Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms. IMPORTANCE: We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1Δ9 and its elongation product C20:1Δ9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.

Pseudomonas aeruginosa kills Staphylococcus aureus in a polyphosphate-dependent manner.

Due to their frequent coexistence in many polymicrobial infections, including in patients with burn or chronic wounds or cystic fibrosis, recent studies have started to investigate the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro co-cultivation conditions, which is mediated by several of P. aeruginosa's virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role for the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP level are less detrimental to S. aureus growth and survival while the gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high level of polyP. We show that the polyP-dependent phenotype could be a direct effect by the biopolymer, as polyP is present in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect through the regulation of one of P. aeruginosa's virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependent and harms S. aureus through membrane damage and the generation of reactive oxygen species, resulting in increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources.

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli.

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is the antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acid side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. Expression of the rcrARB operon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB. The rcrB gene encodes a hypothetical membrane protein, deletion of which substantially increases UPEC's susceptibility to HOCl. However, the mechanism behind protection by RcrB is unclear. In this study, we investigated whether (i) its mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. We provide evidence that RcrB expression is sufficient to protect E. coli from HOCl. Furthermore, RcrB expression is induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation. IMPORTANCE Bacterial infections pose an increasing threat to human health, exacerbating the demand for alternative treatments. Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be equipped with powerful defense systems to fend off the toxic effects of reactive chlorine species. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system toward hypochlorous acid (HOCl) stress and phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli.

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acids side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs), have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. The regulon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB . rcrB encodes the putative membrane protein RcrB, deletion of which substantially increases UPEC's susceptibility to HOCl. However, many questions regarding RcrB's role remain open including whether (i) the protein's mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. Here, we provide evidence that RcrB expression is sufficient to E. coli 's protection from HOCl and induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation. IMPORTANCE: Bacterial infections pose an increasing threat to human health exacerbating the demand for alternative treatment options. UPEC, the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be well equipped with powerful defense systems to fend off the toxic effects of RCS. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system towards HOCl-stress and phagocytosis. Thus, this novel HOCl-stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Body Condition Score in Danish Horses Related to Type, Use, and Training Level: Patterns, Risk, and Protective Factors.

Body condition in horses is a growing concern that has different patterns of development in horses that are above and below the ideal range. This study used professional and para-professional evaluators (veterinarians, farriers, trainers, Danish Equestrian Federation (DEF) officials) who were trained and certified in the use of a modified Henneke scoring system to assign a body condition score (BCS) on a scale of 1-9. Scores of 5-6 are regarded as ideal, and 78.6% of the evaluated horses were in these groups. Only 4.8% of horses were below ideal BCS but 16.5% were above ideal BCS, and this was influenced by type, age, and training. A significant protective effect towards above ideal BCS was shown for horses trained at higher intensities. Cold-blooded horses and traditional ponies had increased risk for being above ideal BCS. Although BCS increased with age, a large proportion of geriatric horses were both above and below ideal BCS. Discipline was not related to BCS. Patterns of BCS distribution for horses attended by different professionals were investigated. Veterinarians attended more horses with BCS above and below ideal values, farriers mostly saw horses that were above ideal BCS, and officials at competitions mainly saw horses with ideal BCS.

A Year at the Forefront of Bacterial Defense Systems Against Neutrophilic Oxidants.

One challenge for invading pathogens represents the exposure to highly microbicidal hypohalous acids (HOX), such as hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Generated at high concentrations by innate immune cells during phagocytosis, HOX kills the engulfed microbes through extensive macromolecular damage. However, microorganisms have evolved strategies to detoxify the oxidants and/or alleviate HOX-mediated damage, which improves their survival during HOX exposure. Many of these defense systems are bacteria-specific and therefore considered potential drug targets. Our minireview highlights recent (July 2021 to November 2022) advances in the field of microbial HOX defense systems and how these systems are regulated. We report recent progress on redox-sensing transcriptional regulators, two-component systems, and σ/anti-σ factors and review how oxidative modifications in these regulatory proteins affect the expression of their target genes. Moreover, we discuss novel studies that describe how HOCl affects the activity of redox-regulated enzymes and highlight mechanisms that bacteria employ to reduce HOSCN.

The Novel Silver-Containing Antimicrobial Potentiates Aminoglycoside Activity Against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments into novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX®, a broad-spectrum silver containing antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX® could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX® on several antibiotic classes. We found that the combination of AGXX® and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX®/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX®/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX®/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.

A Social Media Intervention for Promoting Oral Health Behaviors in Adolescents: A Non-Randomized Pilot Clinical Trial.

