Giardia duodenalis is an intestinal protozoan transmitted through contaminated food and water and is a major cause of diarrheal disease worldwide and has been linked to post-infectious sequelae and growth impairment in children. Quantifying parasite burden in vivo is essential for studying infection dynamics and host responses, yet commonly used methods (stool PCR, antigen detection, and terminal trophozoite counts from segments of the intestine) are limited by intermittent stool shedding, uneven parasite distribution, sampling error, and lack of longitudinal data. We developed a bioluminescent reporter system for the assemblage B strain GS to enable noninvasive tracking of infection in mice without antibiotic pretreatment. Using in vivo bioluminescent imaging (BLI), we observed peak signal in immunocompetent C57BL/6J and BALB/c mice at days 6-8 post-infection, followed by return to baseline by ~day 10, consistent with the self-limiting nature of giardiasis. In immunodeficient mice, radiance increased and persisted over time, demonstrating stable luciferase expression during chronic infection in vivo without continuous antibiotic selection. Whole-animal radiance strongly correlated with direct trophozoite counts from excised intestinal segments (r = 0.915, P < 0.001), validating BLI as a useful alternative for determining parasite burden. Together, these findings establish BLI as a robust platform for longitudinal studies of Giardia infection dynamics and for future applications that may examine host immunity, the microbiota, nutrient-dependent effects, and drug testing.

Giardia duodenalis is an intestinal protozoan parasite common in low- and middle-income countries. Infection is often subclinical, even when it is associated with other pathologies like growth stunting in children. Recent longitudinal cohort studies have found Giardia more frequently in patients with milder symptoms and have even suggested that Giardia reduces rotavirus symptom severity. One potential mechanism for limiting disease severity due to other enteropathogens is the promotion of anti-inflammatory responses that limit pathology. Our lab previously showed that Giardia reduces production of interleukin (IL)-12 by dendritic cells stimulated with Toll-like receptor agonists. In this study, we show that Giardia increases the production of the anti-inflammatory cytokine IL-10 by mouse peritoneal macrophages in response to bacterial lipopolysaccharide. This potentiation is specific to IL-10, as no changes were seen in the production of the proinflammatory cytokine tumor necrosis factor ɑ. Moreover, peritoneal macrophages from mice lacking macrophage galactose-binding lectin, a pathogen recognition receptor that has been previously shown to bind N-acetylgalactosamine, failed to increase IL-10 production after stimulation with Giardia and lipopolysaccharide. Giardia's immunoregulation of the IL-10 response may help us understand the parasite's role in reducing diarrheal severity.

Diarrhoeal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with the protozoan parasite Giardia intestinalis decreases diarrhoeal severity. Here we show a high incidence of asymptomatic Giardia infection in school-aged children from Nigeria. In a mouse model, Giardia induced a Type 2 mucosal immune response, characterized by antigen-specific Th2 cells, IL-25, Type 2 cytokines, and goblet cell hyperplasia. Single-cell RNA sequencing and multiparameter flow cytometry revealed expansion of IL-10-producing Th2 cells, which promoted parasite persistence and protected against Toxoplasma gondii-induced ileitis and dextran sulfate sodium-induced colitis. This protective effect was STAT6 dependent, as IL-4R blockade or STAT6 deficiency impaired IL-10+ Th2 responses, resulting in Th1/Th17-driven tissue damage, inflammation and clearance of Giardia infection. Our findings demonstrate that Giardia reshapes mucosal immunity toward a Type 2 response, facilitating parasitism and conferring mutualistic protection from inflammatory pathologies, highlighting a key role for protists in mucosal defence regulation.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5’-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.

