Experimental Evidence for Manure-Borne Bacteria Invasion in Soil During a Coalescent Event: Influence of the Antibiotic Sulfamethazine.

The fertilization of agricultural soil by organic amendment that may contain antibiotics, like manure, can transfer bacterial pathogens and antibiotic-resistant bacteria to soil communities. However, the invasion by manure-borne bacteria in amended soil remains poorly understood. We hypothesized that this kind of process is both influenced by the soil properties (and those of its microbial communities) and by the presence of contaminants such as antibiotics used in veterinary care. To test that, we performed a microcosm experiment in which four different soils were amended or not with manure at an agronomical dose and exposed or not to the antibiotic sulfamethazine (SMZ). After 1 month of incubation, the diversity, structure, and composition of bacterial communities of the soils were assessed by 16S rDNA sequencing. The invasion of manure-borne bacteria was still perceptible 1 month after the soil amendment. The results obtained with the soil already amended in situ with manure 6 months prior to the experiment suggest that some of the bacterial invaders were established in the community over the long term. Even if differences were observed between soils, the invasion was mainly attributable to some of the most abundant OTUs of manure (mainly Firmicutes). SMZ exposure had a limited influence on soil microorganisms but our results suggest that this kind of contaminant can enhance the invasion ability of some manure-borne invaders.

Widespread Occurrence of Pesticides in Organically Managed Agricultural Soils-the Ghost of a Conventional Agricultural Past?

Pesticides are applied in large quantities to agroecosystems worldwide. To date, few studies assessed the occurrence of pesticides in organically managed agricultural soils, and it is unresolved whether these pesticide residues affect soil life. We screened 100 fields under organic and conventional management with an analytical method containing 46 pesticides (16 herbicides, 8 herbicide transformation products, 17 fungicides, seven insecticides). Pesticides were found in all sites, including 40 organic fields. The number of pesticide residues was two times and the concentration nine times higher in conventional compared to organic fields. Pesticide number and concentrations significantly decreased with the duration of organic management. Even after 20 years of organic agriculture, up to 16 different pesticide residues were present. Microbial biomass and specifically the abundance of arbuscular mycorrhizal fungi, a widespread group of beneficial plant symbionts, were significantly negatively linked to the amount of pesticide residues in soil. This indicates that pesticide residues, in addition to abiotic factors such as pH, are a key factor determining microbial soil life in agroecosystems. This comprehensive study demonstrates that pesticides are a hidden reality in agricultural soils, and our results suggest that they have harmful effects on beneficial soil life.

Ecotoxicological risk assessment of wastewater irrigation on soil microorganisms: Fate and impact of wastewater-borne micropollutants in lettuce-soil system.

The implementation of the new Water Reuse regulation in the European Union brings to the forefront the need to evaluate the risks of using wastewater for crop irrigation. Here, a two-tier ecotoxicological risk assessment was performed to evaluate the fate of wastewater-borne micropollutants in soil and their ecotoxicological impact on plants and soil microorganisms. To this end, two successive cultivation campaigns of lettuces were irrigated with wastewater (at agronomical dose (not spiked) and spiked with a mixture of 14 pharmaceuticals at 10 and 100 µg/L each) in a controlled greenhouse experiment. Over the two cultivation campaigns, an accumulation of PPCPs was observed in soil microcosms irrigated with wastewater spiked with 100 µg/L of PPCPs with the highest concentrations detected for clarithromycin, hydrochlorothiazide, citalopram, climbazole and carbamazepine. The abundance of bacterial and fungal communities remained stable over the two cultivation campaigns and was not affected by any of the irrigation regimes applied. Similarly, no changes were observed in the abundance of ammonium oxidizing archaea (AOA) and bacteria (AOB), nor in clade A of commamox no matter the cultivation campaign or the irrigation regime considered. Only a slight increase was detected in clade B of commamox bacteria after the second cultivation campaign. Sulfamethoxazole-resistant and -degrading bacteria were not impacted either. The irrigation regimes had only a limited effect on the bacterial evenness. However, in response to wastewater irrigation the structure of soil bacterial community significantly changed the relative abundance of Acidobacteria, Chloroflexi, Verrucomicrobia, Beta-, Gamma- and Deltaprotebacteria. Twenty-eight operational taxonomic units (OTUs) were identified as responsible for the changes observed within the bacterial communities of soils irrigated with wastewater or with water. Interestingly, the relative abundance of these OTUs was similar in soils irrigated with either spiked or non-spiked irrigation solutions. This indicates that under both agronomical and worst-case scenario the mixture of fourteen PPCPs had no effect on soil bacterial community.

