Transcriptomic landscape of hepatic lymph nodes, peripheral blood lymphocytes and spleen of swamp buffaloes infected with the tropical liver fluke Fasciola gigantica.

The tropical liver fluke Fasciola gigantica is a parasitic helminth that has been frequently reported to infect mammals, typically involving water buffaloes. In this study, we characterized the tissue transcriptional landscape of buffaloes following infection by F. gigantica. RNAs were isolated from hepatic lymph nodes (hLNs), peripheral blood lymphocytes (pBLs), and spleen at 3-, 42- and 70-days post-infection (dpi), and all samples were subjected to RNA sequencing analyses. At 3 dpi, 2603, 460, and 162 differentially expressed transcripts (DETs) were detected in hLNs, pBLs, and spleen, respectively. At 42 dpi, 322, 937, and 196 DETs were detected in hLNs, pBLs, and spleen, respectively. At 70 dpi, 376, 334, and 165 DETs were detected in hLNs, pBLs, and spleen, respectively. Functional enrichment analysis identified upregulated immune-related pathways in the infected tissues involved in innate and adaptive immune responses, especially in hLNs at 42 and 70 dpi, and pBLs at 3 and 42 dpi. The upregulated transcripts in spleen were not enriched in any immune-related pathway. Co-expression network analysis further identified transcriptional changes associated with immune response to F. gigantica infection. Receiver operating characteristic (ROC) curve analysis showed that 107 genes in hLNs, 32 genes in pBLs, and 36 genes in spleen correlated with F. gigantica load. These findings provide new insight into molecular mechanisms and signaling pathways associated with F. gigantica infection in buffaloes.

Proteomic Profiling of the Liver, Hepatic Lymph Nodes, and Spleen of Buffaloes Infected with Fasciola gigantica.

In the present study, we used an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics technology to characterize the differentially expressed proteins (DEPs) in the liver, hepatic lymph nodes (hLNs), and spleen of buffaloes infected with Fasciola gigantica (F. gigantica). We also used the parallel reaction monitoring (PRM) method to verify the expression levels of the DEPs in the three infected tissues. At three days post-infection (dpi), 225, 1821, and 364 DEPs were detected in the liver, hLNs, and spleen, respectively. At 42 dpi, 384, 252, and 214 DEPs were detected in the liver, hLNs, and spleen, respectively. At 70 dpi, 125, 829, and 247 DEPs were detected in the liver, hLNs, and spleen, respectively. Downregulation of metabolism was prominent in infected livers at all time points, and upregulation of immune responses was marked in the hLNs during early infection (three dpi); however, no changes in the immune response were detected at the late stages of infection (42 and 70 dpi). Compared to the hLNs, there was no significant upregulation in the levels of immune responses in the infected spleen. All the identified DEPs were used to predict the subcellular localization of the proteins, which were related to extracellular space and membrane and were involved in host immune responses. Further PRM analysis confirmed the expression of 18 proteins. These data provide the first simultaneous proteomic profiles of multiple organs of buffaloes experimentally infected with F. gigantica.

iTRAQ-based Quantitative Proteomics Analysis Identifies Host Pathways Modulated during Toxoplasma gondii Infection in Swine.

Toxoplasma gondii is a leading cause of foodborne illness and consumption of undercooked pig meat is a major risk factor for acquiring toxoplasmosis, which causes a substantial burden on society. Here, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify cellular proteins and pathways altered during T. gondii infection in pigs. We also used parallel reaction monitoring-based LC-MS/MS to verify the levels of protein expression of infected spleens and mesenteric lymph nodes (MLNs). At 6 days post-infection (dpi), 156, 391, 170, 292, and 200 differentially expressed proteins (DEPs) were detected in the brain, liver, lung, MLNs and spleen, respectively. At 18 dpi, 339, 351, 483, 388, and 303 DEPs were detected in the brain, liver, lung, MLNs and spleen, respectively. Although proteins involved in immune responses were upregulated in all infected tissues, protein expression signature in infected livers was dominated by downregulation of the metabolic processes. By weighted gene co-expression network analysis, we could further show that all proteins were clustered into 25 co-expression modules and that the pink module significantly correlated with the infection status. We also identified 163 potential anti-T. gondii proteins (PATPs) and provided evidence that two PATPs (HSP70.2 and PDIA3) can reduce T. gondii burden in porcine macrophages in vitro. This comprehensive proteomics analysis reveals new facets in the pathogenesis of T. gondii infection and identifies key proteins that may contribute to the pig's defense against this infection.

