Dirk Werling - Academia.edu (original) (raw)

Papers by Dirk Werling

bioRxiv (Cold Spring Harbor Laboratory), Mar 4, 2024

The role of macrophage (MØ) cellular metabolism and reprogramming during TB infection is of great... more The role of macrophage (MØ) cellular metabolism and reprogramming during TB infection is of great interest due to the influence of Mycobacterium spp. on MØ bioenergetics. Recent studies have shown that M. tuberculosis induces a TLR2-dependent shift towards aerobic glycolysis and metabolic reprogramming, comparable to the established LPS induced pro-inflammatory M1 MØ polarisation. Distinct differences in the metabolic profile of murine and human MØ indicates species-specific differences in bioenergetics. So far, studies examining the metabolic potential of cattle are lacking, thus the basic bioenergetics of bovine and human MØ were explored in response to a variety of innate immune stimuli. Cellular energy metabolism kinetics were measured concurrently for both species on a Seahorse XFe96 platform to generate bioenergetic profiles for the response to the bona-fide TLR2 and TLR4 ligands, FSL-1 and LPS respectively. Despite previous reports of species-specific differences in TLR signalling and cytokine production between human and bovine MØ, we observed similar respiratory profiles for both species. Basal respiration remained constant between stimulated MØ and controls, whereas addition of TLR ligands induced increased glycolysis. In contrast to MØ stimulation with M. tuberculosis PPD, another TLR2 ligand, M. bovis PPD treatment significantly enhanced basal respiration rates and glycolysis only in human MØ. Respiratory profiling further revealed significant elevation of ATP-linked OCR and maximal respiration suggesting a strong OXPHOS activation upon M. bovis PPD stimulation in human MØ. Our results provide an exploratory set of data elucidating the basic respiratory profile of bovine vs. human MØ that will not only lay the foundation for future studies to investigate host-tropism of the M. tuberculosis complex but may explain inflammatory differences observed for other zoonotic diseases.

bioRxiv (Cold Spring Harbor Laboratory), May 31, 2020

Holzapfel et al. TLR5 Evasion by Leptospires the FlaB, but not the FlaA subunits. Altogether, in ... more Holzapfel et al. TLR5 Evasion by Leptospires the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response.

Trends in Parasitology, Nov 1, 2022

Frontiers in Veterinary Science, Feb 22, 2021

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impa... more Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Veterinary Immunology and Immunopathology, Sep 1, 2009

Objective: To describe the clinical characteristics and outcomes in a population of dogs with neg... more Objective: To describe the clinical characteristics and outcomes in a population of dogs with negative-pressure pulmonary edema (NPPE) and to identify the main causes of the disease. To evaluate any associations with morbidity and mortality. Design: Retrospective study. Setting: Three university teaching hospitals and two private referral centers. Animals: Thirty-five client-owned dogs presented with NPPE. Interventions: None Measurements and Main Results: Data collected included patient characteristics, clinical history, clinicopathological abnormalities, radiographic features, treatments and outcome. Median age was 4 months (range 2-90) and median weight was 7.1 kg (range 1.7-37.2). There were many causes of NPPE including leash tugs, near hanging, accidental choking, anatomical obstruction to airflow and purposeful airway obstruction by people. The most common cause of NPPE was accidental choking (40% of cases). Dogs with an anatomical obstruction were older than 24 months. Hypoxemia with an increased alveolar-arterial gradient was common on presentation. The majority of thoracic radiographs (65.7%) showed an alveolar or interstitial pattern in the caudodorsal area as previously described in the literature. Oxygen therapy was administered to 33 (94.3%) dogs. Furosemide was administered to 18 (51.4%) dogs. Median length of hospitalization was 2 days (range 0-14). Twenty-eight (80%) dogs survived to discharge. Seven dogs were mechanically ventilated and only 2 of them (28.6%) 2 survived to discharge. Requirement for mechanical ventilation (p<0.001) was the only parameter associated with mortality. Conclusions: Most cases of NPPE occur in juvenile dogs. Different incidents associated with upper airway obstruction can produce an episode of NPPE. Choking on food or toys and near hanging have not been previously described in the veterinary literature as inciting causes of NPPE. The overall prognosis is good.

