Acute malnutrition (wasting) remains a global public health problem. Ready-to-use therapeutic foods (RUTF) and supplemental foods (RUSF) for treatment of severe acute malnutrition (SAM) and moderate acute malnutrition (MAM), respectively, come in the form of macro- and micronutrient-dense pastes. These lipid-dense treatments provide the energy and nutrients needed to support growth but have significant rates of relapse. Their role in reversing pathophysiological contributors to malnutrition, such as intestinal barrier dysfunction, intestinal dysbiosis, and systemic inflammation have not been evaluated. Traditional lipid-dense RUTFs and RUSFs are rich in pro-inflammatory omega-6 polyunsaturated fatty acids (PUFAs) but low in anti-inflammatory omega-3 PUFAs, which could fuel malnutrition-related pathological inflammation. We reasoned that reduced dietary omega-6 (n-6) PUFAs and increased dietary long-chain omega-3 (n-3) PUFAs found in fish oil would reduce malnutrition-related pathological inflammation. In an established mouse model of MAM, we observed that altering the dietary n-3/n-6 PUFA ratio through replacing dietary corn oil with fish oil improved the diversity and composition of the caecal microbiota, improved intestinal mucosal barrier and immune defence, reduced translocation of bacteria and bacterial lipopolysaccharides (LPS), and dampened systemic inflammation. Furthermore, dietary fish oil protected against weight loss upon systemic challenge with bacterial LPS. The anti-inflammatory effects of dietary fish oil did not compromise host defence against challenge with the intestinal pathogen, Citrobacter rodentium. Collectively, these results suggest that dietary fish oil blunts inflammation that contributes to the pathogenesis of MAM. Inclusion of fish oil in dietary interventions may be beneficial in prevention or reduction of malnutrition-associated inflammation.

The classification of undernutrition-related diabetes as a distinct entity separate from type 1 and type 2 diabetes remains under debate. Here, we show that postnatal undernutrition induces coordinated transcriptional, metabolic, and pathophysiological alterations that mediate the development of undernutrition-related diabetes in young animals. Undernourished mice exhibited hypoglycemia, reduced insulin (P < 0.01), and impaired glucose tolerance (P < 0.05), consistent with pancreatic insufficiency. Micro-computer tomography analysis revealed skeletal fragility, with significant reductions in bone mineralization (P < 0.01), trabecular integrity (P < 0.05), and increased porosity (P < 0.001), as commonly reported in type 1 diabetes. Liver tissue histology revealed substantial triglyceride accumulation (P < 0.001) and histopathological features consistent with hepatic steatosis, including hepatocyte ballooning (P < 0.001). RNA sequencing of liver tissue revealed a gene expression pattern hallmarked by upregulated fatty acid metabolism (P = 0.031) and downregulation of the PI3K/AKT/mTOR signaling pathway (P = 0.05). This metabolic shift was associated with Pparg activation (Z = 2.3) and increased fatty acid oxidation (P < 0.01). Conversely, genes involved in insulin secretion (Z = -3.6) and glucose metabolism (Z < -1.6) were downregulated. Strikingly, transitioning from early undernutrition to a standard diet for 28 days resulted in mild hyperglycemia (P < 0.05), accompanied by persistent hypoinsulinemia due to impaired pancreatic catch-up growth. These findings suggest that early undernutrition fosters a distinct trajectory toward insulin deficiency and undernutrition-related diabetes, while also increasing susceptibility to metabolic comorbidities. These mechanisms could serve as foundational drivers of the double burden of malnutrition, with consequences for both metabolic and skeletal health. NEW & NOTEWORTHY Early-life undernutrition plays a critical role in the development of undernutrition-related diabetes. Undernourished mice exhibited a metabolic shift from the glucose metabolism toward the fatty acid metabolism, leading to bone fragility and hepatic steatosis. Paradoxically, undernutrition-related diabetes emerged after transition to a regular diet due to a persistent compromised insulin production and impaired pancreatic catch-up growth. Early-life undernutrition is a pivotal contributor to metabolic disorders and comorbidities in populations facing the double burden of malnutrition.