Complex and dynamic transcriptional changes allow the helminth Fasciola gigantica to adjust to its intermediate snail and definitive mammalian hosts.

BACKGROUND: The tropical liver fluke, Fasciola gigantica causes fasciolosis, an important disease of humans and livestock. We characterized dynamic transcriptional changes associated with the development of the parasite in its two hosts, the snail intermediate host and the mammalian definitive host. RESULTS: Differential gene transcription analysis revealed 7445 unigenes transcribed by all F. gigantica lifecycle stages, while the majority (n = 50,977) exhibited stage-specific expression. Miracidia that hatch from eggs are highly transcriptionally active, expressing a myriad of genes involved in pheromone activity and metallopeptidase activity, consistent with snail host finding and invasion. Clonal expansion of rediae within the snail correlates with increased expression of genes associated with transcription, translation and repair. All intra-snail stages (miracidia, rediae and cercariae) require abundant cathepsin L peptidases for migration and feeding and, as indicated by their annotation, express genes putatively involved in the manipulation of snail innate immune responses. Cercariae emerge from the snail, settle on vegetation and become encysted metacercariae that are infectious to mammals; these remain metabolically active, transcribing genes involved in regulation of metabolism, synthesis of nucleotides, pH and endopeptidase activity to assure their longevity and survival on pasture. Dramatic growth and development following infection of the mammalian host are associated with high gene transcription of cell motility pathways, and transport and catabolism pathways. The intra-mammalian stages temporally regulate key families of genes including the cathepsin L and B proteases and their trans-activating peptidases, the legumains, during intense feeding and migration through the intestine, liver and bile ducts. While 70% of the F. gigantica transcripts share homology with genes expressed by the temperate liver fluke Fasciola hepatica, gene expression profiles of the most abundantly expressed transcripts within the comparable lifecycle stages implies significant species-specific gene regulation. CONCLUSIONS: Transcriptional profiling of the F. gigantica lifecycle identified key metabolic, growth and developmental processes the parasite undergoes as it encounters vastly different environments within two very different hosts. Comparative analysis with F. hepatica provides insight into the similarities and differences of these parasites that diverged > 20 million years ago, crucial for the future development of novel control strategies against both species.

Metabolomic signature of mouse cerebral cortex following Toxoplasma gondii infection.

BACKGROUND: The protozoan parasite Toxoplasma gondii infects and alters the neurotransmission in cerebral cortex and other brain regions, leading to neurobehavioral and neuropathologic changes in humans and animals. However, the molecules that contribute to these changes remain largely unknown. METHODS: We have investigated the impact of T. gondii infection on the overall metabolism of mouse cerebral cortex. Mass-spectrometry-based metabolomics and multivariate statistical analysis were employed to discover metabolomic signatures that discriminate between cerebral cortex of T. gondii-infected and uninfected control mice. RESULTS: Our results identified 73, 67 and 276 differentially abundant metabolites, which were involved in 25, 37 and 64 pathways at 7, 14 and 21 days post-infection (dpi), respectively. Metabolites in the unsaturated fatty acid biosynthesis pathway were upregulated as the infection progressed, indicating that T. gondii induces the biosynthesis of unsaturated fatty acids to promote its own growth and survival. Some of the downregulated metabolites were related to pathways, such as steroid hormone biosynthesis and arachidonic acid metabolism. Nine metabolites were identified as T. gondii responsive metabolites, namely galactosylsphingosine, arachidonic acid, LysoSM(d18:1), L-palmitoylcarnitine, calcitetrol, 27-Deoxy-5b-cyprinol, L-homophenylalanine, oleic acid and ceramide (d18:1/16:0). CONCLUSIONS: Our data provide novel insight into the dysregulation of the metabolism of the mouse cerebral cortex during T. gondii infection and have important implications for studies of T. gondii pathogenesis.

