Innate immune receptors that form complexes with secondary receptors, activating multiple signall... more Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6′-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macr...
Detection of and response to microbial infections by the immune system depends largely on a famil... more Detection of and response to microbial infections by the immune system depends largely on a family of pattern-recognition receptors called Toll-like receptors (TLRs). These receptors recognize conserved molecular products derived from various classes of pathogens, including Gram-positive and -negative bacteria, DNA and RNA viruses, fungi and protozoa. Recognition of ligands by TLRs leads to a series of signaling events resulting in induction of acute responses necessary to kill the pathogen. TLRs are also responsible for the induction of dendritic cell maturation, which is responsible and necessary for initiation of adaptive immune responses. Although TLRs control induction of adaptive immunity, it is not clear at this point how responses are appropriately tailored by individual TLRs to the advantage of the host.
Editorial on the Research Topic The emerging role of metabolism and metabolic-related receptors o... more Editorial on the Research Topic The emerging role of metabolism and metabolic-related receptors on neutrophil extracellular traps formation Neutrophils play a central role in host innate immunity as first cell line to fight pathogens; however, they are also being responsible for "collateral damage". Neutrophils have a wide repertoire of biological responses, including reactive oxygen species (ROS)dependent and-independent effector mechanisms. Release of neutrophil extracellular traps (NET) has been added as key innate immune reaction. Understanding NET formation has broadened our knowledge regarding innate defence mechanisms as well as pathological mechanisms associated with occurrence of uncontrolled NET. Biological processes associated with neutrophil responses are closely linked to cellular metabolism. Glycolysis seems to be the main route associated with ATP generation, phagocytosis, and NET (1, 2), and the low abundance of mitochondria in neutrophils has led to the assumption that neutrophils exclusively rely on glycolysis for their biological functions (3). However, different metabolic routes are required to fulfil energetic, biosynthetic, and functional requirements of activated neutrophils, including TCA cycle, oxidative phosphorylation (OxPhos), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) (4). These metabolic pathways can generate metabolites that exert second messenger functions or act as ligands of receptors (e.g. purinergic receptors, succinate receptor SUCNR1 and lactate receptor HCA2) (5, 6).
The recent increase in new technologies to analyze host-pathogen interaction has fostered a race ... more The recent increase in new technologies to analyze host-pathogen interaction has fostered a race to develop new methodologies to assess these not only on the cellular level, but also on the tissue level. Due to mouse-other mammal differences, there is a desperate need to develop relevant tissue models that can more closely recapitulate the host tissue during disease and repair. Whereas organoids and organs-on-a-chip technologies have their benefits, they still cannot provide the cellular and structural complexity of the host tissue. Here, precision cut tissue slices (PCTS) may provide invaluable models for complex ex-vivo generated tissues to assess host-pathogen interaction as well as potential vaccine responses in a “whole organ” manner. In this mini review, we discuss the current literature regarding PCTS in veterinary species and advocate that PCTS represent remarkable tools to further close the gap between target identification, subsequent translation of results into clinical s...
Recent evidence suggests that several cattle breeds may be more resistant to infection with the z... more Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present ...
Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever vi... more Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-...
Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV... more Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCVassociated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a “proof of concept” vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of vira...
gd and ab TCR 1 T cells share many properties and their interactions are likely to be coordinated... more gd and ab TCR 1 T cells share many properties and their interactions are likely to be coordinated and regulated. We provide evidence that cattle gd T cells are able to present antigen to CD4 1 T cells. To help elucidate their function gd T cell lines were propagated for extensive character- ization. Cells expressed high levels of MHC class II and production of co-stimulatory molecules as evidenced by the binding of a CTLA-4 fusion protein and synthesis of CD80 transcripts. These properties and the presence of a well-developed endosomal compartment indicated the cells might function as antigen-presenting cells. Resting CD4 1 T cells from calves immunized with ovalbumin or respiratory syncytial virus-antigen proliferated in response to gd T cells pulsed with antigen that appeared to be endocytosed via clathrin-coated pits or specific receptors. J. Leukoc. Biol. 63: 707- 714; 1998.
Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce ... more Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opp...
East Coast Fever (ECF), caused by the tick-borne apicomplexan parasiteTheileria parva, remains on... more East Coast Fever (ECF), caused by the tick-borne apicomplexan parasiteTheileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five differentT. parvaschizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) andSaccharomyces cerevisiaeas an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface ofS. cerevisiae.In vitroanalyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 indu...
Proceedings of the National Academy of Sciences, 2021
Significance Bioelectronic modulation of the autonomic nervous system innervating the spleen repr... more Significance Bioelectronic modulation of the autonomic nervous system innervating the spleen represents a new therapeutic avenue. Studies in rodents suffer from the limitation that stimulation parameters and anatomy are not directly applicable to humans. This work demonstrates the translation of biological mechanisms in a large animal model with similar anatomical, histological, and functional characteristics for derivation of human-relevant parameters. Here, we show the scientific process for translating a bioelectronic medicine to clinical readiness. The results presented can be used in three ways: 1) as a system to demonstrate the species translation of neuroimmune modulation, 2) as an exemplar of how translational models can reveal additional potential mechanisms, and 3) as a general methodology to determine human-relevant stimulation parameters.