BACKGROUND:Acute undifferentiated febrile illness (AUFI) is a challenging clinical condition in tropical regions, caused by a broad range of pathogens. In Villeta municipality, Colombia, data on neglected bacterial causes remain scarce, highlighting the need to expand understanding of the local etiological spectrum. Thus, the aim of the present study was to explore the presence of the neglected pathogens, Bartonella, Borrelia, and Coxiella burnetii, as potential causes of AUFI in Villeta. METHODS:DNA was extracted from whole-blood samples from febrile patients. Quality and purity were assessed spectrophotometrically and by conventional polymerase chain reaction (PCR). Bartonella, Borrelia, and C. burnetii were detected using genus- and species-specific quantitative PCR (qPCR) assays. Bartonella-positive samples were further analyzed by multigene PCRs and sequencing for species identification. Anti-Bartonella and anti-C. burnetii immunoglobulin G (IgG) antibodies were evaluated by indirect immunofluorescence to assess recent or past exposure to these agents. RESULTS:A total of 41 febrile patients were evaluated. Bartonella DNA was detected in 9.8% (4/41) of samples. No Borrelia or C burnetii DNA was detected. Phylogenetic analysis revealed two distinct clades, although none could be assigned to species level. Serological analysis showed anti-Bartonella IgG antibodies in 29.3% (12/41) of cases, with 9.8% (4/41) exhibiting seroconversion. One patient presented both molecular and seroconversion evidence of recent Bartonella infection. None of the patients were seropositive for C. burnetii. CONCLUSIONS:This study provides the first molecular and serological evidence of Bartonella circulation among febrile patients in Villeta, Colombia, revealing genetically distinct lineages and indicating both active and past infections, underscoring its potential role in AUFI.

Spotted fever group (SFG) rickettsioses are emerging zoonotic diseases of increasing relevance in Latin America, yet the specific species involved in human infections remain poorly defined in many endemic regions. This study aimed to determine the most probable antigen among SFG-seroreactive febrile patients from Villeta, Colombia. A panel of 25 convalescent-phase serum samples previously identified as positive for SFG Rickettsia spp. antibodies was analyzed by indirect immunofluorescence assay using antigens of Rickettsia rickettsii, R. amblyommatis and R. parkeri. Antibody titers were compared to identify differential seroreactivity patterns. Overall, 44% (11/25) of the samples showed differential antibody titers against one of the tested antigens. Among these, nine (36%) exhibited higher titers to R. parkeri and two (8%) to R. amblyommatis, while none showed exclusive reactivity to R. rickettsii. The remaining 56% (14/25) presented similar titers across antigens, consistent with indeterminate or cross-reactive SFG responses. Antibody titers ranged from 1:128 to 1:4096, with R. parkeri showing the strongest reactivity. These findings suggest R. parkeri or a highly related Rickettsia species as the predominant probable antigen in Villeta, highlighting its potential role in mild rickettsial infections and emphasizing the need for eco-epidemiological studies to identify local vectors and reservoirs.

BACKGROUND:Acute undifferentiated febrile illness (AUFI) represents a major health challenge in tropical regions due to its wide range of etiologies. In Villeta, Colombia, previous studies investigated common causes such as malaria, arboviral diseases, leptospirosis and rickettsiosis, as well as several neglected bacterial agents. However, some patients remained without an identified etiology, underscoring the need for broader approaches to uncover other potential causes. Therefore, the aim of the present study was to investigate into other potential bacterial causes of AUFI through advanced molecular strategies utilizing 16S rRNA sequencing. METHODS:The study analyzed AUFI patient samples previously screened for fourteen pathogens. The V3-V9 hypervariable region of the 16S rRNA gene was amplified from whole-blood DNA of unresolved cases and sequenced using the Oxford Nanopore GridION platform. Reads were filtered, quality-checked, and taxonomically classified using the SILVA database. RESULTS:Eight samples from individuals without evidence of infection or recent exposure to previously screened pathogens were selected for 16S rRNA sequencing. DNA quality and integrity were confirmed, and enrichment produced high-quality amplicons for all samples. Sequencing generated high-quality reads overwhelmingly dominated by Francisella, representing over 93% of classified reads, followed by Coxiella and Arcobacter. CONCLUSIONS:This study provides the first molecular evidence of Francisella in whole-blood from febrile patients in Colombia. Findings highlight its potential role in AUFI, demonstrate the value of 16S rRNA barcoding, and underscore the need for expanded surveillance of highly neglected bacterial taxa.