Global Transcriptome Profiling of Multiple Porcine Organs Reveals Toxoplasma gondii-Induced Transcriptional Landscapes.

We characterized the porcine tissue transcriptional landscapes that follow Toxoplasma gondii infection. RNAs were isolated from liver, spleen, cerebral cortex, lung, and mesenteric lymph nodes (MLNs) of T. gondii-infected and uninfected (control) pigs at days 6 and 18 postinfection, and were analyzed using next-generation sequencing (RNA-seq). T. gondii altered the expression of 178, 476, 199, 201, and 362 transcripts at 6 dpi and 217, 223, 347, 119, and 161 at 18 dpi in the infected brain, liver, lung, MLNs and spleen, respectively. The differentially expressed transcripts (DETs) were grouped into five expression patterns and 10 sub-clusters. Gene Ontology enrichment and pathway analysis revealed that immune-related genes dominated the overall transcriptomic signature and that metabolic processes, such as steroid biosynthesis, and metabolism of lipid and carboxylic acid, were downregulated in infected tissues. Co-expression network analysis identified transcriptional modules associated with host immune response to infection. These findings not only show how T. gondii infection alters porcine transcriptome in a tissue-specific manner, but also offer a gateway for testing new hypotheses regarding human response to T. gondii infection.

Global serum proteomic changes in water buffaloes infected with Fasciola gigantica.

BACKGROUND: The liver fluke Fasciola gigantica modulates several signaling pathways in infected buffaloes to facilitate its survival and establishment of persistent infection. In response to the parasite invasion, buffaloes activate innate and adaptive immune responses to counter the parasite infection. To detect new proteins that might be involved in the interaction between F. gigantica and the buffaloes, and that also might serve as biomarkers for fasciolosis, we used proteomic techniques to study the serum proteome of buffaloes during F. gigantica infection. Here, we used an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach to identify serum proteins that are differentially expressed in infected buffaloes compared to uninfected control buffaloes. Additionally, we applied a parallel reaction monitoring (PRM) assay to validate specific proteins identified by the iTRAQ method. RESULTS: A total of 313, 459 and 399 proteins were identified at 3, 42 and 70 days post-infection, respectively; of these 92, 93 and 138 were differentially abundant proteins. Some of the identified differentially abundant proteins, including complement factor H related 5, complement component C6, complement component C7, amine oxidase, plasma serine protease inhibitor and lysozyme, are known to be involved in complement system activation, blood coagulation, platelet activation, lymphocyte's adhesion and lysozyme hydrolysis. Analysis of data for all three time points after infection identified six significantly upregulated proteins in infected serum that separated infected and uninfected buffaloes into distinct clusters. Further PRM analysis confirmed the expression of five proteins, namely MHC class I antigen, Beta-2-microglobulin, NID2 protein, Fetuin-B and Fibrinogen gamma-B chain. CONCLUSIONS: These findings provide novel insights into the serum proteomics signature of buffaloes during F. gigantica infection.

Th2-related cytokines are associated with Fasciola gigantica infection and evasion in the natural host, swamp buffalo.

The infection of ruminants by Fasciola spp. always induces a non-protective Th2-type immune response. However, little is known about changes in the local and systemic immune environment during F. gigantica migration in buffalo. In this study, native swamp buffaloes were each infected with 500 viable F. gigantica metacercariae. Mesenteric lymph node (MLN), hepatic lymph node (HLN), spleen, and serum samples were collected from control and infected buffaloes at 3, 10, 28, 42, 70, and 98 days post-infection (DPI). The mRNA expression levels of the Th1- and Th2-related cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, and CD4 were measured during different infection stages in the MLNs, spleens, and HLNs using quantitative real-time PCR (qRT-PCR). Levels of the specific anti-ESP isotype antibodies IgG, IgG1, and IgG2 were used to reflect changes in humoral immunity. The results of this study indicated that swamp buffaloes were susceptible to F. gigantica infection, and that susceptibility to this infection was closely related to the cytokine environment associated with the Th2-type immune response. The MLNs showed a mixed Th1- and Th2-type immune response during the acute infection stages, after which the production of these cytokines returned to normal. Cytokine expression in the HLNs also expressed a mixed Th1- and Th2-type immune response during the early infection stages. When the infection became chronic, the typical Th2 immune response was induced in the HLNs. At the acute infection stages, the spleen exhibited a Th2 immune response. Nevertheless, cytokines associated with the Th1 and Th2 immune responses were upregulated at 98 DPI. In addition, the total IgG and IgG1 of the parasite-specific antibodies increased. This suggested that the Th2-related cytokines and IgG1 induced by F. gigantica infection might mediate successful F. gigantica infection in the natural host, swamp buffalo.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) interacting with buffalo serum of different infection periods by shotgun LC-MS/MS.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.

