Effect of a pre-milking teat foam and a liner disinfectant on the presence of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking

Contamination of raw milk by psychrotrophs can lead to the production of heat-resistant proteases and subsequent spoilage of UHT milk. Therefore, this research communication evaluated the effect of a pre-milking teat disinfectant (active components: L-(+)-lactic acid and salicylic acid) and a liner disinfectant (active components: peracetic acid and hydrogen peroxide) on the number of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking. The teat orifices of 10 cows were sampled using a swabbing procedure before and after treatment with a pre-milking teat disinfectant on six subsequent days. On the teat orifices, there was a small but statistically significant decrease in the psychrotrophic bacterial counts between pre and post dipping. No differences were observed for the mesophilic bacterial counts and proteolytic active counts. Liners were also sampled using swabs pre and post disinfection. No statistically significant decrease in the bacterial counts was observed post liner disinfection, although there was a numerical decrease. Sixty-two percent of the proteolytic psychrotrophs were pseudomonads: 16.5% of which were P. fragi, 14.3% P. lundensis, 10.0% P. fluorescens and 2.9% P. putida. Trinitrobenzenesulfonic acid (TNBS) analysis revealed a wide variety in proteolytic activity (from 0 to 55 mu mol glycine/ml milk) and the presence of high producers. It can be concluded that there was only a minor effect of teat and liner disinfection on the psychrotrophic bacterial counts indicating that the measures presented did not result in a reduction of the targeted bacteria on teat orifices and liners

Exposure of ciprofloxacin-resistant Escherichia coli broiler isolates to subinhibitory concentrations of a quaternary ammonium compound does not increase antibiotic resistance gene transfer

Resistance to antibiotics threatens to become a worldwide health problem. An important attributing phenomenon in this context is that pathogens can acquire antibiotic resistance genes through conjugative transfer of plasmids. To prevent bacterial infections in agricultural settings, the use of veterinary hygiene products, such as disinfectants, has gained popularity and questions have been raised about their contribution to such spreading of antibiotic resistance. Therefore, this study investigated the effect of subinhibitory concentrations of benzalkoniumchloride (BKC), a quaternary ammonium compound (QAC), on the conjugative transfer of antibiotic resistance genes. Five Escherichia coli field strains originating from broiler chickens and with known transferable plasmid-mediated ciprofloxacin resistance were exposed to subinhibitory BKC concentrations: 1/3, 1/10 and 1/30 of the minimum bactericidal concentration. Antibiotic resistance transfer was assessed by liquid mating for 4 h at 25 degrees C using E. coli K12 MG1655 as recipient strain. The transfer ratio was calculated as the number of transconjugants divided by the number of recipients. Without exposure to BKC, the strains showed a ciprofloxacin resistance transfer ratio ranging from 10(-4) to 10(-7). No significant effect of exposure to subinhibitory concentrations of BKC was observed on this transfer ratio

Expression study by real-time quantitative RT-PCR of the Salmonella typhimurium mntH gene

The aim of our study was to compare the mntH expression of different Salmonella Typhimurium strains and other Salmonella serovars with real-time RT-PCR. Following the mntH expression in function of the growth showed that the mntH expression of S. Typhimurium is growth dependent. A strong decrease of the mntH expression is noticed when the growth reaches 1.78 108 CFU/ml. After induction with EDTA or H2O2, variations between different S. Typhimurium strains were observed. For some S. Typhimurium strains a 10 to 20 times higher mntH expression was noticed after H2O2 induction. The EDTA induction was for most strains lower (5 to 10 times) but also variations between different strains were observed. The other Salmonella serovars were strongly induced after H2O2 but not after EDTA induction

Effect of subinhibitory exposure to quaternary ammonium compounds on the ciprofloxacin susceptibility ofEscherichia colistrains in animal husbandry

