Epidemiology of Salmonella enterica subspecies enterica serotypes, isolated from imported, farmed and feral poultry in the Cayman Islands.

Non-typhoidal Salmonellae (NTS) are common foodborne pathogens throughout the world causing acute gastroenteritis. Compared to North America and Europe, there is little information on NTS in the Caribbean. Here we investigated the prevalence and characteristics of NTS present in the local poultry of the Cayman Islands to determine the public health risk. In total, we collected 156 samples. These were made up of boot swabs of 31 broiler farms and 31 layer farms (62 samples), paper bedding from 45 imported chick boxes, and 49 pooled cecum samples from feral chickens, each sample representing 10 individual chickens. Salmonella was isolated using the ISO 6579 protocol and isolates were characterized using Whole Genome Sequencing (WGS) analysis. Eighteen Salmonella isolates were obtained and comprised six S. enterica subspecies enterica serotypes and one subspecies houtenae serotype. Serotypes were: S. Kentucky (n = 9), S. Saintpaul (n = 5), S. Javiana (n = 1), S. Senftenberg (n = 1), S. Poona (n = 1) and S. Agona (n = 1). S. Kentucky strains were all ST152 and clonally related to poultry strains from the United states. S. Saintpaul ST50 strains showed clonality to North American strains. Over half of the strains (n = 11) contained resistance genes to at least two antibiotic groups and five strains were MDR, mainly those from imported day-old chicks. The blaCMY-2 gene was found in S. Kentucky from day-old chicks. Strains from feral poultry had no acquired AMR genes. While serotypes from feral poultry have been identified in human infections, they pose minimal risk due to their low virulence.

Determination of minimum individual cow colostral immunoglobulin G concentration required to provide adequate total immunoglobulin G mass in pooled colostrum fed to Jersey calves.

OBJECTIVE: Quantify the minimum individual cow colostral immunoglobulin G (IgG) concentration required for pooling to achieve adequate transfer of passive immunity in calves. ANIMALS: 201 Jersey cows. METHODS: Colostrum was collected from 28 pools and heat treated before being fed to calves or stored. Parity, total number of cows contributing to the pool, individual cow colostral volume contributions, and total volume of each colostrum pool were recorded. Colostrum IgG concentrations in individual and pooled (pre- and post-heat treatment) samples were analyzed by radial immunodiffusion and Brix refractometry. Colostral IgG concentration of ≥ 50g/L was considered the current recommended dairy industry standard for acceptable colostrum quality. Multivariable models were performed to determine factors affecting pooled colostral IgG concentrations. The minimum colostral IgG concentration required for pooling to achieve the recommended total mass of at least 200g IgG to be fed to a calf was calculated. RESULTS: Total pool volume and the number of cows contributing to the pool were significant factors affecting IgG concentration. Colostrum pools from ≤ 7 cows, with a minimum pool IgG concentration of 70.4 g/L (22.9% Brix) or colostrum pool volume ≤ 40 L, with a minimum pool IgG concentration of 66.2 g/L (21.8% Brix) achieved the recommended total mass of at least 200g IgG in 4L of colostrum. CLINICAL RELEVANCE: When feeding pooled colostrum, IgG concentrations higher than the industry standard of 50 g/L is recommended to reduce the risk of failure of transfer of passive immunity in calves.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Differential Activation of Splenic cDC1 and cDC2 Cell Subsets following Poxvirus Infection of BALB/c and C57BL/6 Mice.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

Sex-differential non-specific effects of adjuvanted and non-adjuvanted rabies vaccines versus placebo on all-cause mortality in dogs (NERVE-Dog study): a study protocol for a randomized controlled trial with a nested case-control study.

BACKGROUND: It has been proposed that childhood vaccines in high-mortality populations may have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these non-specific effects (NSEs) are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. One area of controversy is the reported association of non-live vaccines with increased female mortality. In a previous randomized controlled trial (RCT), we observed that non-live alum-adjuvanted animal rabies vaccine (ARV) was associated with increased female but not male mortality in young, free-roaming dogs. Conversely, non-live non-adjuvanted human rabies vaccine (NRV) has been associated with beneficial non-specific effects in children. Alum adjuvant has been shown to suppress Th1 responses to pathogens, leading us to hypothesize that alum-adjuvanted rabies vaccine in young dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases. In this paper, we present the protocol of a 3-arm RCT comparing the effect of alum-adjuvanted rabies vaccine, non-adjuvanted rabies vaccine and placebo on all-cause mortality in an owned, free-roaming dog population, with causal mediation analysis of the RCT and a nested case-control study to test this hypothesis. METHODS: Randomised controlled trial with a nested case-control study. DISCUSSION: We expect that, among the placebo group, males will have higher mortality caused by higher pathogen loads and more severe disease, as determined by haematological parameters and inflammatory biomarkers. Among females, we expect that there will be no difference in mortality between the NRV and placebo groups, but that the ARV group will have higher mortality, again mediated by higher pathogen loads and more severe disease. We anticipate that these changes are preceded by shifts in key serum cytokine concentrations towards an anti-inflammatory immune response in females. If confirmed, these results will provide a rational basis for mitigation of detrimental NSEs of non-live vaccines in high-mortality populations.