Protective Efficacy Against Acute and Chronic Toxoplasma gondii Infection Induced by Immunization With the DNA Vaccine TgDOC2C.

Toxoplasma gondii is a ubiquitous intracellular apicomplexan parasite that can cause zoonotic toxoplasmosis. Effective vaccines against T. gondii infection are necessary to prevent and control the spread of toxoplasmosis. The present study analyzed the B-linear epitopes of T. gondii DOC2 (TgDOC2) protein and then cloned the C-terminus of the TgDOC2 gene (TgDOC2C) to construct the pVAX-TgDOC2C eukaryotic vector. After intramuscular injection of pVAX-TgDOC2C, immune responses were monitored. Two weeks after the last immunization, the protective effects of pVAX-TgDOC2C against acute and chronic toxoplasmosis were evaluated by challenges with T. gondii RH tachyzoites (genotype I) and PRU cysts (genotype II). The DNA vaccine elicited strong humoral and cellular immune responses with high levels of IgG antibody, IL-2 and IFN-γ production compared to those of the controls. The percentage of CD4+ and CD8+ T cells in mice immunized with pVAX-TgDOC2C was significantly increased compared to that of mice injected with empty pVAX I or PBS. After acute infection with 103 lethal tachyzoites, mice immunized with pVAX-TgDOC2C survived longer (12.5 days) than mice treated with pVAX I (8 days) and PBS (7.5 days). Mice immunized with pVAX-TgDOC2C had significantly less brain cysts (1600.83 ± 284.61) compared to mice immunized with pVAX I (3016.67 ± 153.84) or PBS (3100 ± 246.98). Together, these results demonstrated that TgDOC2C confers protective immunity against T. gondii infection and may be a promising candidate antigen for further development of an effective multicomponent vaccine for veterinary use against toxoplasmosis in livestock animals.

Acute Toxoplasma Gondii Infection in Cats Induced Tissue-Specific Transcriptional Response Dominated by Immune Signatures.

RNA-sequencing was used to detect transcriptional changes in six tissues of cats, seven days after T. gondii infection. A total of 737 genes were differentially expressed (DEGs), of which 410 were up-regulated and 327 were down-regulated. The liver exhibited 151 DEGs, lung (149 DEGs), small intestine (130 DEGs), heart (123 DEGs), brain (104 DEGs), and spleen (80 DEGs)-suggesting tissue-specific transcriptional patterns. Gene ontology and KEGG analyses identified DEGs enriched in immune pathways, such as cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, MAPK signaling pathway, T cell receptor signaling pathway, and the cytosolic DNA sensing pathway. C-X-C motif chemokine 10 (CXCL10) was involved in most of the immune-related pathways. PI3K/Akt expression was down-regulated in all tissues, except the spleen. The genes for phosphatase, indoleamine 2,3-dioxygenase, Hes Family BHLH Transcription Factor 1, and guanylate-binding protein 5, playing various roles in immune defense, were co-expressed across various feline tissues. Multivariate K-means clustering analysis produced seven gene clusters featuring similar gene expression patterns specific to individual tissues, with lung tissue cluster having the largest number of DEGs. These findings suggest the presence of a broad immune defense mechanism across various tissues in cats against acute T. gondii infection.

The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells.

BACKGROUND: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats. METHODS: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0. RESULTS: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-ß) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway. CONCLUSIONS: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.

Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii.

