal samples of chickens were collected at 6 points of the rearing process and the microbiome of these samples profiled. We identified OTUs differences at different taxonomic levels in the fecal community between high and low performing birds within each genetic line, indicating a specificity of the microbial community profiles correlated to performance factors. Using machine-learning methods, we built a classification model that could predict feed conversion performance from the fecal microbial community. With respect to maturation, we found that the fecal microbiome is dynamic in early life but stabilizes after 3 weeks of age independent of lineage. Our results indicate that the fecal microbiome profile can be used to predict feed conversion, but not weight gain in these pedigree lines. From the time series experiments, it appears that these predictions can be evaluated as early as 20 days of age. Our data also indicates that there is a genetic factor for the microbiome profile., Funding for this study was provided by Cobb-Vantress, Inc. Authors RH and RO are commercially affiliated with the funder. The funder provided support in the form of salaries for SDS, and RH and RO, employed by the funder had active roles in the study as stated in the authors’ contributions.

Exploring the microbial diversity of ticks is crucial to understand geographical dispersion and pathogen transmission. Tick microbes participate in many biological processes implicated in the acquisition, maintenance, and transmission of pathogens, and actively promote host phenotypic changes, and adaptation to new environments. The microbial community of Ixodes ventalloi still remains unexplored. In this study, the bacterial microbiota of wild-caught I. ventalloi was characterized using shotgun-metagenomic sequencing in samples from unfed adults collected during December 2013-January 2014 in two locations from Sicily, Italy. The microbiota identified in I. ventalloi was mainly composed of symbiotic, commensal, and environmental bacteria. Interestingly, we identified the genera Anaplasma and Borrelia as members of the microbiota of I. ventalloi. These results advance our information on I. ventalloi microbiota composition, with potential implications in tick-host adaptation, geographic expansion, and vector competence., This work was financially supported by the H2020 Collaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe (COMPARE) Grant 643476. We thank the Juan de la Cierva incorporación grants funded by Spanish Ministry of Economy, and the Program for Teacher Development for the Superior Type (PRODEP, México) for post-doctoral and pre-doctoral fellowship support.

The development of antibiotic resistant pathogens has resulted from the use of sub-therapeutic concentrations of antibiotics delivered in poultry feed. Furthermore, there are a number of consumer concerns regarding the use of antibiotics in food animals including residue contamination of poultry products and antibiotic resistant bacterial pathogens. These issues have resulted in recommendations to reduce the use of antibiotics as growth promoters in livestock in the United States. Unlike conventional production, organic systems are not permitted to use antibiotics. Thus, both conventional and organic poultry production need alternative methods to improve growth and performance of poultry. Herbs, spices, and various other plant extracts are being evaluated as alternatives to antibiotics and some do have growth promoting effects, antimicrobial properties, and other health-related benefits. This review aims to provide an overview of herbs, spices, and plant extracts, currently defined as phytobiotics as potential feed additives.

An innovative metaomics approach integrating metatranscriptomics and metaproteomics was used to characterize bacterial communities in the microbiota of the Lyme borreliosis spirochete vector, Ixodes ricinus (Acari: Ixodidae). Whole internal tissues and salivary glands from unfed larvae and female ticks, respectively were used. Reused I. ricinus RNA-sequencing data for metranscriptomics analysis together with metaproteomics provided a better characterization of tick bacterial microbiota by increasing bacteria identification and support for identified bacteria with putative functional implications. The results showed the presence of symbiotic, commensal, soil, environmental, and pathogenic bacteria in the I. ricinus microbiota, including previously unrecognized commensal and soil microorganisms. The results of the metaomics approach may have implications in the characterization of putative mechanisms by which pathogen infection manipulates tick microbiota to facilitate infection. Metaomics approaches integrating different omics datasets would provide a better description of tick microbiota compositions, and insights into tick interactions with microbiota, pathogens and hosts., This work was financially supported by the H2020 COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe (COMPARE) Grant 643476. MV was supported by the Research Plan of the University of Castilla- La Mancha(UCLM), Spain.