Visceral leishmaniasis (VL) is transmitted by Leishmania-infected sand fly bites and malnutrition is a known risk factor in human VL. Models using sand fly transmission or malnutrition promote parasite dissemination. By investigating features of L. donovani-Lutzomyia longipalpis transmission to malnourished mice, we show that a comparable IL1-β-driven acute inflammation is maintained in malnourished (MN-SF) and well-nourished (WN-SF) sand fly-infected mice. However, parasite dissemination was more pronounced in MN-SF that had a significantly higher acute (P ≤ 0.001) and chronic (P ≤ 0.0001) splenic parasite burden compared to WN-SF. Compared to WN-SF, MN-SF exhibited chronic clinical symptoms (P ≤ 0.0001), neutrophilia (P ≤ 0.001), lymphocytopenia (P ≤ 0.0001), increased heme oxygenase-1 (P ≤ 0.001) and IL17-A (P ≤ 0.0001) levels, dysregulation of liver enzymes, lymph node barrier dysfunction, and augmented dysbiosis, all associated with enhanced VL severity. Combining vector-transmission and malnutrition provides an improved model to study VL pathogenesis and host defense.

BACKGROUND:Scrub typhus, caused by Orientia spp., is a mite-borne disease historically restricted to the Asia-Pacific region. Autochthonous cases have been confirmed in southern Chile, while serological evidence has been reported in Peru, Honduras and Colombia, suggesting wider distribution in Latin America. Identifying recent exposure among febrile patients is essential to evaluate its role in acute undifferentiated febrile illness (AUFI) etiology. AIM:To assess the possible implication of Orientia infection as a cause of AUFI in Villeta, Colombia. METHODS:Between September and December 2021, patients with AUFI were recruited. Whole-blood samples were tested for Orientia DNA by real-time PCR targeting the 16S rRNA gene. Acute and convalescent serum samples were analyzed for IgG antibodies by IFA and ELISA, with paired samples titration to assess seroconversion. Reactive sera were further confirmed by Western blot. RESULTS:Forty-one acute whole-blood samples were tested by qPCR; no Orientia DNA was detected. Serology identified antibodies in 27 % (11/41) of patients: six positive by ELISA, two by IFA, and three by both methods. Based on paired sera, eight cases (20 %) were classified as recent infections, including cases of seroconversion, and three (7 %) as previous exposures. Western blot confirmed specific reactivity against Orientia antigens in all positive samples. CONCLUSIONS:This study provides serological evidence of Orientia circulation among febrile patients in Villeta, Colombia, including clear cases of seroconversion. Western blot validated reactivity against immunodominant proteins. Findings suggest a local scrub typhus-like illness and highlight the need for pathogen isolation and genetic characterization to clarify its contribution to AUFI.

Acute undifferentiated febrile illness (AUFI) is associated with several etiological agents, including vector-borne pathogens. In tropical areas endemic for multiple pathogens, it is difficult to efficiently determine the etiology of AUFI. The goal for this study was to diagnose several arboviruses and other pathogens in patients with AUFI in Yucatan, Mexico, during the final phase of the coronavirus disease 2019 pandemic. A total of 215 patients with AUFI were included, with 170 from the urban area of Merida City and 45 from the rural area of the Molas community. Between December 2022 and December 2023, subjects were enrolled at health facilities, one in the rural area and two in the urban area. Overall, 36.7% (79/215) of AUFI patients were confirmed to have dengue virus (DENV). A significant difference (P <0.05) was found in the prevalence of dengue in the urban area (84.8%) compared with that in the rural area (15.2%). When diagnosed patients were grouped by age, most cases occurred in young adults (17-29 years old). Acute infections caused by Leptospira interrogans (L. interrogans) serovars Bratislava and Australis, Rickettsia spp., and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were also identified in patients with AUFI. In Merida City, five cases of multiple infections were found with dengue, SARS-CoV-2, and L. interrogans serovars Bratislava and Australis. In conclusion, DENV was confirmed as the main causative agent of AUFI in the present study; however, the simultaneous circulation of other relevant endemic causal agents that cause febrile infections in the area is a key point to consider when making differential diagnoses.

