Biology subjects

Werling, D.

Publications and source records attributed to Werling, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Analysis of genetic variation in the bovine Mannose Receptor gene (MRC1), its influence on receptor expression, and a potential association with resistance to bovine tuberculosis

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/734952v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1bffc22org.highwire.dtl.DTLVardef@1422f8aorg.highwire.dtl.DTLVardef@3f3de1org.highwire.dtl.DTLVardef@1f7b960_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Bovine H5N1 influenza viruses have adapted to more efficiently use receptors abundant in cattle

Sustained mammal-to-mammal transmission of high pathogenicity H5N1 avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected in early 2024. Genomic analyses revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identified alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

microbiology↗

bioRxiv2025

Safety and Efficacy of a Novel Glycoengineered Recombinant Vaccine Candidate against Haemonchus contortus in Sheep

Haemonchus contortus is considered the most pathogenic nematode in small ruminants and South American camelids, causing significant production losses and threatening animal welfare worldwide. Compared to the use of anthelmintic drugs, to which resistance is increasing, vaccination is considered a more sustainable control strategy. The safety and efficacy of a novel glycoengineered vaccine produced in Hi5 insect cells was evaluated in a randomized, controlled vaccination-and-challenge trial. For this, 35 male Jura x Lacaune sheep were assigned to five groups (n=7). Three experimental groups were vaccinated three times subcutaneously with 1 ml of either the commercial Barbervax(R) vaccine, the novel glycoengineered vaccine, consisting of a cocktail of five antigens (H11, H11-1, H11-2, H11-4 and GA1), or its non-glycoengineered counterpart before challenge with 5,000 H. contortus third-stage larvae. Clinical assessments, differential blood counts, serum antibody responses and fecal egg counts were evaluated at least once per week within the 16-week trial period. The abomasal worm burden was counted 40 days after challenge. The results of the mixed model showed a lower degree of anemia (mean packed cell volume (PCV) of 26.77%; mean hemoglobin (Hb) of 9.03 g/dl), in sheep vaccinated with the glycoengineered antigen cocktail compared to the unvaccinated group (PCV of 24.34%; mean Hb of 7.84 g/dl) and to the sheep vaccinated with the non-glycoengineered antigens (PCV of 26.11%; mean Hb of 8.53 g/dl). However, the degree of anemia was lower in sheep vaccinated with native antigens (Barbervax(R)) (PCV of 29.10%; mean Hb of 9.84 g/dl). Serum IgG and IgE antibodies were elevated in all vaccinated groups. Sheep vaccinated with the glycoengineered antigens showed a reduction in abomasal worm burden of 25.36% and a reduction of fecal egg shedding of 81.09%, the group vaccinated with non-glycoengineered antigen showed no worm reduction and a fecal egg count reduction of 32.72%. While there was no significant difference in egg shedding between sheep vaccinated with the glycoengineered antigens and sheep vaccinated with Barbervax(R) (98.32%; p = 0.33), the Barbervax(R) group showed a greater reduction in worm burden (86.40%). The results emphasize that glycoengineering of vaccine candidates is essential to achieve protective immunity against H. contortus in sheep and that the novel glycoengineered vaccine produced in insect cells clearly has the potential to reduce fecal egg shedding and worm burden, paving the way for the development of highly effective recombinant vaccines against this and other parasitic worms in the future.

molecular biology↗

bioRxiv2025

Glycoengineering of nematode antigens using insect cells: a promising approach for producing bioactive vaccine antigens of the barber's pole worm Haemonchus contortus

The H11 antigens, located on the intestinal microvilli of Haemonchus contortus, comprise a group of homologous aminopeptidases essential for the parasites digestion of blood meals. Native H11 proteins are promising vaccine antigens, capable of eliciting robust protective immunity against H. contortus in sheep and goats. However, recombinant forms of H11, produced either in conventional expression systems or in transgenic Caenorhabditis elegans, failed to replicate the protective efficacy of the native form, most likely due to two critical factors: improper glycosylation and protein misfolding. To address these limitations, we developed a novel strategy to produce recombinant Haemonchus antigens in glycoengineered insect cells. By introducing three C. elegans genes that alter the native N-glycosylation pathways of Hi5 insect cells we successfully expressed soluble H11 and GA1 antigens featuring nematode-specific glycan epitopes, including tri-fucosylated structures and the Gal{beta}1,4Fuc motif. The glycoengineered H11 proteins retained aminopeptidase activity and stimulated cytokine secretion from ovine peripheral blood mononuclear cells in vitro. These findings establish a platform for producing bioactive vaccine antigens against the parasitic nematode H. contortus.

bioengineering↗

bioRxiv2025

Pre-stimulation of Precision-Cut bovine udder slices with zymosan before LPS exposure indicates indicators for trained immunity