Background Quaternary ammonium compound based disinfectants are commonly used in pig and poultry husbandry to maintain farm hygiene. However, studies have shown that subinhibitory concentrations of these disinfectants may increase antibiotic resistance. Investigation of antibiotic susceptibility is usually assessed via the microbroth dilution method, although this conventional culture-based technique only provides information on the bacteriostatic activity of an antimicrobial agent. Therefore, experiments were performed to investigate the effect of prior benzalkonium chloride (BKC) exposure on the viability of subsequent ciprofloxacin (CIP) treatedEscherichia coli. Results Following CIP treatment, bacterial cell counts were significantly higher after exposure to a subinhibitory BKC concentration than without BKC exposure. The flow cytometric results suggested a BKC-dependent onset of membrane damage and loss of membrane potential. Conclusion Our results indicate a lower bactericidal effect of CIP treatment on BKC-exposedE. coliisolates compared to unexposedE. coliisolates

Repeated disinfectant use in broiler houses and pig nursery units does not affect disinfectant and antibiotic susceptibility in Escherichia coli field isolates

Background Disinfectants are frequently used in animal production to reduce or eliminate the load of infectious agents and parasites in buildings and equipment associated with the housing or transportation of animals. There are growing concerns that the use of disinfectants would select for resistance to antibiotics and disinfectants. The aim of this study was to determine the effect of repeated use of different disinfectants on the disinfectant and antibiotic susceptibility under practical conditions in a broiler and pig pilot farm. Therefore, the susceptibility of Escherichia coli (E. coli) to 14 antibiotics and 4 disinfectants was monitored over a one-year period. Results High (20-50%) to very high (> 50%) resistance levels for ampicillin, sulfamethoxazole, trimethoprim and tetracycline were observed in both animal production types. Disinfectant susceptibility did not change over time and did not depend on the used disinfection product. Compared to in-use concentrations of formaldehyde, benzalkoniumchloride and a peracetic acid - hydrogen peroxide formulation, all E. coli strains remained susceptible indicating that the use of disinfectants did not select for disinfectant resistance. Moreover, no association could be found between the use of disinfectants and antibiotic resistance. Conclusions These findings suggest that repeated use of disinfectants in agricultural environments does not select for antibiotic resistance nor does it reduce disinfectant susceptibility

Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by cross-contaminated feed

Aims: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1mgl(-1)), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an exvivo model of the porcine caecum. Methods and Results: A DOX-resistant, tet(A)-carrying, porcine commensal E.coli strain (EC 682) was cultivated for 6days in the porcine caecum model under different conditions (0, 1 and 4mgl(-1) DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n=454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4mgl(-1) DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4mgl(-1) DOX compared to medium without DOX. Conclusions: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E.coli, and promote the selection of transconjugants in a porcine caecum model. Significance and Impact of the Study: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3

The restricted bone marrow (BM) localisation of multiple myeloma (MM) cells most likely results from a specific homing influenced by chemotactic factors, combined with the proper signals for growth and survival provided by the BM microenvironment. In analogy to the migration and homing of normal lymphocytes, one can hypothesise that the BM homing of MM cells is mediated by a multistep process, involving the concerted action of adhesion molecules and chemokines. In this study, we report that primary MM cells and myeloma derived cell lines (Karpas, LP-1 and MM5.1) express the chemokine receptor CCR2. In addition, we found that the monocyte chemotactic proteins (MCPs) MCP-1, -2 and -3, three chemokines acting as prominent ligands for CCR2, are produced by stromal cells, cultured from normal and MM BM samples. Conditioned medium (CM) from BM stromal cells, as well as MCP-1, -2 and -3, act as chemoattractants for human MM cells. Moreover, a blocking antibody against CCR2, as well as a combination of neutralizing antibodies against MCP-1, -2 and -3, significantly reduced the migration of human MM cells to BM stromal cell CM. The results obtained in this study indicate the involvement of CCR2 and the MCPs in the BM homing of human MM cells. (C) 2003 Cancer Research UK

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut

Abstract Background The importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian β-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds. Results Time-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05). Conclusion Amino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota

Immunoassay for quantification of antigen-specific IgG fucosylation

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021