Here, we report the draft genome sequences of isolates of Anaplasma phagocytophilum, Anaplasma marginale, and Anaplasma ovis. The genomes of A. phagocytophilum (human), A. marginale (cattle), and A. ovis (goat) isolates from the United States were sequenced and characterized. This is the first report of an A. ovis genome sequence., This research was supported by the COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe (COMPARE) grant 643476.

Previously, we reported the use of quinolones in broiler chickens resulted in residues in retail poultry meat obtained from nine districts in the Santiago Province of the Dominican Republic. Residues in poultry products are a concern due to consumer allergies and the potential to develop antibiotic-resistant bacteria. Given the use of quinolones in poultry production and our previous findings in poultry meat, the objective of this study was to evaluate the presence of quinolone residues in eggs. Samples were collected from 48 different farms located in three of the four municipalities (Moca, Cayetano Germosén, and Jamao) of the Espaíllat Province. Each farm was sampled three times between July and September for a total of 144 samples. Samples were evaluated qualitatively and quantitatively for quinolone residues using the Equinox test. Operation systems (cage or floor), seasonality, and location were considered along with egg-producer sizes that were defined as small scale,30,000 eggs per day; medium scale, 30,000 to 60,000 eggs per day; or large scale,60,000 eggs per day. From small-, medium-, and large-scale producers, 69, 50, and 40% of samples were positive for quinolone residues, respectively. A greater number of samples were positive (61%) in floor-laying hen producers compared with those using cages (40%). In the Jamao municipality, 67% of the samples were positive compared with Moca and Cayetano Germosén, where 56 and 25% of samples were positive, respectively. Sampling time had an effect on percent positives: samples collected in July, August, and September were 71, 19, and 63% positive, respectively. Overall, 51% of the samples obtained from eggs produced in the province of Espaíllat were positive for quinolone residues at levels higher than the maximum limits for edible tissue established by the regulatory agencies, including the European Union and U.S. Department of Agriculture. The results obtained from this research confirmed the presence of quinolone residue in eggs, which may present a health risk to some consumers.

Background : Probiotic strains of bacteria can prevent Salmonella from causing disease by preventing the pathogen from colonizing the intestines. Two strains of probiotics, Lactobacillus acidophilius and Pediococcus spp, that were obtained from poultry fecal samples have been shown to be efficacious in poultry. The objective of this study was to determine if these strains of probiotics could prevent salmonellosis in a mouse model. Methods : First, both strains of probiotics were evaluated for in vitro efficacy to inhibit the growth of and interfere with virulence gene regulation in Salmonella enterica. For in vivo efficacy, mice was used which models Typhoid illness. Mice were divided into 2 groups: Control and treatment, Lactobacillus and Pediococcus (LP; 108 Log CFU). Two experiments were conducted. In the first experiment, the mice were treated with LP in water for the first two days of the experiment and challenged with Salmonella at day three. In the second experiment, the LP treatment was given in the water for 10 days and challenge was performed on day 11. In both experiments, at day 20 post-challenge, all mice were sacrificed, intestinal tracts and organs removed and cultured for Salmonella. Results : The probiotic strains inhibited the growth of Salmonella and down-regulation of virulence genes was noted, but dependent on the strain of Salmonella being evaluated. For the in vivo experiment, the probiotics did not afford the mice protection from infection and increasing the length of time the probiotics were administered did not improve the efficacy of the probiotics. Conclusions : It appears that these strains of probiotic bacteria are effective against Salmonella in vitro. However, these isolates did not afford protection from Salmonella infection to mice which may be due to host specifity as these isolates were obtained from poultry. Keywords : Salmonella, Probiotic, Lactobacillus, Pediococcus, Mice