Poor oral hygiene and excessive consumption of soda are among the main drivers of systemic health issues in adolescents in the United States. This non-randomized pilot clinical trial focused on the effects of a health text message system and smartphone-based intervention on adolescent tooth-brushing behavior and dietary choices, with a convenience sample of 94 participants aged 12 to 14 years old. A group of 75 participants agreed to use a tooth-brushing app and received a health text message; the other group of 15 agreed to use the tooth-brushing app, but did not receive a health text message. Saliva specimens were collected directly before and at the end of each experiment; changes in the salivary presence of cariogenic bacteria over the duration of the study were evaluated and compared with the demographics and behavioral variables. Within the text message group, 5% of participants increased the frequency of daily tooth brushing. Within the non-intervention group, 29% of participants increased the frequency of their daily tooth brushing. There were reductions in the total salivary bacteria and total streptococci in both groups (p < 0.001), but no change in the presence of cariogenic Mutans streptococci. Raising adolescents' consciousness of oral health behavior resulted in marginal to moderate improvements to oral hygiene and dietary choices, as well as reductions in total salivary bacteria.

Redox-Mediated Inactivation of the Transcriptional Repressor RcrR is Responsible for Uropathogenic Escherichia coli's Increased Resistance to Reactive Chlorine Species.

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as a critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and is associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non-stress and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis; however, their responses to and defenses from HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared with other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator RcrR. We identified RcrR as a HOCl-responsive transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the derepression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl and phagocytosis indicating that the HOCl-mediated induction of the regulon plays a major role for UPEC's HOCl resistance. IMPORTANCE How do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in noninvasive enteropathogenic E. coli. Our data suggest expression of the target gene rcrB is exclusively responsible for UPEC's increased HOCl tolerance in culture and contributes to UPEC's survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Pre-Competition Oral Findings in Danish Sport Horses and Ponies Competing at High Level.

This study addresses the presence and location of oral lesions in 342 dressage, show jumping, and eventing horses examined at an obligatory veterinary inspection before competing in the Danish National Championship in 2020. Ulcers in the lip commissures were photographed for subsequent pathological analysis. If a lesion was found at the lip commissures on one side, there was an increased risk of finding a similar lesion on the other side (ulcer: p < 0.0001; scarring/depigmentation: p < 0.0001; fissure: p = 0.002; erosion/contusion: p < 0.0001). At the lip commissures, external (cutaneous) ulcers were correlated with internal (mucosal) ulcers (p < 0.0001) and with scarring/depigmentation (p < 0.0001). Both mucosal and cutaneous ulcers were correlated with scarring/depigmentation (p < 0.0001). Erosion/contusion at the lip commissures was associated with similar lesions on the bars (p = 0.0002), and ulcers of the buccal mucosa were associated with ulcers on the bars (p = 0.003). Dental hooks or sharp enamel points on one side were associated with similar lesions on the other side (p < 0.0001). Dental findings were not related to mucosal ulcers or erosion/contusion at the lip commissures but were associated with scarring/depigmentation (p = 0.01).

Assessment of Skin and Mucosa at the Equine Oral Commissures to Assess Pathology from Bit Wear: The Oral Commissure Assessment Protocol (OCA) for Analysis and Categorisation of Oral Commissures.

This study addresses the presence and location of natural pigmentation, potentially pathological changes in pigment, interruptions of the natural lining (scars), roughness, and erosions/contusion (bruising) in and around the corners of the lips of 206 horses presented to a veterinarian for routine preventative dental treatment. After sedation, photographs were taken and later evaluated for the presence of lesions. During the photographic analysis, the Oral Commissure Assessment (OCA) protocol was developed to map precisely the areas of skin and mucosa around the corners of the lips, and the presence of lesions was recorded for each area. Potentially pathological pigment changes occurred more frequently in horses with a higher level of training (p = 0.04) and in light-coloured horses (p = 0.0004), but there was no association with the current use of a bit or the discipline that the horse participated in (p = 0.20). Scars occurred more frequently in horses competing at a higher level. Only two horses had contusions or erosions, five had ulcers, and none showed bleeding; these numbers were too low for statistical analysis. Using the OCA protocol provides a detailed method for categorizing and recording lesions in and around the corners of the lips, including natural vs. potential and/or definite pathological character.

Changes of adenosine deaminase activity in serum and saliva around parturition in sows with and without postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. RESULTS: Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. CONCLUSIONS: Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

Keel bone fractures in Danish laying hens: Prevalence and risk factors.

Keel bone fractures (KBF) in commercial poultry production systems are a major welfare problem with possible economic consequences for the poultry industry. Recent investigations suggest that the overall situation may be worsening. Depending on the housing system, fracture prevalences exceeding 80% have been reported from different countries. No specific causes have yet been identified and this has consequently hampered risk factor identification. The objective of the current study was to investigate the prevalence of KBF in Danish layer hens and to identify risk factors in relation to KBF in all major productions systems, including parent stock production. For risk factor identification, production data from the included flocks was used. In total, 4794 birds from 40 flocks were investigated at end-of-lay. All birds were euthanized on farm and underwent inspection and palpation followed by necropsy. All observations were recorded and subsequently analysed using the SAS statistical software package. In flocks from non-caged systems, fracture prevalence in the range 53%-100%, was observed whereas the prevalence in flocks from enriched cages ranged between 50-98%. Furthermore, often multiple fractures (≥4) were observed in individual birds (range 5-81% of the birds with fractures) depending on the flock. The localization of the fractures at the distal end of the keel bone is highly consistent in all flocks (>96%). Macroscopically the fractures varied morphologically from an appearance with an almost total absence of callus, most frequently observed in caged birds, to large callus formations in and around the fracture lines, which was a typical finding in non-caged birds. Despite being housed under cage-free conditions, parent birds had significantly fewer fractures (all flocks were 60 weeks old) per bird, than other birds from cage-free systems. The body weight at end-of-lay had an effect on the risk of having fractures, heavy hens have significantly fewer fractures at end-of-lay. The older the hens were at onset of lay, the lower was the flock prevalence at end-of-lay. Additionally, the daily egg size at onset of lay was of importance for the risk of developing fractures, the production of heavier eggs initially, resulted in higher fracture prevalence at depopulation. The odds ratio of body weight, (+100 g) was 0.97, age at onset of lay (+1 week) was 0.87 and daily egg weight at onset (+1 gram) was 1.03. In conclusion, the study demonstrated a very high prevalence of KBF in hens from all production systems and identified hen size, age at onset of lay and daily egg weight at onset of lay to be major risk factors for development of KBF in the modern laying hen. Further research regarding this is warranted to strengthen the longevity and enhance the welfare of laying hens.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Lack of evidence of mastitis as a causal factor for postpartum dysgalactia syndrome in sows.

To investigate the prevalence of mastitis in sows suffering from postpartum dysgalactia syndrome (PDS), we examined milk constituents and concentrations of lipopolysaccharides (LPS) obtained from the udder vein (v. epigastrica). As part of a case-cohort study, 109 sows were monitored daily from 60 h antepartum (a.p.) to 36 h postpartum (p.p.). Over time, 38 sows were diagnosed with PDS (PDS+) and were retrospectively matched with 38 healthy sows (PDS-). The study period was divided into 7 smaller time periods (A, B, C, D, E, F, G, H, and E), allowing the studied values, in period B to G, to be compared with period A that served as a baseline, and PDS+ and PDS- sows were compared within the time periods. All sows were subjected to a thorough daily clinical examination and blood was sampled from v. epigastrica for LPS detection. Milk samples were obtained for bacteriological evaluation and detection of N-acetyl-beta-d-glucosaminidase (NAGase), lactate dehydrogenase (LDH), ß-glucuronidase (ß-glu; for evaluation of mastitis), isocitrate (isoC), free glucose, uric acid (UA; for evaluation of the mammary energy status), ß-hydroxybutyrate acid (BHBA; for evaluation of ketosis), and milk urea (for evaluation of the protein status). The results revealed that PDS+ sows had decreased concentrations of UA in milk (P < 0.0001), increased heart rates (P < 0.01), increased mammary edema (P < 0.05), and prolonged capillary refill time in the vulvar mucosa (P < 0.01) compared with PDS- sows. Compared with baseline, feces became more solid 0 to 36 h p.p. (P < 0.0001) and the respiration rate decreased 12 to 24 h p.p. (P < 0.0001) for both PDS+ and PDS- sows. No differences were found between PDS+ and PDS- sows for severe bacterial infections, concentrations of LPS in blood or LDH, NAGase, BHBA, free glucose, isoC, or urea in milk. Concentrations of LPS in blood were not associated with signs of mastitis or edema in the mammary glands. However, a difference over time was seen for redness (P < 0.0001), warmth (P < 0.0001), and hardness (P < 0.05) of the 6 most anterior glands in both PDS+ and PDS- sows from 60 h a.p to 36 h p.p. The PDS- sows had greater concentrations of ß-glu than the PDS+ sows, but no change over time was demonstrated for this marker. In conclusion, signs of mastitis were not consistently linked to PDS in sows. However, the cardiovascular system seemed to be compromised in PDS+ sows and the cause should be investigated to elucidate the pathogenesis of PDS.

Bacterial Defense Systems against the Neutrophilic Oxidant Hypochlorous Acid.