BACKGROUND: Although sexual reproduction of the parasite Toxoplasma gondii exclusively occurs in the cat intestine, knowledge about the alteration of gene expression in the intestine of cats infected with T. gondii is still limited. Here, we investigated the temporal transcriptional changes that occur in the cat intestine during T. gondii infection. METHODS: Cats were infected with 100 T. gondii cysts and their intestines were collected at 6, 12, 18, 24, 72 and 96 hours post-infection (hpi). RNA sequencing (RNA-Seq) Illumina technology was used to gain insight into the spectrum of genes that are differentially expressed due to infection. Quantitative RT-PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing. RESULTS: Our transcriptome analysis revealed 2363 DEGs that were clustered into six unique patterns of gene expression across all the time points after infection. Our analysis revealed 56, 184, 404, 508, 400 and 811 DEGs in infected intestines compared to uninfected controls at 6, 12, 18, 24, 72 and 96 hpi, respectively. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in catalytic activity and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the intestine of infected cats evolve over the course of infection, and the largest difference in the enriched pathways was observed at 96 hpi. The anti-T. gondii defense response of the feline host was mediated by Major Histocompatibility Complex class I, proteasomes, heat-shock proteins and fatty acid binding proteins. CONCLUSIONS: This study revealed novel host factors, which may be critical for the successful establishment of an intracellular niche during T. gondii infection in the definitive feline host.

Differential Brain MicroRNA Expression Profiles After Acute and Chronic Infection of Mice With Toxoplasma gondii Oocysts.

Brain microRNAs (miRNAs) change in abundance in response to Toxoplasma gondii infection. However, their precise role in the pathogenesis of cerebral infection with T. gondii oocyst remains unclear. We studied the abundance of miRNAs in the brain of mice on days 11 and 33 post-infection (dpi) in order to identify miRNA pattern specific to early (11 dpi) and late (33 dpi) T. gondii infection. Mice were challenged with T. gondii oocysts (Type II strain) and on 11 and 33 dpi, the expression of miRNAs in mouse brain was investigated using small RNA (sRNA) sequencing. miRNA expression was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to identify the biological processes, molecular functions, and cellular components, as well as pathways involved in infection. More than 1,500 miRNAs (1,352 known and 150 novel miRNAs) were detected in the infected and control mice. The expression of miRNAs varied across time after infection; 3, 38, and 108 differentially expressed miRNAs (P < 0.05) were detected during acute infection, chronic infection and chronic vs. acute infection, respectively. GO analysis showed that chronically infected mice had more predicted targets of dysregulated miRNAs than acutely infected mice. KEGG analysis indicated that most predicted targets were involved in immune- or disease-related pathways. Our data indicate that T. gondii infection alters the abundance of miRNAs in mouse brain particularly at the chronic stage, probably to fine-tune conditions required for the establishment of a latent brain infection.

Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic disease caused by the intracellular protozoan Toxoplasma gondii; and a major source of infection in humans is via ingestion of T. gondii tissue cysts. Ultimately, the goal of anti-toxoplasmosis vaccines is to elicit a sustainable immune response, capable of preventing formation of the parasite tissue cysts-or, at least, to restrain its growth. In this study, we formulated a cocktail DNA vaccine and investigated its immunologic efficacy as a protection against the establishment of T. gondii cysts in the mouse brain. This multicomponent DNA vaccine, encoded the TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, which play key roles in the pathogenesis of T. gondii infection. Results showed that mice immunized via intramuscular injection three times, at 2-week intervals with this multicomponent DNA vaccine, mounted a strong humoral and cellular immune response, indicated by significantly high levels of total IgG, CD4+ and CD8+ T lymphocytes, and antigen-specific lymphocyte proliferation when compared with non-immunized mice. Immunization also induced a mixed Th1/Th2 response, with a slightly elevated IgG2a to IgG1 ratio. The increased production of proinflammatory cytokines gamma-interferon, interleukin-2, and interleukin-12 (p < 0.0001) correlated with increased expression of p65/RelA and T-bet genes of the NF-κB pathway. However, no significant difference was detected in level of interleukin-4 (p > 0.05). The number of brain cysts in immunized mice was significantly less than those in non-immunized mice (643.33 ± 89.63 versus 3,244.33 ± 96.42, p < 0.0001), resulting in an 80.22% reduction in the parasite cyst burden. These findings indicate that a multicomponent DNA vaccine, encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, shows promise as an immunization strategy against chronic toxoplasmosis in mice, and calls for a further evaluation in food-producing animals.