When prebiotics and other fermentation substrates are delivered to animals as feed supplements, the typical goal is to improve weight gain and feed conversion. In this work, we examined pasture flock chicken cecal contents using next generation sequencing (NGS) to identify and understand the composition of the microbiome when prebiotics and fermentation substrates were supplemented. We generated 16S rRNA sequencing data for 120 separate cecal samples from groups of chickens receiving one of 3 prebiotics or fiber feed additives. The data indicated that respective feed additives enrich for specific bacterial community members and modulate the diversity of the microbiome. We applied synthetic learning in microbial ecology (SLiME) analysis to interpret 16S rRNA microbial community data and identify specific bacterial operational taxonomic units (OTU) that are predictive of the particular feed additives used in these experiments. The results suggest that feed can influence microbiome composition in a predictable way, and thus diet may have indirect effects on weight gain and feed conversion through the microbiome., Research funding was awarded to author SCR by the USDA/CSREES (Cooperative State Research, Education, and Extension Service, Washington DC) National Integrated Food Safety Initiative (NIFSI, Washington DC) grant # 406 2008 51110, USDA CREES Food Safety Consortium grant, the Plum Board (Sacramento, CA), and a donation of FOS and GOS prebiotics from GTC Nutrition Co. (Golden, CO).

The zoonotic pathogen, Campylobacter, is the leading cause of foodborne illness worldwide. It colonizes the intestinal tract of chickens, its primary host. In chickens, this pathogen is considered commensal and does not cause illness or have adverse impacts on production performance. A number of methods aim at controlling Campylobacter in the farm. This chapter discusses the general features and epidemiology of Campylobacter, improving biosecurity in the farm, antimicrobial therapy and increased host resistance, boosting the immune response, and DNA vaccines for preventing campylobacteriosis. It is concluded that, along with biosecurity, preventative treatments for the birds can act synergistically to significantly reduce the risk of contamination.

Due to the significance of the microbiome on human health, much of the current data available regarding microbiome functionality is centered on human medicine. For agriculturally important taxa, the functionality of gastrointestinal bacteria has been studied with the primary goals of improving animal health and production performance. With respect to cattle, the digestive functions of bacteria in cattle are unarguably critical to digestion and positively impact production performance. Conversely, some research suggests that the gastrointestinal microbiome in chickens competes with the host for nutrients and produces toxins that can harm the host resulting in decreased growth efficiency. Concerning many other species including reptiles and cetaceans, some cataloging of fecal bacteria has been conducted, but the functionality within the host remains ambiguous. These taxa could provide interesting gastrointestinal insight into functionality and symbiosis considering the extreme feeding regimes (snakes), highly specialized diets (vampire bats), and living environments (polar bears), which warrants further exploration.

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

Due to the scarcity of water resources in the >Mancha Húmeda> Biosphere Reserve, the use of treated wastewater has been proposed as a solution for the conservation of natural threatened floodplain wetlands. In addition, wastewater treatment plants of many villages pour their effluent into nearby natural lakes. We hypothesized that certain avian pathogens present in wastewater may cause avian mortalities which would trigger avian botulism outbreaks. With the aim of testing our hypothesis, 24 locations distributed in three wetlands, two that receive wastewater effluents and one serving as a control, were monitored during a year. Sediment, water, water bird feces, and invertebrates were collected for the detection of putative avian pathogenic Escherichia coli (APEC), Salmonella spp., Clostridium perfringens type A, and Clostridium botulinum type C/D. Also, water and sediment physicochemical properties were determined. Overall, APEC, C. perfringens, and C. botulinum were significantly more prevalent in samples belonging to the wetlands which receive wastewater. The occurrence of a botulism outbreak in one of the studied wetlands coincided with high water temperatures and sediment 5-day biochemical oxygen demand (BOD5), a decrease in water redox potential, chlorophyll a, and sulfate levels, and an increase in water inorganic carbon levels. The presence of C. botulinum in bird feces before the onset of the outbreak indicates that carrier birds exist and highlights the risk of botulinum toxin production in their carcasses if they die by other causes such as bacterial diseases, which are more probable in wastewater wetlands., This study was supported by the Spanish Ministry of Environment (grants OAPN 035/2009). I. Anza was supported by a JAE PRE grant from Spanish Council of Research (CSIC); D. Vidal was supported with a JAE DOC contract from the CSIC; and S. Díaz-Sánchez held a Ph.D. research grant funded by the Junta de Comunidades de Castilla—La Mancha (JCCM) (AG07). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla—La Mancha and Fondo Social Europeo for his research fellowship (09/02-C).

