Zingiber officinale supplementation suppresses eimeriosis and regulates goblet cell response

Coccidiosis affects both domestic and wild animals and negatively impacting industries worldwide. Medicinal plants are widely used against parasites. Using infected mice with Eimeria papillata, we assessed the anticoccidial impact of Zingiber officinale extract (ZE). The animals in the first group were just given distilled water, while the animals in the second group were given ZE. The parasite's oocysts were infected into the third and fourth groups. The fourth group was given ZE for five days. The oocysts in mice faeces were reduced after treatment with ZE. The total parasitic stages were reduced after treatments by about 50%. Also, gamonts, meronts and oocysts inside the jejunum were decreased after treatment with ZE.The infection caused hypoplasia of goblet cells of jejunum. ZE was able to ameliorate the goblet cells decrease. Behavioral response of animals to infection and treatment was investigated. All of these improvements could be attributed to the existence of active chemical classes of substances identified using infrared spectroscopy. Additional experiments are required to identify the phytochemical compounds in ZE and to understand their fighting mechanism against the parasite.     

Evaluation of nanoselenium and nanogold activities against murine intestinal schistosomiasis

Nanomedicine is one of the most important methods used to treat human diseases including parasitic diseases. Schistosomiasis is a major parasitic disease that affects human health in tropical regions. Whilst Praziquantel is the main classic antischistosomal drug, new drugs are required due to the poor effect of the drug on the parasite juveniles and immature worms, and the emergence of drug resistant strains of Schistosoma. The present study aimed to examine the curative roles of both gold and selenium nanoparticles on jejunal tissues of mice infected with Schistosoma mansoni. Transmission electron microscopy was used for characterization of nanoparticles. Gold nanoparticles of 1 mg/kg mice body weight and selenium nanoparticles 0.5 mg/kg body weight were inoculated separately into mice infected with S. mansoni. The parasite induced a significant decrease in glutathione levels; however, the levels of nitric oxide and malondialdehyde were significantly increased. Additionally, the parasite introduced deteriorations in histological architecture of the jejunal tissue. Treatment of mice with metal nanoparticles reduced the levels of body weight changes, oxidative stress and histological impairment in the jejunal tissue significantly. Therefore, our results revealed the protective role of both selenium and gold nanoparticles against jejunal injury in mice infected with S. mansoni.     

Anthelmintic,anticoccidial and antioxidant activity of Salvadora persica root extracts

Human infection with parasites is still one of the big problems worldwide. Medicinal plants succeeded to overcome a variety of protozoan and helminthic parasites. In this study, Salvadora persica root extracts (SE) were used to treat helminthosis and coccideosis. Three doses were used (200, 100 and 50 mg/ml) to study the anthelmintic activity of S. persica. Allolobophora caliginosa was used as a model worm. Also, Albendazole was used as a reference drug. In order to study the anticoccideal activity of SE, a group of mice were infected with Eimeria papillata sporulated oocysts. Experimental mice were treated with SE (300 mg/Kg) for 5 days. The extract was able to decrease the number of meronts and gamonts of the parasite in jejunum. Also, it regulates the level of glutathione and malondialdehyde and the activity of catalase as well. We conclude that S. persica possesses a powerful Anthelmintic, anticoccidial and antioxidant activity.     

Aptamer Based,Non-PCR,Non-Serological Detection of Chagas Disease Biomarkers in Trypanosoma cruzi Infected Mice

Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite Trypanosoma cruzi (T. cruzi), can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called T. cruzi Excreted Secreted Antigens (TESA), are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of T. cruzi infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of T. cruzi infected mice. An Enzyme Linked Aptamer (ELA) assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44) showed significant and specific binding to TESA as well as T. cruzi trypomastigote extract and not to host proteins or proteins of Leishmania donovani, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of T. cruzi. In mice infected with T. cruzi, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of T. cruzi infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.     

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr^?/? mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.     

Anti-coccidial and anti-apoptotic activities of palm pollen grains on Eimeria papillata-induced infection in mice