Insights into the Function and Horizontal Transfer of Isoproturon Degradation Genes (pdmAB) in a Biobed System.

Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.IMPORTANCE Our study provides novel insights into the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes, and verifies that on-farm biobed systems are hot spots for the evolution of pesticide catabolic traits.

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis.

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

Batf3-Dependent Intestinal Dendritic Cells Play a Critical Role in the Control of Cryptosporidium parvum Infection.

Understanding the protective immune response to Cryptosporidium parvum infection is of critical importance to reduce the widespread impact caused by this disease in young individuals. Here, we analyzed the various subsets of CD103+ and CD103- intestinal dendritic cells (DCs) of wild-type and Batf3-/- neonatal mice at homoeostasis and investigated their role during infection. Neonatal Batf3-/- mice had a low CD103+/CD103- DC ratio, resulting in higher susceptibility to the acute phase of the infection and they could not cure the infection. Early during infection, CD103- DCs of Batf3-/- neonates had a lower ability to produce interleukin-12 than their wild-type littermates and lower levels of interferon-gamma mRNA were detected in the infected mucosa. Amplification of CD103+ DCs in Batf3-/- neonates prior to infectious challenge reduced their susceptibility to infection. CD103+ DCs thus outperform CD103- DCs in controlling C. parvum infections and represent a primary target of host-directed immunotherapies dedicated to neonates.

Paneth Cell Defects Induce Microbiota Dysbiosis in Mice and Promote Visceral Hypersensitivity.

BACKGROUND & AIMS: Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages. METHODS: We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction. RESULTS: Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared with mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E coli during maternal separation and visceral hypersensitivity. CONCLUSIONS: Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.

Identification of the hcb Gene Operon Involved in Catalyzing Aerobic Hexachlorobenzene Dechlorination in Nocardioides sp. Strain PD653.

Nocardioides sp. strain PD653 was the first identified aerobic bacterium capable of mineralizing hexachlorobenzene (HCB). In this study, strain PD653-B2, which was unexpectedly isolated from a subculture of strain PD653, was found to lack the ability to transform HCB or pentachloronitrobenzene into pentachlorophenol. Comparative genome analysis of the two strains revealed that genetic rearrangement had occurred in strain PD653-B2, with a genomic region present in strain PD653 being deleted. In silico analysis allowed three open reading frames within this region to be identified as candidate genes involved in HCB dechlorination. Assays using recombinant Escherichia coli cells revealed that an operon is responsible for both oxidative HCB dechlorination and pentachloronitrobenzene denitration. The metabolite pentachlorophenol was detected in the cultures produced in the E. coli assays. Significantly less HCB-degrading activity occurred in assays under oxygen-limited conditions ([O2] < 0.5 mg liter-1) than under aerobic assays, suggesting that monooxygenase is involved in the reaction. In this operon, hcbA1 was found to encode a monooxygenase involved in HCB dechlorination. This monooxygenase may form a complex with the flavin reductase encoded by hcbA3, increasing the HCB-degrading activity of PD653.IMPORTANCE The organochlorine fungicide HCB is widely distributed in the environment. Bioremediation can effectively remove HCB from contaminated sites, but HCB-degrading microorganisms have been isolated in few studies and the genes involved in HCB degradation have not been identified. In this study, possible genes involved in the initial step of the mineralization of HCB by Nocardioides sp. strain PD653 were identified. The results improve our understanding of the protein families involved in the dechlorination of HCB to give pentachlorophenol.

Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL-1ß and TNFα released by inflammatory monocytes.

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and ß-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c+ inflammatory monocytes to the subepithelial space. TNFα and IL-1ß produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.

The antigen-specific response to Toxoplasma gondii profilin, a TLR11/12 ligand, depends on its intrinsic adjuvant properties.