[EN]: Colibacillosis in different forms is responsible for significant economic losses in the poultry industry worldwide. Escherichia coli strains frequently implicated in poultry disease are designated as avian pathogenic E. coli (APEC). Natural infections and disease due to APEC have been described in wild birds, but not as yet in red-legged partridges. During an outbreak in an experimental partridge farm, 23 of 43 1-day-old chicks belonging to the same batch died. Putative APEC strains were detected and isolated both in cloacal swabs and in tissues originating from the same individuals and from different birds showing similar clinical signs. This is the first study that identifies APEC strains linked to a colibacillosis outbreak in farmed red-legged partridges, and also confirms the importance of farmed partridges as fecal carriers and potential spreaders of APEC.

The era of molecular biology and automation of the Sanger chain-terminator sequencing method has led to discovery and advances in diagnostics and biotechnology. The Sanger methodology dominated research for over 2 decades, leading to significant accomplishments and technological improvements in DNA sequencing. Next-generation high-throughput sequencing (HT-NGS) technologies were developed subsequently to overcome the limitations of this first generation technology that include higher speed, less labor, and lowered cost. Various platforms developed include sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLiD sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress made toward third generation sequencing technologies are being reported, which include Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and even more economically feasible with the eventual goal of obtaining real-time results. These technologies can be directly applied to improve poultry production and enhance food safety. For example, sequence-based (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be carried out. Gut microbialflora/communities of poultry can be sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth. Thus, the purpose of this review is to provide an overview of the principles of these current technologies and their potential application to improve poultry production and food safety as well as public health.

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Two allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, and subAB2, located on a pathogenicity island (PAI) together with tia gene. While subAB1 has been reported to be more frequent among bovine strains, subAB2 has been mainly associated with strains from small ruminants. We investigated the presence of the two variants of subAB among 59 eae-negative STEC from large game animals (deer and wild boar) and their meat and meat products in order to assess the role of other species in the epidemiology of subAB-positive, eae-negative STEC. For this approach, the strains were PCR-screened for the presence of subAB, including the specific detection of both allelic variants, for the presence of saa, tia and sab, and for stx subtyping. Overall, subAB genes were detected in 71.2% of the strains: 84.1% of the strains from deer and 33.3% of the strains from wild boar. Most of them (97.6%) possessed subAB2 and most of these subAB2-positive strains (92.7%) were also positive for tia and negative for saa, suggesting the presence of the subAB2-harbouring PAI. Subtype stx2b was present in most of the strains (67.8%) and a statistically significant association could be established between subAB2 and stx2b. Our results suggest that large game animals, mainly deer, may represent an important animal reservoir of subAB2-positive, eae-negative STEC, and also highlight the risk of human infection posed by the consumption of large game meat and meat products., Sergio Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his fellowship (09/02-C) and the Juan de la Cierva programme from the Ministerio de Economía y Competitividad for his research contract. This study was supported by a grant from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03).

Although wild ruminants have been identified as reservoirs of Shiga-toxin producing Escherichia coli (STEC), little information is available concerning the role of Salmonella spp. and Campylobacter spp. in large game species. We evaluated the presence of these pathogens in faeces (N=574) and carcasses (N=585) sampled from red deer (N=295), wild boar (N=333) and other ungulates (fallow deer, mouflon) (N=9). Animal sampling was done in situ from 33 hunting estates during two hunting seasons. Salmonella spp. and Campylobacter spp. strains associated with human campylobacteriosis were infrequently detected indicating that both pathogens had a limited zoonotic risk in our study area. The overall STEC prevalence in animals was 21% (134/637), being significantly higher in faeces from red deer (90 out of 264). A total of 58 isolates were serotyped. Serotypes O146:H- and O27:H30 were the most frequent in red deer and the majority of isolates from red deer and wild boar were from serotypes previously found in STEC strains associated with human infection, including the serotype O157:H7. The STEC prevalence in red deer faeces was significantly higher with the presence of livestock (p, This study was partially supported by a grant from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03), by Red Española de Investigación en Patología Infecciosa (REIPI RD06/0008/1016-1018) and grants PS09/01273 (Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Gobierno de España), AGL-2008-02129 (Ministerio de Ciencia e Innovación, Gobierno de España) and 09TAL007261PR, 10MRU261023PR and 2007/000044-0 (Xunta de Galicia and The European Regional Development Fund, ERDF). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his fellowship (09/02-C). S. Díaz-Sánchez acknowledges the Junta de Comunidades de Castilla-La Mancha for her PhD research grant (AG/07). D. Vidal acknowledges the Consejo Superior de Investigaciones Científicas for her JAE-Doc contract. A. Mora acknowledges the Ramón y Cajal programme from the Spanish Ministerio de Ciencia e Innovación.