Leptospira is a bacterial genus that includes several pathogenic species related to leptospirosis. In Colombia, leptospirosis is a mandatorily reported disease, widely distributed across the country. In the Villeta municipality, leptospirosis has been identified as an important cause of febrile illness; however, to date, no studies have been performed to identify the circulating species. A genus-specific qualitative qPCR was performed on DNA extracted from febrile patients’ acute-phase whole-blood samples targeting a fragment of the rrs gene. Positive qPCR samples were further amplified for the adk, icdA, LipL32, LipL41, rrs, and secY genes through conventional PCR for sequencing. All high-quality obtained sequences were further assessed through concatenated phylogenetic analysis. A total of 25% (14/56) of febrile patients’ acute blood samples were positive for Leptospira spp. High-quality sequences were obtained for only five genes, and analysis through concatenated phylogeny identified that all sequences clustered within the P1/pathogenic clade; some of them formed a robustly supported clade with Leptospira santarosai, and others were closely related with other Leptospira species but exhibited considerable genetic divergence. We describe the presence of pathogenic Leptospira species among febrile patients from the Villeta municipality and identify L. santarosai and other Leptospira species as causative agents of leptospirosis in the region.

Introduction Acute undifferentiated febrile illnesses (AUFIs) impose a large burden in the tropics. Understanding of AUFI’s epidemiology is limited. Insufficient diagnostic capacity hinders the detection of outbreaks. The lack of interconnection in healthcare systems hinders timely response. We describe a protocol to study the epidemiology and aetiologies of AUFI and pathogen discovery in strategic areas of Latin America (LA).Methods and analysis Global Infectious Diseases Network investigators comprising institutions in Colombia, Dominican Republic, México, Perú and the USA, developed a common cohort study protocol. The primary objective is to determine the aetiologies of AUFI at healthcare facilities in high-risk areas. Data collection and laboratory testing for viral, bacterial and parasitic agents are performed in rural and urban healthcare facilities and partner laboratories. Centralised laboratory and data management cores deploy diagnostic tests and data management tools. Subjects >6 years with fever for <8 days without localised infection are included in the cohort. They are evaluated during the acute and convalescent phases of illness. Study personnel collect clinical and epidemiological information. Blood, urine, nasal or pharyngeal swabs and saliva are collected in the acute phase and blood in convalescent phase. Specimens are banked at −80°C. Malaria, dengue and COVID-19 are tested onsite in the acute phase. The acute-phase serum is PCR tested for dengue, chikungunya, Venezuelan equine encephalitis, Mayaro, Oropouche, Zika, and yellow fever viruses. Paired convalescent and acute serum antibody titters are tested for arbovirus, Leptospira spp, and Rickettsia spp. Serum is used for viral cultures and next-generation sequencing for pathogen discovery. Analysis includes variable distributions, risk factors and regression models. Laboratory results are shared with health authorities and network members.Ethics and dissemination The protocol was approved by local ethics committees and health authorities. The results will be published in peer-reviewed journals. All study results are shared with local and regional health authorities.

Acute undifferentiated febrile illness (AUFI) is the main cause of medical attention in the tropics worldwide. Malaria, arboviral diseases, and leptospirosis are the most important etiologies. These are highly endemic in the Urabá antioqueño, Colombia, being the main causes of fever in several municipalities in this region. However, up-to-date data regarding the infecting species and serotypes are lacking. Thus, we characterized the etiology of AUFI, focusing on malaria, arboviruses, and leptospirosis in this region and the circulating infecting species. An active surveillance was conducted between January and April 2022, and July and October 2023 in two local hospitals in the Urabá antioqueño. Febrile patients were enrolled voluntarily. Malaria, arboviral diseases, and leptospirosis were screened through direct, serological, molecular, and rapid diagnostic methods. Amplicons obtained for dengue virus (DENV) and Leptospira spp. were analyzed through phylogenetic analysis. A total of 184 febrile patients were enrolled. A confirmed etiology was detected in 43.4% of patients from Apartadó and 61.2% from Turbo. Malaria was the most frequent cause in both municipalities, which was caused mainly by Plasmodium falciparum in Apartadó and Plasmodium vivax in Turbo. Dengue virus serotype 1 genotype V, DENV genotype Asian-American, and DENV genotype Cosmopolitan were identified, as well as pathogenic Leptospira species closely related to Leptospira santarosai and Leptospira noguchii. The present study confirms the importance of malaria, dengue fever, and leptospirosis in the Urabá antioqueño. Plasmodium falciparum and P. vivax were identified, as well as two DENV serotypes and three DENV genotypes and two different Leptospira species.