Mastitis in cattle poses a significant health challenge and results in substantial economic losses for the dairy industry. This study aimed to establish precision-cut bovine udder slices (PCBUS) as an in vitro model to explore the potential of stimulating trained immunity in the udder. The goal was to investigate whether this approach could influence the early pathophysiological processes of mastitis and pave the way for developing new therapeutic strategies for udder inflammation in future research. PCBUS remained viable in culture for up to two weeks. When stimulated with E. coli-derived lipopolysaccharide (LPS), zymosan (an inducer of trained immunity), or pre-incubated with zymosan followed by LPS stimulation, the slices exhibited distinct responses in terms of pro-inflammatory cytokine production and lipid mediator profiles. Additionally, cytokine production was influenced by the presence or absence of fetal calf serum (FCS), highlighting the potential limitations of FCS in in vitro studies. While the current experimental setup did not definitively confirm the induction of trained immunity in the bovine udder, it validated the utility of PCBUS as a robust in vitro model for studying bovine udder inflammation. This model offers a promising platform for developing innovative mastitis treatments, particularly given the growing concern over antimicrobial resistance. It also provides a valuable tool for advancing our understanding of immune responses in the bovine udder. By adapting the precision-cut tissue slice technique to bovine udders, this model enables extensive research into new therapeutic approaches and supports basic research efforts to characterize complex pathophysiological processes associated with mastitis.

immunology↗

bioRxiv2024

POLYGENIC DETERMINANTS OF H5N1 ADAPTATION TO BOVINE CELLS

Avian influenza H5N1 clade 2.3.4.4b viruses caused a global panzootic and, unexpectedly, widespread outbreaks in dairy cattle, therefore representing a pandemic threat. To inform effective control strategies, it is critical to determine whether the potential to adapt to bovine cells is a generalised feature of H5N1 viruses, or is specific to clade 2.3.4.4b, or even more restricted to specific genotypes within this clade (e.g., B3.13 and D1.1). Using a large panel of H5N1 viruses representing >60 years of their natural history and other IAV for comparative purposes, we demonstrate that virus adaptation to bovine cells is: (i) highly variable across 2.3.4.4b genotypes, (ii) limited in viruses predating the global expansion of this clade, (iii) determined by the viral internal gene cassette, and (iv) not restricted to udder epithelial cells. Mutations in the PB2 polymerase subunit, particularly M631L, emerge as key determinants of adaptation, although their phenotypic effects are context dependent and have limited enhanced viral polymerase activity in human cells. Bovine B3.13 and some avian genotypes also exhibit enhanced modulation of bovine interferon-induced antiviral responses, determined by at least the viral PB2, nucleoprotein, and the non-structural protein NS1. Our results highlight the polygenic nature of IAV host range and reveal that the potential to cross the species barrier varies during the evolutionary trajectory of H5N1, with some avian viruses more predisposed to spillover than others.

microbiology↗

bioRxiv2024

Multi-omics analysis reveals regime shifts in the gastrointestinal ecosystem in chickens following anticoccidial vaccination and Eimeria tenella challenge

Coccidiosis, caused by Eimeria parasites, poses significant economic and welfare challenges in poultry farming. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and host metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis (MOFA) machine learning model, data on caecal microbiota and host metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.

microbiology↗

bioRxiv2024

M. bovis PPD Enhances Respiratory Bioenergetics of Human vs. Bovine Macrophages

The role of macrophage (MO) cellular metabolism and reprogramming during TB infection is of great interest due to the influence of Mycobacterium spp. on MO 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 MO polarisation. Distinct differences in the metabolic profile of murine and human MO 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 MO 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 MO, we observed similar respiratory profiles for both species. Basal respiration remained constant between stimulated MO and controls, whereas addition of TLR ligands induced increased glycolysis. In contrast to MO stimulation with M. tuberculosis PPD, another TLR2 ligand, M. bovis PPD treatment significantly enhanced basal respiration rates and glycolysis only in human MO. 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 MO. Our results provide an exploratory set of data elucidating the basic respiratory profile of bovine vs. human MO 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. HighlightsO_LISimilar baseline respiratory profiles for human and bovine macrophages C_LIO_LIM. bovis PPD treatment altered metabolic profile only in human MO C_LIO_LIStrong OXPHOS activation upon M. bovis PPD stimulation only in human MO C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/582730v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@e833fborg.highwire.dtl.DTLVardef@acdda6org.highwire.dtl.DTLVardef@10bcbfforg.highwire.dtl.DTLVardef@17ddf31_HPS_FORMAT_FIGEXP M_FIG Created with BioRender (www.biorender.com) by A. Gibson C_FIG

immunology↗

bioRxiv2023

Identification of methylation markers for age and Bovine Respiratory Disease in dairy cattle