Giardia duodenalis is an intestinal protozoan parasite, common in low- and middle-income countries. Infection is frequently subclinical, even when it is associated with other pathologies like growth stunting in children. Recent longitudinal cohort studies have found Giardia more frequently in patients with milder symptoms and have even suggested that Giardia reduces rotavirus symptom severity. One potential mechanism for limiting disease severity due to other enteropathogens is the promotion of anti-inflammatory responses that limit pathology. Our lab previously showed that Giardia reduced production of IL-12 by dendritic cells stimulated with TLR agonists. In this study, we show that Giardia increases the production of the anti-inflammatory cytokine IL-10 by mouse peritoneal macrophages in response to bacterial LPS. This potentiation is specific to IL-10, as no changes were seen in the production of the pro-inflammatory cytokine TNF-□. Moreover, peritoneal macrophages from mice lacking macrophage galactose-binding lectin (MGL1), a pathogen recognition receptor that has been previously shown to bind N-acetylgalactosamine, failed to increase IL-10 production after stimulation with Giardia and LPS. Giardia’ s immunoregulation of the IL-10 response may help us understand the parasite’s role in reducing diarrheal severity.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACTDiarrheal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection withGiardia intestinalisdecreases the severity of diarrhea. Here, we show thatGiardiais highly prevalent in the stools of asymptomatic school-aged children. It orchestrates a Th2 mucosal immune response, characterized by increased antigen-specific Th2 cells, IL-25, Type 2-associated cytokines, and goblet cell hyperplasia.Giardiainfection expanded IL-10-producing Th2 and GATA3+Treg cells that promoted chronic carriage, parasite transmission, and conferred protection againstToxoplasma gondii-induced lethal ileitis and DSS-driven colitis by downregulating proinflammatory cytokines, decreasing Th1/Th17 cell frequency, and preventing collateral tissue damage. Protection was dependent on STAT6 signaling, asGiardia-infected STAT6-/-mice no longer regulated intestinal bystander inflammation. Our findings demonstrate thatGiardiainfection reshapes mucosal immunity toward a Type 2 response, which confers a mutualistic protection against inflammatory disease processes and identifies a critical role for protists in regulating mucosal defenses.

Bluetongue (BT) is a vector-borne disease affecting wild and domestic ruminants in many parts of the world. Although bluetongue virus (BTV) is widespread in ungulates in Africa, available epidemiological information on BT in this continent is limited. This systematic review and meta-analysis aimed to estimate the seroprevalence of BTV and summarize information on associated risk factors in domestic ruminants and camels in Africa. Systematic searches were conducted from the inception of the database to November 2022 on PubMed/MEDLINE, ScienceDirect, Web of Science, and Google/Google Scholar. Forty-four eligible publications were identified, published in the range from 1973 to 2020, and statistically analyzed. The pooled overall seroprevalence of BTV was 45.02% (95% confidence interval [CI]: 36.00-54.00%). The pooled seroprevalence was 49.70% (95% CI: 34.50-65.00%) in cattle, 47.00% (95% CI: 29.90-64.50%) in goats, 40.80% (95% CI: 19.60-63.90%) in camels, and 36.30% (95% CI: 29.00-44.90%) in sheep. The pooled seroprevalence decreased after 1990 and increased again after 2010. The highest pooled overall seroprevalence was found in the southeastern region, and the highest pooled overall seroprevalence was obtained by Competitive Enzyme-Linked Immunosorbent Assay. Finally, the seroprevalence in females (53.30%, 95% CI: 34.80-71.00%) was significantly higher than in males (28.10%, 95% CI: 17.40-40.30%) (p < 0.05). We showed that antibodies against BTV were common in African ruminants and camels. Monitoring the seroprevalence of BTV, as well as systematic and continuous surveillance of the Culicoides population, are encouraged to prevent and control the spread of BT.

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia duodenalis. Acute symptoms can include diarrhea, but infections are often sub-clinical. It is transmitted via ingesting infectious cysts in contaminated food or water. In developing countries, giardiasis is one of the leading causes of growth stunting in children under two years old. Changes in intestinal barrier integrity have been shown to contribute to impaired nutrient absorption, thereby leading to growth stunting. Although intestinal barrier defects have been linked to infection, there is little understanding of barrier repair dynamics post-infection. Previous studies have demonstrated that Giardia infection is associated with dysbiosis in humans and animals. Because Aryl hydrocarbon receptor (AHR) signaling has been shown to promote intestinal repair in other systems, we are exploring the role of AHR in barrier repair following a Giardia infection. We quantified specific microbiome-derived AHR ligands in the plasma of infected C57BL/6 mice and observed a reduction of indole-3-ethanol and indole-3-pyruvic acid 21 days post-infection. Since IL-22 signaling can also promote barrier repair, we quantified IL-22 transcripts by RT-PCR and found significantly increased levels of IL-22 mRNA in infected mice. These data suggest that Giardia can reduce barrier repair through altered production of AHR ligands by the microbiome, but that IL-22 can counter this effect in some situations. This study will give better insight into developing effective dietary interventions to prevent growth stunting in infected children. Supported by grant from NIH AI166467