Non-specific effects of veterinary vaccines: a systematic review.

The benefits of vaccines have been centred on their specific effects on subsequent infections by target pathogens. Recent studies, however, have opened up new insights into additional effects of vaccines known as non-specific effects (NSEs) or heterologous effects of vaccines. While several articles have reviewed epidemiological and immunological evidence for NSEs of vaccines in humans, similar works on veterinary vaccines are scarce. The objective of this paper was to review the findings of published studies on NSEs of vaccines developed or repurposed for use in animals. In total 8412 titles were retrieved from PubMed and CABI databases on the 30th of April 2021. After the final stage of screening, 45 eligible articles were included in the review. Data from these articles were summarised and presented here. In general, most of the vaccines studied in the reviewed articles have beneficial NSEs against multiple pathogens and disease conditions. There were, however, fewe studies reporting detrimental NSEs from both non-live and live vaccines which is in contrast to the currently existing evidence of beneficial NSEs of live vaccines and detrimental NSEs of non-live vaccines. This review may be used as a complement for future review of RCT studies of NSEs of vaccines in animals and provide a useful addition to the evolving understanding of the NSEs of vaccines.

Identification and Antimicrobial Resistance of Dermatophilus congolensis from Cattle in Saint Kitts and Nevis.

Dermatophilosis is a form of dermatitis caused by the bacterium Dermatophilus congolensis. The disease usually presents as localized purulent dermatitis, crusty hair masses or widespread matting of the hair. This condition is most common in domestic ruminants; but it can also affect other wild animals and humans. Antimicrobial therapy is used in many regions to treat clinical dermatophilosis with varying results. In this study, we aimed to assess the antimicrobial susceptibility of D. congolensis isolates. Fifty-two isolates were obtained from animals showing clinical signs of the disease at farms in St. Kitts. The isolates were then confirmed as D. congolensis by phenotypic tests, PCR and MALDI-TOF Mass Spectrometry. Furthermore, minimum inhibitory concentrations (MIC) of 16 antimicrobial agents were determined, using the broth microdilution method. Although most antimicrobials showed MICs in line with published values, the tetracycline results displayed a clear bimodal distribution over the tested range, with most isolates showing low MICs and 6 isolates much higher values (+/- 100-fold increase). These results indicate the presence of acquired tetracycline resistance in D. congolensis on the island of St. Kitts. Whether the current observation has implications for efficacy of treating the disease must be confirmed in further research.

Draft Genome Sequences of 40 Dermatophilus congolensis Isolates from Bovine Dermatophilosis Cases in St. Kitts and Nevis.

Dermatophilus congolensis causes dermatophilosis in cattle, mainly in tropical climates. Despite the economic losses caused by this bacterium, its pathogenic factors are less well understood. We report draft genomes of D. congolensis strains isolated during a dermatophilosis outbreak in cattle in St. Kitts and Nevis. Some isolates contain tet(Z), which is responsible for resistance to tetracyclines.

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors - cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes.

TLR3 provides immediate type I IFN response following entry of stimulatory PAMPs into the CNS, as it is in HSV infection. The receptor plays a vital role in astrocytes, contributing to rapid infection sensing and suppression of viral replication, precluding the spread of virus beyond neurons. The route of TLR3 mobilization culminating in the receptor activation remains unexplained. In this research, we investigated the involvement of various types of endosomes in the regulation of the TLR3 mobility in C8-D1A murine astrocyte cell line. TLR3 was transported rapidly to early EEA1-positive endosomes as well as LAMP1-lysosomes following stimulation with the poly(I:C). Later, TLR3 largely associated with late Rab7-positive endosomes. Twenty-four hours after stimulation, TLR3 co-localized with LAMP1 abundantly in lysosomes of astrocytes. TLR3 interacted with poly(I:C) intracellularly from 1 min to 8 h following cell stimulation. We detected TLR3 on the surface of astrocytes indicating constitutive expression, which increased after poly(I:C) stimulation. Our findings contribute to the understanding of cellular modulation of TLR3 trafficking. Detailed analysis of the TLR3 transportation pathway is an important component in disclosing the fate of the receptor in HSV-infected CNS and may help in the search for rationale therapeutics to control the replication of neuropathic viruses.

