Dexamethasone Modulation of the Interaction between Two Types of Phagocytes of the Holothurian <Emphasis Type="Italic">Eupentacta fraudatrix</Emphasis> (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida)

This study examines the interaction between two types of phagocytes (P1 and P2) of the holothurian Eupentacta fraudatrix and its in vitro modulation by dexamethasone. Our results indicate that inhibition of apoptosis in P1 phagocytes by P2 phagocytes was accompanied by increased activities of antioxidant enzymes and reduced synthesis of interleukin-1α-like substances. We hypothesize that P1-phagocyte-related effects occurred in response to a high level of hydrogen peroxide produced by P2 phagocytes. The reduced anti-apoptotic effect of P2-phagocyte supernatant during prolonged incubation (24 h) was accompanied by a decline in defense reactions in P1 phagocytes due to depletion of antioxidant enzymes (catalase, glutathione reductase, and glutathione transferase). Inhibition of apoptotis in P1 phagocytes associated with upregulation of antioxidant enzyme defense in response to P2 phagocytes preincubated with dexamethasone (100 µM) indicates that P2 phagocytes affect P1 phagocytes via a ROS-associated mechanism. Thus, our data provide evidence that P1 and P2 phagocytes exhibit their maximum activity at different stages of the immune response, thus causing inhibition of activity in target cells during prolonged exposure. Dexamethasone enhances these effects.     

Adhesins and Host Serum Factors Drive Yop Translocation by Yersinia into Professional Phagocytes during Animal Infection

Yersinia delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process requiring several adhesins and host complement. When WT Yptb or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by ΔailΔinvΔyadA but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that Yersinia adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, Yersinia uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.     

Evolutionary Conservation of Divergent Pro-Inflammatory and Homeostatic Responses in Lamprey Phagocytes

In higher vertebrates, phagocytosis plays a critical role in development and immunity, based on the internalization and removal of apoptotic cells and invading pathogens, respectively. Previous studies describe the effective uptake of these particles by lower vertebrate and invertebrate phagocytes, and identify important molecular players that contribute to this internalization. However, it remains unclear if individual phagocytes mediate internalization processes in these ancient organisms, and how this impacts the balance of pro-inflammatory and homeostatic events within their infection sites. Herein we show that individual phagocytes of the jawless vertebrate Petromyzon marinus (sea lamprey), like those of teleost fish and mice, display the capacity for divergent pro-inflammatory and homeostatic responses following internalization of zymosan and apoptotic cells, respectively. Professional phagocytes (macrophages, monocytes, neutrophils) were the primary contributors to the internalization of pro-inflammatory particles among goldfish (C. auratus) and lamprey (P. marinus) hematopoietic leukocytes. However, goldfish showed a greater ability for zymosan phagocytosis when compared to their jawless counterparts. Coupled to this increase was a significantly lower sensitivity of goldfish phagocytes to homeostatic signals derived from apoptotic cell internalization. Together, this translated into a significantly greater capacity for induction of antimicrobial respiratory burst responses compared to lamprey phagocytes, but also a decreased efficacy in apoptotic cell-driven leukocyte homeostatic mechanisms that attenuate this pro-inflammatory process. Overall, our results show the long-standing evolutionary contribution of intrinsic phagocyte mechanisms for the control of inflammation, and illustrate one effective evolutionary strategy for increased responsiveness against invading pathogens. In addition, they highlight the need for development of complementary regulatory mechanisms of inflammation to ensure continued maintenance of host integrity amidst increasing challenges from invading pathogens.     

Phagocytosis of Bacteria Adhering to a Biomaterial Surface in a Surface Thermodynamic Perspective

Bacterial biofilms can increase the pathogenicity of infection and constitute a major problem in modern health-care, especially on biomaterial implants and devices. Biofilms are difficult to eradicate by the host immune system, even with antibiotics, and have been the number one cause of biomaterial implant and device failure for decades. Therefore, it is important to understand how immune cells interact with adhering pathogens. This study firstly aims to develop a simple method to quantify phagocytosis of six different strains of staphylococci adhering on a surface with phase-contrast-microscopy. Phagocytosis of adhering staphylococci to a glass surface by phagocytes was quantified in a parallel plate flow chamber, and expressed as a phagocytosis rate, accounting for the number of adhering staphylococci initially present and for the duration of phagocytosis. Murine macrophages were more effective in clearing staphylococci from a surface than human phagocytes, which require differentiation from their monocyte or promyelocytic state during an experiment. Direct visualization of internalization of a GFP-modified S. aureus strain inside phagocytes confirmed the validity of the method proposed. As a second aim, the differences in phagocytosis rates observed were investigated on a surface thermodynamic basis using measured contact angles of liquids on macroscopic lawns of staphylococci and phagocytes, confirming that phagocytosis of adhering pathogens can be regarded as a surface phenomenon. In addition, surface thermodynamics revealed that phagocytosis of adhering pathogens is determined by an interplay of physical attraction between pathogens and phagocytes and the influence of chemo-attractants. For future studies, these results will help to place in vitro experiments and murine infection models in better perspective with respect to human ones.     

Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.     

Immunomodulatory molecules in the compound ascidian Botryllus schlosseri: evidence from conditioned media

The supernatant from cultures of haemocytes of the compound ascidian Botryllus schlosseri incubated with zymosan (conditioned medium; CM) can enhance yeast phagocytosis by Botryllus blood cells. It contains molecules recognised by antibodies raised against the mammalian pro-inflammatory cytokines IL-1-α and TNF-α which appear as a single band of 60 kDa in immunoblot analysis. The effects on phagocytosis are abolished by the presence of sugars, such as galactose and rhamnose, sharing the same hydroxyl group configuration at C2 and C4. The same sugars also inhibit the haemagglutinating activity of the CM, suggesting the presence of lectins with opsonic activity. With immunoblot analysis, we confirmed the presence, in CM, of B. schlosseri rhamnose-binding lectins (BsRBLs), recently identified and characterised by our team, as a single electrophoretic band of 37 kDa. We had already demonstrated that these molecules are synthesised and secreted by activated phagocytes. Since previous studies have demonstrated that cytotoxic morula cells, and not phagocytes, are the haemocytes responsible for the release of molecules recognised by anti-cytokine antibodies, we propose a new scenario in which morula cells act as sentinels, able to sense foreign molecules and release immunomodulatory factors which induce phagocytes to secrete lectins able to enhance phagocytosis by acting as bridges between foreign particles and phagocyte surfaces.     

Binding and degradation of soluble immunoglobulin aggregates by mouse mononuclear phagocytes—Stimulation by colony-stimulating factor

The binding and degradation of soluble guinea pig IgG₂ aggregates by murine mononuclear phagocytes were studied. Bone marrow mononuclear phagocytes cultured in the presence of an embryonic fibroblast-conditioned medium (CM) degraded the aggregates to a much greater degree than did resident peritoneal macrophages. Binding and degradation by resident peritoneal macrophages were enhanced by culture in the presence of CM.Freshly harvested thioglycollate-induced peritoneal macrophages bound and degraded the aggregates to the same degree as the cultured bone marrow mononuclear phagocytes did. However, the thioglycollate-induced macrophages lost most of these capacities when cultured in vitro without CM. When CM was added to these cultures, the capacity to bind and degrade was restored in a dose-dependent fashion. To obtain the maximum effect, exposure to CM must be maintained for more than 2 days. The effect of CM could be reproduced with purified CSF-1. Taken together the results of this study indicate that Fc receptor expression is modulated by CSF-1.     

Aggregation of Sea Urchin Phagocytes Is Augmented In Vitro by Lipopolysaccharide

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ∼10% of the cells were positive for Sp185/333 proteins. At 24 hr, ∼90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.     

Leishmania enriettii: Immune induction of macrophage activation in an experimental model of immunoprophylaxis in the mouse

This study describes some of the parameters of the cellular immune response elicited in mice by inoculation of the nonpathogenic protozoan parasite, Leishmania enriettii. Incubation in vitro of leishmania-infected mouse peritoneal macrophages with spleen cells from syngeneic leishmania-immune animals resulted in activation of the phagocytes, leading to intracellular parasite destruction. Activation required interaction of sensitized lymphocytes with parasite antigen released or displayed by infected macrophages. The effect was dependent both on the dose of parasites used for in vivo priming and on the number of spleen cells cocultivated with parasitized macrophages. The activating capacity of lymphocytes was abrogated by anti-Thy-1 antiserum treatment and was retained in the effluent cells after nylon-wool separation. Activation was followed by lysis of part of the macrophage monolayer. Destruction of the phagocytes did not appear to result from the activation process per se and may represent a cytotoxic activity of sensitized lymphocytes for macrophages bearing parasite antigen on their surface.     

Effects of human peripheral blood monocytes,monocyte-derived macrophages,and spleen mononuclear phagocytes on Toxoplasma gondii

In vitro effects of human peripheral blood monocytes, peripheral blood monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii were studied. In almost all instances, over 80% of human monocytes and monocyte-derived macrophages infected with Toxoplasma in vitro destroyed the organism. Degeneration of intracellular Toxoplasma was not due to decreased viability of organisms in the challenge inoculum. Human monocytes did not elaborate into the culture medium substances which altered the capacity of Toxoplasma to survive and replicate within mouse macrophages. The early reduction in intracellular Toxoplasma was not affected by inhibitors of various intracellular processes or by diseases associated with altered cellular immunity (sarcoidosis, infectious mononucleosis, or lymphoma.) The Toxoplasma that remained after 6 hr within human monocytes and macrophages multiplied. This multiplication was observed both microscopically and in a radioassay which detects uptake of [³H]uracil or [³H]deoxyuridine into nucleic acids of intracellular Toxoplasma. Intracellular Toxoplasma in monocytes cultured with poly(I:C) or in monocyte-derived macrophages cultured with lymphokines showed decreased uptake of radiolabeled precursors into nucleic acids of intracellular Toxoplasma. Treatment of monocytes with endotoxin did not alter nucleic acid synthesis of surviving intracellular Toxoplasma. These results suggest that human mononuclear phagocytes in peripheral blood and in tissue (spleen) have the capacity to eliminate a large percentage of the Toxoplasma that they ingest or that invade them. The inhibition of nucleic acid synthesis of remaining Toxoplasma by exposure of monocyte-derived macrophages to lymphokines suggests that lymphocyte products may be important for elimination of the Toxoplasma that remain and multiply within a small proportion of mononuclear phagocytes.     