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

Background The mechanisms underlying the clinical outcome disparity during human infection with Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite’s genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis. Methods The genetic heterogeneity of G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal–Wallis non-parametric test with a post hoc Dunn test. Results We found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of G. duodenalis , whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite’s genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers. Conclusions These findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections. Graphical Abstract

Giardia duodenalis is a common intestinal pathogen transmitted via consumption of contaminated water. In addition to causing intestinal distress symptoms, Giardia infection is one of the top 5 infectious causes of growth stunting in children under two. Growth faltering is a complex pathology facilitated by changes in the intestinal barrier and enzymatic deficiencies. While infection is widely associated with barrier damage in vitro, in animal models, and in patient studies, the mechanisms behind this process are not well understood. Since PAR2 has a prominent role in increased intestinal permeability in Celiac disease, we are focusing on the involvement of the protease activated receptor 2 (PAR2) in giardiasis-facilitated intestinal defects. We are using human intestinal cell lines, murine intestinal organoids, and a mouse infection model to address the role of PAR2. Co-culture of Giardia and Caco2 cells results in altered tight junction morphology as revealed by staining for ZO-1; these changes are reduced by treatment with a PAR2 antagonist. Effects of PAR2 signaling on barrier function are being assessed by measuring transepithelial electrical resistance (TEER) in Caco2 cells and PAR2+/− and PAR2−/− organoids cultured on transwell filters. Additionally, while C57BL/6 mice fed a low-protein diet exhibit reduced weight gain when infected with Giardia, mice lacking PAR2 gain weight normally when infected. These data indicate that PAR2 signaling contributes to intestinal permeability defects and growth faltering. Identifying parasite factors that activate PAR2 will provide novel targets for development of pharmaceuticals and vaccines to improve child growth. AI166467

Giardia duodenalis is one of the world's most common intestinal parasites, with an estimated 280 million cases globally each year. Infections can cause diarrhea and cramps, although they are often subclinical. Interestingly, Giardia can have a protective effect and reduce the incidence of fatal diarrhea in co-infections with other enteric pathogens. In this study, we investigate the anti-inflammatory response to Giardia via the macrophage galactose lectin-type 1 (MGL1) receptor. Our reasons for choosing this receptor are three-fold: it is expressed on macrophages and some dendritic cells and plays a role in immune response to other pathogens, it has been shown to induce anti-inflammatory effects in the DSS colitis model, and it demonstrably binds galactose-containing carbohydrates, like the N-Acetylgalactosamine (GalNAc) present in the cell wall of Giardia cysts. We show that peritoneal macrophages isolated from wildtype C57BL/6 mice produce markedly more of the anti-inflammatory cytokine IL-10 when stimulated with lipopolysaccharide (LPS) and Giardia extract compared to LPS alone. The potentiated IL-10 response to Giardia is lost in peritoneal macrophages isolated from MGL1 receptor knockout mice or in extracts centrifuged at 10,000 X g. Elucidating this anti-inflammatory pathway not only promises a better understanding of giardiasis, which disproportionately affect lower-middle income communities. also might present opportunities to mimic the parasite in the treatment of a wide array of inflammatory diseases. Supported by a grant from NIH (R15 AI109591).

Clostridium thermocellum is a desirable biocatalyst for biohydrogen production, with a native ability to simultaneously saccharify cellulose and to metabolize released cellodextrins for hydrogen production. During fermentation with C. thermocellum, partial pressures of two gases - CO2 and H2 - are critical drivers of overall reaction kinetics. Biohydrogen production is enhanced by maintaining a low hydrogen partial pressure, while high concentrations of dissolved CO2 promote microbial biomass synthesis. Our study evaluates the inherent trade-offs between hydrogen stripping and inorganic carbon supply for optimized biohydrogen synthesis. We find that nitrogen sparging at low flow rates increases hydrogen production when compared with an equivalent nitrogen overlay, but that high rates of nitrogen sparging inhibit cell growth and hydrogen production. Decreasing dissolved hydrogen partial pressure via nitrogen sparging also lowers the production of reduced metabolites, including lactate and ethanol. To address potential stripping of inorganic carbon from the production medium, we supplemented CO2 to the sparging gas and co-optimized for gas flow rate and for the CO2 fraction of the sparging gas. Total hydrogen production increased from 50 mmol center dot L- 1 in the base condition, when the bioreactor was sparged with 0.1 LPM of pure nitrogen, to 181.3 mmol center dot L-1 when sparged with 1.3 LPM of 33 % CO2, demonstrating that biohydrogen production is highly sensitive to both parameters. Fine sensitivity of biohydrogen production to sparging conditions highlights the critical importance of bioreactor design and operation to achieve maximum H2 removal without compromising inorganic carbon supply to bacterial central metabolism.

community effort, and we invite comments, criticisms, and suggested edits.

