Genomic analysis of Salmonella enterica from Metropolitan Manila abattoirs and markets reveals insights into circulating virulence and antimicrobial resistance genotypes.

The integration of next-generation sequencing into the identification and characterization of resistant and virulent strains as well as the routine surveillance of foodborne pathogens such as Salmonella enterica have not yet been accomplished in the Philippines. This study investigated the antimicrobial profiles, virulence, and susceptibility of the 105 S. enterica isolates from swine and chicken samples obtained from slaughterhouses and public wet markets in Metropolitan Manila using whole-genome sequence analysis. Four predominant serovars were identified in genotypic serotyping, namely, Infantis (26.7%), Anatum (19.1%), Rissen (18.1%), and London (13.3%). Phenotypic antimicrobial resistance (AMR) profiling revealed that 65% of the isolates were resistant to at least one antibiotic, 37% were multidrug resistant (MDR), and 57% were extended-spectrum ß-lactamase producers. Bioinformatic analysis revealed that isolates had resistance genes and plasmids belonging to the Col and Inc plasmid families that confer resistance against tetracycline (64%), sulfonamide (56%), and streptomycin (56%). Further analyses revealed the presence of 155 virulence genes, 42 of which were serovar-specific. The virulence genes primarily code for host immune system modulators, iron acquisition enzyme complexes, host cell invasion proteins, as well as proteins that allow intracellular and intramacrophage survival. This study showed that virulent MDR S. enterica and several phenotypic and genotypic AMR patterns were present in the food chain. It serves as a foundation to understand the current AMR status in the Philippines food chain and to prompt the creation of preventative measures and efficient treatments against foodborne pathogens.

Conservation of S20 as an Ineffective and Disposable IFNγ-Inducing Determinant of Plasmodium Sporozoites Indicates Diversion of Cellular Immunity.

Despite many decades of research to develop a malaria vaccine, only one vaccine candidate has been explored in pivotal phase III clinical trials. This candidate subunit vaccine consists of a portion of a single Plasmodium antigen, circumsporozoite protein (CSP). This antigen was initially identified in the murine malaria model and shown to contain an immunodominant and protective CD8+ T cell epitope specific to the H-2K d (BALB/c)-restricted genetic background. A high-content screen for CD8+ epitopes in the H2K b /D b (C57BL/6)-restricted genetic background, identified two distinct dominant epitopes. In this study, we present a characterization of one corresponding antigen, the Plasmodium sporozoite-specific protein S20. Plasmodium berghei S20 knockout sporozoites and liver stages developed normally in vitro and in vivo. This potent infectivity of s20(-) sporozoites permitted comparative analysis of knockout and wild-type parasites in cell-based vaccination. Protective immunity of irradiation-arrested s20(-) sporozoites in single, double and triple immunizations was similar to irradiated unaltered sporozoites in homologous challenge experiments. These findings demonstrate the presence of an immunogenic Plasmodium pre-erythrocytic determinant, which is not essential for eliciting protection. Although S20 is not needed for colonization of the mammalian host and for initiation of a blood infection, it is conserved amongst Plasmodium species. Malarial parasites express conserved, immunogenic proteins that are not required to establish infection but might play potential roles in diverting cellular immune responses.

Mycobacterium tuberculosis whole genome sequencing provides insights into the Manila strain and drug-resistance mutations in the Philippines.