The present work aimed to study the effect of palm pollen extract (PPE) as an anticoccidial and anti-apoptotic modulator during the course of murine intestinal Eimeria papillata infection. The fact that PPE has an anticoccidial efficacy against intestinal E. papillata infection in mice has been clarified by the reduction of faecal output of oocysts on day five post infection from 49.5 × 10³ to 34 × 10³ oocyst/g. Moreover, the number of intracellular eimerian stages of zygots and developing oocysts decreased by about 89% and that of schizonts and gamonts to 42% and 72%, respectively. E. papillata infection also induced an increase in the number of apoptotic cells from 17.5 to 122.8 apoptotic nuclei/10 villous crypt units (VCU). In addition, it caused a state of systemic inflammatory response as revealed by an elevation in levels of the pro-inflammatory biomarkers, inducible nitric oxide synthase (iNOs) and tumor necrosis factor alpha (TNF-α) from 5.3 and 78.3 to 33 pmol ml^?1 and 96.3 pg ml^?1 in blood, respectively, with concurrent duplication in the total leucocytic number. Upon treatment of infected mice with the aqueous PPE, the activity of iNOs was reduced by 55% and the level of TNF-α was decreased by 30%. Moreover, the total leucocytic count was significantly reduced from 9.05 × 10³ to 7.8 × 10³ cells/mm³. Based on our results, PPE showed both anti-coccidial, anti-inflammatory and anti-apoptotic activities. So it can be used in developing new herbal medicine against animal coccidiosis and may be suitable agent for treating eimeriosis associated inflammatory response.     

Effect of gold nanoparticles on mice splenomegaly induced by schistosomiasis mansoni

Schistosomiasis is still one of the main parasitic diseases that affect human health in tropical regions. Whilst praziquantel (PZQ) is the main classic antischistosomal drug, the need for new drugs is still a must due to the low effectiveness of the drug on the schistosome young worms, and the evolving of PZQ resistant strains. Nanotechnology is one of the most important recent and current methods used to treat human diseases including parasitic ones. Therefore, the present study aimed to examine the curative role of gold nanoparticles (GNPs) on splenic tissue of mice infected with Schistosoma mansoni Sambon, 1907. High-resolution transmission electron microscopy was used for characterization of nanoparticles (NP). GNPs of 1 mg/kg mice body weight were inoculated into mice infected with S. mansoni. The parasite caused deteriorations in histological architecture of the spleen tissue, and splenomegaly. Additionally, the parasite induced a significant reduction in splenic tissue glutathione levels; however, the concentrations of nitric oxide and malondialdehyde were significantly increased. Treatment of mice with GNPs reduced the extent of histological impairment and oxidative stress in spleen tissue. Therefore, our results demonstrate the protective role of GNPs against splenic damage in mice infected with S. mansoni.     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.     

The Goblet Cell Is the Cellular Source of the Anti-Microbial Angiogenin 4 in the Large Intestine Post Trichuris muris Infection

Background

Mouse angiogenin 4 (Ang4) has previously been described as a Paneth cell–derived antimicrobial peptide important in epithelial host defence in the small intestine. However, a source for Ang4 in the large intestine, which is devoid of Paneth cells, has not been defined.

Methodology/Principal Findings

Analysis was performed on Ang4 expression in colonic tissue by qPCR and immunohistochemistry following infection with the large intestine dwelling helminth parasite Trichuris muris. This demonstrated an increase in expression of the peptide following infection of resistant BALB/c mice. Further, histological analysis of colonic tissue revealed the cellular source of this Ang4 to be goblet cells. To elucidate the mechanism of Ang4 expression immunohistochemistry and qPCR for Ang4 was performed on colonic tissue from T. muris infected mouse mutants. Experiments comparing C3H/HeN and C3H/HeJ mice, which have a natural inactivating mutation of TLR4, revealed that Ang4 expression is TLR4 independent. Subsequent experiments with IL-13 and IL-4 receptor alpha deficient mice demonstrated that goblet cell expression of Ang4 is controlled either directly or indirectly by IL-13.

Conclusions

The cellular source of mouse Ang4 in the colon following T. muris infection is the goblet cell and expression is under the control of IL-13.     

MAP Kinase Phosphatase-2 Plays a Key Role in the Control of Infection with Toxoplasma gondii by Modulating iNOS and Arginase-1 Activities in Mice

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2^−/− mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2^−/− mice did not, however, demonstrate defective type-1 responses compared with MKP-2^+/+ mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2^+/+, but not MKP-2^−/−, mice was nitric oxide (NO) dependent as infected MKP-2^+/+, but not MKP-2^−/− mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2^−/− but not MKP-2^+/+ mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2^−/− mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.     

Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease.     

Splenic natural killer-cell activity in mice infected with Leishmania donovani

Several strains of inbred mice were infected with the protozoan parasite Leishmania donovani, and, at several points during the infection, spleens of groups of these mice were tested for natural killer (NK)-cell activity vs lymphoma target cells in vitro and were evaluated for parasite burdens. Generally, elevated followed by normal (compared to uninfected control mice) or subnormal NK responses occurred as the result of infection. Elevated NK responses were not accompanied by high circulating levels of interferon, yet infected mice responded to an injection of an interferon inducer with interferon production as great as control mice. No consistent correlations among susceptibility phenotype to L. donovani infection, spontaneous NK activity phenotype, and infection-induced NK activation/depression patterns were detected among the various strains of mice.     