Tuberculosis, Sep 1, 2007

Members of the Mycobacterium tuberculosis complex share a high level of genetic identity, however... more Members of the Mycobacterium tuberculosis complex share a high level of genetic identity, however pathogenic ability appears to display host restriction. Interestingly M. tuberculosis, the primary cause of tuberculosis in humans, is non-pathogenic in cattle. Conversely Mycobacterium bovis, the cause of tuberculosis in cattle, is also responsible for a proportion of tuberculosis cases in humans. We hypothesise that differences in the abilities of M. bovis and M. tuberculosis to cause pathogenesis in cattle will be reflected in their interactions with bovine antigen presenting cells. To analyse the importance of host species in mycobacterial infection, bovine antigen presenting cells were infected with bovine or human mycobacterial strains. Levels of nitric oxide and tumour necrosis factor production, markers of antimicrobial activity, were found to be associated with a specific mycobacterial strain, and varied between cell subsets.

Journal of Comparative Pathology, Jul 1, 2023

Parasite Immunology, Dec 1, 2015

Previously, vaccination of cattle with Escherichia coli‐expressed bovine lungworm paramyosin (EcP... more Previously, vaccination of cattle with Escherichia coli‐expressed bovine lungworm paramyosin (EcPMY) adjuvanted with Quil A resulted in considerable reduction in worm burden and larvae shedding (Strube et al., 2015). To further evaluate the protective potential of PMY, cattle vaccination trials were performed using either E. coli‐ (EcPMY) or Pichia pastoris‐expressed PMY (PpPMY) with different adjuvants (Matrix‐Q™ or Quil A). Combinations EcPMY+Matrix‐Q™ (trial 1), PpPMY+Matrix‐Q™ (trial 2) and PpPMY+Quil A (trial 3) were tested against challenge infections with 2000 Dictyocaulus viviparus larvae. Even though GM worm burden and larvae shedding was lower in almost all vaccinated groups, there were high variations between individuals hampering significant differences. However, in all vaccinated groups, lungworms were significantly shorter compared with those in controls. In vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant (r)PMY revealed no significant proliferation following vaccinations or challenge infection. All vaccinated cattle showed a significant rise in specific antibodies, particularly IgG and its subclass IgG1, and detected the native lungworm PMY in immunoblots starting 2 weeks after the first vaccination. The use of a different rPMY‐adjuvant combination or combined vaccination with additional recombinant antigens might be a promising future approach towards a new vaccine against lungworms in cattle.

Veterinary Immunology and Immunopathology, Nov 1, 1994

The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of capr... more The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined. Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929. These results indicate that CAEV infection changes caprine monocyte cytokine responsivity.

Veterinary Immunology and Immunopathology, Sep 1, 2007

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the ... more A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9,

Frontiers in Veterinary Science, Feb 26, 2020

Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. L... more Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. Live coccidiosis vaccines for chickens have been available for almost 70 years, but the requirement to formulate blends of oocysts from multiple Eimeria species makes vaccine production costly and logistically demanding. A multivalent vaccine that does not require chickens for its production and can induce protection against multiple Eimeria species is highly desirable. However, despite the identification and testing of many vaccine candidate antigens, no recombinant coccidiosis vaccine has been developed commercially. Currently, assessment of vaccine efficacy against Eimeria, and the disease coccidiosis, can be done only through in vivo vaccination and challenge experiments but the design of such studies has been highly variable. Lack of a "standard" protocol for assessing vaccine efficacy makes comparative evaluations very difficult, complicating vaccine development, and validation. The formulation and schedule of vaccination, the breed of chicken and choice of husbandry system, the species, strain, magnitude, and timing of delivery of the parasite challenge, and the parameters used to assess vaccine efficacy all influence the outcomes of experimental trials. In natural Eimeria infections, the induction of strong cell mediated immune responses are central to the development of protective immunity against coccidiosis. Antibodies are generally regarded to be of lesser importance. Unfortunately, there are no specific immunological assays that can accurately predict how well a vaccine will protect against coccidiosis (i.e., no "correlates of protection"). Thus, experimental vaccine studies rely on assessing a variety of post-challenge parameters, including assessment of pathognomonic lesions, measurements of parasite replication such as oocyst output or quantification of Eimeria genomes, and/or measurements of productivity such as body weight gain and feed conversion rates. Understanding immune responses to primary and secondary infection can inform on the most appropriate immunological assays. The discovery of new antigens for different Eimeria species and the development of new methods of vaccine antigen delivery necessitates a more considered approach to assessment of novel vaccines with robust, repeatable study design. Careful consideration of performance and welfare factors that are genuinely relevant to chicken producers and vaccine manufacturers is essential.