Immune response and protective effect against chronic Toxoplasma gondii infection induced by vaccination with a DNA vaccine encoding profilin.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals. T. gondii profilin (TgPF) plays a crucial role in parasite motility and host cell invasion, and has shown promise against toxoplasmosis. DNA vaccine was considered to elicit effective humoral and cell-mediated immunity against T. gondii infection. The objective of the present study was to evaluate the immunogenicity of TgPF in mice using a DNA vaccination strategy. METHODS: A DNA vaccine (pVAX-PF) encoding TgPF gene was constructed and then was intramuscularly injected into mice with and without a plasmid encoding IL-15 (pVAX-IL-15). The immune responses in immunized Kunming mice including lymphocyte proliferation, levels of cytokines, antibody titers and T lymphocyte subclasses were analyzed. The protective efficacy against chronic T. gondii infection was observed at 4 weeks post-infection with the cyst-forming PRU strain of T. gondii (Genotype II). RESULTS: EitherpVAX-PF with or without pVAX-IL-15 could elicit higher level of IgG and IgG2a antibodies and produce strong cellular immune responses in the immunized mice. The brain cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 (1843 ± 215.7) and pVAX-PF (1897 ± 337.8) were reduced 40.82% and 39.08%, respectively, compared to that in mice received nothing (3114 ± 168.8), and the differences were statistically significant (P < 0.0001). However, the T. gondii cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 were not statistically significantly different compared to that in mice immunized with pVAX-PF alone [t(10) = 0.33, P > 0.05]. CONCLUSIONS: The present study indicated that TgPF could be a promising vaccine candidate against chronic toxoplasmosis, which can be further used to develop multi-epitope vaccine formulations in food-producing animals against T. gondii infection.

Characterization of the complete mitochondrial genome of Marshallagia marshalli and phylogenetic implications for the superfamily Trichostrongyloidea.

Marshallagia marshalli (Nematoda: Trichostrongylidae) infection can lead to serious parasitic gastroenteritis in sheep, goat, and wild ruminant, causing significant socioeconomic losses worldwide. Up to now, the study concerning the molecular biology of M. marshalli is limited. Herein, we sequenced the complete mitochondrial (mt) genome of M. marshalli and examined its phylogenetic relationship with selected members of the superfamily Trichostrongyloidea using Bayesian inference (BI) based on concatenated mt amino acid sequence datasets. The complete mt genome sequence of M. marshalli is 13,891 bp, including 12 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. All protein-coding genes are transcribed in the same direction. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes supported the monophylies of the families Haemonchidae, Molineidae, and Dictyocaulidae with strong statistical support, but rejected the monophyly of the family Trichostrongylidae. The determination of the complete mt genome sequence of M. marshalli provides novel genetic markers for studying the systematics, population genetics, and molecular epidemiology of M. marshalli and its congeners.

Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica.

Infection of ruminants and humans with Fasciola gigantica is attracting increasing attention due to its economic impact and public health significance. However, little is known of innate immune responses during F. gigantica infection. Here, we investigated the expression profiles of genes involved in Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways in buffaloes infected with 500F. gigantica metacercariae. Serum, liver and peripheral blood mononuclear cell (PBMC) samples were collected from infected and control buffaloes at 3, 10, 28, and 70days post infection (dpi). Then, the levels of 12 cytokines in serum samples were evaluated by ELISA. Also, the levels of expression of 42 genes, related to TLRs and NLRs signaling, in liver and PBMCs were determined using custom RT2 Profiler PCR Arrays. At 3 dpi, modest activation of TLR4 and TLR8 and the adaptor protein (TICAM1) was detected. At 10 dpi, NF-κB1 and Interferon Regulatory Factor signaling pathways were upregulated along with activation of TLR1, TLR2, TLR6, TLR10, TRAF6, IRF3, TBK1, CASP1, CD80, and IFNA1 in the liver, and inflammatory response with activated TLR4, TLR9, TICAM1, NF-κB1, NLRP3, CD86, IL-1B, IL-6, and IL-8 in PBMCs. At 28 dpi, there was increase in the levels of cytokines along with induction of NLRP1 and NLRP3 inflammasomes-dependent immune responses in the liver and PBMCs. At 70 dpi, F. gigantica activated TLRs and NLRs, and their downstream interacting molecules. The activation of TLR7/9 signaling (perhaps due to increased B-cell maturation and activation) and upregulation of NLRP3 gene were also detected. These findings indicate that F. gigantica alters the expression of TLRs and NLRs genes to evade host immune defenses. Elucidation of the roles of the downstream effectors interacting with these genes may aid in the development of new interventions to control disease caused by F. gigantica infection.