Background:Co-inhibitory receptors (immune checkpoints) regulate activated immune cells. Their expression on T cells can limit host defense. We hypothesized that chronic Leishmania donovani infection in patients with visceral leishmaniasis (VL) leads to expression of co-inhibitory receptors that could be markers of treatment response and clinical outcome. Method:A prospective cohort of 21 subjects with VL (7 with HIV coinfection) and 10 controls was established to measure T-cell expression of co-inhibitory receptors (PD-1, Tim-3, LAG-3, CTLA-4, and TIGIT) by flow cytometry in discarded remnants of diagnostic splenic or bone marrow aspirates and peripheral blood collected before and after treatment. Plasma levels of soluble co-inhibitory proteins (sPD-1, sTim-3, sLAG-3, and sCTLA-4) and selected cytokines were determined by immunoassay. Results:Expression of co-inhibitory receptors in peripheral blood T cells generally reflected findings in spleen and bone marrow aspirates. PD-1 and Tim-3 were upregulated in CD4+ T cells in HIV-negative and HIV-positive subjects with VL compared to controls. CD8+ T cells from HIV-negative subjects with VL displayed a similar pattern. Plasma levels of sPD-1 and sTim-3 were also greater in VL patients than controls. CD8+ and CD4+ T cells coexpressing PD-1 and Tim-3 showed considerable decline with treatment. Mortality in HIV-negative VL patients was associated with increased CD8+ T cells coexpressing Tim-3 and PD-1, triple-positive CD4+ and CD8+ T cells (PD-1+Tim-3+LAG-3+), and elevated sLAG3. Conclusions:Tim-3 and PD-1 expression on CD4+ and CD8+ T cells, and increased plasma sLAG-3, were markers of treatment response and clinical outcome in patients with VL.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Background:Acute undifferentiated febrile illness (AUFI) is one of the leading causes of illness in tropical regions. Although malaria is the most important cause, other pathogens such as Dengue (DENV), Leptospira and recently, Coronavirus Disease 2019 (COVID-19) have gained importance. In Colombia, few studies aimed to identify the etiology of AUFI. Most of them performed in Apartadó and Villeta municipalities, identifying the active circulation of several pathogens. Thus, we conducted a cross-sectional study in these municipalities to characterize the etiologies of AUFI during COVID-19 pandemic. Methods:An active surveillance was conducted between September and December 2021 in local hospitals of Apartadó and Villeta municipalities. Febrile patients were enrolled after voluntarily agreeing to participate in the study. Ten different etiologies were evaluated through direct, serological, molecular and rapid diagnostic methods. Results:In Apartadó a confirmed etiology was found in 60% of subjects, DENV (25%) being the most frequent, followed by leptospirosis (16.7%), malaria (10%), COVID-19 (8.3%), spotted fever group (SFG) rickettsiosis (6.7%) and Chikungunya (1.7%). In Villeta, a specific etiology was confirmed in 55.4% of patients, of which SFG rickettsiosis (39.3%) was the most frequent, followed by leptospirosis (21.4%), DENV (3.6%) and malaria (1.8%). No cases due to Mayaro, Yellow Fever, Oropouche and Venezuelan Equine Encephalitis viruses were detected. Conclusion:We confirm the relevance of dengue fever, leptospirosis, SFG rickettsiosis, COVID-19 and malaria as causes of AUFI in the municipality of Apartadó, and highlight the great importance of SFG rickettsiosis as the main cause of AUFI in the municipality of Villeta.

Ethiopia is among the countries with a high leishmaniasis burden. In this retrospective review, we aimed to determine hematological and clinical features associated with initial poor treatment outcomes of visceral leishmaniasis (VL) patients. The majority of VL cases in this study had leucopenia (94.3%), thrombocytopenia (87.1%), and anemia (85.9%). HIV coinfection was present in 7.0% (n = 23) of VL cases. At the center, VL patients without HIV coinfection were treated with sodium stibogluconate and paromomycin combination, whereas HIV coinfected cases were treated with AmBisome and miltefosine combination therapy. End-of-treatment cure rates among HIV-positive and HIV-negative visceral leishmaniasis cases, respectively, were 52.2% and 96.9%. Case fatality rates were 34.8% and 2.7% in HIV-positive and HIV-negative cases, respectively. Overall, non-survivors in this study were more likely to have HIV (55.0% vs. 4.1%, p < 0.001), sepsis (15.0% vs. 1.4%, p = 0.019), and dyspnea (40.0% vs. 2.7%, p < 0.001) at admission. In this regard, particular attention to the management of superimposed disease conditions at admission, including sepsis, HIV, and dyspnea, is needed to improve VL patients’ treatment outcomes. The inadequacy of the current treatments, i.e., AmBisome and miltefosine combination therapy, for HIV coinfected visceral leishmaniasis patients requires further attention as it calls for new treatment modalities.