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14⁺ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P₃ antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP⁺ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP⁺ cells represented more significant populations in tissues of Cd14^−/− than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14⁺ phagocytes.Subject terms: Phospholipids, Cell death and immune response     

Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.     

Lipase activity in the hemolymph of Biomphalaria glabrata (Mollusca) challenged with bacterial lipids

The levels of lipase activity in both the cellular and serum constituents of the hemolymph of Biomphalaria glabrata that had been challenged in vitro to beat-killed and sonicated Bacillus megaterium as well as samples challenged with live B. megaterium were ascertained. There were no significant alterations in the levels of enzyme activity in both cells and serum of the samples that had been challenged with sonicated bacteria; however, there was a signficant elevation in the enzyme activity associated with both the cells and serum of hemolymph that had been challenged with live bacteria. It has been concluded that live B. megaterium can stimulate hypersynthesis of lipase, a lysosomal enzyme, in phagocytes of B. glabrata and that this enzyme subsequently is released into serum. Consequently, the hydrolysis of lipid constituents of bacteria could theoretically occur in serum as well as within phagocytes.     

Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.     

A Naturally Occurring Single Nucleotide Polymorphism in the Salmonella SPI-2 Type III Effector srfH/sseI Controls Early Extraintestinal Dissemination

CD18 expressing phagocytes associated with the gastro-intestinal (GI) epithelium can shuttle Salmonella directly into the bloodstream within a few minutes following microbial ingestion. We have previously demonstrated that Salmonella controls the CD18 pathway to deeper tissue, manipulating the migratory properties of infected cells as an unappreciated component of its pathogenesis. We have observed that one type III effector, SrfH (also called SseI) that Salmonella secretes into infected phagocytes manipulates the host protein TRIP6 to stimulate their migration. Paradoxically, SrfH was shown in another study to subvert a different host protein, IQGAP1, in a manner that inhibits the productive motility of such cells, perhaps to avoid interactions with T cells. Here, we resolve the discrepancy. We report that one naturally occurring allele of srfH promotes the migration of infected phagocytes into the bloodstream, while another naturally occurring allele that differs by only a single nucleotide polymorphism (SNP) does not. This SNP determines if the protein contains an aspartic acid or a glycine residue at position 103 and may determine if SrfH binds TRIP6. SrfH Gly103 is a rare allele, but is present in the highly invasive strain Salmonella enterica serovar Typhimurium UK-1 (stands for universal killer). It is also present in the genome of the only sequenced strain belonging to the emerging pandemic Salmonella enterica serovar 4, [5],12,i:-, which is frequently associated with septicemia. Finally, we present evidence that suggests that Gifsy-2, the bacteriophage upon which srfH resides, is present in a clinical isolate of the human-specific pathogen, Salmonella enterica serovar Typhi. These observations may have interesting implications for our understanding of Salmonella pathogenesis.     

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.     

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non‐professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.     

The Cell Wall Fraction from Fonsecaea pedrosoi Stimulates Production of Different Profiles of Cytokines and Nitric Oxide by Murine Peritoneal Cells In Vitro

Chromoblastomycosis is a chronic, suppurative and granulomatous mycosis whose main etiologic agent is the fungus Fonsecaea pedrosoi. The severity of chromoblastomycosis clinical manifestations correlates with the Th1 or Th2 immune response, and an efficient cellular immune response depends on the interaction between immune cells and the cell wall of the fungi, which is able to promote this activation. The objective of this study was to analyze the influence of cell wall fractions of Fonsecaea pedrosoi on the activation of peritoneal phagocytes obtained from mice. Our results revealed that after 4 h of inoculation with fungal cell wall components, there was a cell migration predominantly comprised of neutrophils followed, after 72 h, by migration of the macrophages. After 4 h, the F2 fraction caused increased production of nitric oxide in phagocytes, but this effect was not observed in the phagocytes after 72 h. The F1 fraction stimulated production of IL-12 in cells that migrated after 72 h, while the inactivated fungus and the F2 fraction led to production of IL-10. The F2 fraction decreased the rate of phagocytosis and increased the production of IL-10. Our results suggest that the F2 fraction and its components caused an important disruption of microbicidal mechanisms negatively modulating the immune response and favoring the persistence of the fungus.     

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.