Neutrophils kill invading microbes and therefore represent the first line of defense of the innate immune response. Activated neutrophils assemble NADPH oxidase to convert substantial amounts of molecular oxygen into superoxide, which, after dismutation into peroxide, serves as the substrate for the generation of the potent antimicrobial hypochlorous acid (HOCl) in the phagosomal space. In this minireview, we explore the most recent insights into physiological consequences of HOCl stress. Not surprisingly, Gram-negative bacteria have evolved diverse posttranslational defense mechanisms to protect their proteins, the main targets of HOCl, from HOCl-mediated damage. We discuss the idea that oxidation of conserved cysteine residues and partial unfolding of its structure convert the heat shock protein Hsp33 into a highly active chaperone holdase that binds unfolded proteins and prevents their aggregation. We examine two novel members of the Escherichia coli chaperone holdase family, RidA and CnoX, whose thiol-independent activation mechanism differs from that of Hsp33 and requires N-chlorination of positively charged amino acids during HOCl exposure. Furthermore, we summarize the latest findings with respect to another bacterial defense strategy employed in response to HOCl stress, which involves the accumulation of the universally conserved biopolymer inorganic polyphosphate. We then discuss sophisticated adaptive strategies that bacteria have developed to enhance their survival during HOCl stress. Understanding bacterial defense and survival strategies against one of the most powerful neutrophilic oxidants may provide novel insights into treatment options that potentially compromise the ability of pathogens to resist HOCl stress and therefore may increase the efficacy of the innate immune response.

Hormonal and metabolic indicators before and after farrowing in sows affected with postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) in sows is difficult to diagnose and the pathogenesis is obscure. Hormonal changes related to the disease are often difficult to distinguish from those found in the normal transition period from gestation to lactation. The study aimed to investigate metabolic and hormonal changes related to PDS with the goal of identifying potential biomarkers in sows suffering from PDS (PDS+). Selected biomarkers were examined by comparing 38 PDS+ sows with 38 PDS negative (PDS-) sows. The sows were sampled every 24 h from 60 h ante partum (a.p.) to 36 h post partum (p.p.). RESULTS: Compared to the baseline (60 to 36 h a.p.), cortisol in serum and saliva and fasting blood glucose concentrations increased in PDS+ as well as PDS- sows. C-peptide decreased relative to the baseline in PDS+ sows, and prolactin and 8-epi prostaglandin F2 alpha (8-epi-PGF2α) decreased in PDS- sows. Concentrations of cortisol in serum and saliva, salivary chromogranin A (CgA), fasting blood glucose, C-peptide, and 8-epi-PGF2α differed significantly between PDS+ and PDS- sows, with levels of cortisol in serum and saliva, salivary CgA, and 8-epi-PGF2α in serum being different in the two groups already before parturition. Concentrations of salivary CgA were significantly lower in PDS- sows than in PDS+ sows during the entire study period. CONCLUSIONS: The results suggest that salivary CgA, cortisol and serum 8-epi-PGF2α may potentially serve as early diagnostic indicators for PDS. The consistently higher salivary CgA concentration in PDS+ sows compared to PDS- sows may indicate that homeostatic disturbances are present between 36 to 60 h before parturition in sows developing PDS. The higher serum and saliva cortisol concentration in PDS+ sows compared to PDS- sows could reflect an early sign of inflammation or stress. The significantly lower C-peptide in PDS+ sows compared to PDS- sows may reflect a lower food intake. Our results contribute to the understanding of the pathogenesis of PDS, and the homeostatic disturbances detected before parturition warrants further investigation. The diagnostic potential of the markers identified in this study should be investigated further in a larger population of sows.

The use of phoxim and bendiocarb for control of fleas in farmed mink (Mustela vison).

BACKGROUND: Fleas (Ceratophyllus sciurorum) are common on farmed mink in Denmark. When present, the fleas have a negative impact on the health of the farmed mink and are of nuisance for farm staff. Severe infestations of fleas cause anemia, poor growth and may result in death of mink kits. Changed behavior of the dams is also observed. Further it has been demonstrated that the fleas are vectors of Aleutian disease virus. Flea control is based on use of a few insecticides and resistance has been reported against permethrin. There is thus a need for new flea control products. In this blinded, randomized clinical trial according to GCP standard, phoxim spray and bendiocarb powder for flea control on mink farms were investigated. RESULTS: Both the phoxim spray solution and bendiocarb powder were found to be efficient for the control of C. sciurorum fleas on farmed mink. Phoxim treatments reduced the number of fleas by 98.4% and the bendiocarb treatments reduced the number of fleas by 99.0% in the mink nest boxes when compared to counts in controls. No clinical signs were observed post treatment. CONCLUSIONS: The study demonstrated that phoxim sprayed on the animals and the use of bendiocarb powder in the nest box material were highly efficient for the control of the C. sciurorum fleas on farmed mink. Both products were safe to use at the recommended dose rate. Both compounds are recommended to be integrated in a new farm management plan suggested here.