People are infected with Leishmania donovani when the parasite is deposited in the dermis during the blood meal of the sand fly vector. Most infected people develop a subclinical latent infection, but some develop progressive visceral leishmaniasis. Malnutrition is a risk factor for the development of active VL. We previously demonstrated increased parasite dissemination from the skin to visceral organs in a murine model of malnutrition. Here we investigated the mechanism of early parasite dissemination. After delivery of L . donovani to the skin, we found enhanced capture of parasites by inflammatory monocytes and neutrophils in the skin of malnourished mice. However, parasite dissemination in malnourished mice was driven primarily by infected inflammatory monocytes, which showed increased CCR7 expression, greater intrinsic migratory capacity, and increased trafficking from skin to spleen. PGE 2 production, which was increased at the site of skin infection, increased monocyte CCR7 expression and promoted CCR7-related monocyte-mediated early parasite dissemination in malnourished mice. Parasite dissemination in monocytes was reduced by inhibition of PGE 2 , knockdown or silencing of CCR7 in monocytes, and depletion of inflammatory monocytes through administration of diphtheria toxin to CSFR1-DTR transgenic mice that have monocyte-specific DT receptor expression. CCR7-driven trafficking of infected inflammatory monocytes through the lymph node was accompanied by increased expression of its ligands CCL19 and CCL21. These results show that the CCR7/PGE 2 axis is responsible for the increased trafficking of L . donovani -infected inflammatory monocytes from the skin to the spleen in the malnourished host. Undernutrition and production of PGE 2 are potential targets to reduce the risk of people developing VL. Nutritional interventions that target improved immune function and reduced PGE 2 synthesis should be studied in people at risk of developing VL.

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection. Key features • Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice. • Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry. • Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice. • Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children.

Acute malnutrition, or wasting, is implicated in over half of all deaths in children under five and increases risk of infectious disease. Studies in humans and preclinical models have demonstrated that malnutrition is linked to an immature intestinal microbiota characterized by increased prevalence of Enterobacteriaceae. Observational studies in children with moderate acute malnutrition (MAM) have also observed heightened systemic inflammation and increased circulating bacterial lipopolysaccharides (LPS; endotoxin). However, the mechanisms that underpin the systemic inflammatory state and endotoxemia, and their pathophysiological consequences, remain uncertain. Understanding these pathophysiological mechanisms is necessary to design targeted treatments that will improve the unacceptable rate of failure or relapse that plague current approaches. Here we use a mouse model of MAM to investigate the mechanisms that promote inflammation in the malnourished host. We found that mice with MAM exhibited increased systemic inflammation at baseline, increased translocation of bacteria and bacterial LPS, and an exaggerated response to inflammatory stimuli. An exaggerated response to bacterial LPS was associated with increased acute weight loss. Remarkably, intestinal inflammation and barrier dysfunction was found in the cecum and colon. The cecum showed a dysbiotic microbiota with expansion of Gammaproteobacteria and some Firmicutes, and contraction of Bacteroidetes. These changes were paralleled by an increase in fecal LPS bioactivity. The inflammatory phenotype and weight loss was modulated by oral administration of non-absorbable antibiotics that altered the proportion of cecal Gammaproteobacteria. We propose that the heightened inflammation of acute malnutrition is the result of changes in the intestinal microbiota, intestinal barrier dysfunction in the cecum and colon, and increased systemic exposure to LPS.

Texas is a geographically large state with large human and livestock populations, many farms, a long coastal region, and extreme fluctuations in weather. During the last 15 years, the state of Texas has frequently suffered disasters or catastrophes causing extensive morbidity and economic loss. These disasters often have complicated consequences requiring multi-faceted responses. Recently, an interdisciplinary network of professionals from multiple academic institutions has emerged to collaborate in protecting Texas and the USA using a One Health approach. These experts are training the next generation of scientists in biopreparedness; increasing under-standing of pathogens that cause repetitive harm; developing new therapeutics and vaccines against them; and developing novel surveillance approaches so that emerging pathogens will be detected early and thwarted before they can cause disastrous human and economic losses. These academic One Health partnerships strengthen our ability to protect human and animal health against future catastrophes that may impact the diverse ecoregions of Texas and the world.