Dynamic expression of cytokine and transcription factor genes during experimental Fasciola gigantica infection in buffaloes.

BACKGROUND: Determining the mechanisms involved in the immune-pathogenesis of the tropical liver fluke, Fasciola gigantica, is crucial to the development of any effective therapeutic intervention. Here, we examined the differential gene expression of cytokines and transcription factors in the liver of F. gigantica-infected buffaloes, over the course of infection. METHODS: Water buffaloes (swamp type) were infected orally with 500 F. gigantica encysted metacercariae. Liver tissue samples were collected 3, 10, 28, 42, 70 and 98 days post-infection (dpi). Levels of gene expression of nine cytokines (IFN-γ, TGF-ß, IL-1ß, IL-4, IL-6, IL-10, IL-12B, IL-13 and IL-17A) and four transcription factors (T-bet, GATA-3, Foxp3 and ROR-γτ) were determined using quantitative real-time PCR (qRT-PCR). We evaluated any correlation between gene expression of these immune-regulatory factors and the severity of liver pathology. RESULTS: Histopathological examination revealed that cellular infiltration, hemorrhage and fibrosis without calcification in the liver parenchyma of infected buffaloes, increased over the course of infection. This progressive pathology was attributed to dysregulated and excessive inflammatory responses induced by infection. The early infection phase (3-10 dpi) was marked by a generalized immunosuppression and elevated TGF-ß expression in order to facilitate parasite colonization. A mixed Th1/Th2 immune response was dominant from 28 to 70 dpi, to promote parasite survival while minimizing host tissue damage. During late infection (98 dpi), the response was biased towards Th1/Treg in order to inhibit the host's Th2 protective response and promote chronic infection. Both IL-10 and IL-17A and the Th17/Treg balance, played key roles in mediating the inflammatory and immunoregulatory mechanisms in the liver during chronic fasciolosis. CONCLUSIONS: Our data showed distinct CD4+ T helper (Th) polarization and cytokine dysregulation in response to F. gigantica infection in water buffaloes over the course of infection. Characterizing the temporal expression profiles for host immune genes during infection should provide important information for defining how F. gigantica adapts and survives in the liver of buffaloes and how host immune responses influence F. gigantica pathogenicity.

Resistance to Chronic Toxoplasma gondii Infection Induced by a DNA Vaccine Expressing GRA16.

Toxoplasma gondii can infect all warm-blooded animals including human beings. T. gondii dense granule protein 16 (TgGRA16) as a crucial virulence factor could modulate the host gene expression. Here, a DNA vaccine expressing TgGRA16 was constructed to explore the protective efficacy against T. gondii infection in Kunming mice. The immune responses induced by pVAX-GRA16 were also evaluated. Mice immunized with pVAX-GRA16 could elicit higher levels of specific IgG antibody and strong cellular response compared to those in controls. The DNA vaccination significantly increased the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) and the percentages of CD4+ and CD8+ T cells in mice. After lethal challenge, mice immunized with pVAX-GRA16 (8.4 ± 0.78 days) did not show a significant longer survival time than that in controls (7.1 ± 0.30 days) (p > 0.05). However, in chronic toxoplasmosis model (administration of 10 brain cysts of PRU strain orally), numbers of tissue cysts in mice immunized with pVAX-GRA16 were significantly reduced compared to those in controls (p < 0.05) and the rate of reduction could reach 43.89%. The results indicated that the TgGRA16 would be a promising vaccine candidate for further development of effective epitope-based vaccines against chronic T. gondii infection in mice.