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection., Sergio Sánchez acknowledges the Juan de la Cierva programme from the Ministerio de Economía y Competitividad for his research contract. This study was supported by grants from the Fondo de Investigación Sanitaria (grant FIS G03-025-COLIRED-O157), from the Junta de Extremadura and FEDER (grants 3PR05A009-IIIPlan Regional de Investigación and PRI09A022-Plan Regional de Investigación 2009) and from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03).

Fallow deer (Dama dama) are widely distributed as natural or naturalised populations, as well as in game parks and deer farms. We used 157 fallow deer sampled in populations considered to be Mycobacterium tuberculosis complex (MTC) free and 73 Mycobacterium bovis-infected fallow deer confirmed postmortem by culture to evaluate the diagnostic performance of two tests for the detection of anti-mycobacterial antibodies: the dual path platform (DPP) VetTB assay and the bovine purified protein derivative (bPPD) ELISA. We also compared their sensitivity with that of the skin test, analyzed the effect of haemolysis degree on the antibody detection and described the relationship between the test readings and presence/absence of gross tuberculosis (TB) compatible lesions. Sensitivity of bPPD ELISA was 51% at a specificity of 96%. Depending on the cut-off value selected, the sensitivity of DPP VetTB ranged from 62 to 71%, while its specificity was 88-95%. In the subgroup of M. bovis-infected deer for which the skin test data were available (33 of 73); this method detected 76% of culture-positive animals, although the specificity of the intradermal test was not determined in this study. When the DPP VetTB and skin test data were combined, the resulting sensitivity obtained in this sub-group of M. bovis-infected deer increased to 97%. Gross pathology identified TB compatible lesions (TBL) in 89% culture-confirmed fallow deer. The infected animals with visible lesions had significantly higher readings in the DPP VetTB, but not in the bPPD ELISA. Only high levels of haemolysis decreased antibody test sensitivity and this effect was more evident for the bPPD ELISA. The results allowed inferring a number of management recommendations for rapid detection of MTC infection in live fallow deer and in surveys on hunter-harvested cervids., This is a contribution to MCINN Plan Nacional Research grant AGL2008-03875 and FEDER, and to EU grant TB-STEP 212414. Studies on diseases shared between domestic animals and wildlife are also supported by grants and contracts from INIA and Ministerio de Medio Ambiente y Medio Rural y Marino (SDGSPP), and Grupo Santander – Fundación Marcelino Botín.

Red-legged partridges (Alectoris rufa) are a significant part of the culture, diet and income for many people in central and southern Spain. Due to declining populations in the wild, intensive farming is common and 4 million juvenile partridges are released each autumn. Intensive management and high densities result in high prevalence of enteric disease and the use of antimicrobials as preventive measures on partridge farms and prior to restocking in the wild. We determined the occurrence of avian pathogenic Escherichia coli (APEC), and screened phenotypic resistance of E. coli against enrofloxacin, gentamicin and cefotaxim in farmed, restocked and wild partridges. Prevalence of APEC in farmed and restocked red-legged partridges was significantly higher than in natural populations. Phenotypic resistance against both gentamicin and enrofloxacin was significantly more frequent in farmed (75%) and restocked (43%) partridges than in wild partridges, while most E. coli isolated from natural populations were susceptible to all three antimicrobials tested (65%). This indicates that farmed and restocked partridges carry APEC that could be a reason for disease outbreaks on farms, and that E. coli carried by farmed and restocked partridges can acquire resistance to frequently used antimicrobials, thus being a concern for the environment, wild birds and consumers. Management in farms and restocking procedures may create a hazard not only for spreading APEC, but also as a potential source of resistant E. coli in the environment., This study was funded by Junta de Comunidades de Castilla-La Mancha (JCCM), Spain (project reference: PAC08-0296-7771) and is also a contribution to project Ag2008-02504GAN funded by the Spanish Ministry for Science and Innovation. Sandra Díaz-Sánchez holds a PhD research grant funded by JCCM (AG07). Dr S. Sánchez acknowledges the Consejería de Educación y Ciencia of the JCCM and Fondo Social Europeo for his research fellowship (09/02-C). The authors also gratefully acknowledge Dr S. Morabito (Istituto Superiore di Sanita, Rome, Italy) for providing E. coli control strains used in this study.