Metabolomic Profiling of Faecal Extracts from Cryptosporidium parvum Infection in Experimental Mouse Models

Cryptosporidiosis is a gastrointestinal disease in humans and animals caused by infection with the protozoan parasite Cryptosporidium. In healthy individuals, the disease manifests mainly as acute self-limiting diarrhoea, but may be chronic and life threatening for those with compromised immune systems. Control and treatment of the disease is challenged by the lack of sensitive diagnostic tools and broad-spectrum chemotherapy. Metabolomics, or metabolite profiling, is an emerging field of study, which enables characterisation of the end products of regulatory processes in a biological system. Analysis of changes in metabolite patterns reflects changes in biochemical regulation, production and control, and may contribute to understanding the effects of Cryptosporidium infection in the host environment. In the present study, metabolomic analysis of faecal samples from experimentally infected mice was carried out to assess metabolite profiles pertaining to the infection. Gas-chromatography mass spectrometry (GC-MS) carried out on faecal samples from a group of C. parvum infected mice and a group of uninfected control mice detected a mean total of 220 compounds. Multivariate analyses showed distinct differences between the profiles of C. parvum infected mice and uninfected control mice,identifying a total of 40 compounds, or metabolites that contributed most to the variance between the two groups. These metabolites consisted of amino acids (n = 17), carbohydrates (n = 8), lipids (n = 7), organic acids (n = 3) and other various metabolites (n = 5), which showed significant differences in levels of metabolite abundance between the infected and uninfected mice groups (p < 0.05). The metabolites detected in this study as well as the differences in abundance between the C. parvum infected and the uninfected control mice, highlights the effects of the infection on intestinal permeability and the fate of the metabolites as a result of nutrient scavenging by the parasite to supplement its streamlined metabolism.     

Influence of Parasite Load on Renal Function in Mice Acutely Infected with Trypanosoma cruzi

Background

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Despite the vast number of studies evaluating the pathophysiological mechanisms of the disease, the influence of parasite burden on kidney lesions remains unclear. Thus, the main goal of this work was to evaluate the effect of T. cruzi infection on renal function and determine whether there was a correlation between parasite load and renal injury using an acute experimental model of the disease.

Methodology/Principal Findings

Low, medium and high parasite loads were generated by infecting C57BL/6 mice with 300 (low), 3,000 (medium) or 30,000 (high) numbers of “Y” strain trypomastigotes. We found that mice infected with T. cruzi trypomastigotes show increased renal injury. The infection resulted in reduced urinary excretion and creatinine clearance. We also observed a marked elevation in the ratio of urine volume to kidney and body weight, blood urea nitrogen, chloride ion, nitric oxide, pro- and anti-inflammatory cytokines and the number of leukocytes in the blood and/or renal tissues of infected mice. Additionally, we observed the presence of the parasite in the cortical/medullary and peri-renal region, an increase of inflammatory infiltrate and of vascular permeability of the kidney. Overall, most renal changes occurred mainly in animals infected with high parasitic loads.

Conclusions/Significance

These data demonstrate that T. cruzi impairs kidney function, and this impairment is more evident in mice infected with high parasitic loads. Moreover, these data suggest that, in addition to the extensively studied cardiovascular effects, renal injury should be regarded as an important indicator for better understanding the pan-infectivity of the parasite and consequently for understanding the disease in experimental models.     

Plasmodium chabaudi-infected mice spleen response to synthesized silver nanoparticles from Indigofera oblongifolia extract

Malaria is a worldwide serious-threatening infectious disease caused by Plasmodium and the parasite resistance to antimalarial drugs has confirmed a significant obstacle to novel therapeutic antimalarial drugs. In this article, we assessed the antioxidant and anti-inflammatory activity of nanoparticles prepared from Indigofera oblongifolia extract (AgNPs) against the infection with Plasmodium chabaudi caused in mice spleen. AgNPs could significantly suppress the parasitaemia caused by the parasite to approximately 98% on day 7 postinfection with P. chabaudi and could improve the histopathological induced spleen damage. Also, AgNPs were able to increase the capsule thickness of the infected mice spleen. In addition, the AgNPs functioned as an antioxidant agent that affects the change in glutathione, nitric oxide and catalase levels in the spleen. Moreover spleen IL1β, IL-6 and TNF-α-mRNA expression was regulated by AgNPs administration to the infected mice. These results indicated the anti-oxidant and the anti-inflammatory protective role of AgNPs against P. chabaudi-induced spleen injury.