Veterinary Immunology and Immunopathology, 2003

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive ... more Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, which is mediated by B and T lymphocytes, recognises pathogens by rearranged high af®nity receptors. However, the establishment of adaptive immunity is often not rapid enough to eradicate microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defense mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors (PRR). Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns (PAMP) to TLR induces the production of reactive oxygen and nitrogen intermediates (ROI and RNI), pro-in¯ammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this review, we will summarize the discovery and the critical roles of the TLR family in host defense, brie¯y allude to signaling mechanisms mediating the response to TLR ligands, and will provide an update on current knowledge regarding the ligand speci®city of these receptors and their role in immunity of domestic animals, particularly cattle.

Advances in Animal Biosciences, 2011

Introduction The relatively high saturated fatty acid (SFA) content in milk fat has raised critic... more Introduction The relatively high saturated fatty acid (SFA) content in milk fat has raised criticism in the past since it is assumed to be associated with negative effects on human health, especially due to the presence of C12:0, C14:0 and C16:0. On the other hand, monounsaturated fatty acids (MUFA), such as c9 C18:1 (oleic; OA) and t11 C18:1 (vaccenic; VA) and polyunsaturated fatty acids (PUFA), such as n-3 and n-6 groups and c9t11 conjugated linoleic acid (CLA9) found in milk, have been linked to beneficial effects on human health (Haug et al. 2007). c9c12c15 C18:3 (ALN) and c9c12 C18:2 (LA) are the main n-3 and n-6 FA in milk respectively. Differences in milk fatty acid composition between different dairy management systems in UK, such as conventional, organic and low input, have been reported in other studies (Butler et al. 2008; Ellis et al. 2006). Grazing intake, silage and sward composition, forage:concentrate ratio and oilseed supplementation, all factors that widely vary between management systems, can be responsible for milk fat compositional differences (Dewhurst et al. 2006). In a previous study, a stronger effect on milk fatty acid composition was found when the differences on grazing intake between management systems were more extreme. The comparison between low input and conventional milk showed higher differences in milk fatty acid composition than when conventional milk was compared with organic (Butler et al. 2008). The aim of this study was to investigate and explain possible differences in milk fatty acid composition between farms under different management practices in the North East of England. Material and methods Milk from the bulk tank of 20 farms in the North East of England, representing 4 different management systems, was collected every 8 weeks for 10 month period. Conventional systems are characterized by an average of 41% of their diet as concentrates, with conserved forage fed while animals are fully housed during winter but access to ryegrass pasture when conditions allowed in summer. In organic systems, cows graze in ryegrass/clover swards, usually between April and October, and are housed in winter, with an average 22% concentrate supplementation. On intensive farms, cows are milked 3 times per day, nutrition is consistent throughout the year and is based on silage, which in that case may also contain maize silage, and 49% concentrates. Farms that apply robotic milking were selected to have the same feeding practices as conventional farms in order to investigate the effect of milking procedure. Analysis of FA methyl esters was performed with a Gas Chromatography system (Shimadzu, GC-2014, Japan) using a Varian CP-SIL 88 fused silica capillary column (100m x 0.25mmID x 0.2µm film thickness). Peaks were identified using a 39 FAME and CLA isomer standards. Analysis of variance (ANOVA) using linear mixed effects model (LME) was used to analyze results in R statistical environment using "Management system" (conventional, organic, intensive, robotic) and "sampling month" (7 sampling months over one year) as fixed factors and farm number as random factor. Results Compared to conventional milk, organic milk showed significantly higher concentrations of C14:0, ALN, n-3 and n-3:n-6 ratio while milk from intensive farms showed higher milk n-6 concentrations. When organic milk was compared to milk from intensive farms, significantly higher concentrations of C14:0, VA, CLA9, ALN, n-3, higher n-3:n-6 ratio and significantly lower concentrations of LA were found. Robotic milking farms showed significantly higher milk concentrations of C12:0 and C14:0 and lower concentrations of CLA9 than conventional farms. * *** ANOVA P-values refer to the overall effect of management system and bold values indicate significant differences compared with milk from conventional farms. Significances were declared at ***: P<0.001, *: P<0.05, †: 0.05<P<0.10, ns: P>0.05. Different superscripts within the same column indicate differences between the reported management systems. Conclusions The high content of ALN in organic milk and LA in milk from intensive farms can be attributed to the increased grazing intake and the use of maize silage and by-products respectively. Many differences can be explained by production intensity and the proportion of grazing in DMI, hence CLA9 and VA concentrations of milk were higher in organic milk only when compared with intensively produced milk. For relatively smaller differences in grazing intake (18% DMI in conventional, 38% DMI in organic), the CLA9 content of milk was similar, regardless the dairy management system. The lower CLA9 content of milk from farms with robotic milking should be further investigated since differences in nutrition, that could possibly affect the CLA9 content of milk between those and conventional farms, were limited.