A total of 142 samples of game meat and ready-to-eat meat products from red deer and wild boar were analysed in order to assess the presence of Shiga toxin-producing Escherichia coli (STEC). Shiga-toxin encoding genes (stx genes) were detected by PCR in 36 (25.4%) of the samples and STEC was isolated from 8 (5.6%) of the same samples. None of the samples tested positive for E. coli O157:H7. Four different serotypes were found among the 8 STEC isolates, with serotype O27:H30 being predominant (62.5%, 5/8). The PCR assay indicated the presence of the stx2 gene in all of the STEC isolates and further subtyping resulted in detection of three different subtypes: stx2a, stx2b and stx2g. The only stx1-positive isolate was further subtyped as stx1c. The ehxA gene was detected in 3 (37.5%) of the isolates and none of them contained the eae gene. All STEC isolates were sensitive to the 13 antibiotics tested. Some isolates possessed serotypes and virulence gene profiles previously associated with STEC infections in humans. The isolation of a STEC strain carrying the stx2. a subtype from a ready-to-eat meat product from deer suggests the role of these products as a potential source of STEC infections in humans., This study was supported by a grant from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for this fellowship (09/02-C). S. Díaz-Sánchez acknowledges the Junta de Comunidades de Castilla-La Mancha for her PhD research grant (AG/07). D. Vidal acknowledges the Consejo Superior de Investigaciones Científicas for her JAE-Doc contract.

For hunting purposes, several millions of red-legged partridges (Alectoris rufa) are released each year in Spain, and these releases have the potential to introduce new parasites and disease into wild populations. We studied the prevalence of Escherichia coli, Campylobacter sp. and Salmonella sp. in the intestinal flora of red-legged partridges from three different husbandry groups: farm-reared, restocked and natural populations. Prevalence of E. coli was significantly higher in farm-reared (45%, p = 0.01) and restocked partridges (60%, p < 0.001) than in wild ones (6%, p > 0.05). The prevalence of Campylobacter sp. (23%, 100 out of 444) did not differ significantly between these three husbandry groups, and Salmonella sp. was only detected in a group of partridge chicks on one of the farms studied (0.9%, 5 out of 544). These results suggest that farm-reared and restocked partridges can act as carriers of these three enteropathogens and highlight a potential risk of transmission to natural populations via the releases of farm-reared partridges. However, future investigations are needed regarding the relation of the isolated bacteria with zoonotic strains and dissemination of antibiotic resistant microorganisms, especially E. coli, and to better evaluate the effect that these three enteropathogens have on partridge health and on the success of restocking with farm-reared birds., This study was funded by Junta de Comunidades de Castilla La Mancha (JCCM, Spain; project reference: PAC08-0296-7771) and is also a contribution to project Ag2008-02504GAN funded by the Spanish Ministry for Science and Innovation. Capture of partridges from natural populations was carried out under projects PII1I09-0271-5037, PAI06- 0112 and PAII1C09-0227-0104. Sandra Diaz-Sanchez holds a PhD research grant funded by Junta de Comunidades de Castilla–La Mancha (JCCM) (AG07).

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity., This work was financially supported by INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), project FAU2006-00019-C03-00 and by Department of Environment, Territorial Planning, Agriculture and Fisheries of the Basque Government. P. Acevedo was supported by a Beatriu de Pinós fellowship funded by Comissionat per a Universitats i Recerca del Departament d'Innovació, Universitats i Empresa, of the Generalitat de Catalunya and the COFUND Programme-Marie Curie Actions under 7th Marc Programme of the European Community.