The Philippines has a high incidence of tuberculosis disease (TB), with an increasing prevalence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains making its control difficult. Although the M. tuberculosis "Manila" ancient lineage 1 strain-type is thought to be prevalent in the country, with evidence of export to others, little is known about the genetic diversity of circulating strains. By whole genome sequencing (WGS) 178 isolates from the Philippines National Drug Resistance Survey, we found the majority (143/178; 80.3%) belonged to the lineage 1 Manila clade, with the minority belonging to lineages 4 (European-American; n = 33) and 2 (East Asian; n = 2). A high proportion were found to be multidrug-resistant (34/178; 19.1%), established through highly concordant laboratory drug susceptibility testing and in silico prediction methods. Some MDR-TB isolates had near identical genomic variation, providing potential evidence of transmission. By placing the Philippine isolates within a phylogeny of global M. tuberculosis (n > 17,000), we established that they are genetically similar to those observed outside the country, including a clade of Manila-like strain-types in Thailand. An analysis of the phylogeny revealed a set of ~200 SNPs that are specific for the Manila strain-type, and a subset can be used within a molecular barcode. Sixty-eight mutations known to be associated with 10 anti-TB drug resistance were identified in the Philippine strains, and all have been observed in other populations. Whilst nine putative streptomycin resistance conferring markers in gid (8) and rrs (1) genes appear to be novel and with functional consequences. Overall, this study provides an important baseline characterisation of M. tuberculosis genetic diversity for the Philippines, and will fill a gap in global datasets and aid the development of a nation-wide database for epidemiological studies and clinical decision making. Further, by establishing a molecular barcode for detecting Manila strains it will assist with the design of diagnostic tools for disease control activities.

Mosquito and human hepatocyte infections with Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

BACKGROUND: Human ovale malaria is caused by the two closely related species, Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. METHODS: Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. RESULTS: In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands and used to successfully infect cultured human hepatocytes. CONCLUSIONS: This protocol provides a method for the utilisation of pretreatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of loop-activated amplification as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

The ecology of immune state in a wild mammal, Mus musculus domesticus.

The immune state of wild animals is largely unknown. Knowing this and what affects it is important in understanding how infection and disease affects wild animals. The immune state of wild animals is also important in understanding the biology of their pathogens, which is directly relevant to explaining pathogen spillover among species, including to humans. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Making an immune response is costly, and many factors (such as age, sex, infection status, and body condition) have individually been shown to constrain or promote immune responses. But, whether or not these factors affect immune responses and immune state in wild animals, their relative importance, and how they interact (or do not) are unknown. Here, we have investigated the immune ecology of wild house mice-the same species as the laboratory mouse-as an example of a wild mammal, characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we show how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identify the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals' immune state, while factors such as microparasite infection and season are comparatively unimportant. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal.

An in vitro assay to measure antibody-mediated inhibition of P. berghei sporozoite invasion against P. falciparum antigens.

A large research effort is currently underway to find an effective and affordable malaria vaccine. Tools that enable the rapid evaluation of protective immune responses are essential to vaccine development as they can provide selection criteria to rank order vaccine candidates. In this study we have revisited the Inhibition of Sporozoite Invasion (ISI) assay to assess the ability of antibodies to inhibit sporozoite infection of hepatocytes. By using GFP expressing sporozoites of the rodent parasite P. berghei we are able to robustly quantify parasite infection of hepatocyte cell lines by flow cytometry. In conjunction with recently produced transgenic P. berghei parasites that express P. falciparum sporozoite antigens, we have been able to use this assay to measure antibody mediated inhibition of sporozoite invasion against one of the lead malaria antigens P. falciparum CSP. By combining chimeric rodent parasites expressing P. falciparum antigens and a flow cytometric readout of infection, we are able to robustly assess vaccine-induced antibodies, from mice, rhesus macaques and human clinical trials, for their functional ability to block sporozoite invasion of hepatocytes.

The comparative immunology of wild and laboratory mice, Mus musculus domesticus.

The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.

Host Resistance to Plasmodium-Induced Acute Immune Pathology Is Regulated by Interleukin-10 Receptor Signaling.

The resolution of malaria infection is dependent on a balance between proinflammatory and regulatory immune responses. While early effector T cell responses are required for limiting parasitemia, these responses need to be switched off by regulatory mechanisms in a timely manner to avoid immune-mediated tissue damage. Interleukin-10 receptor (IL-10R) signaling is considered to be a vital component of regulatory responses, although its role in host resistance to severe immune pathology during acute malaria infections is not fully understood. In this study, we have determined the contribution of IL-10R signaling to the regulation of immune responses during Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM). We show that antibody-mediated blockade of the IL-10R during P. berghei ANKA infection in ECM-resistant BALB/c mice leads to amplified T cell activation, higher serum gamma interferon (IFN-γ) concentrations, enhanced intravascular accumulation of both parasitized red blood cells and CD8+ T cells to the brain, and an increased incidence of ECM. Importantly, the pathogenic effects of IL-10R blockade during P. berghei ANKA infection were reversible by depletion of T cells and neutralization of IFN-γ. Our findings underscore the importance of IL-10R signaling in preventing T-cell- and cytokine-mediated pathology during potentially lethal malaria infections.