Intranasal vaccinations are becoming more important in both human and animal medicine to generate... more Intranasal vaccinations are becoming more important in both human and animal medicine to generate a localized IgA immune response not seen with parenteral vaccinations. This localized IgA response is more effective at reducing pathogen load on the mucosal surface of a potential host. One prerequisite for a successful nasal vaccination is the need to understand the distribution pattern of the nebulized vaccine, which requires an understanding the volume of the nares as well as the mucosal surface area. The exact mucosal surface area of ruminant nares has not yet been investigated. The aim of this concept study is to provide a detailed breakdown of a new method of volumetric rendering that can be used to calculate the volume and mucosal surface area of ruminant nares from computed tomographic images. The program Seg 3D was used to perform semi-automatic segmentation of a CT scan of a 9-month-old lamb head. Threshold segmentation and manual segmentation were used in combination to sele...
Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like recep... more Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vi...
Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where hos... more Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where host genetics is thought to contribute to susceptibility or resistance. One of the genes implicated is the SLC11A1 gene, that encodes for the natural resistanceassociated macrophage protein 1 (NRAMP1). The aim of this study was to identify SLC11A1 polymorphisms and to investigate any resulting functional differences in NRAMP1 expression that might be correlated with resistance/susceptibility to M. bovis infection. Sequencing of the SLC11A1 gene in cDNA isolated from Brown Swiss, Holstein Friesian, and Sahiwal cattle identified five single nucleotide polymorphisms (SNPs) in the coding region, but only one of these (SNP4, c.1066C>G, rs109453173) was present in all three cattle breeds and therefore warranted further investigation. Additionally, variations of 10, 11, and 12 GT repeats were identified in a microsatellite (MS1) in the SLC11A1 3 UTR. Measurement of NRAMP1 expression in bovine macrophages by ELISA showed no differences between cells generated from the different breeds. Furthermore, variations in the length of the MS1 microsatellite did not impact on NRAMP1 protein expression as analyzed by luciferase reporter assay. However, further analysis of the ELISA data identified that the presence of the alternative G allele at SNP4 was associated with increased expression of NRAMP1 in bovine macrophages. Since NRAMP1 has been shown to influence the survival of intracellular pathogens such as M. bovis through the sequestering of iron, it is possible that cattle expressing the alternative G allele might have an increased resistance to bTB through increased NRAMP1 expression in their macrophages.
Innate immune receptors that form complexes with secondary receptors, activating multiple signall... more Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6′-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macr...
Detection of and response to microbial infections by the immune system depends largely on a famil... more Detection of and response to microbial infections by the immune system depends largely on a family of pattern-recognition receptors called Toll-like receptors (TLRs). These receptors recognize conserved molecular products derived from various classes of pathogens, including Gram-positive and -negative bacteria, DNA and RNA viruses, fungi and protozoa. Recognition of ligands by TLRs leads to a series of signaling events resulting in induction of acute responses necessary to kill the pathogen. TLRs are also responsible for the induction of dendritic cell maturation, which is responsible and necessary for initiation of adaptive immune responses. Although TLRs control induction of adaptive immunity, it is not clear at this point how responses are appropriately tailored by individual TLRs to the advantage of the host.
Editorial on the Research Topic The emerging role of metabolism and metabolic-related receptors o... more Editorial on the Research Topic The emerging role of metabolism and metabolic-related receptors on neutrophil extracellular traps formation Neutrophils play a central role in host innate immunity as first cell line to fight pathogens; however, they are also being responsible for "collateral damage". Neutrophils have a wide repertoire of biological responses, including reactive oxygen species (ROS)dependent and-independent effector mechanisms. Release of neutrophil extracellular traps (NET) has been added as key innate immune reaction. Understanding NET formation has broadened our knowledge regarding innate defence mechanisms as well as pathological mechanisms associated with occurrence of uncontrolled NET. Biological processes associated with neutrophil responses are closely linked to cellular metabolism. Glycolysis seems to be the main route associated with ATP generation, phagocytosis, and NET (1, 2), and the low abundance of mitochondria in neutrophils has led to the assumption that neutrophils exclusively rely on glycolysis for their biological functions (3). However, different metabolic routes are required to fulfil energetic, biosynthetic, and functional requirements of activated neutrophils, including TCA cycle, oxidative phosphorylation (OxPhos), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) (4). These metabolic pathways can generate metabolites that exert second messenger functions or act as ligands of receptors (e.g. purinergic receptors, succinate receptor SUCNR1 and lactate receptor HCA2) (5, 6).