Potential use of a canine whole blood culture system to evaluate the immune response to Leptospira.

In susceptible hosts, protection from Leptospira infection is mediated by the innate immune response at the point of entry and humoral immunity. Thus, identifying and segregating the initial host response at the representative host-pathogen interface is needed to understand the typical outcomes of Leptospira infection, clearance, persistence, or disease. An in vitro whole blood culture system to study the overall immune response using pathogenic and non-pathogenic Leptospira strains was explored in this study. Using an ELISA, increased IL-8, TNF alpha, and IL-1 in blood samples stimulated with pathogenic and nonpathogenic Leptospira compared to unstimulated controls were detected. In RT2 Profiler PCR Array assays, consistent upregulation of 22 genes and downregulation of 25 genes were observed. Few of the notable upregulated genes included BPI, CCL3, CXCL2, IL-6, IL-8, TLR1, TLR2, TLR6, and TNF and downregulated genes included, LBP, LYZ, MPO, MYD88. IFNß was upregulated in samples treated with pathogenic Leptospira and IL-1ß was upregulated in samples treated with nonpathogenic Leptospira. Toll- like Receptor signaling and expression of pattern recognition receptors were two of the five prominent canonical pathways observed. Individual deconvolution of each of the specific and significant pathways observed in this study may improve the understanding of the pathogenesis of this important zoonotic agent. The use of this system in conjunction with whole transcriptome analysis in a larger population, may unveil the robust nature of host/Leptospira interaction.

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells.

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

Mitochondrial Heat Shock Response Induced by Ectromelia Virus is Accompanied by Reduced Apoptotic Potential in Murine L929 Fibroblasts.

Poxviruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxviruses on mtHsps has never been studied. We used highly infectious Moscow strain of ectromelia virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.

Ectromelia virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process.

BACKGROUND: Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. RESULTS: Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. CONCLUSIONS: Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells.

Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes.

Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-ß, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-ß by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer's disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.

Bacterial and viral pathogen-associated molecular patterns induce divergent early transcriptomic landscapes in a bovine macrophage cell line.

BACKGROUND: Pathogens stimulate immune functions of macrophages. Macrophages are a key sentinel cell regulating the response to pathogenic ligands and orchestrating the direction of the immune response. Our study aimed at investigating the early transcriptomic changes of bovine macrophages (Bomacs) in response to stimulation with CpG DNA or polyI:C, representing bacterial and viral ligands respectively, and performed transcriptomics by RNA sequencing (RNASeq). KEGG, GO and IPA analytical tools were used to reconstruct pathways, networks and to map out molecular and cellular functions of differentially expressed genes (DE) in stimulated cells. RESULTS: A one-way ANOVA analysis of RNASeq data revealed significant differences between the CpG DNA and polyI:C-stimulated Bomac. Of the 13,740 genes mapped to the bovine genome, 2245 had p-value ≤0.05, deemed as DE. At 6 h post stimulation of Bomac, poly(I:C) induced a very different transcriptomic profile from that induced by CpG DNA. Whereas, 347 genes were upregulated and 210 downregulated in response to CpG DNA, poly(I:C) upregulated 761 genes and downregulated 414 genes. The topmost DE genes in poly(I:C)-stimulated cells had thousand-fold changes with highly significant p-values, whereas in CpG DNA stimulated cells had 2-5-fold changes with less stringent p-values. The highest DE genes in both stimulations belonged to the TNF superfamily, TNFSF18 (CpG) and TNFSF10 (poly(I:C)) and in both cases the lowest downregulated gene was CYP1A1. CpG DNA highly induced canonical pathways that are unrelated to immune response in Bomac. CpG DNA influenced expression of genes involved in molecular and cellular functions in free radical scavenging. By contrast, poly(I:C) highly induced exclusively canonical pathways directly related to antiviral immune functions mediated by interferon signalling genes. The transcriptomic profile after poly(I:C)-stimulation was consistent with induction of TLR3 signalling. CONCLUSION: CpG DNA and poly(I:C) induce different early transcriptional landscapes in Bomac, but each is suited to a specific function of macrophages during interaction with pathogens. Poly(I:C) influenced antiviral response genes, whereas CpG DNA influenced genes important for phagocytic processes. Poly(I:C) was more potent in setting the inflammatory landscape desirable for an efficient immune response against virus infection.

Ectromelia virus induces tubulin cytoskeletal rearrangement in immune cells accompanied by a loss of the microtubule organizing center and increased α-tubulin acetylation.

Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line.

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission⁻fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Long actin-based cellular protrusions as novel evidence of the cytopathic effect induced in immune cells infected by the ectromelia virus.

The aim of the study was to evaluate the influence of ectromelia virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination.