Sandra Díaz-Sánchez

Prevalence of Escherichia coli, Salmonella sp. and Campylobacter sp. in the intestinal flora of farm-reared, restocked and wild red-legged partridges (Alectoris rufa): is restocking using farm-reared birds a risk?

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.

Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. in large game animals intended for consumption: Relationship with management practices and livestock influence

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<0, 01) where high densities of red deer (p<0.001) were present. To the best of our knowledge, this is the first study reporting the occurrence of Salmonella spp. and STEC in carcasses of large game animals. Furthermore, this study confirmed by pulsed-field gel electrophoresis (PFGE) that cross contamination of STEC during carcass dressing occurred, implying the likelihood of these pathogens entering into the food chain.

Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

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.

Detection and characterization of Shiga toxin-producing Escherichia coli in game meat and ready-to-eat meat products.

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 stx2a 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.

Eutrophication and Bacterial Pathogens as Risk Factors for Avian Botulism Outbreaks in Wetlands Receiving Effluents from Urban Wastewater Treatment Plants

ABSTRACT 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.

Ecological factors driving avian influenza virus dynamics in Spanish wetland ecosystems.

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.

Occurrence of avian pathogenic Escherichia coli and antimicrobial-resistant E. coli in red-legged partridges (Alectoris rufa): sanitary concerns of farming

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.

Integrated metatranscriptomics and metaproteomics for the characterization of bacterial microbiota in unfed Ixodes ricinus

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.

Biotic and abiotic factors shape the microbiota of wild-caught populations of the arbovirus vector Culicoides imicola

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.

Draft Genome Sequences of Anaplasma phagocytophilum, A. marginale, and A. ovis Isolates from Different Hosts

ABSTRACT 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.