Long-lived memory B-cell responses following BCG vaccination.

The role of T-cells in immunity against Mycobacterium tuberculosis (M. tuberculosis) infection has been extensively studied, however, that of B-cells still remains comparatively unexplored. In this study, we determined the presence and frequencies of mycobacteria-specific memory B-cells (MBCs) in peripheral blood from clinically healthy, Bacillus Calmette Guerin (BCG) vaccinated (n = 79) and unvaccinated (n = 14) donors. Purified protein derivative (PPD)-specific MBCs were present in most donors (both vaccinated and unvaccinated) but their frequencies were significantly higher in vaccinated than in unvaccinated donors. MBCs specific for other mycobacterial antigens [antigen-85A (Ag85A), antigen-85B (Ag85B), 6 kDalton early secretory antigenic target (ESAT-6) and the 10 kDalton-culture filtrate protein (CFP-10)] were less prevalent than those recognising PPD. Furthermore, PPD-specific MBCs were detected in BCG vaccinated donors without ESAT-6 and CFP-10 specific responses. Together, these results indicate that BCG vaccination induces long-lived MBC responses. Similar patterns of response were seen when we examined mycobacteria-specific antibody and T-cell responses in these donors. Our data show for the first time that BCG vaccination elicits long-lived mycobacteria-specific MBC responses in healthy individuals, suggesting a more substantial role of B-cells in the response to BCG and other mycobacterial infections than previously thought.

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain.

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.

Antigen-specific IL-2 secretion correlates with NK cell responses after immunization of Tanzanian children with the RTS,S/AS01 malaria vaccine.

RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-γ, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-γ(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.

The breadth, but not the magnitude, of circulating memory B cell responses to P. falciparum increases with age/exposure in an area of low transmission.

BACKGROUND: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired. METHODS: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells (MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot. FINDINGS AND CONCLUSIONS: The proportion of individuals with malaria-specific MBC and antibodies, and the average number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence of malaria exposure (defined as a positive response to at least one malarial antigen either by ELISA or ELISPOT), the median number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating memory cell pool for malaria antigens is of similar size to that for other antigens.

Heterogeneous and tissue-specific regulation of effector T cell responses by IFN-gamma during Plasmodium berghei ANKA infection.

IFN-γ and T cells are both required for the development of experimental cerebral malaria during Plasmodium berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4(+) and CD8(+) T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN-γ(-/-) mice during P. berghei ANKA infection. The expansion of splenic CD4(+) and CD8(+) T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ, but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN-γ(-/-) mice; however, the migration to, and accumulation of, effector CD4(+) and CD8(+) T cells in the lung, liver, and brain was altered in IFN-γ(-/-) mice. Interestingly, activation and accumulation of T cells in various nonlymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN-γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended on active IFN-γ-dependent environmental signals--leading to T cell apoptosis--rather than upon intrinsic alterations in T cell programming. To our knowledge, this is the first study to fully investigate the role of IFN-γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.

Short-lived IFN-γ effector responses, but long-lived IL-10 memory responses, to malaria in an area of low malaria endemicity.

Immunity to malaria is widely believed to wane in the absence of reinfection, but direct evidence for the presence or absence of durable immunological memory to malaria is limited. Here, we analysed malaria-specific CD4+ T cell responses of individuals living in an area of low malaria transmission in northern Thailand, who had had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. CD4+ T cell effector memory (CD45RO+) IFN-γ (24 hours ex vivo restimulation) and cultured IL-10 (6 day secretion into culture supernatant) responses to malaria schizont antigens were detected only in malaria-exposed subjects and were more prominent in subjects with long-lived antibodies or memory B cells specific to malaria antigens. The number of IFN-γ-producing effector memory T cells declined significantly over the 12 months of the study, and with time since last documented malaria infection, with an estimated half life of the response of 3.3 (95% CI 1.9-10.3) years. In sharp contrast, IL-10 responses were sustained for many years after last known malaria infection with no significant decline over at least 6 years. The observations have clear implications for understanding the immunoepidemiology of naturally acquired malaria infections and for malaria vaccine development.