Agonists that activate Toll-like receptors (TLR) are potential vaccine adjuvants. In particular, Toxoplasma gondii profilin (TgPRF) is recognized by TLR11/12 to generate an inflammatory response. Unlike most TLR ligands, TgPRF is also a protein and can therefore simultaneously induce innate and adaptive immune responses. We found that variations in the conformation of TgPRF can affect its ability to induce a TLR11/12-dependent inflammatory response. The secreted recombinant T. gondii (S2-profilin), produced by Schneider 2 cells, has lost its ability to generate an IL-12 response. Reduction of the intramolecular disulfide bonds in S2-profilin rescued the TLR11/12-dependent IL-12 response. Immunization of mice with reduced S2-profilin induced strong cellular and humoral responses compared to mice immunized with unreduced S2-profilin. A mixed Th1/Th2 response was induced with both S2-profilins. However, a more polarized Th1-type response, which was consistent with the IgG2a-polarized humoral response, was observed with reduced S2-profilin. In conclusion, the intrinsic adjuvant properties of TgPRF had significant consequences on the immune response against TgPRF.

Synthesis and evaluation of the anticoccidial activity of trifluoropyrido[1,2-a]pyrimidin-2-one derivatives.

Screening of our chemical library to discover new molecules exhibiting in vitro activity against the invasion of host cells by Eimeria tenella revealed a lead compound with an IC50 of 15µM. Structure-activity relationship studies were conducted with 34 newly synthesized compounds to identify more active molecules and enhance in vitro activity against the parasite. Four compounds were more effective in inhibiting MDBK cell invasion in vitro than the lead compound.

s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil.

The Arthrobacter sp. strain AK-YN10 is an s-triazine pesticide degrading bacterium isolated from a sugarcane field in Central India with history of repeated atrazine use. AK-YN10 was shown to degrade 99 % of atrazine in 30 h from media supplemented with 1000 mg L(-1) of the herbicide. Draft genome sequencing revealed similarity to pAO1, TC1, and TC2 catabolic plasmids of the Arthrobacter taxon. Plasmid profiling analyses revealed the presence of four catabolic plasmids. The trzN, atzB, and atzC atrazine-degrading genes were located on a plasmid of approximately 113 kb.The flagellar operon found in the AK-YN10 draft genome suggests motility, an interesting trait for a bioremediation agent, and was homologous to that of Arthrobacter chlorophenolicus. The multiple s-triazines degradation property of this isolate makes it a good candidate for bioremediation of soils contaminated by s-triazine pesticides.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.

Intestinal CD103+ dendritic cells are key players in the innate immune control of Cryptosporidium parvum infection in neonatal mice.

Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.

The early intestinal immune response in experimental neonatal ovine cryptosporidiosis is characterized by an increased frequency of perforin expressing NCR1(+) NK cells and by NCR1(-) CD8(+) cell recruitment.

Cryptosporidium parvum, a zoonotic protozoan parasite, causes important losses in neonatal ruminants. Innate immunity plays a key role in controlling the acute phase of this infection. The participation of NCR1+ Natural Killer (NK) cells in the early intestinal innate immune response to the parasite was investigated in neonatal lambs inoculated at birth. The observed increase in the lymphocyte infiltration was further studied by immunohistology and flow cytometry with focus on distribution, density, cellular phenotype related to cytotoxic function and activation status. The frequency of NCR1+ cells did not change with infection, while their absolute number slightly increased in the jejunum and the CD8+/NCR1- T cell density increased markedly. The frequency of perforin+ cells increased significantly with infection in the NCR1+ population (in both NCR1+/CD16+ and NCR1+/CD16- populations) but not in the NCR1-/CD8+ population. The proportion of NCR1+ cells co-expressing CD16+ also increased. The fraction of cells expressing IL2 receptor (CD25), higher in the NCR1+/CD8+ population than among the CD8+/NCR1- cells in jejunal Peyer's patches, remained unchanged during infection. However, contrary to CD8+/NCR1- lymphocytes, the intensity of CD25 expressed by NCR1+ lymphocytes increased in infected lambs. Altogether, the data demonstrating that NK cells are highly activated and possess a high cytotoxic potential very early during infection, concomitant with an up-regulation of the interferon gamma gene in the gut segments, support the hypothesis that they are involved in the innate immune response against C. parvum. The early significant recruitment of CD8+/NCR1- T cells in the small intestine suggests that they could rapidly drive the establishment of the acquired immune response.