The recent increase in new technologies to analyze host-pathogen interaction has fostered a race ... more The recent increase in new technologies to analyze host-pathogen interaction has fostered a race to develop new methodologies to assess these not only on the cellular level, but also on the tissue level. Due to mouse-other mammal differences, there is a desperate need to develop relevant tissue models that can more closely recapitulate the host tissue during disease and repair. Whereas organoids and organs-on-a-chip technologies have their benefits, they still cannot provide the cellular and structural complexity of the host tissue. Here, precision cut tissue slices (PCTS) may provide invaluable models for complex ex-vivo generated tissues to assess host-pathogen interaction as well as potential vaccine responses in a “whole organ” manner. In this mini review, we discuss the current literature regarding PCTS in veterinary species and advocate that PCTS represent remarkable tools to further close the gap between target identification, subsequent translation of results into clinical s...
Recent evidence suggests that several cattle breeds may be more resistant to infection with the z... more Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present ...
Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever vi... more Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-...
Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV... more Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCVassociated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a “proof of concept” vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of vira...
gd and ab TCR 1 T cells share many properties and their interactions are likely to be coordinated... more gd and ab TCR 1 T cells share many properties and their interactions are likely to be coordinated and regulated. We provide evidence that cattle gd T cells are able to present antigen to CD4 1 T cells. To help elucidate their function gd T cell lines were propagated for extensive character- ization. Cells expressed high levels of MHC class II and production of co-stimulatory molecules as evidenced by the binding of a CTLA-4 fusion protein and synthesis of CD80 transcripts. These properties and the presence of a well-developed endosomal compartment indicated the cells might function as antigen-presenting cells. Resting CD4 1 T cells from calves immunized with ovalbumin or respiratory syncytial virus-antigen proliferated in response to gd T cells pulsed with antigen that appeared to be endocytosed via clathrin-coated pits or specific receptors. J. Leukoc. Biol. 63: 707- 714; 1998.
Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce ... more Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opp...
East Coast Fever (ECF), caused by the tick-borne apicomplexan parasiteTheileria parva, remains on... more East Coast Fever (ECF), caused by the tick-borne apicomplexan parasiteTheileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five differentT. parvaschizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) andSaccharomyces cerevisiaeas an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface ofS. cerevisiae.In vitroanalyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 indu...
Proceedings of the National Academy of Sciences, 2021
Significance Bioelectronic modulation of the autonomic nervous system innervating the spleen repr... more Significance Bioelectronic modulation of the autonomic nervous system innervating the spleen represents a new therapeutic avenue. Studies in rodents suffer from the limitation that stimulation parameters and anatomy are not directly applicable to humans. This work demonstrates the translation of biological mechanisms in a large animal model with similar anatomical, histological, and functional characteristics for derivation of human-relevant parameters. Here, we show the scientific process for translating a bioelectronic medicine to clinical readiness. The results presented can be used in three ways: 1) as a system to demonstrate the species translation of neuroimmune modulation, 2) as an exemplar of how translational models can reveal additional potential mechanisms, and 3) as a general methodology to determine human-relevant stimulation parameters.
Intranasal vaccinations are becoming more important in both human and animal medicine to generate... more Intranasal vaccinations are becoming more important in both human and animal medicine to generate a localized IgA immune response not seen with parenteral vaccinations. This localized IgA response is more effective at reducing pathogen load on the mucosal surface of a potential host. One prerequisite for a successful nasal vaccination is the need to understand the distribution pattern of the nebulized vaccine, which requires an understanding the volume of the nares as well as the mucosal surface area. The exact mucosal surface area of ruminant nares has not yet been investigated. The aim of this concept study is to provide a detailed breakdown of a new method of volumetric rendering that can be used to calculate the volume and mucosal surface area of ruminant nares from computed tomographic images. The program Seg 3D was used to perform semi-automatic segmentation of a CT scan of a 9-month-old lamb head. Threshold segmentation and manual segmentation were used in combination to sele...
Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like recep... more Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vi...
Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where hos... more Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where host genetics is thought to contribute to susceptibility or resistance. One of the genes implicated is the SLC11A1 gene, that encodes for the natural resistanceassociated macrophage protein 1 (NRAMP1). The aim of this study was to identify SLC11A1 polymorphisms and to investigate any resulting functional differences in NRAMP1 expression that might be correlated with resistance/susceptibility to M. bovis infection. Sequencing of the SLC11A1 gene in cDNA isolated from Brown Swiss, Holstein Friesian, and Sahiwal cattle identified five single nucleotide polymorphisms (SNPs) in the coding region, but only one of these (SNP4, c.1066C>G, rs109453173) was present in all three cattle breeds and therefore warranted further investigation. Additionally, variations of 10, 11, and 12 GT repeats were identified in a microsatellite (MS1) in the SLC11A1 3 UTR. Measurement of NRAMP1 expression in bovine macrophages by ELISA showed no differences between cells generated from the different breeds. Furthermore, variations in the length of the MS1 microsatellite did not impact on NRAMP1 protein expression as analyzed by luciferase reporter assay. However, further analysis of the ELISA data identified that the presence of the alternative G allele at SNP4 was associated with increased expression of NRAMP1 in bovine macrophages. Since NRAMP1 has been shown to influence the survival of intracellular pathogens such as M. bovis through the sequestering of iron, it is possible that cattle expressing the alternative G allele might have an increased resistance to bTB through increased NRAMP1 expression in their macrophages.
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Papers by Dirk Werling