Measures of immune function of wild mice, Mus musculus.

The immune function of wild animals has been rather little studied. Wild animals' immune function may differ from that of laboratory bred animals because of their different environments. This idea follows from the concept of resource partitioning in which animals distribute scarce resources to all aspects of life, including to costly immune responses. A logical extension of this idea is that there may be substantial interindividual variation in the immune function of wild animals. To begin to investigate this, we compared the immune function of a laboratory bred mouse strain (C57BL/6, a widely used mouse strain that makes potent immune responses) and wild caught Mus musculus. We found that by most measures of immune function, the wild caught mice had greater immune function. Specifically, wild mice had greater concentrations and more avid antigen-specific IgG responses, as well as higher concentrations of total IgG and IgE, compared with those laboratory bred mice. Moreover, flow cytometric analysis showed a comparatively greater overall level of activation of the cells of the immune system in wild mice. Lastly, we observed that immune function was substantially more variable among wild caught mice than among the laboratory bred mice. The next research challenge is to understand which aspects of an individual animal's life determine its immune function.

Essential role for IL-27 receptor signaling in prevention of Th1-mediated immunopathology during malaria infection.

Successful resolution of malaria infection requires induction of proinflammatory immune responses that facilitate parasite clearance; however, failure to regulate this inflammation leads to immune-mediated pathology. The pathways that maintain this immunological balance during malaria infection remain poorly defined. In this study, we demonstrate that IL-27R-deficient (WSX-1(-/-)) mice are highly susceptible to Plasmodium berghei NK65 infection, developing exacerbated Th1-mediated immune responses, which, despite highly efficient parasite clearance, lead directly to severe liver pathology. Depletion of CD4(+) T cells---but not CD8(+) T cells---prevented liver pathology in infected WSX-1(-/-) mice. Although WSX-1 signaling was required for optimal IL-10 production by CD4(+) T cells, administration of rIL-10 failed to ameliorate liver damage in WSX-1(-/-) mice, indicating that additional, IL-10-independent, protective pathways are modulated by IL-27R signaling during malaria infection. These data are the first to demonstrate the essential role of IL-27R signaling in regulating effector T cell function during malaria infection and reveal a novel pathway that might be amenable to manipulation by drugs or vaccines.

Natural immunization against malaria: causal prophylaxis with antibiotics.

Malaria remains the most prevalent vector-borne infectious disease and has the highest rates of fatality. Current antimalarial drug strategies cure malaria or prevent infections but lack a sustained public health impact because they fail to expedite the acquisition of protective immunity. We show that antibiotic administration during transmission of the parasite Plasmodium berghei results in swift acquisition of long-lived, life cycle-specific protection against reinfection with live sporozoites in mice. Antibiotic treatment specifically inhibits the biogenesis and inheritance of the apicoplast in Plasmodium liver stages, resulting in continued liver-stage maturation but subsequent failure to establish blood-stage infection. Exponential expansion of these attenuated liver-stage merozoites from a single sporozoite induces potent immune protection against malaria. If confirmed in residents of malaria-endemic areas, periodic prophylaxis with safe and affordable antibiotics may offer a powerful shortcut toward a needle-free surrogate malaria immunization strategy.

Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.

There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF(-/-), IFN-gamma(-/-), IL-12(-/-) and RAG-1(-/-) malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses.

Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease.

Cerebral malaria is a life-threatening complication of malaria infection. The pathogenesis of cerebral malaria is poorly defined and progress in understanding the condition is severely hampered by the inability to study in detail, ante-mortem, the parasitological and immunological events within the brain that lead to the onset of clinical symptoms. Experimental murine models have been used to investigate the sequence of events that lead to cerebral malaria, but there is significant debate on the merits of these models and whether their study is relevant to human disease. Here we review the current understanding of the parasitological and immunological events leading to human and experimental cerebral malaria, and explain why we believe that studies with experimental models of CM are crucial to define the pathogenesis of the condition.