Prediction of the Fate of Organic Compounds in the Environment From Their Molecular Properties: A Review.

A comprehensive review of quantitative structure-activity relationships (QSAR) allowing the prediction of the fate of organic compounds in the environment from their molecular properties was done. The considered processes were water dissolution, dissociation, volatilization, retention on soils and sediments (mainly adsorption and desorption), degradation (biotic and abiotic), and absorption by plants. A total of 790 equations involving 686 structural molecular descriptors are reported to estimate 90 environmental parameters related to these processes. A significant number of equations was found for dissociation process (pKa), water dissolution or hydrophobic behavior (especially through the KOW parameter), adsorption to soils and biodegradation. A lack of QSAR was observed to estimate desorption or potential of transfer to water. Among the 686 molecular descriptors, five were found to be dominant in the 790 collected equations and the most generic ones: four quantum-chemical descriptors, the energy of the highest occupied molecular orbital (EHOMO) and the energy of the lowest unoccupied molecular orbital (ELUMO), polarizability (α) and dipole moment (µ), and one constitutional descriptor, the molecular weight. Keeping in mind that the combination of descriptors belonging to different categories (constitutional, topological, quantum-chemical) led to improve QSAR performances, these descriptors should be considered for the development of new QSAR, for further predictions of environmental parameters. This review also allows finding of the relevant QSAR equations to predict the fate of a wide diversity of compounds in the environment.

Poly(I:C)-induced protection of neonatal mice against intestinal Cryptosporidium parvum infection requires an additional TLR5 signal provided by the gut flora.

The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c(+) cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen.

Ongoing functional evolution of the bacterial atrazine chlorohydrolase AtzA.

Triazine herbicides such as atrazine and simazine which were heavily used in the latter half of the twentieth century constituted a rich new source of nitrogen for soil microbes. An atzA dechlorinase active against both atrazine and simazine was isolated from various soil bacteria from diverse locations in the mid 1990s. We have surveyed the atzA genes from eight triazine-degrading Aminobacter aminovorans strains isolated from French agricultural soils recurrently exposed to triazines in 2000. Six amino acid differences from the original isolate were each found in more than one of the A. aminovorans strains. Three of these in particular (V92L, A170T and A296T) were recovered from a majority of the isolates and from locations separated by up to 900 km, so may reflect ongoing selection for the new function. Two of the latter (A170T and A296T) were indeed found to confer higher specificity for simazine, albeit not atrazine, and greater affinity for a metal ion required for activity, than did the original variant. In contrast, we found that ongoing maintenance of the original atzA-containing isolate in laboratory culture for 12 years in a medium containing high concentrations of atrazine has led to the fixation of another amino acid substitution that substantially reduces activity for the triazines. The high concentrations of atrazine in the medium may have relaxed the selection for a highly efficient triazine dechlorinase activity, and that there is some, as yet uncharacterised, counter selection against the activity of this enzyme under these conditions.

Deleterious intestinal inflammation in neonatal mice treated with TLR2/TLR6 agonists.

By providing innate immune modulatory stimuli, the early life immune system can be enhanced to increase resistance to infections. Activation of innate cell surface receptors called pattern recognition receptors (PRRs) by TLR ligands is one promising approach that can help to control infections as described for listeriosis and cryptosporidiosis. In this study, the effect of TLR2/TLR1 and TLR2/TLR6 agonists was compared when injected into neonatal mice. Surprisingly, the stimulation of TLR2/TLR6 led to the death of the neonatal mice which was not observed in adult mice. The TLR2/TLR6 agonist administration induced higher systemic and intestinal inflammation both in adult and neonatal mice when compared to TLR2/TLR1 agonist. The mortality of neonatal mice was IFN-γ dependent and involved the intestinal production of IL-22 and IL-17A. This study clearly demonstrates that targeting TLRs as new control strategy of neonatal infections has to be used with caution. Depending on its heterodimeric form, the TLR2 stimulation can induce adverse effects more or less severe relying on the age-related immune functions of the host.