Methylation profiles of animals is known to differ by age and disease status. Bovine respiratory disease (BRD), a complex infectious disease, primarily affects calves and has significant impact on animal welfare and the cattle industry, predominantly from production losses. BRD susceptibility is multifactorial, influenced by both environmental and genetic factors. We investigated the epigenetic profile of BRD susceptibility in calves and age-related methylation differences between healthy calves and adult dairy cows using Reduced Representation Bisulfite Sequencing (RRBS). We identified 3,452 genes within differentially methylated regions between calves and adults. Functional analysis revealed enrichment of developmental pathways including cell fate commitment and tissue morphogenesis. Between healthy and BRD affected calves, 964 genes were identified within differentially methylated regions. Immune and vasculature regulatory pathways were enriched and key candidates in BRD susceptibility involved in complement cascade regulation, vasoconstriction and respiratory cilia structure and function were identified.

genetics↗

bioRxiv2023

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand

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 macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognize endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

immunology↗

bioRxiv2022

Defining the genes required for survival of Mycobacterium bovis in the bovine host offers novel insights into the genetic basis of survival of pathogenic mycobacteria

Tuberculosis has severe impacts in both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human adapted species, and Mycobacterium bovis, the animal adapted species is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models, however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study we utilise transposon insertion sequencing in M. bovis to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, PDIM synthesis, mycobactin synthesis and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis, phoPR is required by M. bovis in vivo despite the known defect in signalling through this system. Comparison to studies performed in glycerol adapted species such as M. bovis BCG and M. tuberculosis suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon), oxidation (hsd) and detoxification (cyp125). We report good correlation with existing mycobacterial virulence functions, but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. ImportanceThis is the first report of the genetic requirements of an animal adapted member of the MTBC in a natural host. M. bovis has devastating impacts in cattle and bovine tuberculosis is a considerable economic, animal welfare and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identifies several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.

microbiology↗

bioRxiv2021

A novel whole yeast-based subunit oral vaccine against Eimeria tenella in chickens

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 opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).

immunology↗

bioRxiv2021

Probing differences in gene essentiality between the human and animal adapted lineages of the Mycobacterium tuberculosis complex using TnSeq

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

microbiology↗

bioRxiv2021

Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis than others. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Frisian (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 taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses containing H326Q were found to be partially recovered compared to human TLR2. The creation of human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific response to mycobacterial antigens, but furthermore, that there are clear differences between the response seen in different cattle breeds, which may contribute to their enhanced or reduced susceptibility to mycobacterial infection.

immunology↗

bioRxiv2021

Targeted next-generation sequencing of Candidate Regions Identified by GWAS Revealed SNPs Associated with IBD in GSDs

Canine Inflammatory bowel disease (IBD) is a chronic multifactorial disease, resulting from complex interactions between the intestinal immune system, microbiota and environmental factors in genetically predisposed dogs. Previously, we identified several single nucleotide polymorphisms (SNP) and regions on chromosomes (Chr) 7, 9, 11 and 13 associated with IBD in German shepherd dogs (GSD) using GWAS and FST association analyses. Here, building on our previous results, we performed a targeted next-generation sequencing (NGS) of a two Mb region on Chr 9 and 11 that included 14 of the newly identified candidate genes, in order to identify potential functional SNPs that could explain these association signals. Furthermore, correlations between genotype and treatment response were estimated. Results revealed several SNPs in the genes for canine EEF1A1, MDH2, IL3, IL4, IL13 and PDLIM, which, based on the known function of their corresponding proteins, further our insight into the pathogenesis of IBD in dogs. In addition, several pathways involved in innate and adaptive immunity and inflammatory responses (i.e. T helper cell differentiation, Th1 and Th2 activation pathway, communication between innate and adaptive immune cells and differential regulation of cytokine production in intestinal epithelial cells by IL-17A and IL-17F), were constructed involving the gene products in the candidate regions for IBD susceptibility. Interestingly, some of the identified SNPs were present in only one outcome group, suggesting that different genetic factors are involved in the pathogenesis of IBD in different treatment response groups. This also highlights potential genetic markers to predict the response in dogs treated for IBD.

genetics↗

bioRxiv2021

Development of a yeast-based vaccine for Theileria parva infection in cattle

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria 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 different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length native Tp2 and Tp9 as well as fragments of native Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNy responses using PBMCs from ITM-animals, while Tp2 and Tp9 induced IFNy responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induce Tp2 specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cows will further contribute to the development of a yeast-based vaccine for ECF.

immunology↗

bioRxiv2021

Bovine neutrophils release extracellular traps and cooperate with macrophages in Mycobacterium avium subsp. paratuberculosis clearance

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1{beta} and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1{beta} and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

immunology↗

bioRxiv2020

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. However, the host and pathogen determinants driving host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed both blood-derived primary human and bovine macrophages (hM{phi} or bM{phi}, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hM{phi} whereas both Mbv and Mtb efficiently replicate in bM{phi}. Specifically, we show that out of the four infection combinations, only the infection of bM{phi} with Mbv promoted the formation of multinucleated cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bM{phi} promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC formation in the contrasting pathology between Mtb and Mbv for the bovine host, and identify MPB70 from Mbv and extracellular vesicles from bM{phi} as mediators of this process.

microbiology↗