Lignin is the most abundant renewable source of aromatics on earth, and conversion of it to chemicals and fuels is needed to build an economically viable renewable biofuels industry. Biological routes to converting lignin to fuels and chemicals involve depolymerizing lignin using lignin-degrading enzymes that catalyze the breaking of ether and carbon-carbon bonds in the phenolic and non-phenolic subunits of lignin. Laccases are a crucial class of lignin-degrading enzymes and are copper-containing enzymes capable of oxidizing electron-rich organic substrates such as lignin using molecular oxygen as an electron acceptor. The genome of Cerrena unicolor was recently added to the JGI MycoCosm database and has eight laccases. Two of these laccases, designated Lc1 and Lc2, predicted to have the highest likelihood for successful expression in soluble, active form were selected for characterization. Lc1 and Lc2, which share 65% sequence identity, were heterologously expressed in Komagataella pastoris (formerly Pichia pastoris), allowing characterization and comparison of their purified forms. Lc1 and Lc2 had half-lives of 16 min and 185 min at 60°C, respectively, and, based on molecular dynamics simulations, the longer half-life of Lc2 was due to an increased number and persistence of salt bridges compared to Lc1. Using model lignin-like dimers and a nanostructure-initiator mass spectrometry assay to quantify catalysis of specific bond-breaking events, both Lc1 and Lc2 had their highest activity at pH 3 and in combination with syringaldehyde as a mediator, with Lc1 having a higher catalytic efficiency of β-O-4' ether and C-C bond breaking. This comparative study demonstrates the diversity, including thermostability differences, of laccases from the same fungus, and improves our understanding of laccase catalyzed breaking of bonds commonly found in lignin, which will facilitate the developing this important class of enzymes for applications in the conversion of lignin to valuable bioproducts.

We report on complete genome sequences of five Pseudomonas soil isolates that are capable of metabolizing pentose sugars and aromatic monomers. These complete genome sequence data provide insight into possible alternative hosts for the production of biofuels and bio-based chemicals from lignocellulosic feedstock.

laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

Some of the largest scale chemical processes for the production of important commodity chemicals and fuels involve an H2-containing gas mixture as an intermediate [e.g. ammonia, urea, methanol, Fischer-Tropsch (F-T) diesel, and hydrotreatment of crude petroleum to refined fuels]. The successful development of a model H2-oxidizing chemoautotrophic host could expand the range of fuels and chemicals produced from H2 and CO2 intermediates in the chemical, oil, and gas sectors in the near term, as well as from renewable and waste-derived sources of these gases that are expected to greatly expand in coming years. Among non-photosynthetic bacteria that can utilize H2 and CO2, Cupriavidus necator (C. necator, formerly Ralstonia eutropha), is the best studied. C. necator is an excellent microbial host for the production of a variety of chemicals because it grows extremely quickly to very high cell densities autotrophically on H2 and CO2, is genetically tractable, and has the ability to accumulate polymers, such as polyhydroxybutyrate, at industrial levels. Despite having great potential as a platform bioproduction host, genetic tools are limited, making metabolic engineering of this organism slow and laborious. In this CRADA project, we developed a number of genetic tools for C. necator: 1) Improvement of C. necator genetic transformation efficiency 2) Integration of heterologous genes into the C. necator chromosome and development of promoter library 3) Development of graded RBSs to control heterologous protein expression 4) Use of RBSs to demonstrate fatty alcohol production in C. necator 5) Demonstration of CRISPR-Cas9 gene editing in C. necator

•Giardia infection alters the microbiome, generally including an increase in Prevotella and a decrease in Bacteroides.•Bile salts and their metabolites mediate several effects of the microbiome on Giardia and vice versa.•Giardia modulates innate immune responses during single and co-infections.